MEANS AND METHODS FOR PRODUCING NEISSERIA MENINGITIDIS CAPSULAR POLYSACCHARIDES OF LOW DISPERSITY

Abstract

The present invention relates to in vitro methods for producing Neisseria meningitidis capsular polysaccharides which have a defined length. The present invention also relates to compositions comprising at least one capsule polymerase, at least one donor carbohydrate and at least one acceptor carbohydrate, wherein the ratio of donor carbohydrate to acceptor carbohydrate is a ratio from 10:1 to 400:1. Moreover, the present invention provides truncated versions of the capsule polymerases of Neisseria meningitidis serogroups A and X. Also provided herein are pharmaceuticals, in particular vaccines, comprising the synthetic capsular polysaccharides of Neisseria meningitidis which have a defined length. Furthermore, the invention provides for methods for the production of said vaccines.

Description

[0001] The present invention relates to in vitro methods for producing Neisseria meningitidis capsular polysaccharides which have a defined length. The present invention also relates to compositions comprising at least one capsule polymerase, at least one donor carbohydrate and at least one acceptor carbohydrate, wherein the ratio of donor carbohydrate to acceptor carbohydrate is a ratio from 10:1 to 400:1. Moreover, the present invention provides truncated versions of the capsule polymerases of Neisseria meningitidis serogroups A and X. Also provided herein are pharmaceuticals, in particular vaccines, comprising the synthetic capsular polysaccharides of Neisseria meningitidis which have a defined length. Furthermore, the invention provides for methods for the production of said vaccines.

[0002] Bacterial meningitidis is a serious threat to global health. It is estimated that each year bacterial meningitidis accounts for 170,000 deaths worldwide (WHO, http://www.who.int/nuvi/meningitidis/en/). Despite the availability of potent antimicrobial agents, case-fatality rates are high (10-40%) and survivors frequently suffer from sequelae such as neurologic disability or limb loss and deafness (Van Deuren et al., Clin Micriobiol Rev 2000; 13(1): 144-166; Kaper et al., Nat Rev Microbiol 2004, 2(2): 123-140). Neisseria meningitidis (Nm) is one of the most important causative agents of bacterial meningitidis because of its potential to spread in epidemic waves (Kaper et al., Nat Rev Microbiol 2004, 2(2): 123-140; Rosenstein et al., N Eng J Med 2001, 344(18): 1378-1388). Crucial virulence determinants of disease causing Nm species are their extracellular polysaccharide capsules that are essential for meningococcal survival in human serum (Vogel et al., Infect Immun 1997, 65(10): 4022-4029). Based on antigenic variation of these polysaccharides at least twelve different serogroups of Nm have been identified (A, B, C, E29, H, I, K, L, W-135, X, Y and Z), but only six (A, B, C, W-135, Y and X) account for virtually all cases of disease; see also Frosch, M., VOGEL, U. (2006) "Structure and genetics of the meningococcal capsule." In Handbook of Meningococcal Disease. Frosch, M., Maiden, M. C. J. (eds). Weinheim: Wiley-VCH.

[0003] Serogroup A (NmA) and C (NmC) are the main causes of meningococcal meningitidis in sub-Saharan Africa, while serogroups B (NmB) and C are the major disease causing isolates in industrialized countries. However, serogroups W-135 (NmW-135) and Y (NmY) are becoming increasingly prevalent. For NmW-135, this is most explicitly evidenced by the 2002 epidemic in Burkina Faso with over 13,000 cases and more than 1,400 deaths (Connolly et al., Lancet 2004, 364(9449): 1974-1983; WHO, Epidemic and Pandemic Alert and Response (EPR) 2008). In contrast, NmY is gaining importance in the United States where its prevalence increased from 2% during 1989-1991 to 37% during 1997-2002 (Pollard et al., J Paediatr Child Health 2001, 37(5): S20-S27). However, also the previously only sporadically found serogroup X (NmX) appeared with high incidence in Niger and caused outbreaks in Kenya and Uganda (Biosier et al., Clin Infect Dis 2007, 44(5): 657-663; Lewis, WHO Health Action in Crisis 1, 6 2006).

[0004] The serogroups A, B, C, 29E, H, I, K, L, W-135, X, Y and Z are well known in the art and are described, e.g., in Frosch, M., VOGEL, U. (2006) loc. cit. The capsular polysaccharides (CPS) of all serogroups are negatively charged linear polymers. Serogroup B and C are encapsuled in homoplymeric CPS composed of sialic acid (Neu5Ac) moieties that are linked by either .alpha.-2-8 glycosidic linkages in serogroup B or by .alpha.-2-9 linkages in serogroup C (Bhattacharjee et al., J Biol Chem 1975, 250(5): 1926-1932). Serogroup W-135 and Y both are heteropolymers. They are composed of either galactose/Neu5Ac repeating units [.fwdarw.6)-.alpha.-D-Glcp-(1.fwdarw.4)-.alpha.-Neu5Ac-(2.fwdarw.].sub.n in serogroup W-135 or glucose/Neu5Ac repeating units [.fwdarw.6)-.alpha.-D-Galp-(1.fwdarw.4)-.alpha.-Neu5Ac-(2.fwdarw.].sub.n in serogroup Y (Bhattacharjee et al., Can J Biochem 1976, 54(1): 1-8). The CPS of NmA and NmX do not contain Neu5Ac moieties, but are instead built from N-Acetyl-mannosamine 1-phosphate [.fwdarw.6)-.alpha.-D-ManpNAc-(1.fwdarw.OPO.sub.3.fwdarw.].sub.n or N-Acetyl-glucosamine 1-phosphate [.fwdarw.6)-.alpha.-D-GlcpNAc-(1.fwdarw.OPO.sub.3.fwdarw.].sub.n repeating units, respectively (Bundle et al., Carbohydr Res 1973, 26(1): 268-270; Bundle et al., J Biol Chem 1974, 249(15): 4797-4801); Bundle et al., J Biol Chem 1974, 249(7): 2275-2281; Jennings et al., J Infect Dis 1977, 136 Suppl: S78-S83).

[0005] The CPS of disease causing Nm are attractive vaccine candidates and polysaccharide or polysaccharide-conjugate vaccines are available for serogroups A, C, Y, and W-135 (Broker et al., Minerva Med 2007, 98(5):575-589). Recently, also vaccines for serogroup X have been described (Micoli, P. Proc Natl Acad Sci USA. (2013) 110: 19077-82).

[0006] Key enzymes in the CPS biosynthesis are membrane associated capsule polymerases. Candidate genes have been identified for all six disease causing serogroups (Frosch et al., Proc Natl Acad Sci USA 1989, 86(5): 1669-1673; Claus et al., Mol Gen Genet 1997, 257(1): 28-34; Tzeng et al., Infect Immun 2003, 71(2): 6712-6720). However, our knowledge of enzymology or structure-function relations of those important enzymes is still very limited.

[0007] Until now, polysaccharide production for neisserial vaccines still requires fermentation of Neisseria meningitidis with subsequent multistep purification of the polysaccharides from the culture medium. In these conventional vaccine production processes, the obtained capsule polysaccharides are mostly long polymers. However, for vaccine production, the oligosaccharides ranging in size between degree of polymerization (DP) DP10 to DP60 are needed. Therefore, after purification of the capsule polysaccharides from the culture medium, the obtained polysaccharides have to be fragmented and the fragments have to be characterized and sorted by length. Foremost the biohazard associated with mass production of pathogenic Neisseria stains, but also the technological platforms needed to produce homogenous oligosaccharide fractions for vaccine production and their functionalization for coupling to carrier proteins (e.g. an inactive form of Diphteria-toxin or tetanus toxoid), make the conventional vaccine production processes cost intensive and time consuming. Moreover, due to the complexity of the vaccine production chain it can hardly be carried out in developing countries where most of the Nm infections occur. In addition, these conventional vaccine production processes are at risk for contaminations by neisserial toxins, media components or chemicals required for subsequent purification procedures.

[0008] Thus, the technical problem underlying the present invention is the provision of means and methods which lead to a more convenient production process of vaccines against Neisseria meningitidis.

[0009] The technical problem has been overcome by the methods of the present invention for producing capsular polysaccharides of Neisseria meningitidis in vitro which have a defined length and carry functional groups at the reducing end for carrier protein coupling as will be detailed below. In particular, the herein provided means and methods lead to a vaccine production process which is less expensive and less time consuming as compared to the processes of the prior art. Furthermore, these convenient processes as provided herein can also be carried out in the developing world. Accordingly, the technical problem is solved by the provision of the embodiments as defined in the claims.

[0010] Accordingly, the present invention relates to an in vitro method for producing Neisseria meningitidis capsular polysaccharides which have a defined length, said method comprising the steps:

[0011] (a) incubating at least one capsule polymerase with at least one donor carbohydrate and at least one acceptor carbohydrate; wherein the ratio of donor carbohydrate to acceptor carbohydrate is a ratio from 10:1 to 400:1; and

[0012] (b) isolating the resulting capsular polysaccharides, wherein the capsule polymerase is the capsule polymerase of Neisseria meningitidis serogroup A or a truncated version of the capsule polymerase of Neisseria meningitidis serogroup X.

[0013] The present invention solves the above identified technical problem since, as documented herein below and in the appended Examples, it was surprisingly found that capsule polysaccharides of Neisseria meningitidis (Nm) serogroups A and X which have a uniform and defined length can be produced in vitro.

[0014] The inventive means and methods have the advantageous property that the biohazard in association with large scale Nm cultures and the significant coasts for purifying capsular polysaccharides which have the desired length can be avoided. Moreover, the inventive methods provide the perspective that vaccine production chains can be established also in areas that are lacking advanced infrastructural conditions. In particular, the present invention provides for a pyrogen-free, enzyme catalyzed in vitro synthesis production of capsular polysaccharides which have a defined length.

[0015] More specifically, the inventors of the present invention surprisingly identified that the capsular polymerase (CP) of Nm serogroup A (CP-A) can be regulated by the ratio of donor to acceptor saccharides. More specifically, in context of the present invention it has been found that this capsular polymerase can be regulated via the donor-acceptor ratio such that it produces capsular polysaccharides with a uniform and defined length. This finding is of high relevance as for vaccine production, neisserial capsular polysaccharides with a uniform and defined length are required. In addition, regulation of the capsule polymerases of Nm serogroup A by the donor-acceptor ratio is highly unexpected as the full length capsule polymerases of Nm serogroups X, Y and W-135 cannot be easily regulated by donor-acceptor ratio.

[0016] Furthermore, the inventors of the present invention astonishingly found that, when terminally truncated, also the capsule polymerase of Nm serogroup X (CP-X) can be regulated via the donor-acceptor ratio in order to produce capsular polysaccharides with a uniform and defined length. Moreover, as shown in the illustrating appended examples, this terminally truncated version of the capsule polymerase of Nm serogroup X has the additional advantage that it can also be regulated by the reaction time. Thus, as shown in the appended examples, by choosing a suitable reacting time (e.g. 3 to 45 minutes), the terminally truncated version of the capsule polymerase of Nm serogroup X can be forced to produce capsular polysaccharides with a uniform and defined length of DP10 to DP60. This length is needed for effective vaccine production. It is noted that the inventive truncated (e.g. terminally truncated) capsular polymerase of NmX has the additional advantage that it has a significantly improved activity as compared to its full length counterpart. This is a surprising finding as the prior art has shown that truncated versions of the capsule polymerase of Nm serogroup B (CP-B) have the same or even less activity as compared to the full length CP-B (Keys, Analytical Biochemistry 427, 60-68 (2012)).

[0017] Thus, the present invention paves the way for efficient, economical and fast in vitro production methods for Nm capsule polysaccharides, which are suitable for vaccine development. Furthermore, in context of the present invention two important features of the capsule polymerase of NmA (also called CP-A or CsaB) have been identified: (i) the minimal efficient acceptor is the dimer and (ii) the reducing end phosphate group can be extended with rather large chemical groups (such as decyl-ester groups). This latter finding bears the perspective that chain elongation can be primed with reagents of very high purity and functional groups that facilitate conjugation of glycans to carrier proteins in the vaccine production chain.

[0018] Moreover, in context of the present invention it was further found that an O-acetyltransferase (e.g. the CsaC of NmA) can be applied in the inventive in vitro production methods and directly effecting O-acetylation of the produced capsular polysaccharides. This is of advantage as usually for vaccine production Nm capsular polysaccharides must be O-acetylated. In addition, O-acetylation has also an effect on the immunogenicity of the produced capsular polysaccharides. Natural capsule polysaccharides of NmA are O-acetylated in position 3-O and to a minor extend in position 4-O of ManNAc (Gudlavalleti, Carbohydr. Res. (2006) 341: 557-562). It has been shown that capsular polysaccharides of NmA are immunogenic only if they are O-acetylated (Berry et al., 2002 Infection and immunity 70 (7), 3707-3713). As demonstrated in the appended examples, during in vitro synthesis of CPS an O-acetyltransferase (such as the CsaC of NmA) can be directly added to the mixture comprising the capsule polymerase, the donor carbohydrate(s) and the acceptor carbohydrate(s). Thus, in vitro production and O-acetylation can be performed in a one-pot reaction. Accordingly, the O-acetyltransferase may be used in step (a) of the herein described in vitro method for producing Nm capsular polysaccharides. In addition or alternatively, O-acetylation may be performed as an additional step after the synthesis of the Nm capsular polysaccharides. Thus, in the herein described in vitro method for producing Nm capsular polysaccharides, O-acetylation may be performed after step (a) in the additional step (a').

[0019] Accordingly, the present invention provides for effective in vitro methods for producing exactly the type of Nm capsular polysaccharides which is required for vaccine production (i.e. capsular polysaccharides which have a defined length and which are O-acetylated). Thereby, the previously used cost- and time-intensive vaccine production processes can be avoided.

[0020] In vitro methods for producing synthetic capsular polysaccharides of Neisseria meningitidis have been described in the prior art (WO 2011/023764 A1). However, the prior art does not disclose how to generate synthetic Nm capsular polysaccharides which have a uniform and defined length and the prior art does not disclose that an O-acetyltransferase can be applied in the in vitro production of capsular polysaccharides to obtain O-acetylated capsular polysaccharides.

[0021] By contrast, in the methods of the present invention, isolated Nm capsular polymerases are regulated by the donor-acceptor ratio and/or by the reaction time to allow for the production of Nm capsular polysaccharides which have a uniform and defined length. Accordingly, the invention provides for methods for producing Nm capsular polysaccharides, wherein the capsular polymerases are regulated by the donor-acceptor ratio and/or the reaction time so as to produce capsular polysaccharides which have a uniform and defined length.

[0022] Moreover, a further advantageous property of the methods of the invention is that they can be combined with an O-acetyltransferase to generate O-acetylated capsular polysaccharides which can directly be used for vaccine production.

[0023] Thus, the present invention relates to an in vitro method for producing Neisseria meningitidis capsular polysaccharides which have a defined length, said method comprising the steps:

[0024] (a) incubating at least one capsule polymerase with at least one donor carbohydrate and at least one acceptor carbohydrate; wherein the ratio of donor carbohydrate to acceptor carbohydrate is a ratio from about 10:1 to about 400:1; and

[0025] (b) isolating the resulting capsular polysaccharides, wherein the capsule polymerase is the capsule polymerase of Neisseria meningitidis serogroup A or a truncated version of the capsule polymerase of Neisseria meningitidis serogroup X, and wherein in step (a) the corresponding capsular polysaccharide is produced. Said production method is an in vitro production method and allows for isolation of capsular polysaccharides from the incubation mixture as defined in step (a); i.e. this isolation allows for the separation of the produced capsular polysaccharides from the capsule polymerase(s), the donor carbohydrate(s) and/or the acceptor carbohydrate(s).

[0026] The appended examples demonstrate that capsular polysaccharides which have the desired length of DP10 to DP60 can be produced by using CP-A or a terminally truncated version of CP-X and a ratio of donor carbohydrate to acceptor carbohydrate which is approximately between 10:1 and 400:1. Therefore, in the herein described methods, if the length of the Nm capsular polysaccharides is regulated via the ratio of donor carbohydrate to acceptor carbohydrate, than the donor:acceptor ratio is 10:1 to 400:1 (e.g. 20:1 to 400:1). For example, the ratio may be 10:1, 20:1, 40:1, 60:1, 80:1, 100:1, 150:1, 200:1, 220:1, 240:1, 250:1, 300:1, 350:1 or 400:1. In particular aspects of the invention also other donor acceptor ratios, e.g. ratios from 2:1 to 2000:1 may be used.

[0027] If CP-A is used in the in vitro methods described herein, than the ratio of donor carbohydrate to acceptor carbohydrate is preferably from 10:1 to 240:1, and more preferably from 10:1 to 80:1, and even more preferably from 10:1 to 75:1, and even more preferably from 20:1 to 75:1, and most preferably from 20:1 to 60:1 (e.g. 40:1 to 60:1 or 50:1). The term "CP-A" includes a polypeptide which has the amino acid sequence of SEQ ID NO: 9 or 10 and a polypeptide which has at least 80% (preferably at least 90%) sequence identity to SEQ ID NO: 9 (CP-A wildtyp/full-length) or to SEQ ID NO: 10 (.DELTA.N69-CP-A).

[0028] If the terminally truncated CP-X is used and if it is regulated via the ratio of donor carbohydrate to acceptor carbohydrate (and not via the reaction time) then the following ratios are preferably used. For example, if the capsule polymerase is a polypeptide which has the amino acid sequence of SEQ ID NO: 28 or which has at least 80% (preferably at least 90%) sequence identity to SEQ ID NO: 28 (.DELTA.C99-CP-X), then the ratio of donor carbohydrate to acceptor carbohydrate is preferably from 10:1 to 80:1, more preferably from 20:1 to 60:1, even more preferably from 20:1 to 50:1, and most preferably from 33:1 to 50:1. In addition, if the capsule polymerase is a polypeptide which has the amino acid sequence of SEQ ID NO: 25 or which has at least 80% (preferably at least 90%) sequence identity to SEQ ID NO: 25 (.DELTA.N58-CP-X), then the ratio of donor carbohydrate to acceptor carbohydrate is preferably from 10:1 to 80:1, more preferably from 20:1 to 50:1, and most preferably from 20:1 to 33:1 (e.g. 33:1). Furthermore, if the capsule polymerase is a polypeptide which has the amino acid sequence of SEQ ID NO: 29 or which has at least 80% (preferably at least 90%) sequence identity to SEQ ID NO: 29 (.DELTA.N58.DELTA.C99-CP-X), then the ratio of donor carbohydrate to acceptor carbohydrate is preferably from 50:1 to 400:1, more preferably from 80:1 to 400:1, even more preferably from 100:1 to 400:1, and most preferably from 100:1 to 200:1.

[0029] All capsule polymerases which are applied in the herein described methods are functional. For example, a polypeptide which has at least 80% (preferably at least 90%) sequence identity to any one of the sequences of SEQ ID NOs: 9, 10, 25, 28, and 29 is considered to be functional, i.e. to have the same function as the polypeptide which has the sequence of SEQ ID NO: 9, 10, 25, 28, or 29, respectively. In particular, the function of a polypeptide having the sequence of SEQ ID NO: 9 or 10 is the ability to transfer ManNAc-1P (or a derivative thereof) from UDP-ManNAc (or a derivative thereof) onto an acceptor carbohydrate. Preferably, at least 80% of the acceptor carbohydrates are polysaccharides with a DP of .gtoreq.4. In addition, the function of a polypeptide having the sequence of any one of SEQ ID NOs: 25, 28, and 29 is the ability to transfer GlcNAc-1P (or a derivative thereof) from UDP-GlcNAc (or a derivative thereof) onto an acceptor carbohydrate.

[0030] If (in the herein described in vitro methods for producing Nm capsular polysaccharides (Nm CPS) which have a defined length) the length of the produced capsular polysaccharides is regulated via the donor:acceptor ratio, then 50-100 nM of the capsular polymerase may be used. The amount of donor carbohydrate may be 1-100 mM, e.g. 5-10 mM. The amount of acceptor carbohydrate is adjusted to the amount of donor carbohydrate so as to result in the desired donor:acceptor ratio ratio. For example, the amount of acceptor carbohydrate may be 1-1000 .mu.M, e.g. 10-40 .mu.M. Preferably, at least 80% of the acceptor carbohydrates are polysaccharides with a DP of .gtoreq.4.

[0031] For example, if in the herein described methods for producing Nm polysaccharides with a defined length the CP-A is used, then 1-100 nM (preferably 70-100 nM) of CP-A, 10 mM donor carbohydrate (e.g. a dimer of ManNAc-1-phosphate), and an amount of acceptor carbohydrate (e.g. purified capsular polysaccharides of NmA) resulting in a ratio of donor carbohydrate to acceptor carbohydrate which is from 10:1 to 400:1, preferably from 10:1 to 240:1, more preferably from 10:1 to 80:1, even more preferably from 10:1 to 75:1, even more preferably from 20:1 to 75:1, and most preferably from 20:1 to 60:1 (e.g. 40:1 to 60:1 or 50:1) may be used. In a particular example of the herein described methods for producing Nm polysaccharides with a defined length, 70-100 nM of CP-A, 10 mM donor carbohydrate (e.g. UDP-ManNAc; or UDP-GlcNAc, wherein in step (a) the capsule polymerase is further incubated with the UDP-GlcNAc-epimerase), and an amount of acceptor carbohydrate (e.g. purified capsular polysaccharides of NmA of which at least 80% have a DP of .gtoreq.4) resulting in a ratio of donor carbohydrate to acceptor carbohydrate which is from 20:1 to 60:1 is used. The term "CP-A" includes a polypeptide having the amino acid sequence as shown in SEQ ID NO: 9 (CP-A wildtyp/full-length) or SEQ ID NO: 10 (.DELTA.N69-CP-A) and a polypeptide with an amino acid sequence having at least 80% (preferably at least 90%) sequence identity to SEQ ID NO: 9 or to SEQ ID NO: 10 and being functional.

[0032] In another particular example of the herein described methods, the length of the produced Nm polysaccharides is regulated via the donor:acceptor ratio and 50-70 nM of a polypeptide having the amino acid sequence of SEQ ID NO: 25 (.DELTA.N58-CP-X), 28 (.DELTA.C99-CP-X), or 29 (.DELTA.N58.DELTA.C99-CP-X), or having an amino acid sequence which is at least 80% (preferably at least 90%) identical to SEQ ID NO: 25, 28, or 29 and being functional; 10 mM donor carbohydrate (e.g. UDP-GlcNAc), and an amount of acceptor carbohydrate (e.g. purified capsular polysaccharides of NmX of which at least 80% have a DP of .gtoreq.4) resulting in the desired ratio of donor carbohydrate to acceptor carbohydrate is used.

[0033] In another example of the invention, the length of the Nm CPS is regulated by the reaction time, and 50-70 nM capsular polymerase (e.g. .DELTA.C99-CP-X or .DELTA.N58.DELTA.C99-CP-X), 10 mM donor carbohydrate (e.g. UDP-GlcNAc) and 10-30 .mu.M acceptor carbohydrate (e.g. purified capsular polysaccharides of NmX of which at least 80% have a DP of .gtoreq.4) is used.

[0034] Step (a) of the herein provided in vitro method may be performed by incubating the capsule polymerase, the donor carbohydrate and the acceptor carbohydrate in a reaction buffer (e.g. a buffer comprising 50 mM Tris pH 8.0 and 20 mM MgCl.sub.2). The incubation in step (a) may be performed at a temperature range between 20.degree. C. and 40.degree. C., preferably between 25.degree. C. and 37.degree. C., more preferably between 30 and 37.degree. C.

[0035] In context of the present invention it has surprisingly and unexpectedly been found that in the herein described methods, if the length of the Nm capsular polysaccharides is regulated via the ratio of donor carbohydrate to acceptor carbohydrate (and not via the reaction time) and if the CP-A or a C-terminally truncated version of the CP-X is used (e.g. .DELTA.C99-CP-X), then the donor:acceptor ratio (i.e. the donor:acceptor quotient) reflects the avDP (i.e. the length of the major species within the produced capsular polysaccharides). For example, if the donor:acceptor ratio is 15:1, then the CP-A or the C-terminally truncated CP-X produces capsular polysaccharides which have an avDP15. Or, if the donor:acceptor ratio is 50:1, then the CP-A or the C-terminally truncated CP-X produces capsular polysaccharides which have an avDP50 (see, e.g., FIG. 1A, 2C, 2F). Or, in other words, if the donor:acceptor ratio is 15:1 (or 50:1, respectively), then the major species (i.e. the main components) of the produced capsular polysaccharides have a length around DP15 (or DP50, respectively). Or, in other words, the product pool has an avDP15 (or avDP50, respectively) and is narrowly distributed around the major species having a DP of 15 (or 50, respectively). In line with this, if the donor:acceptor ratio is from 15:1 to 20:1, then the major species of the produced capsular polysaccharides have a length of avDP15 to avDP20.

[0036] Accordingly, the present invention relates to an in vitro method for producing Neisseria meningitidis capsular polysaccharides which have a defined length, said method comprising the steps:

[0037] (a1) incubating at least one capsule polymerase with at least one donor carbohydrate and at least one acceptor carbohydrate; and thereby producing capsule polysaccharides, wherein at least 60% (preferably at least 80% and most preferably at least 90%) of the produced capsular polysaccharides have a length or in other words a degree of polymerisation corresponding to the ratio of donor carbohydrate to acceptor carbohydrate -20/+30 repeating units; and

[0038] (b) isolating the resulting capsular polysaccharides, wherein the capsule polymerase is the CP-A or a C-terminally truncated version of the CP-X. The C-terminally truncated version of the CP-X may be a polypeptide which comprises:

[0039] (i) an amino acid sequence encoded by a nucleic acid molecule which comprises the nucleic acid sequence of SEQ ID NO: 20 (.DELTA.C99-CP-X); or a nucleic acid sequence having at least 80% (preferably at least 90%) identity to the nucleic acid sequence of SEQ ID NO: 20 and encoding a functional polypeptide; wherein the function comprises the ability to transfer GlcNAc-1P from UDP-GlcNAc onto an acceptor carbohydrate; or

[0040] (ii) the amino acid sequence of SEQ ID NO: 28 (.DELTA.C99-CP-X); or an amino acid sequence having at least 80% (preferably at least 90%) identity to SEQ ID NO: 28 and being functional, wherein the function comprises the ability to transfer GlcNAc-1P from UDP-GlcNAc onto an acceptor carbohydrate.

[0041] As indicated, in the above described method, the produced capsular polysaccharides comprise as a main component capsular polysaccharides having a DP corresponding to about the ratio of donor carbohydrate to acceptor carbohydrate. Preferably, at least 80% of the acceptor carbohydrates are polysaccharides with a DP of .gtoreq.4. Thus, the length of the produced capsular polysaccharides can easily be regulated via the donor:acceptor ratio. One aspect of the invention relates to the above described method, wherein the donor:acceptor ratio is from 15:1 to 20:1, and wherein at least 60% (preferably 80%, most preferably 90%) of the produced capsular polysaccharides have a length, or in other words, a degree of polymerisation corresponding to the ratio of donor carbohydrate to acceptor carbohydrate -20/+30 repeating units.

[0042] Herein the term "Neisseria meningitidis capsular polysaccharides which have a defined length" means that the produced capsular polysaccharides have a defined DP (degree of polymerization) or a defined avDP (average degree of polymerization), e.g. a DP between 10 and 60 or an avDP between 15 and 20. The term "Neisseria meningitidis capsular polysaccharides which have a defined length" preferably means that at least 60% (more preferably at least 80%, most preferably at least 90%) of the produced capsular polysaccharides have a length between DP10 and DP60. For example, the DP may be between DP20 and DP60. The DP indicates the number of monosaccharide-units which built up the capsular polysaccharide. For example, a DP of 10 means that the capsular polysaccharide consists of 10 monosaccharide-units and a DP of 60 means that the capsular polysaccharide consists of 60 monosaccharide-units. Accordingly, a NmX capsular polysaccharide having DP10 means that the capsular polysaccharide consists of 10 GlcNAc-1P repeating units. Acidic hydrolysis used to produce oligosaccharide fragments form long polysaccharide chains, results in fragments of different lengths, which in subsequent chromatographic steps can be separated into size classes. The average DP (avDP) describes the dispersion of chains with one class and can be calculated according to established protocols as described, e.g. in Berti (2012) Vaccine 30 (45), 6409-6415. Or, in other words, the avDP describes the average length of a polysaccharide pool. Alternative ways to determine the DP of oligosaccharides are high percentage polyacrylamide gel electrophoresis or HPLC-anion-exchange chromatography as demonstrated in the appended examples. The avDP may be determined by the protocol described in Berti (2012) 30(45): 6409-15., which includes .sup.31P NMR and High Performance Anionic Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD). For vaccine production usually capsular polysaccharides having an avDP of 15 to 20 is used. The product pool of polysaccharides with an avDP of 15 to 20 consists of polysaccharides having a DP of 10 to 60. The herein provided in vitro methods have the advantage that capsule polysaccharides with the length which is desired for vaccine production (i.e. DP10 to DP60) can directly be produced. Herein the term "Neisseria meningitidis capsular polysaccharides which have a defined length" also means that the produced capsular polysaccharides have a low dispersity (i.e. a narrow product distribution).

[0043] In the above described in vitro method, if the capsule polymerase of Nm serogroup A is used, than capsule polysaccharides of Nm serogroup A are produced. Analogously, if a truncated version of the capsule polymerase of Nm serogroup X is used, that capsule polysaccharides of Nm serogroup X are produced.

[0044] As described herein and illustrated in the appended examples, one aspect of the present invention relates to the identification of terminally truncated versions of the capsule polymerase of Nm serogroup X (CP-X, also designated as CsxA). These terminally truncated proteins have the advantage that they have an increased activity as compared to the full length CP-X. A further advantageous property of these terminally truncated proteins is that they can be regulated by the ratio of donor carbohydrates to acceptor carbohydrates such that they produce capsular polysaccharides which have a uniform and defined length. This is surprising since the full length CP-X cannot be regulated by the donor-acceptor ratio. Furthermore, in context of the present invention it has astonishingly been found that these terminally truncated versions of the CP-X can also be regulated by the reaction time so as to produce capsular polysaccharides which have a uniform and defined length. More specifically, the time how long the terminally truncated CP-X is incubated with the donor carbohydrates (and, optionally, the acceptor carbohydrates) regulates the length of the produced capsule polysaccharides. Accordingly, an incubation time can be chosen which results in the production of capsule polysaccharides of NmX which have the length which is desired for vaccine production (i.e. DP10 to DP60). Accordingly, one embodiment of the invention relates to an in vitro method for producing Neisseria meningitidis capsular polysaccharides which have a defined length, said method comprising the steps:

[0045] (a) incubating at least one capsule polymerase with at least one donor carbohydrate and at least one acceptor carbohydrate; wherein the incubation time ranges from 3 to 45 minutes; and

[0046] (b) isolating the resulting capsular polysaccharides, wherein the capsule polymerase is a truncated version of the capsule polymerase of Neisseria meningitidis serogroup X.

[0047] Or, in other words, the invention relates to an in vitro method for producing Neisseria meningitidis capsular polysaccharides which have a defined length, said method comprising the steps:

[0048] (a) incubating at least one capsule polymerase with at least one donor carbohydrate and at least one acceptor carbohydrate; wherein the incubation time ranges from about 3 to about 45 minutes; and

[0049] (b) isolating the resulting capsular polysaccharides, wherein the capsule polymerase is a truncated version of the capsule polymerase of Neisseria meningitidis serogroup X, and wherein in step (a) the corresponding capsular polysaccharide is produced. Said production method is an in vitro production method and allows for isolation of capsular polysaccharides from the incubation mixture as defined in step (a); i.e. this isolation allows for the separation of the produced capsular polysaccharides from the capsule polymerase(s), the donor carbohydrate(s) and/or the acceptor carbohydrate(s).

[0050] Due to the distributive nature of the elongation mechanism used by the truncated CP-X, the reaction can be stopped at any time whereby the resulting avDP depends on the reaction time. Therefore, if the truncated version of the CP-X is regulated via the reaction time, then the ratio of donor carbohydrate to acceptor carbohydrate is not relevant. For example, the ratio of donor carbohydrate to acceptor carbohydrate can range from 1:1 to 50000:1, preferably from 10:1 to 10000:1 [Hier wurde entsprechend der Anspruche 10:1 to 10000:1 gewahlt] (e.g. from 200:1 to 10000:1, or more preferably from 100:1 to 8000:1).

[0051] In context of the present invention it has surprisingly been found that the herein described capsule polymerases are active even if they are coupled to a solid phase. Immobilizing a component on a solid phase has the advantage that it reduces labor in the isolating step (b). More specifically, immobilizing a component to a solid phase omits further purification steps to remove the produced capsular polysaccharides from the reaction mixture of step (a). Thus, in the herein provided in vitro methods for producing Nm capsular polysaccharides, one of the components may be immobilized (i.e. bound/coupled) on a solid phase. For example, the donor carbohydrate or the capsule polymerase may be immobilized on a solid phase. The solid phase may be column, such as a His-Trap column (i.e. a column having His-Trap beads). In a particular aspect of the inventive in vitro methods, the capsule polymerase is immobilized on a solid phase. For example, a polypeptide which has the amino acid sequence of SEQ ID NO: 29 (.DELTA.N58.DELTA.C99-CP-X) or which has at least 80% (preferably at least 90%) sequence identity to SEQ ID NO: 29 and being functional may be immobilized on a solid phase.

[0052] It is noted that the capsule polymerase of Nm serogroup X is capable of a de novo synthesis of capsular polysaccharides. Or, in other words, the CP-X is able to synthesize capsular polysaccharides in the absence of acceptor carbohydrates (Fiebig, Glycobiology (2014) 24:150-8). Thus, if the terminally truncated version of CP-X is regulated by the reaction time, the presence of acceptor carbohydrates is optional. Accordingly, one aspect of the invention relates to an in vitro method for producing Nm capsular polysaccharides of NmX which have a defined length, said method comprising the steps:

[0053] (a) incubating a terminally truncated version of the CP-X with at least one donor carbohydrate (and, optionally, with at least one acceptor carbohydrate); wherein the incubation time ranges from 3 to 45 minutes; and

[0054] (b) isolating the resulting capsular polysaccharides.

[0055] As described herein and illustrated in the appended examples, the inventive terminally truncated versions of the capsule polymerase of Nm serogroup X can be regulated by both, the donor-acceptor ratio and the reaction time (i.e. the time how long the terminally truncated version of the CP-X is incubated with the donor saccharides and the acceptor saccharides).

[0056] As mentioned above, if the truncated version of the CP-X is regulated via the reaction time, then the ratio of donor carbohydrate to acceptor carbohydrate is not relevant. Thus, as also demonstrated in the appended Examples, if the length of the Nm capsular polysaccharides is regulated by the reaction time, then higher donor-acceptor ratios can be used (to produce Neisseria meningitidis capsular polysaccharides which have a defined length) as compared to the method wherein the length of the Nm capsular polysaccharides is regulated via the donor-acceptor ratio (and not via the reaction time). Accordingly, one aspect of the invention relates to an in vitro method for producing Neisseria meningitidis capsular polysaccharides which have a defined length, said method comprising the steps:

[0057] (a) incubating at least one capsule polymerase with at least one donor carbohydrate and at least one acceptor carbohydrate; wherein

[0058] (i) the ratio of donor carbohydrate to acceptor carbohydrate is a ratio from 10:1 to 10000:1 [Hier wurde entsprechend der Anspruche 10:1 to 10000:1 gewahlt], and

[0059] (ii) the incubation time ranges from 3 to 45 minutes; and

[0060] (b) isolating the resulting capsular polysaccharides, wherein the capsule polymerase is a truncated version of the capsule polymerase of Neisseria meningitidis serogroup X.

[0061] Or, in other words, the invention relates to an in vitro method for producing Neisseria meningitidis capsular polysaccharides which have a defined length, said method comprising the steps:

[0062] (a) incubating at least one capsule polymerase with at least one donor carbohydrate and at least one acceptor carbohydrate; wherein

[0063] (i) the ratio of donor carbohydrate to acceptor carbohydrate is a ratio from about 10:1 to about 10000:1, and

[0064] (ii) the incubation time ranges from about 3 to about 45 minutes; and

[0065] (b) isolating the resulting capsular polysaccharides, wherein the capsule polymerase is a truncated version of the capsule polymerase of Neisseria meningitidis serogroup X; and wherein in step (a) the corresponding capsular polysaccharide is produced. Said production method is an in vitro production method and allows for isolation of capsular polysaccharides from the incubation mixture as defined in step (a); i.e. this isolation allows for the separation of the produced capsular polysaccharides from the capsule polymerase(s), the donor carbohydrate(s) and/or the acceptor carbohydrate(s).

[0066] Or, in other words, the invention relates to an in vitro method for producing Nm capsular polysaccharides of NmX which have a defined length, said method comprising the steps:

[0067] (a) incubating a terminally truncated version of CP-X with at least one donor carbohydrate and at least one acceptor carbohydrate; wherein

[0068] (i) the ratio of donor carbohydrate to acceptor carbohydrate is a ratio from 10:1 to 10000:1, and

[0069] (ii) the incubation time ranges from 3 to 45 minutes; and

[0070] (b) isolating the resulting capsular polysaccharides.

[0071] The appended examples demonstrate that capsular polysaccharides of NmX which have the desired length of DP10 to DP60 can be produced by using a terminally truncated version of CP-X and a ratio of donor carbohydrate to acceptor carbohydrate which is approximately between 10:1 and 400:1. The appended Examples also demonstrate that higher donor-acceptor ratios (e.g., 200:1 or 8000:1) can be used to produce Nm capsule polysaccharides which have a defined length, if the terminally truncated version of the CP-X is regulated via the reaction time (i.e. if the reaction time is from 3 to 45 min).

[0072] Accordingly, if the length of the Nm capsular polysaccharides is regulated by the reaction time, then the ratio of donor carbohydrate to acceptor carbohydrate is not relevant. For example, the donor:acceptor ratio can be from 10:1 to 10000:1, preferably from 100:1 to 8000:1 (e.g. from 200:1 to 1000:1)

[0073] For example, if the terminally truncated CP-X is regulated by the reaction time, then the ratio of donor carbohydrate to acceptor carbohydrate of 10:1, 20:1, 40:1, 60:1, 80:1, 100:1, 120:1, 140:1, 160:1, 180:1, 200:1, 220:1, 240:1, 250:1, 300:1, 350:1, 400:1, 450:1, 500:1, 550:1, 600:1, 650:1, 700:1, 750:1, 800:1, 850:1, 900:1, 950:1, 1000:1, 2000:1, 3000:1, 4000:1, 5000:1, 6000:1, 7000:1, 8000:1, 9000:1, or 10000:1 may be used.

[0074] In the herein described and provided in vitro methods, if the length of the produced capsule polysaccharides in regulated via the reaction time, it is envisaged that the truncated version of the CP-X is used in a concentration ranging from 20 to 500 nM, preferably from 20 to 200 nM, or more preferably from 50 to 100 nM (e.g. 50 nM). In addition, it is envisaged herein that said truncated version of the CP-X is incubated with a donor concentration of 1 to 100 mM, preferably of 1 to 50 mM, more preferably of 2 to 20 mM, even more preferably of 5 to 10 mM, or most preferably of 10 mM UDP-GlcNAc; and with an acceptor concentration resulting in a donor to acceptor ratio from 10:1 to 10000:1, preferably from 100:1 to 8000:1, (e.g. from 200:1 to 1000:1) and that the reaction time ranges from 3 to 45 minutes, more preferably from 5 to 30 minutes, or most preferably from 5 to 10 minutes. Preferably, at least 80% of the acceptor carbohydrates are polysaccharides with a DP of .gtoreq.4.

[0075] Or, in other words, if the length of the Nm capsular polysaccharides is regulated by the reaction time, it is envisaged that the concentration of the truncated version of the CP-X (e.g. a capsule polymerase having at least 80% (preferably at least 90%) identity to any one of the sequences of SEQ ID NOs: 25, 28 and 29 and being functional) ranges from 20 to 500 nM, preferably from 20 to 200 nM, or more preferably from 50 to 100 nM (e.g. 50 nM). In addition, it is envisaged herein that said truncated version of the CP-X is incubated with a donor concentration of 1 to 100 mM, preferably of 1 to 50 mM, more preferably of 2 to 20 mM, even more preferably of 5 to 10 mM, or most preferably of 10 mM UDP-GlcNAc; and with an acceptor concentration resulting in a donor to acceptor ratio from 10:1 to 10000:1, preferably from 100:1 to 8000:1 (e.g. 200:1 to 1000:1); and that the reaction time ranges from 3 to 45 minutes. Preferably, at least 80% of the acceptor carbohydrates are polysaccharides with a DP of .gtoreq.4.

[0076] As indicated above, if the length of the Nm capsular polysaccharides is regulated by the reaction time, then the reaction time ranges (i.e. is) from 3 to 45 minutes. Preferably, the reaction time ranges from 5 to 30 minutes, more preferably from 5 to 10 minutes (e.g. 5 or 10 min). For example, if the capsule polymerase is a polypeptide which has at least 80% (preferably at least 90%) sequence identity to SEQ ID NO: 28 (.DELTA.C99-CP-X) and being functional, then the reaction time ranges preferably from 5 to 10 minutes, and is most preferably 5 minutes. In addition, if the capsule polymerase is a polypeptide which has at least 80% (preferably at least 90%) sequence identity to SEQ ID NO: 25 (.DELTA.N58-CP-X) and being functional, then the reaction time ranges preferably from 5 to 30 minutes. Furthermore, if the capsule polymerase is a polypeptide which has at least 80% (preferably at least 90%) sequence identity to SEQ ID NO: 29 (.DELTA.N58.DELTA.C99-CP-X) and being functional, then the reaction time ranges preferably from 5 to 30 minutes and is most preferably 10 minutes.

[0077] In the inventive in vivo methods, the temperature in the incubation step (a) may be from 20.degree. C. to 40.degree. C., preferably from 25.degree. C. to 37.degree. C., more preferably from 30.degree. C. to 37.degree. C., and most preferably 37.degree. C.

[0078] As indicated above, if the length of the Nm CPS is regulated by the reaction time, the reaction time may be 3-45 min, preferably 5-30 minutes. For example, the reaction time may be 3 min, 5 min, 10 min, 15 min, 20 min, 25 min 30 min, 35 min, 40 min or 45 min. In addition, if the length of the Nm CPS is regulated by the reaction time, it is preferred that a C- and N-terminally truncated version of the CP-X (such as .DELTA.N58.DELTA.C99-CP-X) is used. This truncated version of the CP-X may be a polypeptide having the amino acid sequence of SEQ ID NO: 29 or a polypeptide having an amino acid sequence which has at least 80% homology to SEQ ID NO: 29 and being functional.

[0079] If the length of the Nm CPS is regulated by the ratio of donor carbohydrate to acceptor carbohydrate, then the reaction time may be, e.g., 4-7 hours.

[0080] In step (b) of the herein described in vitro methods for producing Nm CPS which have a defined length (i.e. which have low dispersity in terms of length), the resulting CPS are isolated. Isolation of the synthesized CPS may be performed, e.g., by anion exchange chromatography (AEC). For example, the produced CPS may be purified via AEC by using a MonoQ HR5/5 column (Pharmacia biotech) at a flow rate of 1 mL/min and a linear sodium chloride gradient or by High Performance Anionic Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD) as described (Berti et al. 2012 Vaccine. (2012) 30:6409-15).

[0081] In accordance with the present invention, the ingredients which are used in the herein described methods for producing Nm CPS can be packed together as a composition. Accordingly, one aspect of the invention relates to a composition comprising:

[0082] (i) at least one capsule polymerase;

[0083] (ii) at least one donor carbohydrate; and

[0084] (iii) at least one acceptor carbohydrate, wherein the ratio of donor carbohydrate to acceptor carbohydrate is a ratio from 10:1 to 10000:1 (e.g. 20:1 to 1000:1), and wherein the capsule polymerase is the capsule polymerase of Neisseria meningitidis serogroup A or a truncated capsule polymerase of Neisseria meningitidis serogroup X.

[0085] The skilled person understands that, depending on the exactness of the measurements, the exact ratio can slightly deviate from 10:1 to 10000:1. Accordingly, the methods of the invention can also be performed with a ratio of about 10:1 to 10000:1. Thus, one aspect of the invention relates to a composition comprising:

[0086] (i) at least one capsule polymerase;

[0087] (ii) at least one donor carbohydrate; and

[0088] (iii) at least one acceptor carbohydrate, wherein the ratio of donor carbohydrate to acceptor carbohydrate is a ratio from about 10:1 to about 10000:1 (e.g. about 20:1 to about 1000:1), and wherein the capsule polymerase is the capsule polymerase of Neisseria meningitidis serogroup A or a truncated capsule polymerase of Neisseria meningitidis serogroup X.

[0089] For example, in the herein provided compositions, 20 nM to 500 .mu.M (preferably 20 to 200 nM, more preferably 50 to 100 nM, e.g. 50-70 nM) of the capsular polymerase (e.g. .DELTA.C99-CP-X, .DELTA.N58.DELTA.C99-CP-X or .DELTA.N58-CP-X, or a polypeptide having at least 80% (preferably at least 90%) identity to any one of SEQ ID NOs: 25, 28 and 29 and being functional) may be used. The amount of donor carbohydrate (e.g. UDP-GlcNAc) may be 1-100 mM, preferably 1 to 50 mM, more preferably 2 to 20 mM (e.g. 5-10 mM). The acceptor carbohydrate may be purified capsular polysaccharides of NmX. Preferably, the amount of the acceptor carbohydrate is adjusted to result in a ratio of donor carbohydrate to acceptor carbohydrate of 10:1 to 10000:1, preferably of 100:1 to 8000:1 (e.g. of 200:1 to 1000:1). For example, the amount of the acceptor carbohydrate may be 1-1000 .mu.M, e.g. 10-40 .mu.M.

[0090] In the herein provided compositions, the ratio of donor carbohydrate to acceptor carbohydrate is 10:1 to 10000:1, e.g. 20:1, 40:1, 60:1, 80:1, 100:1, 150:1, 200:1, 220:1, 240:1, 250:1, 300:1, 350:1, 400:1, 450:1, 500:1, 550:1, 600:1, 650:1, 700:1, 750:1, 800:1, 850:1, 900:1, 950:1, 1000:1, 2000:1, 3000:1, 4000:1, 5000:1, 6000:1, 7000:1, 8000:1, 9000:1 or 10000:1. For example, the donor-acceptor-ratio may be from 2:1 to 2000:1. One aspect of the invention relates to the herein described composition, wherein the ratio of donor carbohydrate to acceptor carbohydrate is a ratio from 20:1 to 240:1.

[0091] The composition of the invention may be suitable for storage (e.g. storage under cold temperatures to prevent that the synthesis of capsular polysaccharides starts). For instance, the composition may be suitable for storage in 4.degree. C., 0.degree. C., -10.degree. C., -20.degree. C. or -80.degree. C.

[0092] The inventive compositions and in vitro methods comprise "at least one" capsule polymerase. Therefore, the inventive compositions and in vitro methods can comprise several capsular polymerases of different types in one reaction mixture (e.g. CP-A together with the terminally truncated CP-X). However, it is preferred that the inventive compositions and in vitro methods comprise only capsule polymerases of one type (e.g. only CP-A or only the terminally truncated version of CP-X).

[0093] The in vitro methods of the invention may be realized by using an appropriate kit. Accordingly, another embodiment of the invention relates to a kit for carrying out the in vitro methods of the invention, comprising the above described composition. The embodiments disclosed in connection with the in vitro method of the present invention apply, mutatis mutandis, to the composition and the kit of the present invention.

[0094] Advantageously, the kit of the present invention further comprises, optionally (a) reaction buffer(s), storage solutions, wash solutions and/or remaining reagents or materials required for the conduction of the assays as described herein. Furthermore, parts of the kit of the invention can be packaged individually in vials or bottles or in combination in containers or multicontainer units. These vials/bottles/containers or multicontainers may, in addition to the capsule polymerases and donor carbohydrates and acceptor carbohydrates as described herein, comprise preservatives or buffers for storage. In addition, the kit may contain instructions for use.

[0095] The composition of the present invention or the kit of the present invention may be advantageously used for carrying out the in vitro methods as described herein (i.e. for producing Nm capsular polysaccharides of NmX which have a defined length). The manufacture of the kit of the present invention follows preferably standard procedures which are known to the person skilled in the art.

[0096] In the methods and compositions of the invention, the capsule polymerase of NmA (CP-A also designated as CsaB) may be used. The DNA sequence of the wild type full length CP-A is shown herein as SEQ ID NO: 1. The DNA sequence of the full length CP-A which is optimized for expression in E. coli (i.e. a codon optimized DNA sequence) is shown herein as SEQ ID NO: 2. The polypeptide resulting from the expression of the wild type or codon optimized CP-A polynucleotide is identical. The amino acid sequence of the full length CP-A is given in SEQ ID NO: 9.

[0097] Of note, within the DNA sequence of the CP-A, the inventors of the present invention identified an additional ATG start codon and cloned the corresponding truncation .DELTA.N69-CP-A (which starts from the alternative ATG). The designation ".DELTA.N69-CP-A" means that 69 amino acids are lacking at the N-terminus of the CP-A protein. Herein, ".DELTA.N69-CP-A" is also designated as "dN69-CsaB". The polynucleotide sequence of a codon optimized version of .DELTA.N69-CP-A is shown in SEQ ID NO: 3. The polynucleotide sequence of the wild type .DELTA.N69-CP-A is shown in SEQ ID NO: 46. The amino acid sequence of the expressed wild type or codon optimized .DELTA.N69-CP-A is identical and in SEQ ID NO: 10. Surprisingly, .DELTA.N69-CP-A is advantageous over the full length CP-A. In particular, as demonstrated in the appended illustrative examples, while increased degradation and concomitantly reduced expression was seen for the codon optimized full length CP-A, the codon optimized .DELTA.N69-CP-A is well expressed and no degradation is detectable. In addition, as shown in the appended examples, by using the radioincorporation assay as described in Fiebig, Glycobiology (2014) 24:150-8, it was demonstrated the .DELTA.N69-CP-A protein is active (i.e. is capable of producing NmA capsular polysaccharides). Furthermore, the appended examples also show that the .DELTA.N69-CP-A and the full length CP-A show identical activity profiles. This is surprising since several other tested truncated versions of CP-A (i.e. .DELTA.N97-CP-A, .DELTA.N167-CP-A, .DELTA.N235-CP-A, .DELTA.C45-CP-A and .DELTA.C25-CP-A) were demonstrated to be not active. It is mentioned that herein, in particular in the appended examples, "A" is also designated as "d" or "delta".

[0098] Accordingly, one aspect of the present invention relates to a truncated version of the CP-A (i.e. .DELTA.69-CP-A), wherein said truncated version of the CP-A is the polypeptide of any one of (a) to (f):

[0099] (a) a polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 3 or 46;

[0100] (b) a polypeptide comprising the amino acid sequence of SEQ ID NO: 10;

[0101] (c) a polypeptide encoded by a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 10 or of a functional fragment thereof, wherein the function comprises the ability to transfer ManNAc-1P or a derivative thereof from UDP-ManNAc or a derivative thereof onto an acceptor carbohydrate;

[0102] (d) a polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule hybridizing under stringent conditions to the complementary strand of a nucleic acid molecule as defined in (a) or (c) and encoding a functional polypeptide; or a functional fragment thereof, wherein the function comprises the ability to transfer ManNAc-1P or a derivative thereof from UDP-ManNAc or a derivative thereof onto an acceptor carbohydrate;

[0103] (e) a polypeptide having at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, or even more preferably at least 99% homology to the polypeptide of any one of (a) to (d), whereby said polypeptide is functional; or a functional fragment thereof, wherein the function comprises the ability to transfer ManNAc-1P or a derivative thereof from UDP-ManNAc or a derivative thereof onto an acceptor carbohydrate; and

[0104] (f) a polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule being degenerate as a result of the genetic code to the nucleotide sequence of a nucleic acid molecule as defined in (a), (c), and (d).

[0105] As indicated above, the herein provided truncated version of the CP-A comprises the ability to transfer ManNAc-1P or a derivative thereof from UDP-ManNAc or a derivative thereof onto an acceptor carbohydrate (i.e. onto the hydroxyl group of C6 of a capsular polysaccharide from NmA or a derivative thereof). Accordingly, this function can be the ability to transfer ManNAc-1P from UDP-ManNAc onto an acceptor carbohydrate. The acceptor carbohydrate may be extended/activated at the reducing end or the reducing end phosphate. In addition, the size of the produced capsular polysaccharides can be controlled by the ratio of donor and acceptor carbohydrate when the ratio is in the range of 10:1 to 400:1, preferably from 10:1 to 240:1, more preferably from 10:1 to 80:1, even more preferably from 10:1 to 75:1, even more preferably from 20:1 to 75:1, and most preferably from 20:1 to 60:1 (e.g. 40:1 to 60:1 such as 50:1). Within this range, only one product distribution corresponding in size to the calculated donor-acceptor ratio is obtained at reaction endpoints.

[0106] The transfer of ManNAc-1P (or a derivative thereof) from UDP-ManNAc (or a derivative thereof) onto an acceptor carbohydrate may be performed by incubating a truncated version of the CP-A (i.e. .DELTA.N69-CP-A) with UDP-ManNAc (or a derivative thereof) and an acceptor carbohydrate (e.g. a tetramer or a dimer consisting of ManNAc-1P units linked together by phosphodiester bonds). Whether a transfer from ManNAc-1P (or a derivative thereof) from UDP-ManNAc (or a derivative thereof) onto an acceptor carbohydrate has occurred can be tested, e.g. by high percentage PAGE and developed by alcian blue/silver staining (Fiebig, Glycobiology (2014) 24:150-8). in the presence of a size marker or by .sup.31P NMR monitoring the characteristic phosphodiester signal, or by High Performance Anionic Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD) as described (Berti Vaccine (2012) 30:6409-15).

[0107] A derivative of ManNAc-1P may be ManNAc-3-O-Ac or ManNAc-4-O-Ac or ManNAc-3,4-di-O-Ac. A derivative of UDP-ManNAc may be UDP-ManNAc-3-O-Ac or UDP-ManNAc-4-O-Ac or UDP-ManNAc-3,4-di-O-Ac.

[0108] One aspect of the present invention relates to the above described truncated version of the CP-A (i.e. .DELTA.N69-CP-A), wherein the function comprises the ability to transfer ManNAc-1P (or a derivative thereof) from UDP-ManNAc (or a derivative thereof) onto an acceptor carbohydrate, wherein the truncated version of the CP-A shows the same activity profile as the full length CP-A. An activity profile means that elongated products are of similar or identical dispersity.

[0109] The full length CP-A, the codon optimized CP-A, the .DELTA.N69-CP-A or the codon optimized .DELTA.N69-CP-A may be used in the herein described in vitro methods for producing Nm capsular polysaccharides or compositions. Accordingly, one embodiment of the present invention relates to the in vitro method of the invention or the composition of the invention, wherein the capsule polymerase of Neisseria meningitidis serogroup A is the polypeptide of any one of (a) to (f):

[0110] (a) a polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule having the nucleic acid sequence of any one of SEQ ID NO: 1 to 3;

[0111] (b) a polypeptide comprising the amino acid sequence of SEQ ID NO: 9 or 10;

[0112] (c) a polypeptide encoded by a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 9 or 10, or of a functional fragment thereof;

[0113] (d) a polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule hybridizing under stringent conditions to the complementary strand of a nucleic acid molecule as defined in (a) or (c) and encoding a functional polypeptide; or a functional fragment thereof;

[0114] (e) a polypeptide having at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98% or most preferably at least 99% identity to the polypeptide of any one of (a) to (d), whereby said polypeptide is functional; or a functional fragment thereof; and

[0115] (f) a polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule being degenerate as a result of the genetic code to the nucleotide sequence of a nucleic acid molecule as defined in (a), (c), and (d).

[0116] A particular embodiment of the present invention relates to the in vitro method of the invention or the composition of the invention, wherein the capsule polymerase of Neisseria meningitidis serogroup A is the polypeptide of any one of (a) to (f):

[0117] (a) a polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule having the nucleic acid sequence of any one of SEQ ID NO: 1 to 3;

[0118] (b) a polypeptide comprising the amino acid sequence of SEQ ID NO: 9 or 10;

[0119] (c) a polypeptide encoded by a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 9 or 10, or of a functional fragment thereof, wherein the function comprises the ability to transfer ManNAc-1P or a derivative thereof from UDP-ManNAc or a derivative thereof onto an acceptor carbohydrate;

[0120] (d) a polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule hybridizing under stringent conditions to the complementary strand of a nucleic acid molecule as defined in (a) or (c) and encoding a functional polypeptide; or a functional fragment thereof, wherein the function comprises the ability to transfer ManNAc-1P or a derivative thereof from UDP-ManNAc or a derivative thereof onto an acceptor carbohydrate;

[0121] (e) a polypeptide having at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98% or most preferably at least 99% identity to the polypeptide of any one of (a) to (d), whereby said polypeptide is functional; or a functional fragment thereof, wherein the function comprises the ability to transfer ManNAc-1P or a derivative thereof from UDP-ManNAc or a derivative thereof onto an acceptor carbohydrate; and

[0122] (f) a polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule being degenerate as a result of the genetic code to the nucleotide sequence of a nucleic acid molecule as defined in (a), (c), and (d).

[0123] As indicated, the function of the above under items (a) to (f) described capsule polymerase of Neisseria meningitidis serogroup A comprises the ability to transfer ManNAc-1P or a derivative thereof from UDP-ManNAc or a derivative thereof onto an acceptor carbohydrate. Preferably, at least 80% of the acceptor carbohydrates are polysaccharides with a DP of .gtoreq.4. For example, the acceptor (or at least 80% of the acceptor carbohydrates) may be DP4, DP5 or DP6; preferably the acceptor (or at least 80% of the acceptor carbohydrates) is a tetramer or pentamer of ManNAc-1P with a free reducing end.

[0124] As described, the full length CP-A, the codon optimized CP-A or the codon optimized .DELTA.N69-CP-A (i.e. the polypeptide described above under items (a) to (f)) may be used in the herein described in vitro methods for producing Nm capsular polysaccharides or in the herein described compositions. For example, if one of these capsule polymerases is used in the in vitro methods or compositions described herein, than the ratio of donor carbohydrate to acceptor carbohydrate is from 10:1 to 400:1, preferably from 10:1 to 240:1, and more preferably from 10:1 to 80:1, and even more preferably from 10:1 to 75:1, even more preferably from 20:1 to 75:1, and most preferably from 20:1 to 60:1 (e.g. 40:1 to 60:1 such as 50:1).

[0125] In a prioritized aspect of the invention, the codon optimized .DELTA.N69-CP-A is used in the herein described in vitro methods for producing Nm CPS which have a defined length or in the herein described compositions.

[0126] The inventors of the present invention surprisingly identified truncated versions of the capsule polymerase of Nm serogroup X (CP-X). For example, the CP-X-fragment .DELTA.N67.DELTA.C99-CP-X (wherein 67 amino acids are lacking at the N-terminus and 99 amino acids are lacking at the C-terminus) was demonstrated to be functional (i.e. able to transfer GlcNAc-1P (or a derivative thereof) from UDP-GlcNAc (or a derivative thereof) onto an acceptor carbohydrate. Preferably, at least 80% of the acceptor carbohydrates are polysaccharides with a DP of .gtoreq.4. For example, the acceptor (or at least 80% of the acceptor carbohydrates) may be DP4, DP5, or DP6 (see, e.g., FIG. 3E); preferably, the acceptor (or at least 80% of the acceptor carbohydrates) is a tetramer or pentamer of GlcNAc-1P with a free reducing end as shown in FIG. 3. The DNA sequence of .DELTA.N67.DELTA.C99-CP-X is provided herein as SEQ ID NO: 23 and the amino acid sequence is shown herein as SEQ ID NO: 31. In context of the present invention it is expected that the C-terminally truncated .DELTA.C136-CP-X (wherein 136 amino acids are lacking at the C-terminus) is active, as several C-terminal mutations (except of a mutation at position 351) do not affect activity of the CP-X (unpublished data, see also FIG. 6C). In addition also the fragment .DELTA.N83.DELTA.C136-CP-X (wherein 83 amino acids are lacking at the N-terminus and 136 amino acids are lacking at the C-terminus) is considered to be active because the CP-A is still active if it carries a mutation at amino acid 250 (in the N-terminus) which corresponds to amino acid 84 in CP-X.

[0127] As demonstrated in the appended examples, the inventors of the present invention surprisingly found that, when terminally truncated, the CP-X can be regulated via the donor-acceptor ratio such that it produces CPS with a uniform and defined length. Furthermore, these terminally truncated versions of the CP-X have the additional advantage that they can also be regulated by the reaction time. Moreover, the inventive terminally truncated versions of CP-X have the superior property that they have a significantly improved activity as compared to the full length CP-X. The DNA sequence of the full length CP-X is shown herein as SEQ ID NO: 16 and the amino acid sequence of the full length CP-X is shown herein as SEQ ID NO: 24.

[0128] The illustrative appended examples demonstrate several truncated versions of the CP-X which are capable of producing NmX capsular polysaccharides. For example, the proteins .DELTA.N58-CP-X (wherein 58 amino acids are lacking at the N-terminus of CP-X and which comprises approximately 88% of the full length CP-X), .DELTA.C99-CP-X (wherein 98 amino acids are lacking at the C-terminus of CP-X and which comprises approximately 80% of the full length CP-X) and .DELTA.N58.DELTA.C99-CP-X (wherein 58 amino acids are lacking at the N-terminus and 98 amino acids are lacking at the C-terminus and which comprises approximately 68% of the full length CP-X) have been demonstrated to be functional capsular polysaccharides. Herein, in particular in the appended examples, ".DELTA.N58-CP-X", ".DELTA.C99-CP-X" and ".DELTA.N58.DELTA.C99-CP-X" are also designated as "dN58-CsxA", "dC99-CsxA" and "dN58dC99-CsxA", respectively. Regarding the designation ".DELTA.C99" it is noted that this designation has been chosen as the DNA sequence which was used to generate the .DELTA.C99-CP-X construct ends with codon 387 of the CP-X sequence. However, this DNA sequence was cloned to have an XhoI restriction site (CTCGAG) at the 3' end. The codon CTC within this site encodes leucine. Amino acid 388 of wild type CP-X is also a leucine (however encoded by CTT). Therefore, in the proteins .DELTA.C99-CP-X and .DELTA.N58.DELTA.C99-CP-X, 98 (instead of 99) amino acids are lacking at the C-terminus. In this regard it is further noted that if the herein provided polynucleotide encoding .DELTA.C99-CP-X (i.e. SEQ ID NO: 20) is expressed in a host cell, then the resulting polypeptide has the sequence as given herein in SEQ ID NO: 28. Analogously, if the herein provided polynucleotide encoding .DELTA.N58.DELTA.C99-CP-X (i.e. SEQ ID NO: 21) is expressed in a host cell, then the resulting polypeptide has the sequence as given herein in SEQ ID NO: 29. Herein, in particular in the appended examples, ".DELTA.C99-CP-X" and ".DELTA.N58.DELTA.C99-CP-X" are also designated as "dC99-CsxA" and "dN58dC99-CsxA", respectively.

[0129] As mentioned, the inventors of the present invention surprisingly found that truncated versions of the CP-X have a considerably higher activity as compared to the full length CP-X. More specifically, by using a radioincorporation assay, the appended illustrative examples demonstrate that truncated versions of the CP-X (e.g. .DELTA.N58-CP-X, .DELTA.C99-CP-X or .DELTA.N58.DELTA.C99-CP-X) are more active (i.e. assemble more saccharide monomers to capsule polysaccharides) as compared to the full length CP-X. In the herein described and provided in vitro methods and compositions, the truncated version of the CP-X may preferably be a terminally truncated version of the CP-X. Accordingly, a further embodiment of the present invention relates to a truncated version of the CP-X, which is a polypeptide comprising:

[0130] (a) an amino acid sequence encoded by a nucleic acid molecule which is a terminally truncated version of the nucleic acid sequence of SEQ ID NO: 16 and comprises maximal 50-95% of the nucleic acid sequence of SEQ ID NO: 16;

[0131] (b) an amino acid sequence which is a terminally truncated version of the amino acid sequence of SEQ ID NO: 24 and comprises maximal 50-95% of the amino acid sequence of SEQ ID NO: 24;

[0132] (c) a polypeptide encoded by a nucleic acid molecule encoding a polypeptide having an amino acid sequence which is a terminally truncated version of the amino acid sequence of SEQ ID NO: 24 and comprises maximal 50-95% of the amino acid sequence of SEQ ID NO: 24;

[0133] (d) a polypeptide encoded by a nucleic acid molecule hybridizing under stringent conditions to the complementary strand of a nucleic acid molecule as defined in (a) or (c) and encoding a functional polypeptide;

[0134] (e) a polypeptide having at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98% or most preferably at least 99% identity to the polypeptide of any one of (a) to (d), whereby said polypeptide is functional; or

[0135] (f) a polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule being degenerate as a result of the genetic code to the nucleotide sequence of a nucleic acid molecule as defined in (a), (c) or (d).

[0136] The function of the terminally truncated version of the CP-X comprises the ability to transfer GlcNAc-1P from UDP-GlcNAc onto an acceptor carbohydrate. Preferably, at least 80% of the acceptor carbohydrates are polysaccharides with a DP of .gtoreq.4. For example, the acceptor (or at least 80% of the acceptor carbohydrates) may be DP4, DP5, or DP6; preferably, the acceptor (or at least 80% of the acceptor carbohydrates) is a tetramer or pentamer of GlcNAc-1P with a free reducing end as shown in FIG. 3. The terminally truncated version of CP-X which is applied herein has also the ability to transfer derivatives of GlcNAc-1P from derivatives of UDP-GlcNAc onto an acceptor carbohydrate. Thus, the function of the terminally truncated version of the CP-X comprises the ability to transfer GlcNAc-1P or a derivative thereof from UDP-GlcNAc or a derivative thereof onto an acceptor carbohydrate. Therefore, the present invention also relates to a truncated version of the CP-X, which is a polypeptide comprising:

[0137] (a) an amino acid sequence encoded by a nucleic acid molecule which is a terminally truncated version of the nucleic acid sequence of SEQ ID NO: 16 and comprises maximal 50-95% of the nucleic acid sequence of SEQ ID NO: 16;

[0138] (b) an amino acid sequence which is a terminally truncated version of the amino acid sequence of SEQ ID NO: 24 and comprises maximal 50-95% of the amino acid sequence of SEQ ID NO: 24;

[0139] (c) a polypeptide encoded by a nucleic acid molecule encoding a polypeptide having an amino acid sequence which is a terminally truncated version of the amino acid sequence of SEQ ID NO: 24 and comprises maximal 50-95% of the amino acid sequence of SEQ ID NO: 24;

[0140] (d) a polypeptide encoded by a nucleic acid molecule hybridizing under stringent conditions to the complementary strand of a nucleic acid molecule as defined in (a) or (c) and encoding a functional polypeptide; wherein the function comprises the ability to transfer GlcNAc-1P or a derivative thereof from UDP-GlcNAc or a derivative thereof onto an acceptor carbohydrate;

[0141] (e) a polypeptide having at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98% or most preferably at least 99% identity to the polypeptide of any one of (a) to (d), whereby said polypeptide is functional; wherein the function comprises the ability to transfer GlcNAc-1P or a derivative thereof from UDP-GlcNAc or a derivative thereof onto an acceptor carbohydrate; or

[0142] (f) a polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule being degenerate as a result of the genetic code to the nucleotide sequence of a nucleic acid molecule as defined in (a), (c) or (d).

[0143] As described above, the herein provided terminally truncated version of the CP-X comprises maximal 50-95% of the full length CP-X. It is envisaged, that the terminally truncated version of the CP-X comprises minimal 50% of the full length CP-X and maximal 95% of the full length CP-X. For example, the terminally truncated CP-X of the invention may comprise 50-95%, 55-90%, 60-85% or 65-80% of the full length CP-X. For example, the terminally truncated CP-X may comprise 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or the full length CP-X.

[0144] The herein provided terminally truncated version of the CP-X comprises the ability to transfer GlcNAc-1P (or a derivative thereof) from UDP-GlcNAc (or a derivative thereof) onto an acceptor carbohydrate (i.e. onto the hydroxyl group of C4 of a capsular polysaccharide from NmX (or a derivative thereof)). The acceptor carbohydrate may be extended/activated at the reducing end or the reducing end phosphate. Preferably, at least 80% of the acceptor carbohydrates are polysaccharides with a DP of .gtoreq.4. For example, the acceptor (or at least 80% of the acceptor carbohydrates) may be DP4, DP5, or DP6; preferably, the acceptor (or at least 80% of the acceptor carbohydrates) is a tetramer or pentamer of GlcNAc-1P with a free reducing end as shown in FIG. 3. In addition, the size of the produced capsular polysaccharides can be controlled by the ratio of donor and acceptor carbohydrate when the ratio is in the range of 10:1-400:1. Within this range, only one product distribution with a low dispersity (compared to full-length CP-X) is obtained at reaction endpoints. More specifically, within this range, only one product distribution with a low dispersity is obtained during all time points of the reaction.

[0145] The transfer GlcNAc-1P (or a derivative thereof) from UDP-GlcNAc (or a derivative thereof) onto an acceptor carbohydrate may be performed by incubating a truncated version of the CP-X (e.g. .DELTA.C99-CP-X, .DELTA.N58-CP-X or .DELTA.N58.DELTA.C99-CP-X) with UDP-GlcNAc (or a derivative thereof) and an acceptor carbohydrate and in reaction buffer (50 mM Tris pH 8.0, and 20 mM MgCl.sub.2, and optionally 1-2 mM (e.g. 1 mM) DTT) and incubated for 1 h--over night (preferably for 4-7 hours) at 25-37.degree. C. (preferably between 30 and 37.degree. C.) (Fiebig et al., 2014; Glycobiology. 2014 February; 24(2):150-8). Preferably, at least 80% of the acceptor carbohydrates are polysaccharides with a DP of .gtoreq.4. For example, the acceptor (or at least 80% of the acceptor carbohydrates) may be DP4, DP5, or DP6; preferably, the acceptor (or at least 80% of the acceptor carbohydrates) is a tetramer or pentamer of GlcNAc-1P with a free reducing end as shown in FIG. 3.

[0146] Whether a transfer from GlcNAc-1P (or a derivative thereof) from UDP-GlcNAc (or a derivative thereof) onto an acceptor carbohydrate has occurred can be tested, e.g. by high percentage PAGE and developed by alcian blue/silver staining (Fiebig et al., Glycobiology. 2014 February; 24(2):150-8) in the presence of a size marker or by .sup.31P NMR monitoring the characteristic phosphodiester signal (Fiebig et al., Glycobiology. 2014 February; 24(2):150-8), or by High Performance Anionic Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD) as described (Berti et al. Vaccine. 2012 Oct. 5; 30(45):6409-15; Fiebig et al., Glycobiology. 2014 February; 24(2):150-8).

[0147] In context of the present invention it was surprisingly found that the C-terminally truncated CP-X as well as the C- and N-terminally truncated CP-X can be regulated via the donor-acceptor ratio and/or via the reaction time so as to produce capsular polysaccharides that have a uniform and defined length. In particular, the appended examples show that the truncated proteins .DELTA.C99-CP-X and .DELTA.N58.DELTA.C99-CP-X can be regulated via the donor-acceptor ratio or via the reaction time such that they produce capsular polysaccharides which have a uniform and defined length. Accordingly, one aspect the invention relates to a terminally truncated version of the CP-X, which is a C-terminally or a C- and N-terminally truncated version of the full length CP-X.

[0148] Thus, the invention relates to the herein provided terminally truncated version of the CP-X, comprising:

[0149] (a) an amino acid sequence which is a C-terminally or C- and N-terminally truncated version of the amino acid sequence of SEQ ID NO: 24 and comprises maximal 50-95% of the amino acid sequence of SEQ ID NO: 24;

[0150] (b) a polypeptide encoded by a nucleic acid molecule encoding a polypeptide having an amino acid sequence which is a C-terminally or C- and N-terminally truncated version of the amino acid sequence of SEQ ID NO: 24 and comprises maximal 50-95% of the amino acid sequence of SEQ ID NO: 24; or

[0151] (c) a polypeptide having at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98% or most preferably at least 99% identity to the polypeptide of (a) or (b), whereby said polypeptide is functional; wherein the function comprises the ability to transfer GlcNAc-1P or a derivative thereof from UDP-GlcNAc or a derivative thereof onto an acceptor carbohydrate.

[0152] In one aspect of the present invention, the terminally truncated version of the CP-X which is described and provided herein is the construct .DELTA.N58-CP-X, .DELTA.C99-CP-X or .DELTA.N58.DELTA.C99-CP-X.

[0153] Thus, the invention relates to the herein provided terminally truncated version of the CP-X, comprising

[0154] (a) a polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule having the nucleic acid sequence of any one of SEQ ID NO: 17, 20 or 21;

[0155] (b) a polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 25, 28 or 29;

[0156] (c) a polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule hybridizing under stringent conditions to the complementary strand of a nucleic acid molecule as defined in (a) and encoding a functional polypeptide; wherein the function comprises the ability to transfer GlcNAc-1P or a derivative thereof from UDP-GlcNAc or a derivative thereof onto an acceptor carbohydrate;

[0157] (d) a polypeptide having at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98% or most preferably at least 99% identity to the polypeptide of any one of (a) to (c), whereby said polypeptide is functional; wherein the function comprises the ability to transfer GlcNAc-1P or a derivative thereof from UDP-GlcNAc or a derivative thereof onto an acceptor carbohydrate; and

[0158] (e) a polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule being degenerate as a result of the genetic code to the nucleotide sequence of a nucleic acid molecule as defined in (a) or (c).

[0159] Accordingly, in one aspect the invention relates to the terminally truncated version of the CP-X comprising:

[0160] (i) an amino acid sequence encoded by a nucleic acid molecule which comprises the nucleic acid sequence of SEQ ID NO: 17, 20 or 21; or a nucleic acid sequence having at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98% or most preferably at least 99% identity to the nucleic acid sequence of any one of SEQ ID NO: 17, 20 or 21 and encoding a functional polypeptide; wherein the function comprises the ability to transfer GlcNAc-1P or a derivative thereof from UDP-GlcNAc or a derivative thereof onto an acceptor carbohydrate; or

[0161] (ii) the amino acid sequence of SEQ ID NO: 25, 28 or 29; or an amino acid sequence having at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98% or most preferably at least 99% identity to any one of SEQ ID NOs: 25, 28 or 29 and being functional, wherein the function comprises the ability to transfer GlcNAc-1P or a derivative thereof from UDP-GlcNAc or a derivative thereof onto an acceptor carbohydrate.

[0162] A particular embodiment of the invention relates to the above described terminally truncated version of the CP-X, wherein the function comprises the ability to transfer GlcNAc-1P or a derivative thereof from UDP-GlcNAc or a derivative thereof onto an acceptor carbohydrate; wherein the produced polymers have a DP between 10 and 60 or an avDP between 15 and 20, when the ratio of donor carbohydrate to acceptor carbohydrate is in the range of 10:1 to 400:1. Preferably, at least 80% of the acceptor carbohydrates are polysaccharides with a DP of .gtoreq.4. For example, the acceptor (or at least 80% of the acceptor carbohydrates) may be DP4, DP5, or DP6; preferably, the acceptor (or at least 80% of the acceptor carbohydrates) is a tetramer or pentamer of GlcNAc-1P with a free reducing end as shown in FIG. 3.

[0163] As described, the terminally truncated version of the CP-X (i.e. the polypeptide described above under items (i) and (ii)) may be used in the herein described in vitro methods for producing Nm capsular polysaccharides or in the herein described compositions.

[0164] For example, if the terminally truncated CP-X is regulated via the ratio of donor carbohydrate to acceptor carbohydrate (and not via the reaction time) then the following preferred ratios can produce a product distribution with a low dispersity. Or, in other words, if the terminally truncated version of the CP-X is regulated via the ratio of donor carbohydrate to acceptor carbohydrate, then the following preferred ratios can produce capsular polysaccharides of which at least 60%, (preferably at least 80%) have a defined length between DP10 and DP60 or between avDP15 and avDP20. Moreover, the following capsule polymerases and ratios are preferably used in the compositions described herein.

[0165] For example, if the capsule polymerase is a polypeptide which has at least 80% (preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98% or most preferably at least 99%) sequence identity to a polypeptide encoded by a nucleic acid molecule which comprises the nucleic acid sequence of SEQ ID NO: 20 (.DELTA.C99-CP-X) and being functional, then the ratio of donor carbohydrate to acceptor carbohydrate is preferably from 10:1 to 80:1, more preferably from 20:1 to 60:1, even more preferably from 20:1 to 50:1, and most preferably from 33:1 to 50:1. In addition, if the capsule polymerase is a polypeptide which has at least 80% (preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98% or most preferably at least 99%) sequence identity to a polypeptide encoded by a nucleic acid molecule which comprises the nucleic acid sequence of SEQ ID NO: 17 (.DELTA.N58-CP-X) and being functional, then the ratio of donor carbohydrate to acceptor carbohydrate is preferably from 10:1 to 80:1, more preferably from 20:1 to 50:1, and most preferably from 20:1 to 33:1 (e.g. 33:1). Furthermore, if the capsule polymerase is a polypeptide which has at least 80% (preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98% or most preferably at least 99%) sequence identity to a polypeptide encoded by a nucleic acid molecule which comprises the nucleic acid sequence of SEQ ID NO: 21 (.DELTA.N58.DELTA.C99-CP-X) and being functional, then the ratio of donor carbohydrate to acceptor carbohydrate is preferably from 50:1 to 400:1, more preferably from 80:1 to 400:1, even more preferably from 100:1 to 400:1, and most preferably from 100:1 to 200:1. In addition, if the capsule polymerase is a polypeptide which comprises an amino acid sequence which has at least 80% (preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98% or most preferably at least 99%) sequence identity to SEQ ID NO: 28 (.DELTA.C99-CP-X) and being functional, then the ratio of donor carbohydrate to acceptor carbohydrate is preferably from 10:1 to 80:1, more preferably from 20:1 to 60:1, even more preferably from 20:1 to 50:1, and most preferably from 33:1 to 50:1. Furthermore, if the capsule polymerase is a polypeptide which comprises an amino acid sequence which has at least 80% (preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98% or most preferably at least 99%) sequence identity to SEQ ID NO: 25 (.DELTA.N58-CP-X) and being functional, then the ratio of donor carbohydrate to acceptor carbohydrate is preferably from 10:1 to 80:1, more preferably from 20:1 to 50:1, and most preferably from 20:1 to 33:1 (e.g. 33:1). Moreover, if the capsule polymerase is a polypeptide which comprises an amino acid sequence which has at least 80% (preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98% or most preferably at least 99%) sequence identity to SEQ ID NO: 29 (.DELTA.N58.DELTA.C99-CP-X) and being functional, then the ratio of donor carbohydrate to acceptor carbohydrate is preferably from 50:1 to 400:1, more preferably from 80:1 to 400:1, even more preferably from 100:1 to 400:1, and most preferably from 100:1 to 200:1.

[0166] In context of the invention the terminally truncated version of the CP-X may also be a polypeptide comprising the construct .DELTA.N65.DELTA.C10-CP-X (which is shown in SEQ ID NOs: 22 and 30) or .DELTA.N67.DELTA.C99-CP-X (which is shown in SEQ ID NOs: 23 and 31) which have been demonstrated to be active (data not shown) and which can be regulated via the donor-acceptor ratio or via the reaction time such that they produce capsular polysaccharides with a uniform and defined length.

[0167] Accordingly, the terminally truncated version of the CP-X of the present invention may be a polypeptide comprising the amino acid of any one of SEQ ID NOs: 25, 28, 29, 30 or 31. Also encompassed by the present invention are terminally truncated versions of the CP-X comprising the amino acid sequence of any one of SEQ ID NOs: 25, 28, 29, 30 or 31 wherein one, two, three or more amino acid residues are added, deleted or substituted. The polypeptides may have the function of the terminally truncated version of the CP-X (i.e. the ability to transfer GlcNAc-1P or a derivative thereof from UDP-GlcNAc or a derivative thereof onto an acceptor carbohydrate). Preferably, at least 80% of the acceptor carbohydrates are polysaccharides with a DP of .gtoreq.4. The amino acid sequence of the polypeptides may be at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and most preferably at least 99% identical to SEQ ID NO: 25, 28, 29, 30 or 31. The polypeptides may have the function of a truncated version of the CP-X. Preferably, the function comprises the ability to transfer GlcNAc-1P (or a derivative thereof) from UDP-GlcNAc (or a derivative thereof) onto an acceptor carbohydrate; wherein at least 60% (preferably at least 80%) of the produced polymers have a DP between 10 and 60 or an avDP between 15 and 20, when the ratio of donor carbohydrate to acceptor carbohydrate is in the range of 10:1 to 400:1. Preferably, at least 80% of the acceptor carbohydrates are polysaccharides with a DP of .gtoreq.4. For example, the acceptor (or at least 80% of the acceptor carbohydrates) may be DP4, DP5, or DP6; preferably, the acceptor (or at least 80% of the acceptor carbohydrates) is a tetramer or pentamer of GlcNAc-1P with a free reducing end as shown in FIG. 3.

[0168] The herein described terminally truncated versions of the CP-X may be linked (e.g. operatively linked) to additional, heterologous sequences. E.g. the nucleic acid molecule encoding a truncated version of the CP-X may be linked to additional, heterologous nucleic acid sequences in a recombinant nucleic acid molecule. For example, said additional nucleic acid sequence may be a coding gene.

[0169] The term "recombinant nucleic acid molecule" relates to nucleic acid molecules originating from a different genetic context and combined by molecular biological methods. Here, the term "different genetic context" relates to genomes from different species, varieties or individuals or different positions within a genome. Recombinant nucleic acid molecules can contain not only natural sequences but also sequences, which, compared to the natural ones are mutated or chemically modified or else, the sequences are altogether newly synthesized sequences.

[0170] As indicated above, the terminally truncated version of the CP-X may be linked to an additional heterologous molecule. Said additional heterologous molecule may be a nucleic acid molecule which is preferably operatively linked to a truncated version of the CP-X. Said additional heterologous nucleic acid molecule may originate from a different genetic context than the truncated version of the CP-X. Non-limiting examples of additional heterologous molecules comprise in particular marker molecules, like luciferase, galactosidase, GFP, EGFP, DsRed, etc. or tag-molecules, like Flag-tags, CBP and others. In a particular aspect of the invention, said additional heterologous molecule is a tag, such as a StrepII-tag, a thrombin-tag, a his-tag, a MBP-tag or a S3N10-tag. In a further aspect of the invention, said additional heterologous molecule is a nucleic acid molecule encoding a restriction site (e.g. a restriction site of the enzymes BamHI/BglII, XhoI or NdeI). It is also envisaged that the truncated CP-X of the invention is linked to several additional sequences (e.g. several tags or several restriction sites). Yet, as detailed below, also sequences which optimize (e.g. enhance) the expression of the terminally truncated CP-X may be operatively linked to the nucleic acid sequence of the truncated CP-X. Such a sequence may be a particular promoter sequence. A suitable promoter for the expression of the terminally truncated version of the CP-X may be, e.g., Tac-, T7-, or Lacpromoter.

[0171] One embodiment of the present invention relates to a nucleic acid molecule encoding the herein described truncated version of the CP-X. The invention also relates to a vector comprising said nucleic acid molecule. The present invention further relates to vectors containing a nucleic acid molecule of the present invention encoding a terminally truncated version of the CP-X. The present invention relates also to a vector comprising the nucleic acid construct encoding the herein described truncated version of the CP-X.

[0172] The term "vector" relates to circular or linear nucleic acid molecules which can autonomously replicate in host cells into which they are introduced. The "vector" as used herein particularly refers to plasmids, cosmids, viruses, bacteriophages and other vectors commonly used in genetic engineering. In a preferred embodiment, the vectors of the invention are suitable for the transformation of cells, like fungal cells, cells of microorganisms such as yeast or prokaryotic cells. In a particularly preferred embodiment such vectors are suitable for stable transformation of bacterial cells, for example to express the truncated version of the CP-X of the present invention.

[0173] Accordingly, in one aspect of the invention, the vector as provided is an expression vector. Generally, expression vectors have been widely described in the literature. As a rule, they may not only contain a selection marker gene and a replication-origin ensuring replication in the host selected, but also a promoter, and in most cases a termination signal for transcription. Between the promoter and the termination signal there is preferably at least one restriction site or a polylinker which enables the insertion of a nucleic acid sequence/molecule desired to be expressed.

[0174] It is to be understood that when the vector provided herein is generated by taking advantage of an expression vector known in the prior art that already comprises a promoter suitable to be employed in context of this invention (for example expression of a truncated version of the CP-X as described herein above) the nucleic acid construct is inserted into that vector in a manner that the resulting vector comprises only one promoter suitable to be employed in context of this invention. The skilled person knows how such insertion can be put into practice. For example, the promoter can be excised either from the nucleic acid construct or from the expression vector prior to ligation.

[0175] A non-limiting example of the vector of the present invention is the plasmid vector pET22b comprising a nucleic acid construct of the present invention. Further examples of vectors suitable to comprise a nucleic acid construct of the present invention to form the vector of the present invention are known in the art and are, for example other vectors for bacterial expression systems such as vectors of the pET series (Novagen) or pQE vectors (Qiagen).

[0176] In an additional embodiment, the present invention relates to a host cell comprising the herein described vector. In particular, the invention relates to a host cell comprising a nucleic acid construct encoding a terminally truncated version of the CP-X and/or the vector of the present invention. Preferably, the host cell of the present invention may be a prokaryotic cell, for example, a bacterial cell. As a non limiting example, the host cell of the present invention may be Escherichia coli. The host cell provided herein is intended to be particularly useful for generating the terminally truncated version of the CP-X of the present invention.

[0177] Generally, the host cell of the present invention may be a prokaryotic or eukaryotic cell, comprising a nucleic acid construct of the invention or the vector of the invention or a cell derived from such a cell and containing a nucleic acid construct of the invention or the vector of the invention. In a preferred embodiment, the host cell is genetically modified with a nucleic acid construct of the invention or the vector of the invention in such a way that it contains the nucleic acid construct of the present invention integrated into the genome. For example, such host cell of the invention, but also the host cell of the invention in general, may be a bacterial, yeast, or fungus cell.

[0178] In one particular aspect, the host cell of the present invention is capable to express or expresses a terminally truncated version of the CP-X as defined herein and as illustrative characterized in SEQ ID NOs: 17, 20, 21, 22 or 23, for example in SEQ ID NO: 21. An overview of examples of different corresponding expression systems to be used for generating the host cell of the present invention is for instance contained in Methods in Enzymology 153 (1987), 385-516, in Bitter et al. (Methods in Enzymology 153 (1987), 516-544), in Sawers et al. (Applied Microbiology and Biotechnology 46 (1996), 1-9), Billman-Jacobe (Current Opinion in Biotechnology 7 (1996), 500-4), Hockney (Trends in Biotechnology 12 (1994), 456-463), and in Griffiths et al., (Methods in Molecular Biology 75 (1997), 427-440).

[0179] The transformation or genetically engineering of the host cell with a nucleic acid construct of the invention or the vector according to the invention can be carried out by standard methods, as for instance described in Sambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, N.Y., USA; Methods in Yeast Genetics, A Laboratory Course Manual, Cold Spring Harbor Laboratory Press, 1990.

[0180] The nucleic acid molecules, vectors, host cells and polypeptides described herein may be used for producing (i.e. synthesizing) Nm capsular polysaccharides which have a defined length.

[0181] As mentioned, in the herein provided in vitro methods for producing Nm CPS which have a defined length or in the compositions of the invention, the terminally truncated version of the CP-X as described herein may be used.

[0182] As for vaccine production the Nm CPS have preferably a DP of 10 to 60 (i.e. an avDP of 15 to 20), it is preferred that the Nm CPS which are produced by the herein described in vitro methods have this size. Advantageously, if in the herein described in vitro methods the CP-A or a terminally truncated version of the CP-X is used, then the produced Nm CPS have a DP or 10 to 60 (i.e. an avDP of 15 to 20), if the donor-to-acceptor-ratio is in the range from 10:1 to 400:1. In addition, if in herein described in vitro methods the truncated version of the CP-X is used, then the produced Nm CPS have a DP of 10 to 60 (i.e. an avDP of 15 to 20) if the incubation time ranges from 3 to 45 minutes. Thus, one aspect of the invention relates to the herein provided in vitro methods, wherein the capsular polysaccharides have a DP of 10 to 60 or an avDP of 15 to 20. In a preferred aspect of the herein provided in vitro methods, at least 60% (more preferably at least 80%) of the capsular polysaccharides have a DP of 10 to 60 or an avDP of 15 to 20. In addition, a further embodiment of the invention relates to the in vitro methods of the invention or the composition of the invention, wherein

[0183] (i) the ratio of donor carbohydrate to acceptor carbohydrate is a ratio from 20:1 to 80:1; and the capsule polymerase is either a polypeptide comprising maximal 80% of the amino acid sequence of SEQ ID NO: 24 (CP-X) and comprising the amino acid sequence of SEQ ID NO: 28 (.DELTA.C99-CP-X), or a polypeptide comprising the amino acid sequence of SEQ ID NO: 9 (CP-A); or

[0184] (ii) the ratio of donor carbohydrate to acceptor carbohydrate is a ratio from 100:1 to 400:1; and the capsule polymerase is a polypeptide comprising maximal 68% of the amino acid sequence of SEQ ID NO: 24 (CP-X) and comprising the amino acid sequence of SEQ ID NO: 29 (.DELTA.N58.DELTA.C99-CP-X).

[0185] In item (i) of the above described embodiment, the ratio of donor carbohydrate to acceptor carbohydrate may be 50:1.

[0186] According to the inventive in vitro method, the donor carbohydrate which is contacted with at least one purified capsule polymerase (CP) may further be activated during step (a). Thus, one embodiment of the invention relates to the in vitro methods of the invention, wherein said donor carbohydrate is activated or wherein said donor carbohydrate is activated during step (a). Said activation during step (a) may be performed by contacting said donor carbohydrate with an activating enzyme. In addition, during this in step (a) of the inventive in vitro methods, said donor carbohydrate may be further contacted with PEP and/or at least one activating nucleotide. Analogously, in the herein provided composition said donor carbohydrate may be activated. In the methods or in the composition of the invention, said donor carbohydrate may be activated by linkage to an activating nucleotide. Said activating nucleotide may be, e.g. CMP, CDP, CTP, UMP, UDP, TDP, AMP or UTP, preferably UDP.

[0187] For example, the activation may be mediated by linkage of an activating nucleotide such as CMP, UDP, TDP or AMP. Preferably, the activating nucleotide is UDP. The activation of a carbohydrate by linkage of a nucleotide may be catalyzed by several activating enzymes which are known in the art. Such activating enzymes may be incubated with the at least one donor carbohydrate, at least one acceptor carbohydrate and the at least one CP during step (a) of the in vitro method provided herein. UDP-ManNAc is preferably synthesized from UDP-GlcNAc using the enzyme UDP-GlcNAc-epimerase. In SEQ ID NO: 32, the nucleotide sequence of UDP-GlcNAc-epimerase cloned from Neisseria meningitidis serogroup A is shown, the corresponding polypeptide sequence of UDP-GlcNAc-epimerase is shown in SEQ ID NO: 33.

[0188] One particular embodiment of the present invention relates to the herein provided in vitro methods or the herein provided composition, wherein the capsule polymerase is a truncated version of the capsule polymerase of Neisseria meningitidis serogroup X and wherein at least one donor carbohydrate is UDP-GlcNAc. However, another embodiment of the present invention relates to the herein provided in vitro methods or the herein provided composition, wherein the capsule polymerase is a truncated version of the capsule polymerase of Neisseria meningitidis serogroup X and wherein at least one donor carbohydrate is GlcNAc-1-P.

[0189] In a further embodiment of the herein described in vitro methods or compositions the capsule polymerase is the capsule polymerase of Neisseria meningitidis serogroup A and at least one donor carbohydrate is UDP-ManNAc. The sugar building block UDP-ManNAc is commercially not available. Therefore, a further advantage of the present invention is that UDP-ManNAc can be synthesized (during the inventive in vitro method for producing Nm capsular polysaccharides) from cheap UDP-GlcNAc. Thus, another embodiment of the present invention relates to the inventive in vitro methods or the inventive composition, wherein the capsule polymerase is the capsule polymerase of Neisseria meningitidis serogroup A and wherein at least one donor carbohydrate is UDP-GlcNAc.

[0190] In context of the present invention, UDP-ManNAc is preferably synthesized from UDP-GlcNAc using the enzyme UDP-GlcNAc-epimerase. In SEQ ID NO: 32, the nucleotide sequence of UDP-GlcNAc-epimerase cloned from Neisseria meningitidis serogroup A is shown, the corresponding polypeptide sequence of UDP-GlcNAc-epimerase is shown in SEQ ID NO: 33. Thus, one aspect of the invention relates to the herein provided in vitro methods, wherein in step (a) the capsule polymerase is further incubated with the UDP-GlcNAc-epimerase. In addition, one embodiment of the invention relates to the herein provided composition, further comprising the UDP-GlcNAc-epimerase. For example, in the in vitro methods or compositions of the present invention, the UDP-GlcNAc-epimerase (e.g. CsaA of NmA) may be used in a concentration of 1-2000 nmol, e.g. 10 nmol.

[0191] In another embodiment of the inventive in vitro methods and compositions, the capsular polymerase (CP) may be a truncated version of the CP-X or a functional derivative thereof and at least one donor carbohydrate may be GlcNAc-1-phosphate. Said donor carbohydrate GlcNAc-1-phosphate may be further contacted with at least one nucleotide and/or phosphoenolpyruvate (PEP) and auxiliary enzymes. Said nucleotide can be, e.g., UMP, UDP or UTP. Said donor carbohydrate GlcNAc-1-phosphate may further be activated during incubation with the terminally truncated CP-X. In accordance with the herein presented in vitro method, this activation may yield the activated sugar nucleotide UDP-GlcNAc.

[0192] Thus, the CP to be applied in the means and methods described herein may be a truncated version of CP-X or a functional derivative thereof and at least one donor carbohydrate may be UDP-GlcNAc or a derivative thereof. Examples for derivatives of UDP-GlcNAc may be compounds that are alkylated or hydroxylated or that comprise additional functional groups, such as carboxylic acids, azides, amides, acetyl groups or halogen atoms; see also "Carbohydrate chemistry" Volumes 1-34, Cambridge [England], Royal Society of Chemistry, loc. cit.

[0193] Generally, in context of the present invention, the saccharides described herein may also be labelled forms of these saccharides. For example, the saccharides may be labelled radioactively, such as [.sup.14C] or [.sup.3H]. Such labelling may be inter alia useful in diagnostic applications and uses of the saccharides described herein. Such diagnostic applications and uses will be further described herein below.

[0194] In accordance with the inventive method, the terminally truncated version of the CP-X is contacted with at least one donor carbohydrate and with at least one acceptor carbohydrate during the incubation step (a) of the in vitro method presented herein. Said acceptor carbohydrate may be oligomeric or polymeric CPS of Neisseria meningitidis serogroup X (CPSX), and/or a carbohydrate structure containing terminal GlcNAc residues such as hyaluronic acid, heparin, heparin sulphate or protein-linked oligosaccharides.

[0195] In the herein provided methods for producing Nm capsular polysaccharides which have a defined length, it is preferred that the acceptor carbohydrate has a DP.gtoreq.4. Or, in other words, acceptor carbohydrates with a length of DP.gtoreq.4 are particularly preferred in the herein described methods for producing Nm capsular polysaccharides which have a defined length. Normally, the acceptor carbohydrates are purified capsular polysaccharides of the respective serogroup and have different length. However, in the herein provided methods for producing capsular polysaccharides with a defined length, and in the herein provided compositions, it is preferred that at least 80% of the acceptor carbohydrates are polysaccharides with a DP of .gtoreq.4. The acceptor carbohydrates may also consist of polysaccharides with a DP of .gtoreq.4. For example, if the CP-A is used, then the acceptor (or at least 80% of the acceptor carbohydrates) is preferably DP4, DP5, or DP6; more preferably the acceptor (or at least 80% of the acceptor carbohydrates) is a tetramer or pentamer of ManNAc-1P with a free reducing end. If a terminally truncated version of the CP-X is used, then the acceptor (or at least 80% of the acceptor carbohydrates) is preferably DP4, DP5, or DP6; more preferably, the acceptor (or at least 80% of the acceptor carbohydrates) is a tetramer or pentamer of GlcNAc-1P with a free reducing end as shown in FIG. 3.

[0196] An example of the present invention relates to the in vitro method for producing Nm capsular polysaccharides of NmX which have a defined length, said method comprising the steps:

[0197] (a) incubating a truncated version of the CP-X with UDP-GlcNAc and hydrolysed capsular polysaccharides of NmX; wherein the ratio of UDP-GlcNAc to hydrolysed capsular polysaccharides of NmX is a ratio from 10:1 to 400:1 (e.g. 20:1 to 400:1); and

[0198] (b) isolating the resulting capsular polysaccharide.

[0199] A further example of the present invention relates to the in vitro method for producing Nm capsular polysaccharides of NmX which have a defined length, said method comprising the steps:

[0200] (a) incubating a truncated version of the CP-X with UDP-GlcNAc and hydrolysed capsular polysaccharides of NmX; wherein the incubation time ranges from 3 to 45 minutes; and

[0201] (b) isolating the resulting capsular polysaccharide.

[0202] Another example of the present invention relates to the in vitro method for producing Nm capsular polysaccharides of NmX which have a defined length, said method comprising the steps:

[0203] (a) incubating a truncated version of the CP-X with UDP-GlcNAc and hydrolysed capsular polysaccharides of NmX; wherein

[0204] (i) the ratio of donor carbohydrate to acceptor carbohydrate is a ratio from 10:1 to 10000 (e.g. 200:1 to 1000:1), and

[0205] (ii) the incubation time ranges from 3 to 45 minutes; and

[0206] (b) isolating the resulting capsular polysaccharide.

[0207] In the above examples also other combinations of activated or non-activated donor carbohydrates and acceptor carbohydrates as described herein can be applied. Such other combinations do not defer from the gist of the present invention.

[0208] In one embodiment of the present in vitro method, the CP to be used is CP-A or a functional derivative thereof and at least one donor carbohydrate may be UDP-ManNAc or a derivative thereof. Examples for derivatives of UDP-ManNAc may be compounds that are alkylated or hydroxylated or that comprise additional functional groups such as carboxylic acids, azides, amides, acetyl groups or halogen atoms; see also "Carbohydrate chemistry" Volumes 1-34: monosaccharides, disaccharides, and specific oligosaccharides, Reviews of the literature published during 1967-2000, Cambridge (England), Royal Society of Chemistry.

[0209] In another embodiment of the in vitro method described herein, the CP is CP-A or a functional derivative thereof and at least one donor carbohydrate is ManNAc-1-phosphate or a derivative thereof. Examples for derivatives of ManNAc-1-phosphate ManNAc-3-O-Ac or ManNAc-4-O-Ac or ManNAc-3,4-di-O-Ac. In the herein provided in vitro method, said donor carbohydrate ManNAc-1-phosphate (ManNAc-1-P) may be contacted with at least one nucleotide and/or phosphoenolpyruvate (PEP) and auxiliary enzymes during step (a) of the in vitro method. Said nucleotide can be, e.g., UMP, UDP and UTP. Said donor carbohydrate ManNAc-1-phosphate may be activated during incubation with CP-A. In accordance with the herein presented in vitro method, this activation may yield the activated sugar nucleotide UDP-ManNAc, or its derivatives UDP-ManNAc-3-O-Ac or UDP-ManNAc-4-O-Ac or UDP-ManNAc-3,4-di-O-Ac. Thus, one aspect of the invention relates to the herein provided in vitro methods, wherein the capsule polymerase is the capsule polymerase of Neisseria meningitidis serogroup A and wherein at least one donor carbohydrate is ManNAc-1-P.

[0210] In the herein provided in vitro method, CP-A or a functional derivative thereof is contacted with at least one donor carbohydrate and with an acceptor carbohydrate during the incubation step (a) of the inventive in vitro method. In accordance with the inventive in vitro method presented herein, the acceptor carbohydrate may be oligomeric or polymeric CPS of Neisseria meningitidis serogroup A (CPSA) and/or a carbohydrate structure containing terminal GlcNAc or ManNAc residues such as hyaluronic acid, heparin, heparin sulphate or protein-linked oligosaccharides. As described above, in the herein provided in vitro methods, the acceptor carbohydrate of CP-A may be the cheap and easily commercially available UDP-GlcNAc, as the UDP-GlcNAc-epimerase can be used to convert UDP-GlcNAc into UDP-ManNAc.

[0211] An example of the present invention relates to the in vitro method for producing Nm capsular polysaccharides of NmA which have a defined length, said method comprising the steps:

[0212] (a) incubating CP-A with UDP-GlcNAc, UDP-GlcNAc-epimerase and hydrolysed capsular polysaccharides of NmA; wherein the ratio of UDP-GlcNAc to hydrolysed capsular polysaccharides of NmA is a ratio from 10:1 to 400:1 (e.g. 20:1 to 400:1); and

[0213] (b) isolating the resulting capsular polysaccharide.

[0214] A further example of the present invention relates to the in vitro method for producing Nm capsular polysaccharides of NmA which have a defined length, said method comprising the steps:

[0215] (a) incubating CP-A with UDP-ManNAc and hydrolysed capsular polysaccharides of NmA; wherein the ratio of UDP-ManNAc to hydrolysed capsular polysaccharides of NmA is a ratio from 10:1 to 400:1 (e.g. 20:1 to 400:1); and

[0216] (b) isolating the resulting capsular polysaccharide.

[0217] Again, the skilled person readily understands that also other combinations of activated or non-activated donor carbohydrates and acceptor carbohydrates as described herein can be applied. Such other combinations do not defer from the gist of the present invention.

[0218] As described herein and illustrated in the appended examples, the acceptor carbohydrate is preferred by the CP (e.g. CP-A) if it is presented in non-acetylated form. Moreover, the acceptor is even more preferred if presented in non-acetylated, de-phosphorylated form. Accordingly, one embodiment of the present invention relates to the herein provided in vitro methods or the herein provided composition, wherein the acceptor carbohydrate is non-acetylated and/or dephosphorylated, preferably non-acetylated and dephosphorylated.

[0219] Within a CPS obtainable by the inventive method described hereinabove, one or more carbohydrates of the CPS-subunits may be derivatized and may contain, for example, additional functional groups such as acetyl groups, amino groups, alkyl groups, hydroxyl groups, carboxylic acids, azides, amides, or halogen atoms; see also "Carbohydrate chemistry" Volumes 1-34: monosaccharides, disaccharides, and specific oligosaccharides, Reviews of the literature published during 1967-2000, Cambridge (England), Royal Society of Chemistry. Thus, in one embodiment of the inventive in vitro method or composition, the acceptor carbohydrate carries one or more additional functional groups at its reducing end. Said additional functional groups, may be, e.g., acetyl groups, carboxylic acids, azides, amides, or halogen atoms; see also "Carbohydrate chemistry" Volumes 1-34, Cambridge [England], Royal Society of Chemistry, loc. cit.

[0220] One embodiment of the present invention relates to the herein provided in vitro method or the herein provided composition, wherein said acceptor carbohydrate is capsule polysaccharide of Neisseria meningitidis serogroup A or X or a carbohydrate structure containing terminal GlcNAc residues. Said carbohydrate structure containing terminal GlcNAc-residues may be hyaluronic acid, heparin sulphate, heparan sulphate or protein-linked oligosaccharides.

[0221] As demonstrated in the appended examples, the inventors of the present invention analyzed the minimal length of the priming acceptor for CP-A. Therefore, synthetic compounds which are shown in FIG. 25 which vary not only in length, but also with respect to O-acetylation (compounds 2, 4, 6 are 3-O-acetylated) and reducing end modifications have been used. In compounds 1, 2, 5, 6 the reducing ends are occupied by a decyl-phosphate-ester, while a methyl group (OMe) is present in the compounds 3 and 4. As evident from the appended examples, the activity of the codon optimized .DELTA.N69-CP-A did not go beyond background (no acceptor) with compounds 1-4, but, intriguingly, steeply increased with disaccharides carrying a decyl-phosphate-ester at the reducing end (compounds 5, 6) (FIG. 25). With the non-acetylated compound 5, activity values similar to those obtained with the optimized acceptor CPSA.sub.hyd(deOAc)-deP were measured. Interestingly, O-acetylation of compound 5 (resulting in compound 6) reduced the quality of the acceptor. Based on these data, the minimal acceptor recognized by CP-A or a derivative of CP-A (such as .DELTA.N69-CP-A) can be defined as the dimer of ManNAc-1P units linked together by phosphodiester bonds. The presence of a phosphodiester at the reducing end seems obligatory, because compounds ending with OMe groups are not used by the capsule polymerase.

[0222] Thus, in context of the present invention, a dimer of ManNAc-1P carrying a phosphodiester at the reducing end was identified as minimal acceptor for CP-A or derivatives of CP-A (such as .DELTA.N69-CP-A).

[0223] Accordingly, the inventors of the present invention identified two important features of CP-A: (i) the minimal efficient acceptor is a dimer and (ii) the reducing end phosphate group can be extended with rather large chemical groups (such as decyl-ester). This latter finding bears the perspective that chain elongation can be primed with reagents of very high purity and functional groups that facilitate conjugation of glycans to carrier proteins in the vaccine production chain (Costantino (2011) Expert opinion on drug discovery 6 (10), 1045-1066; Bardotti (2008) Vaccine 26 (18), 2284-2296). Besides decyl-ester, chain elongation may be primed with a number of functional groups that allow conjugation to carrier proteins, e.g., Alkyl-azides, Alkyl-amindes or sulfhydryl-reagents chemical functions as described in Costantino et al., 2011 Expt. Opin. Drug. Discov. 6:1053; Table 2.

[0224] Accordingly, one embodiment of the present invention relates to the in vitro method of the invention or the composition of the invention, wherein said acceptor carbohydrate is a dimer of ManNAc carrying a phosphodiester at the reducing end. In this embodiment of the present invention, the capsule polymerase may be the capsule polymerase of Neisseria meningitidis serogroup A. Moreover, the phosphate group at the reducing end may be extended with alkyl-azides, alkyl-amindes or sulfhydryl-groups and chemical functions as described in Costantino (2011) Expt. Opin. Drug. Discov. 6:1053; Table 2. For example, in the herein provided in vitro methods or composition, the acceptor carbohydrate may be a disaccharide carrying a decyl-phosphate-ester at the reducing end. As mentioned, in this embodiment of the present invention, the capsule polymerase may be the capsule polymerase of Neisseria meningitidis serogroup A.

[0225] If in the herein described in vitro methods or compositions the acceptor is a dimer of ManNAc carrying a phosphodiester at the reducing end (which may be extended with large chemical groups such as a decyl-ester), then the capsular polymerase may be CP-A or a derivative of CP-A (such as .DELTA.N69-CP-A).

[0226] The capsule polymerase (CP) which is incubated with the at least one donor carbohydrate in the presented in vitro methods and compositions may be purified. Said CP (or a functional fragment or derivative thereof) may be isolated from Neisseria meningitidis lysates or recombinantly produced. Recombinant production of a CP (or a functional fragment or derivative thereof) may be performed by transferring the DNA coding for the CP (or the functional fragment or derivative thereof) into a vector and using a host (e.g. eukaryotic host cells such as insect cells or yeast or prokaryotic host cells such as Escherichia coli) to express the CP (or the functional fragment or derivative thereof). After expression, the CP (or the functional fragment or derivative thereof) may be purified from the cell lysate. The purification may be carried out following the instructions of the Qiaexpressionist (http://www.qiagen.com/literature/render.aspx?id=128, Qiagen) for bacterial protein purification or by using BD Bioscience Baculo Gold instruction (Cat. No. 560138) for insect cell protein production and purification. For instance, the CP (or the functional fragment or derivative thereof) may be produced by purifying the recombinant CP (or the functional fragment or derivative thereof) from bacteria via his-tag purification following the instructions of the Qiaexpressionist. A CP (or a functional fragment or derivative thereof) may also be synthetically or chemically produced. Therefore solid phase peptide synthesis (SPPS) may be applied.

[0227] The donor carbohydrates which are used in the herein provided means and methods may be obtained from commercial sources or may be chemically synthesized (Wolf, Chemistry (2009) 15:7656-64; Wolf, Eur J Cell Biol. (2010) 89:63-75. The acceptor carbohydrates which are used in the inventive means and methods may be obtained by purifying them from Neisseria meningitidis cultures and can be chemically synthesized as described in the attached manuscript (Black (2010) Synthesis of structures corresponding to the capsular polysaccharide of Neisseria meningitidis group A. 4th Baltic Meeting on Microbial Carbohydrates, Abstracts, p. 56 (abstract 5), Hyytiala Forestry Field Station, Finland; Black (2011) Towards synthetic glycoconjugates based on the structure of Neisseria meningitidis group A capsular polysaccharide. 16th European Carbohydrate Symposium, Abstracts, p. 61 (OL-02), Sorrento, Italy; Nikolaev (2011) From phosphosaccharide chemistry to potential anti-parasite and anti-bacterial carbohydrate vaccines. Carbohydrate Gordon Research Conference, Abstracts, p. 4, Colby College, Waterville, Me., USA.). One embodiment of the invention relates to the herein provided in vitro methods or composition, wherein said acceptor carbohydrate is purified.

[0228] As mentioned, the acceptor carbohydrate which is contacted with the donor carbohydrate and the CP may be purified according to the in vitro method described herein. If said acceptor carbohydrate is oligomeric or polymeric CPS of Neisseria meningitidis, it may be hydrolysed. Thus, one aspect of the invention relates to the herein provided in vitro methods or composition, wherein said acceptor capsule polysaccharide is hydrolysed.

[0229] It is known in the art that capsule polysaccharides of NmA are immunogenic only if O-acetylated (Berry (2002) Infection and immunity 70 (7), 3707-3713). Therefore, in the herein described in vitro methods for producing capsular polysaccharides an O-acetyltransferase can be used which is able to perform this modification in a nature identical form. Accordingly, one embodiment of the invention relates to the herein provided in vitro method, further comprising O-acetylation of the produced capsule polysaccharides. Said O-acetylation may be performed by contacting the produced capsule polysaccharides with an O-acetyltransferase. Also provided herein is the composition of the invention, further comprising an O-acetyltransferase.

[0230] The O-acetyltransferase which is used in the inventive methods and compositions may be, e.g. the CsaC of NmA. Accordingly, one aspect of the invention relates to the in vitro methods of the invention or the composition of the invention, wherein, wherein the O-acetyltransferase is the polypeptide of any one of (a) to (f):

[0231] (a) a polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 53;

[0232] (b) a polypeptide comprising the amino acid sequence of SEQ ID NO: 54;

[0233] (c) a polypeptide encoded by a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 54 or of a functional fragment thereof;

[0234] (d) a polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule hybridizing under stringent conditions to the complementary strand of a nucleic acid molecule as defined in (a) or (c) and encoding a functional polypeptide; or a functional fragment thereof;

[0235] (e) a polypeptide having at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98% or most preferably at least 99% identity to the polypeptide of any one of (a) to (d), whereby said polypeptide is functional; or a functional fragment thereof; and

[0236] (f) a polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule being degenerate as a result of the genetic code to the nucleotide sequence of a nucleic acid molecule as defined in (a), (c), and (d).

[0237] The function of this O-acetyltransferase comprises the ability to transfer Acetyl-groups from the donor Acetyl-Coenzyme A onto hydroxyl-groups of UDP-ManNAc or oligo- and polymeric structures consisting of ManNAc-1-phosphate units linked together by phosphodiester linkages. Or, in other words this O-acetlytransferase catalyses the formation of an ester-bond between an Acetyl-group and a hydroxyl-group of ManNAc-1-phosphate containing molecule. Thus, one aspect of the invention relates to the in vitro methods of the invention or the composition of the invention, wherein the O-acetyltransferase is the polypeptide of any one of (a) to (f):

[0238] (a) a polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 53;

[0239] (b) a polypeptide comprising the amino acid sequence of SEQ ID NO: 54;

[0240] (c) a polypeptide encoded by a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 54 or of a functional fragment thereof, wherein the function comprises the ability to transfer Acetyl-groups from the donor Acetyl-Coenzyme A onto hydroxyl-groups of UDP-ManNAc or oligo- and polymeric structures consisting of ManNAc-1-phosphate units linked together by phosphodiester linkages;

[0241] (d) a polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule hybridizing under stringent conditions to the complementary strand of a nucleic acid molecule as defined in (a) or (c) and encoding a functional polypeptide; or a functional fragment thereof, wherein the function comprises the ability to transfer Acetyl-groups from the donor Acetyl-Coenzyme A onto hydroxyl-groups of UDP-ManNAc or oligo- and polymeric structures consisting of ManNAc-1-phosphate units linked together by phosphodiester linkages;

[0242] (e) a polypeptide having at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98% or most preferably at least 99% identity to the polypeptide of any one of (a) to (d), whereby said polypeptide is functional; or a functional fragment thereof, wherein the function comprises the ability to transfer Acetyl-groups from the donor Acetyl-Coenzyme A onto hydroxyl-groups of UDP-ManNAc or oligo- and polymeric structures consisting of ManNAc-1-phosphate units linked together by phosphodiester linkages; and

[0243] (f) a polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule being degenerate as a result of the genetic code to the nucleotide sequence of a nucleic acid molecule as defined in (a), (c), and (d).

[0244] In context of the present invention, production and O-acetylation of capsular polysaccharides (e.g. of capsular polysaccharides of NmA) may be performed by using a two-step protocol or in a one-pot reaction.

[0245] For example, the O-acetylation of capsular polysaccharides may be performed by incubating in vitro synthesized capsular polysaccharides (e.g. 1 mg) in the presence of an O-acetyltransferase (e.g. 1.2 nmol CsaC of NmA) in a total volume of 0.5 mL. The reaction may be performed in reaction buffer (e.g. a buffer comprising 25 mM Tris pH 7.5 and 50 mM NaCl), started by the addition of an equimolar amount of Acetyl-CoA (e.g. 14 mM final) and allowed to proceed for 4 h at 37.degree. C. Obviously, this protocol may be up or down scale depending on the needed amount of O-acetylated capsular polysaccharides.

[0246] As mentioned above, capsule polysaccharides of NmA are only immunogenic if they are O-acetylated. In addition, natural capsule polysaccharides of NmA are O-acetylated in positions 3 and 4 of ManNAc. Thus, if the herein provided in vitro methods are combined with O-acetylation of the produced capsule polysaccharides, then the used capsule polymerase may be CP-A. Thus, one aspect of the invention relates to the herein provided in vitro methods or composition, wherein the capsule polymerase is the capsule polymerase of Neisseria meningitidis serogroup A. The capsule polysaccharides produced by the inventive in vitro methods may be O-acetylated in positions 3 and 4 of ManNAc.

[0247] As described herein, the capsule polysaccharides (e.g. the O-acetylated capsular polysaccharides) which are produced by the herein described methods may be used for glycoconjugate vaccine production. Thus, one embodiment of the present invention relates to a method for producing a vaccine comprising the herein provided in vitro method. Said method for producing a vaccine may further comprise covalently attaching the produced capsular polysaccharides to a carrier molecule. Said carrier molecule may be tetanus toxoid or CRM.sub.197 (Cross-Reacting Material 197), an inactive form of diphteria-toxin (Micoli et al., 2013; PNAS).

[0248] Thus, the invention relates to a method for the production of a vaccine against a strain genus Neisseria comprising the steps of:

[0249] (a) In vitro production of (a) Nm capsule polysaccharide(s) as defined above; and

[0250] (b) combining said Nm capsule polysaccharide(s) with a pharmaceutically acceptable carrier.

[0251] Accordingly, the invention relates to a method for the production of a vaccine against a strain or strains of the genus Neisseria, in particular N. meningitidis by combining (a) Nm capsular polysaccharide(s) of the invention with a biologically acceptable carrier.

[0252] Another embodiment of the present invention also relates to a capsular polysaccharide which has been produced by the in vitro method of the present invention. The CPS obtainable by the in vitro methods described hereinabove are useful as pharmaceuticals, e.g., as vaccines. In particular, the herein described CPS are advantageous as vaccines in the prophylaxis and treatment of diseases caused by Neisseria meningitidis, such as neisserial meningitidis. Accordingly, one embodiment of the present invention relates to the capsular polysaccharide of the invention for use as a medicament.

[0253] As mentioned, the synthetic CPS obtainable by the inventive in vitro method may be used as vaccines. In a preferred embodiment of the present invention, they are used in vaccination of a human subject. Also disclosed is the use of the CPS obtainable by the inventive in vitro method for the preparation of a vaccine. In a specific embodiment of the present invention, the CPS obtainable by the in vitro methods described herein are used as vaccines against meningococcal meningitidis caused by Neisseria meningitidis serogroup A or X. Thus, the medicaments disclosed herein may be useful in pharmaceutical and vaccination purposes, i.e. for the treatment of Neisseria-induced diseases or the vaccination against these pathogens. Therefore, one embodiment of the present invention relates to a vaccine comprising the capsular polysaccharide of the invention, optionally further comprising a pharmaceutically acceptable carrier. Also provided herein is the capsular polysaccharide of the invention or the vaccine of the invention for use in vaccination of a subject. Accordingly, the present invention provides for a method for treating and/or preventing a meningococcal meningitidis comprising the administration of (a) capsular polysaccharide(s) of the present invention or the vaccine(s) of the present invention to a subject in need of such a treatment. Preferably said subject is a human patient.

[0254] A "patient" or "subject" for the purposes of the present invention includes both humans and other animals, particularly mammals, and other organisms. Thus, the methods are applicable to both human therapy and veterinary applications. In the preferred embodiment the patient is a mammal, and in the most preferred embodiment the patient is human being. As indicated, the herein provided capsular polysaccharide(s) or vaccine(s) may be used for the vaccination against meningococcal meningitidis caused by Neisseria meningitidis serogroup A or X.

[0255] The medicaments provided herein are pharmaceutical compositions and may comprise the CPS of the present invention. The pharmacological compositions may further comprise antibodies specifically directed against the CPS of the present invention. Such CPS as well as the antibodies directed against the same may be used, inter alia, in vaccination protocols, either alone or in combination. Therefore, the pharmaceutical compositions of the present invention which comprise the CPS of the invention or antibodies directed against these CPS, may be used for pharmaceutical purposes such as effective therapy of infected humans or animals and/or, preferably for vaccination purposes. Accordingly, the present invention relates to pharmaceutical compositions comprising the CPS as described herein and/or antibodies or antibody fragments against the CPS as described herein and, optionally, a pharmaceutically acceptable carrier. In context with the present invention, the pharmaceutical compositions described herein may be used, inter alia, for the treatment and/or prevention of Neisseria-induced diseases and/or infections. Preferably, the pharmaceutical composition of the invention is used as a vaccine as will be further described herein below.

[0256] As mentioned, the present invention also relates to pharmaceutical compositions comprising the capsular polysaccharides described herein. Said capsular polysaccharides may be isolated but it is also envisaged that these capsular polysaccharides are to be used in context with other structures, e.g., toxins, adjuvants and the like. Such toxins may, inter alia, function as carriers for the capsular polysaccharides produced by the herein described methods. Numerous methods have been developed to link oligosaccharides covalently to carriers (Lit: (a) Vince Pozsgay, Oligosaccharide-protein conjugates as vaccine candidates against bacteria, Advances in Carbohydrate Chemistry and Biochemistry, Academic Press, 2000, Volume 56, Pages 153-199, (b) Jennings, H. J., R. K. Sood (1994) Synthetic glycoconjugates as human vaccines; in Lee, Y. C. R. T. Lee (eds): Neoglycoconjugates. Preparation and Applications. San Diego, Academic Press, pp 325-371, (c) Pozsgay, V.; Kubler-Kielb, J., Conjugation Methods toward Synthetic Vaccines, Carbohydrate-Based Vaccines, American Chemical Society, Jul. 2, 2008, 36-70); (D) Carl E. Frasch, Preparation of bacterial polysaccharide-protein conjugates: Analytical and manufacturing challenges, Vaccine, In Press, Corrected Proof, Available online 24 Jun. 2009, ISSN 0264-410X, DOI: 10.1016/j.vaccine.2009.06.013.) One example is the covalent coupling of the synthetic CPS molecules provided herein to protein amino-groups by means of reductive amination. As indicated above, for producing a vaccine, the herein produced CPS may be coupled to tetanus toxoid or other carrier proteins to generate a conjugate vaccine.

[0257] Thus, the capsular polysaccharide produced by the herein described methods may preferably used as a vaccine, i.e. the compounds provided herein can be employed for the vaccination of a subject. Such a subject may be a mammal and, in a particular embodiment, a human being. The vaccines provided herein are particularly useful in the vaccination against Neisseria.

[0258] The medicament/pharmaceutical composition (e.g. the vaccine) of the present invention comprises the CPS produced by the in vitro methods of the invention and may further comprise a pharmaceutically acceptable carrier, excipient and/or diluent. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc. Compositions comprising such carriers can be formulated by well known conventional methods. These pharmaceutical compositions can be administered to the subject at a suitable dose. Administration of the suitable compositions may be effected by different ways, e.g., by intravenous, intraperitoneal, subcutaneous, intramuscular, topical, intradermal, intranasal or intrabronchial administration. The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. The pharmaceutical composition of the present invention, particularly when used for vaccination purposes, may be employed at about 0.01 .mu.g to 1 g CPS per dose, or about 0.5 .mu.g to 500 .mu.g CPS per dose, or about 1 .mu.g to 300 .mu.g CPS per dose. However, doses below or above this exemplary ranges are envisioned, especially considering the aforementioned factors. Administration of the suitable compositions may be effected by different ways, e.g., by intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration. However, in particular in the pharmaceutical intervention of the present invention, Neisseria infections can demand an administration to the side of infection, like the brain. Progress can be monitored by periodic assessment. The compositions of the invention may be administered locally or systemically. Administration will generally be parenterally, e.g., intravenously. The compositions of the invention may also be administered directly to the target site, e.g., by biolistic delivery to an internal or external target site or by catheter to a site in an artery. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. Furthermore, the pharmaceutical composition of the invention may comprise further agents such as interleukins and/or interferons depending on the intended use of the pharmaceutical composition. The pharmaceutical composition (e.g. the vaccine) of the invention may also be administered as co-therapy together with at least one other active agent. This at least other active agent may be a medicament which is conventionally used as adjuvant or for preventing and/or treating Nm infections. The other active agent may be, e.g., a vaccine, an antibiotic, an anti-inflammatory agent, an interleukin or an interferon.

[0259] In a preferred embodiment of the present invention, the pharmaceutical composition as defined herein is a vaccine.

[0260] Vaccines may be prepared, inter alia, from one or more CPS as described herein, or from one or more antibodies as described herein, i.e. antibodies against the CPS as disclosed herein. Accordingly, in context with the present invention, vaccines may comprise one or more CPS as described herein and/or one or more antibodies, fragments of said antibodies or derivatives of the antibodies of the invention, i.e. antibodies against the CPS as disclosed herein.

[0261] The CPS or the antibodies, fragments or derivatives of said antibodies of the invention which are used in a pharmaceutical composition may be formulated, e.g., as neutral or salt forms. Pharmaceutically acceptable salts, such as acid addition salts, and others, are known in the art. Vaccines can be, inter alia, used for the treatment and/or the prevention of an infection with pathogens, e.g. Neisseria, and are administered in dosages compatible with the method of formulation, and in such amounts that will be pharmacologically effective for prophylactic or therapeutic treatments.

[0262] A vaccination protocol can comprise active or passive immunization, whereby active immunization entails the administration of an antigen or antigens (like the capsule polysaccharides of the present invention or antibodies directed against these CPS) to the subject/patient in an attempt to elicit a protective immune response. Passive immunization entails the transfer of preformed immunoglobulins or derivatives or fragments thereof (e.g., the antibodies, the derivatives or fragments thereof of the present invention, i.e. specific antibodies directed against the CPS obtained by the means and methods provided herein) to a subject/patient. Principles and practice of vaccination and vaccines are known to the skilled artisan, see, for example, in Paul, "Fundamental Immunology" Raven Press, New York (1989) or Morein, "Concepts in Vaccine Development", ed: S. H. E. Kaufmann, Walter de Gruyter, Berlin, N.Y. (1996), 243-264; Dimitriu S, editor. "Polysaccharides in medicinal application"; New York: Marcel Dekker, pp 575-602. Typically, vaccines are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in or suspension in liquid prior to injection may also be prepared. The preparation may be emulsified or the protein may be encapsulated in liposomes. The active immunogenic ingredients are often mixed with pharmacologically acceptable excipients which are compatible with the active ingredient. Suitable excipients include but are not limited to water, saline, dextrose, glycerol, ethanol and the like; combinations of these excipients in various amounts also may be used. The vaccine may also contain small amounts of auxiliary substances such as wetting or emulsifying reagents, pH buffering agents, and/or adjuvants which enhance the effectiveness of the vaccine. For example, such adjuvants can include aluminum compositions, like aluminumhydroxide, aluminumphosphate or aluminumphosphohydroxide (as used in "Gen H-B-Vax.RTM." or "DPT-Impfstoff Behring"), N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-DMP), N-acetyl-nornuramyl-L-alanyl-D-isoglutamine (CGP 11687, also referred to as nor-MDP), N-acetylmuramyul-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'2'-dipalmitoyl-s- n-glycero-3-hydroxyphaosphoryloxy)-ethylamine (CGP 19835A, also referred to as MTP-PE), MF59 and RIBI (MPL+TDM+CWS) in a 2% squalene/Tween-80.RTM. emulsion.

[0263] The vaccines are usually administered by intravenous or intramuscular injection. Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations. For suppositories, traditional binders and carriers may include but are not limited to polyalkylene glycols or triglycerides. Oral formulation include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. These compositions may take the form of solutions, suspensions, tables, pills, capsules, sustained release formulations or powders and contain about 10% to about 95% of active ingredient, preferably about 25% to about 70%.

[0264] Vaccines are administered in a way compatible with the dosage formulation, and in such amounts as will be prophylactically and/or therapeutically effective. The quantity to be administered generally is in the range of about 0.01 .mu.g to 1 g antigen per dose, or about 0.5 .mu.g to 500 .mu.g antigen per dose, or about 1 .mu.g to 300 .mu.g antigen per dose (in the present case CPS being the antigen), and depends upon the subject to be dosed, the capacity of the subject's immune system to synthesize antibodies, and the degree of protection sought. Precise amounts of active ingredient required to be administered also may depend upon the judgment of the practitioner and may be unique to each subject. The vaccine may be given in a single or multiple dose schedule. A multiple dose is one in which a primary course of vaccination may be with one to ten separate doses, followed by other doses given at subsequent time intervals required to maintain and/or to reinforce the immune response, for example, at one to four months for a second dose, and if required by the individual, a subsequent dose(s) after several months. The dosage regimen also will be determined, at least in part, by the need of the individual, and be dependent upon the practitioner's judgment. It is contemplated that the vaccine containing the immunogenic compounds of the invention may be administered in conjunction with other immunoregulatory agents, for example, with immunoglobulins, with cytokines or with molecules which optimize antigen processing, like listeriolysin.

[0265] For diagnosis and quantification of pathogens like Neisseria, pathogenic fragments, their derivatives, their (poly)peptides (proteins), their polynucleotides, etc. in clinical and/or scientific specimens, a variety of immunological methods, as well as molecular biological methods, like nucleic acid hybridization assays, PCR assays or DNA Enzyme Immuno Assays (DEIA; Mantero et al., Clinical Chemistry 37 (1991), 422-429) have been developed and are well known in the art. In this context, it should be noted that the nucleic acid molecules of the invention may also comprise PNAs, modified DNA analogs containing amide backbone linkages. Such PNAs are useful, inter alia, as probes for DNA/RNA hybridization. The proteins of the invention may be, inter alia, useful for the detection of anti-pathogenic (like, e.g., anti-bacterial or anti-viral) antibodies in biological test samples of infected individuals. It is also contemplated that antibodies of the invention and compositions comprising such antibodies of the invention may be useful in discriminating acute from non-acute infections. The CPS as provided herein can also be used in diagnostic settings, for example as "standards", in, e.g., chromatographic approaches. Therefore, the present CPS can be used in comparative analysis and can be used either alone or in combination to diagnostic methods known in the art.

[0266] The diagnostic compositions of the invention optionally comprise suitable means for detection. The CPS as disclosed and described herein as well as specific antibodies or fragments or derivatives thereof directed or raised specifically against these CPS are, for example, suitable for use in immunoassays in which they can be utilized in liquid phase or bound to a solid phase carrier. Solid phase carriers are known to those in the art and may comprise polystyrene beads, latex beads, magnetic beads, colloid metal particles, glass and/or silicon chips and surfaces, nitrocellulose strips, membranes, sheets, animal red blood cells, or red blood cell ghosts, duracytes and the walls of wells of a reaction tray, plastic tubes or other test tubes. Suitable methods of immobilizing nucleic acids, (poly)peptides, proteins, antibodies, microorganisms etc. on solid phases include but are not limited to ionic, hydrophobic, covalent interactions and the like. Examples of immunoassays which can utilize said proteins, antigenic fragments, fusion proteins, antibodies or fragments or derivatives of said antibodies of the invention are competitive and non-competitive immunoassays in either a direct or indirect format. Commonly used detection assays can comprise radioisotopic or non-radioisotopic methods. Examples of such immunoassays are the radioimmunoassay (RIA), the sandwich (immunometric assay) and the Western blot assay. Furthermore, these detection methods comprise, inter alia, IRMA (Immune Radioimmunometric Assay), EIA (Enzyme Immuno Assay), ELISA (Enzyme Linked Immuno Assay), FIA (Fluorescent Immuno Assay), and CLIA (Chemioluminescent Immune Assay). Other detection methods that are used in the art are those that do not utilize tracer molecules. One prototype of these methods is the agglutination assay, based on the property of a given molecule to bridge at least two particles.

[0267] The CPS of the invention can be bound to many different carriers. Examples of well-known carriers include glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amyloses, natural and modified celluloses, polyacrylamides, agaroses, and magnetite. The nature of the carrier can be either soluble or insoluble for the purposes of the invention.

[0268] A variety of techniques are available for labeling biomolecules. Theses techniques are well known to the person skilled in the art and comprise, inter alia, covalent coupling of enzymes or biotinyl groups, iodinations, phosphorylations, biotinylations, random priming, nick-translations, tailing (using terminal transferases) or labeling of carbohydrates. Such techniques are, e.g., described in Tijssen, "Practice and theory of enzyme immuno assays", Burden, R H and von Knippenburg (Eds), Volume 15 (1985), "Basic methods in molecular biology"; Davis L G, Dibmer M D; Battey Elsevier (1990), Mayer et al., (Eds) "Immunochemical methods in cell and molecular biology" Academic Press, London (1987), or in the series "Methods in Enzymology", Academic Press, Inc., or in Fotini N. Lamari, Reinhard Kuhn, Nikos K. Karamanos, "Derivatization of carbohydrates for chromatographic, electrophoretic and mass spectrometric structure analysis", Journal of Chromatography B, Volume 793, Issue 1, Derivatization of Large Biomolecules, (2003), Pages 15-36.

[0269] Detection methods comprise, but are not limited to, autoradiography, fluorescence microscopy, direct and indirect enzymatic reactions, etc.

[0270] The CPS described herein may be detected by methods known in the art as well as described and exemplified herein. For example, an ELISA (Enzyme-linked immunosorbent assay) based method described herein may be used for the detection and quantification of the chimeric CPS described herein. In this context, the chimeric CPS described herein may be immobilized by an antibody or other binding molecule, such as a lectine or similar, contacting one part or building block of the chimeric CPS. Detection of a second part or building block of the chimeric CPS described herein can be achieved by, e.g., contacting with an antibody or other binding molecule as described herein which is labeled for further detection or a secondary antibody or other binding molecule as described which is labeled for further detection.

[0271] As indicated above, the present invention further relates to antibodies specifically binding to the synthetic CPS obtainable by the in vitro methods described herein. The term "antibody" herein is used in the broadest sense and specifically encompasses intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments, so long as they exhibit the desired biological activity. Also human, humanized, camelized or CDR-grafted antibodies are comprised.

[0272] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e. the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be constructed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler, G. et al., Nature 256 (1975) 495, or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). "Antibody fragments" comprise a portion of an intact antibody. In context of this invention, antibodies specifically recognize CPS obtainable by the in vitro method described herein. Antibodies or fragments thereof as described herein may also be used in pharmaceutical and medical settings such as vaccination/immunization, particularly passive vaccination/immunization.

[0273] The term "antibody" includes antigen-binding portions, i.e., "antigen binding sites," (e.g., fragments, subsequences, complementarity determining regions (CDRs)) that retain capacity to bind an antigen (such as CPS produced by the herein described methods), comprising or alternatively consisting of, for example, (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward; 1989; Nature 341; 544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Antibody fragments or derivatives further comprise F(ab')2, Fv or scFv fragments or single chain antibodies.

[0274] The antibodies of the present invention may be used for vaccinating against, treating and/or diagnosing meningococcal meningitidis caused by Neisseria meningitidis serogroup A or X.

[0275] The term "carbohydrate" as used herein comprises building blocks such as saccharides and sugars in any form as well as aldehydes and ketones with several hydroxyl groups added. A carbohydrate may contain one or more of said building blocks linked via covalent bonds such as glycosidic linkages. A carbohydrate may be of any length, i.e. it may be monomeric, dimeric, trimeric or multimeric. A carbohydrate may also contain one or more building blocks as side chains linked to the main chain via covalent bonds. A carbohydrate may also contain one or more activated saccharides such as nucleotide sugars. Examples of nucleotide sugars are UDP-GlcNAc, UDP-ManNAc, UDP-GlcUA, UDP-Xyl, GDP-Man and GDP-Fuc.

[0276] Herein, the term "nucleic acid molecule" or "polynucleotide" refers to DNA or RNA or hybrids thereof or any modification thereof that is known in the state of the art (see, e.g., U.S. Pat. No. 5,525,711, U.S. Pat. No. 4,711,955, U.S. Pat. No. 5,792,608 or EP 302175 for examples of modifications). The polynucleotide sequence may be single- or double-stranded, linear or circular, natural or synthetic. For instance, the polynucleotide sequence may be genomic DNA, cDNA, mRNA, antisense RNA, ribozymal or a DNA encoding such RNAs or chimeroplasts (Gamper, Nucleic Acids Research, 2000, 28, 4332-4339). Said polynucleotide sequence may be in the form of a plasmid or of viral DNA or RNA. For example, the present invention relates to a nucleic acid molecule having the nucleotide sequence of any one of SEQ ID NOs: 1-8, 16-23 and 32. The present invention also encompasses nucleic acid molecules comprising the nucleic acid molecule of any one of SEQ ID NO: 1-8, 16-23 and 32 wherein one, two, three or more nucleotides are added, deleted or substituted. Such a nucleic acid molecule may encode a polypeptide being active (i.e. functional). The term "activity", "functionality" or "being functional" as used herein refers in particular to the ability of having substantially the same function as the non-modified polynucleotide.

[0277] The present invention further relates to nucleic acid molecules which are complementary to the nucleic acid molecules described above. Also encompassed are nucleic acid molecules which are able to hybridize to nucleic acid molecules described herein. A nucleic acid molecule of the present invention may also be a fragment of the nucleic acid molecules described herein. Particularly, such a fragment is a functional fragment, which means that the fragment has the same function as the non-modified polynucleotide.

[0278] The term "hybridization" or "hybridizes" as used herein in context of nucleic acid molecules/polynucleotieds/DNA sequences may relate to hybridizations under stringent or non-stringent conditions. If not further specified, the conditions are preferably stringent. Said hybridization conditions may be established according to conventional protocols described, for example, in Sambrook, Russell "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Laboratory, N.Y. (2001); Ausubel, "Current Protocols in Molecular Biology", Green Publishing Associates and Wiley Interscience, N.Y. (1989), or Higgins and Hames (Eds.) "Nucleic acid hybridization, a practical approach" IRL Press Oxford, Washington D.C., (1985). The setting of conditions is well within the skill of the artisan and can be determined according to protocols described in the art. Thus, the detection of only specifically hybridizing sequences will usually require stringent hybridization and washing conditions such as 0.1.times.SSC, 0.1% SDS at 65.degree. C. Non-stringent hybridization conditions for the detection of homologous or not exactly complementary sequences may be set at 6.times.SSC, 1% SDS at 65.degree. C. As is well known, the length of the probe and the composition of the nucleic acid to be determined constitute further parameters of the hybridization conditions. Variations in the above conditions may be accomplished through the inclusion and/or substitution of alternate blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.

[0279] In accordance to the invention described herein, low stringent hybridization conditions for the detection of homologous or not exactly complementary sequences may, for example, be set at 6.times.SSC, 1% SDS at 65.degree. C. As is well known, the length of the probe and the composition of the nucleic acid to be determined constitute further parameters of the hybridization conditions.

[0280] Hybridizing nucleic acid molecules also comprise fragments of the above described molecules. Furthermore, nucleic acid molecules which hybridize with any of the aforementioned nucleic acid molecules also include complementary fragments, derivatives and allelic variants of these molecules. Additionally, a hybridization complex refers to a complex between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary G and C bases and between complementary A and T bases; these hydrogen bonds may be further stabilized by base stacking interactions. The two complementary nucleic acid sequences hydrogen bond in an antiparallel configuration. A hybridization complex may be formed in solution (e.g., Cot or Rot analysis) or between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., membranes, filters, chips, pins or glass slides to which, e.g., cells have been fixed). The terms complementary or complementarity refer to the natural binding of polynucleotides under permissive salt and temperature conditions by base-pairing. For example, the sequence "A-G-T" binds to the complementary sequence "T-C-A". Complementarity between two single-stranded molecules may be "partial", in which only some of the nucleic acids bind, or it may be complete when total complementarity exists between single-stranded molecules. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, which depend upon binding between nucleic acids strands.

[0281] The term "hybridizing sequences" preferably refers to sequences which display a sequence identity of at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98% and most preferably at least 99% identity with a nucleic acid sequence as described above.

[0282] The polypeptides and nucleic acid molecule provided herein preferably show a homology, determined by sequence identity, of at least 80%, more preferably of at least 85%, even more preferably of at least 90%, even more preferably of at least 95%, even more preferably of at least 96%, even more preferably of at least 97%, even more preferably of at least 98, and most preferably of at least 99% identity to a sequence as shown in any one of SEQ ID NOs: 1-3, 9, 10, 16, 17, 20-25 or 28-33 as defined herein above.

[0283] The polypeptides and nucleic acid molecules to be employed in accordance with this invention preferably show a homology, determined by sequence identity, to the sequences indicated under any one of SEQ ID NOs: 1-3, 9, 10, 16, 17, 20-25 or 28-33, preferably over the entire length of the sequences compared. The homologous sequences are preferably fragments having a length of at least 100, more preferably at least 200, more preferably at least 300, more preferably at least 400 and more preferably at least 500, more preferably at least 600, more preferably at least 700, more preferably at least 800 and most preferably at least 900 nucleotides which have an identity of at least 80%, more preferably of at least 85%, even more preferably of at least 90%, even more preferably of at least 95%, even more preferably of at least 96%, even more preferably of at least 97%, even more preferably of at least 98, and most preferably of at least 99% with the sequence shown under any one of SEQ ID NOs: 1-3, 9, 10, 16, 17, 20-25 or 28-33, respectively.

[0284] If two sequences which are to be compared with each other differ in length, sequence identity preferably relates to the percentage of the (amino acid or nucleotide) residues of the shorter sequence which are identical with the (amino acid or nucleotide) residues of the longer sequence. Sequence identity can be determined conventionally with the use of computer programs such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive Madison, Wis. 53711), CLUSTALW computer program (Thompson; 1994; Nucl Acids Res; 2; 4673-4680) or FASTDB (Brutlag; 1990; Comp App Biosci; 6; 237-245). Bestfit utilizes the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2 (1981), 482-489, in order to find the segment having the highest sequence identity between two sequences. When using Bestfit or another sequence alignment program to determine whether a particular sequence has for instance 95% identity with a reference sequence of the present invention, the parameters are preferably so adjusted that the percentage of identity is calculated over the entire length of the reference sequence and that homology gaps of up to 5% of the total number of the nucleotides or amino acids in the reference sequence are permitted. When using Bestfit, the so-called optional parameters are preferably left at their preset ("default") values. The deviations appearing in the comparison between a given sequence and the above-described sequences of the invention may be caused for instance by addition, deletion, substitution, insertion or recombination. Such a sequence comparison can preferably also be carried out with the program "fasta20u66" (version 2.0u66, September 1998 by William R. Pearson and the University of Virginia; see also W. R. Pearson, Methods in Enzymology 183 (1990), 63-98, appended examples and http://workbench.sdsc.edu/). For this purpose, the "default" parameter settings may be used.

[0285] The in vitro methods described herein may also be used for automated high-throughput approaches using robotic liquid dispensing workstations. Generally in such high-throughput approaches a plurality of assay mixtures are run in parallel. These mixtures may have the same or different concentrations of the respective ingredients. Typically, one of these mixtures serves as a negative control, i.e. at zero concentration or below the limits of assay detection.

[0286] These and other embodiments are disclosed and obvious to a skilled person and embraced by the description and the Examples of the present invention. Additional literature regarding one of the above-mentioned methods, means and uses, which can be applied within the meaning of the present invention can be obtained from the prior art, for instance in public libraries, e.g. with the use of electronic means. For this purpose, public data bases, such as "Medline", can be accessed via the internet, for instance under the address http://www.ncbi.nlm.nih.gov/PubMed/medline.html. Additional data bases and addresses are known to a skilled person and can be taken from the internet, for instance under the address http://www.lycos.com. An overview of sources and information regarding patents or patent applications in biotechnology is given in Berks, TIBTECH 121 (1994), 352-364.

[0287] A number of documents including patent applications, manufacturer's manuals and scientific publications are cited herein. The disclosure of these documents, while not considered relevant for the patentability of this invention, is herewith incorporated by reference in its entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

[0288] The present invention is further described by reference to the following non-limiting figures and examples.

[0289] The Figures show:

[0290] FIG. 1. The donor:acceptor ratio influences the product distribution and the product length of CsaB- and CsxA-reactions in a different manner

[0291] By using CP-A (i.e. CsaB), the product distribution as well as the product length can be controlled via the donor:acceptor ratio. By using full length CP-X (i.e. CsxA), the product distribution as well as the product length cannot be controlled via the donor:acceptor ratio. Here two product pools are produced, one having small and the other having large polymers.

[0292] A: CPSA distribution obtained after CsaB (in particular .DELTA.N69-CP-A) reactions in the presence of 2 mM UDP-GlcNAc and varying amounts of short CPSA oligos (DP1-DP10). It is mentioned that, in pilot experiments, a similar product length control by varying the donor-acceptor ratio was seen for the full length CP-A. The designation "expected DP" indicates the major species of the produced capsular polysaccharides (i.e. the main component) and also specifies the donor-acceptor ratio (i.e. the donor:acceptor quotient). Thus, the concentrations of the acceptor CPSX range from 40-4 .mu.M. Accordingly, from left to right, the used ratios of donor carbohydrate to acceptor carbohydrate are 50:1, 75:1, 100:1, 200:1 and 500:1.

[0293] B: Purification of CsxA truncations.

[0294] C: Design of CsaB and CsaX truncations. Active truncations are indicated by and asterisk *.

[0295] D: CPSX distribution obtained after CsxA reactions in the presence of 5 mM UDP-GlcNAc and varying amounts of short CPSX oligos (DP1-DP7). The fact that the highest amount of acceptor sugar is not detectable in the gel indicates that all detected molecule are synthesised by CsxA. The marker consists of polymer of the size which is used in vaccine production.

[0296] E: CPSX distribution obtained after CsxA reactions in the presence of 5 mM UDP-GlcNAc and varying amounts of long CPSX oligos (DP10-DP70). The marker consists of polymer of the size which is used in vaccine production.

[0297] FIG. 2. The donor:acceptor ratio influences the product distribution and the product length of the truncated version of the CsxA

[0298] By using a truncated version of CP-X (i.e. dC99-CsxA or dN58dC99-CsxA), the product distribution as well as the product length can be controlled via the donor:acceptor ratio. The designation "expected DP" indicates the major species (i.e. the main component) of the produced capsular polysaccharides and also specifies the donor-acceptor ratio (i.e. the donor:acceptor quotient). For example, an "expected DP" of 20, 33, 40, 50, 64, 75, 80, 100, 160, 200, 400, or 500 means a ratio of donor carbohydrate to acceptor carbohydrate of 20:1, 33:1, 40:1, 50:1, 64:1, 75:1, 80:1, 100:1, 160:1, 200:1, 400:1, or 500:1.

[0299] A/B: CPSX distribution obtained after dN58-CsxA reactions in the presence of 5 mM UDP-GlcNAc and varying amounts of short CPSX oligos (DP1-DP7).

[0300] C/D: CPSX distribution obtained after dC99-CsxA reactions in the presence of 5 mM UDP-GlcNAc and varying amounts of short CPSX oligos (DP1-DP7).

[0301] E: CPSX distribution obtained after dN58dC99-CsxA reactions in the presence of 5 mM UDP-GlcNAc and varying amounts of short CPSX oligos (DP1-DP7).

[0302] F: CPSA distribution obtained after dN69-CsaB reactions in the presence of 2 mM UDP-GlcNAc and varying amounts of short CPSA oligos (DP1-DP10).

[0303] FIG. 3. Determination of the minimal acceptor of full length CsxA and truncated CsxA

[0304] A: Purification of single CPSX oligos.

[0305] B-D: Finding the minimal acceptor of CsxA full-length and CsxA truncations.

[0306] FIG. 4. The reaction time influences the product distribution and the product length of the truncated versions of the CsxA

[0307] By using constant donor-acceptor ratios and truncated versions of the CsxA (i.e. dC99-CsxA, dN58-CsxA or dC99dN58-CsxA) the avDP of the produced products can be controlled via the reaction time. 50 nM of capsule polymerase have been used. The marker indicates length from DP10 to DP60.

[0308] A: Analysis of the CPSX distribution obtained at different time-points during a CsxA reaction in the presence of 10 mM UDP-GlcNAc and DP7. The ratio of donor carbohydrate to acceptor carbohydrate is approximately 8000:1.

[0309] B: Analysis of the CPSX distribution obtained at different time-points during a dN58-CsxA reaction in the presence of 10 mM UDP-GlcNAc and DP7. The ratio of donor carbohydrate to acceptor carbohydrate is approximately 8000:1.

[0310] C: Analysis of the CPSX distribution obtained at different time-points during a dC99-CsxA reaction in the presence of 10 mM UDP-GlcNAc and DP7. The ratio of donor carbohydrate to acceptor carbohydrate is approximately 8000:1.

[0311] D: Analysis of the CPSX distribution obtained at different time-points during a dN58dC99-CsxA reaction in the presence of 10 mM UDP-GlcNAc and DP7. The ratio of donor carbohydrate to acceptor carbohydrate is approximately 8000:1.

[0312] E: Analysis of the CPSX distribution obtained at different time-points during a dC99-CsxA reaction in the presence of 10 mM UDP-GlcNAc and DP5. The ratio of donor carbohydrate to acceptor carbohydrate is approximately 200:1.

[0313] F: D: Analysis of the CPSX distribution obtained at different time-points during a dN58dC99-CsxA reaction in the presence of 10 mM UDP-GlcNAc and DP7. The ratio of donor carbohydrate to acceptor carbohydrate is approximately 200:1. A calibration showed that the product pool in the 20' fraction has an avDP of 15 and the product pool in the 20' fraction has an avDP of 20.

[0314] FIG. 5. The oligomerisation status of CP-A (CsaB; upper panel) and CP-X (CsxA; lower panel) was analysed by size exclusion and different salt concentrations. CP-A elutes in the void volume with 25 mM NaCl and elutes as a broad smeared peak in the presence of 500 mM salt, indicating that the full length enzyme forms aggregates in solution that are distorted but not dissociated into monomers in the presence of 500 mM salt.

[0315] In case of CP-X (CsxA) the C-terminal truncation (dC99) is not sufficient to prevent aggregate formation at low salt concentrations, however, the protein migrates with an apparent molecular mass resembling the trimer if 500 mM salt are present. In contrast the N-terminal truncation (dN58) prevents larger aggregate formation. The mutant dN58 similar to dN58dC99 migrates with an apparent molecular mass that suggest dimer formation already at low salt concentrations (25 mM NaCl). The dimers formed seem to be stable also in the presence of 500 mM salt.

[0316] FIG. 6. A: schematic representation of CP-A (upper bar) and CP-X (lower bar) illustrating the conserved motives existing in these proteins and positions were point mutations have been introduced. Point mutants with reduced- or no activity are marked with one and double star (*), respectively. B: All protein variants could be expressed as recombinant proteins and purified in high quality. C: A radioactive incorporation assay was used to determine the enzymatic activity.

[0317] FIG. 7. Multi-sequence alignment including the bacterial capsule polymerases that are hexosylphosphate transferases identified a novel conserved asparagine-residue (N324 in CP-X) outside of the conserved motifs. Based on a hypothetic model of CP-X (top) N324 may be involved in the coordination of the catalytically important divalent cation (large gray ball in the middle). Replacement of this asparagine by alanine in CP-A and CP-X inactivated enzymatic activity. Remarkably, the respective enzyme isolated from non-pathogenic NmL had no activity neither as wildtype nor after mutation of this position.

[0318] FIG. 8. Fluorescently labelled primers for the separate testing of hexosyl- and sialyltransferase activity.

[0319] Starting from 4-MU-Sia, monovalent forms of SiaD.sub.W-135/SiaD.sub.Y were used in iterative steps to generate labelled acceptors suited to separately assay hexosyl- and sialyltransferase activity in the chimeric capsule polymerases of NmW-135 and NmY. Exemplarily shown are the acceptors with Gal as hexose. For simplicity DP--degree of polymerization--is used to describe the different 4-MU-labelled saccharides.

[0320] FIG. 9. Chromatographic behaviour of 4-MU-labelled oligomers in anion exchange chromatography.

[0321] In consecutive enzymatic reactions 4-MU-Sia (4-MU-DP1) was elongated to 4-MU-DP4. Before the start of a new reaction 0.01% of the sample was loaded onto a CarboPac.RTM. PA-100 column and products eluted with a curved NaNO.sub.2 gradient as shown. MU fluorescence was detected at 375 nm. Overlays of chromatograms obtained with the monovalent forms of SiaD.sub.W-135 (A) and SiaD.sub.Y (B) are shown. Baseline separation of peaks allowed the determination of precise retention times. Of note, the chromatograms show that the conversion of 4-MU-DP1 to 4-MU-DP2 was incomplete under the conditions used, while all other that reactions proceeded to completion.

[0322] FIG. 10. Time lapse recording of the SiaD.sub.W-135 reaction.

[0323] (A) SiaD.sub.W-135 was primed with 1 mM 4-MU-Sia-Gal-Sia (4-MU-DP3) in the presence of 2 mM UDP-Gal and 2 mM CMP-Sia. At indicated time points, product profiles were recorded by HPLC-FD. (B) Enlarged display of the HPLC-FD profile obtained at 40 min and labelling of individual DPs. (C) Normed and weighted peak areas were calculated for each time point and used to construct progress curves. Progress curves obtained in three independent experiments (R1-R3) are shown and document the high reproducibility of the reaction. Peaks marked red represent 4-MU-DP1 and 4-MU, which represent contaminants in the 4-MU-DP3 pool, whereby 4-MU is already present in the starting solution of the substrate 4-MU-Sia and represents the hydrolysis of the substrate. Both peaks remain unchanged over the entire reaction, confirming a step increase in acceptor quality from DP2 to DP3.

[0324] FIG. 11. Use of fluorescent primers to monitor the reaction profiles of single point mutant enzymes

[0325] Reaction profiles of the monovalent point mutants NmW-135-(E307A)-His.sub.6 and NmW-135-(S972A)-His.sub.6 were recorded in the presence of both donor sugars using the appropriate or false fluorescent primer. Reactions were run over 30 min with samples taken after 15 sec and 30 min. A: NmW-135-(E307A)-His.sub.6 with 4-MU-DP2. The only reaction product formed is 4-MU-DP3. B: NmW-135-(E307A)-His.sub.6 primed with 4-MU-DP3. No product is formed. C: NmW-135-(S972A)-His.sub.6 primed with 4-MU-DP2. No product was formed. D: NmW-135-(S972A)-His.sub.6 primed with 4-MU-DP3. The only product formed is 4-MU-DP4.

[0326] FIG. 12. Schematic representation of the capsule polymerases from NmW-135 and NmY and illustration of deletion mutants made thereof.

[0327] A: Schematic illustration of the full length capsule polymerases NmW-135 and NmY. The GT-B folds predicted to comprise hexosyl- and sialyltransferase domain are highlighted in green and purple, respectively. The linker region connecting the GT-B folded domains is shown in black. Mutants made in the course of this project were named according the introduced deletion. All proteins were expressed with N-terminal His.sub.6-tag. B: Western blot analysis of expressed proteins as indicated. Lysates prepared from transformed BL21(DE3) cells were separated into soluble and insoluble fraction and after separation on 10% SDS-PAGE analysed by western blotting using an anti-penta-His antibody.

[0328] FIG. 13. Product Profiles of the reactions obtained with C.DELTA.639-His.sub.6 and with N.DELTA.398-His.sub.6.

[0329] Reaction profiles of the truncates variants NmW-135-(C.DELTA.639)-His.sub.6 and NmW-135-(N.DELTA.398)-His.sub.6 were recorded in the presence of both donor sugars using the corresponding acceptor. Reactions were run over 30 min with samples taken after 15 sec and 30 min. A: NmW-135-(C.DELTA.639)-His.sub.6 with 4-MU-DP3 catalyze the transfer of UDP-Gal by creating 4-MU-DP4. B: NmW-135-(N.DELTA.398)-His.sub.6 primed with 4-MU-DP2 catalyze the transfer of CMP-Sia by producing 4-MU-DP3.

[0330] FIG. 14. Product profiles of N.DELTA.562 and N.DELTA.609 constructs.

[0331] Reaction profiles of the truncates variants NmW-135-(N.DELTA.562)-His.sub.6 and NmW-135-(N.DELTA.609)-His.sub.6 were recorded in the presence of both donor sugars using the corresponding acceptor. Reactions were run over 30 min with samples taken after 15 sec and 30 min. A: NmW-135-(N.DELTA.562)-His.sub.6 with 4-MU-DP2 catalyze the transfer of CMP-Sia by creating 4-MU-DP3. B: NmW-135-(N.DELTA.609)-His.sub.6 primed with 4-MU-DP2 catalyze the transfer of CMP-Sia by producing 4-MU-DP3.

[0332] FIG. 15. Quantification of enzymatic activity obtained with mutants.

[0333] The enzymatic activity of mutant forms was determined in an HPLC-based assay using fractions of purified enzymes. The HexTF catalysis is shown under A which were incubated with 4-MU-DP3 and the SiaTF catalysis is indicated under B which were incubated with 4-MU-DP2. Values represent means of three independent experiments.

[0334] FIG. 16. Secondary structure-based alignment predicted for SiaD.sub.W-135 and SiaD.sub.Y.

[0335] The sequence alignment was performed using the ClustalW2 alignment and secondary structure motif predictions The symbols below the sequence illustrate .beta.-strands (black arrows) and .alpha.-helices (lines). Positions where truncations were introduced are indicated with arrows above the sequences. The selected sequence accession numbers from the GenBank are Y13970 (SiaD.sub.W-135) and Y13969 (SiaD.sub.Y).

[0336] FIG. 17. Mechanisms of polysaccharide synthesis. Polymerization occurs by repeated transfer of a sugar residue (Neu5Ac) from a nucleotide sugar precursor (CMP-Neu5Ac) onto an acceptor substrate (DMB-DP3).

[0337] The mechanism of elongation and product distribution are determined by the nature and extent of interaction with the growing polysaccharide chain. Schematic HPLC profiles of the product distributions are shown at right (DP, degree of polymerization). (a) An extended acceptor binding site confers an increased affinity for longer chains which occupy more of the binding subsites, resulting in uneven chain elongation and a skewed product distribution. For highly processive elongation, the acceptor is retained through many transfer cycles and no intermediates between short and long chains will be observed (top, right). Strict nonprocessive elongation involves dissociation of the enzyme-acceptor complex after each sugar transfer, therefore, all intermediates can be observed in the product distribution (bottom, right). In practice, the precise mechanism is often unclear because the product distributions of less processive and nonprocessive enzymes are indistinguishable16. (b) In the absence of an extended acceptor binding site, the enzyme interacts only with the growing end of the polysaccharide, elongation is nonprocessive and distributive, resulting in uniform elongation of all acceptors. The product profile has a narrow, Poissonian distribution (right).

[0338] FIG. 18. Drifted polySTs exhibit diverse patterns of chain elongation.

[0339] Clones were sorted into three categories based on their average product dispersity. For each category, the reaction time course (top left), HPLC product profiles at three reaction time points (bottom), and an extract from Supplementary Table 1 (top right) are displayed for four representative clones. (a) High dispersity clones (mean dispersity >1.2) exhibit an exacerbated kinetic lag phase, maximum reaction rates up to 10 times greater than the initial rate, and product profiles strongly skewed towards longer chains. (b) Medium dispersity clones (mean dispersity of 1.1-1.2) exhibit a mild lag phase and less skewed product distributions. (c) Low dispersity clones (mean dispersity <1.1) have no lag phase (initial rate is the maximum reaction rate) and uniformly elongate chains resulting in narrow product distributions. Results for all 51 neutral drift clones are given in Supplementary Data Set 2 and Supplementary Table 1.

[0340] FIG. 19. Distribution of mutations across the polyST sequence.

[0341] Sequencing of 122 `neutral` variants from the first round of screening revealed 163 mutations across the .DELTA.25polyST.sub.NmB sequence. Here the mutations are plotted on an alignment of the four bacterial polySTs from Neisseria meningitidis serogroup B (NmB), Neisseria meningitidis serogroup C (NmC), Escherichia coli K1 (EcK1) and Escherichia coli K92 (EcK92). Similarities of homology are shown in different gray scales. Numbering above the alignment indicates residue number in the polyST.sub.NmB sequence. Of note is that the mutations are unevenly distributed across the sequence with targeted positions and short clusters showing high mutability.

[0342] FIG. 20. Single amino acid exchanges alter the mode of chain elongation. (a) Reaction time course for single mutatnts which exhibit either an exacerbated (top) or reduced (bottom) kinetic lag phase compared to the .sub..DELTA.25polyST.sub.NmB (wild type) reference sequence (dotted line). Error bars indicate the standard error of three separate enzyme preparations (from different colonies) analysed in a single experiment. (b) Time lapse HPLC product profiles throughout the polymerization reaction of each of the clones. The .sub..DELTA.25polyST.sub.NmB reference chormatograms (top) are displayed for comparison, and chain lengths are indicated (DP, degree of polymerization; R.T., retention time). Enzymes with an exacerbated lag phase show a broadening of the product distribution specifically after synthesis of chains >DP10, which corresponds in time to the increase in reaction rate observed in the reaction time courses. Enzymes with a reduced lag phase show more uniform chain elongation, and considerably less broadening of the product distribution.

[0343] FIG. 21: Schematic representation of the chromosomal locus (cps) of NmA. Products of genes forming region A are involved in the synthesis of the capsule polysaccharide and are serogroup-specific. For more information, see text [adapted from Harrison et al. (2013)].

[0344] FIG. 22: (A) Coomassie stained SDS-PAGE of purified StrepII-CsaA-His.sub.6 (left panel) and CsaC-His.sub.6 (right panel). (B) To select the construct most suited for the production of active recombinant CsaB the wildtype and a codon optimized (CsaB.sub.co) version of the CsaB sequence were cloned (full length or after N-terminal truncation, .DELTA.69) to produce proteins with tags on either both (StrepII and His.sub.6) or only one end (His.sub.6) as indicated. Transformed bacteria were lysed, separated into soluble (s) and insoluble (i) fraction and fractions separately run on 10% PAGE. After transfer onto nitrocellulose, the blot was developed with an anti-penta-His antibody. (C) Soluble fractions were used to measure CsaB activity in a radioactive incorporation assay. (D) Coomassie stained gel demonstrating the purification result for .DELTA.69-CsaB.sub.co-His.sub.6 (IMAC; immobilized metal ion affinity chromatography; SEC size exclusion chromatography).

[0345] FIG. 23: Schematic representation of the multi-enzyme assay used to continuously follow CsaB activity.

[0346] FIG. 24: (A) The chemical properties of the primers used to test the acceptor preference of .DELTA.69-CsaB.sub.co-His.sub.6 are displayed. (B) .DELTA.69-CsaB.sub.co-His.sub.6 activity was followed using the spectrophotometric assay in the presence of CPSA.sub.hyd of avDP6 and avDP15, in either native O-acetylated form (CPSA.sub.hyd(OAc)) or after de-O-acetylation (CPSA.sub.hyd(deOAc)). Because earlier studies showed that the non-reducing ends in CPSA.sub.hyd are phosphorylated, samples were additionally tested before and after phosphatase treatment (deP). Samples designated with .sub.(deOAc)deP were subject of de-acetylation and de-phosphorylation.

[0347] FIG. 25: Derivatives of ManNAc ending at the reducing end with a methyl group (compounds 3, 4) or a phospho-decyl-ester (compounds 1, 2, 5, 6) were synthesized and used to prime the .DELTA.69-CsaB.sub.co-His.sub.6 reaction in the continues spectrophotometric assay. The dimer of ManNAc-1P carrying a phosphodiester at the reducing end was identified as minimal acceptor. Importantly, compound 5 showed acceptor quality identical to CPSA.sub.hyd(deOAc)-deP and O-acetylation (compound 6) reduced the acceptor quality to approximately the same extend as seen for CPSA.sub.hyd.

[0348] FIG. 26. (A) Products synthesized in the CsaA/CsaB reaction in the presence of UDP-GlcNAc and CPSA.sub.hyd of avDP6 were analysed by high percentage PAGE and a combined alcian blue/silver staining. Long chains were produced in the presence of all reactants (reaction) and, though in small amounts, also in control-1 were no priming oligosaccharides were added. The production of long CPSA-chains in control-1 argues for the capacity of CsaB to start chains de novo. (B) Corresponding .sup.1H- and .sup.31P NMR analysis. (C) HPLC analysis of products obtained in reactions were the ratio between CsaA:CsaB was varied as indicated. This experiment clearly shows that UDP-formation is a side activity of CsaA, which can be prevented if the CsaB concentration is equal or higher than the concentration of CsaA. UMP, UDP and UDP-GlcNAc/UDP-ManNAc were detected at 280 nm and CPSA at 224 nm.

[0349] FIG. 27. (A) In vitro synthesized CPSA (CPSA.sub.iv) was separated from all contaminating reaction products using anion exchange chromatography with the indicated sodium chloride gradient. (B) Purified CPSAiv after O-acetylation (CPSA.sub.iv/OAc was re-chromatographed under the same conditions resulting in a well separated product peak, which in dot (C) was recognized by mAb 932. (D) Corresponding .sup.1H NMR analysis of the produced CPSA in comparison to CPSA from natural source.

[0350] FIG. 28. (A) In vitro synthesis of O-acetylated and non-O-acetylated CPSA using all enzymes CsaA-C in a one pot reaction. To control product formation, substrates and enzymes were added as indicated. After overnight incubation, products were displayed on high percentage PAGE by a combined alcian blue/silver staining. Long chains were synthesized in all reactions containing CsaA, CsaB and UDP-GlcNAc. (B) Products obtained in the reaction where CsaC and acetyl-CoA were present, were detected with mAb 932 specifically directed against the CPSA.sub.OAc.

[0351] FIG. 29. Schematic representation of the truncated CsxA constructs. (A) Full-length, (B) .DELTA.N58 truncation, (C) .DELTA.C99 truncation.

[0352] FIG. 30. Truncation studies. (A) 10% SDS-PAGE followed by Coomassie staining of the soluble (LF) and the insoluble (UF) fraction of lysed BL21(DE3)Gold expressing the constructs as indicated. (B) Corresponding western blot developed against the His.sub.6-tag. (C) Normalized activities from soluble fractions of full-length (wildtyp) and truncated constructs.

[0353] FIG. 31. (A) Analytical size exclusion chromatography in low-salt buffer (25 mM NaCl). (B) Analytical size exclusion chromatography in high-salt buffer (500 mM NaCl). (C) Schematic representation of the constructs tested in A, B and D. (D) Enzymatic activity of the constructs measured using and adaption of the multi-enzyme assay published by Freiberger et al. (2007, Molecular Microbiology 65, 1258-1275).

[0354] FIG. 32. Elongation of an acceptor carbohydrate having DP5 by using 50 nM .DELTA.C99-CP-X (A) or 50 nM .DELTA.N58.DELTA.C99-CP-X (B). The reaction times are indicated at the top. The used marker is avDP15, which is the material which is (coupled to a toxoid) often used in vaccines. The marker indicates length from DP10 to DP60. The donor acceptor ratio is approximately 200:1. As can be seen .DELTA.N58.DELTA.C99-CP-X (B) produces capsular polysaccharides with a lower degree of dispersity as compared to .DELTA.C99-CP-X (A).

[0355] FIG. 33. The capsule polymerase of NmW-135 cannot be regulated via the donor-acceptor ratio. Extension of the synthetic acceptor 4-Mu-Sia-Gal-Sia by CP-W-135 is shown. The concentration of CMP-Sia and UDP-Gal is 2 mM. The designation "ratio" specifies the donor-acceptor-ratio. Even at a ratio of 4 the synthesized polymers are rather long (approximately >200 repeating units).

[0356] FIG. 34. (A) The amount of acceptor was experimentally determined in over-night reactions by incubating .DELTA.N58.DELTA.C99 CsxA in the presence of 10 mM UDP-GlcNAc and varying amounts of DP2-DP10. (B) The progress of the reaction could be determined following the consumption of UDP-GlcNAc and the corresponding production of UMP by HPLC-AEC. (C) The obtained CPSX pool is well in the range of the avDP15 material which is commonly used for vaccine production showing that .DELTA.N58.DELTA.C99 CsxA is active and remains distributive even if coupled to a solid phase (His-Trap beads). (D) The fact that .DELTA.N58.DELTA.C99 CsxA can be eluted from the column after the reaction had been performed demonstrates that the construct was coupled to the solid phase during the whole period of the reaction.

[0357] The Examples illustrate the invention.

EXAMPLE 1

Biochemical Characterisation of the Capsule Polymerases of NmA (CsaB) and NmX (CsxA)

Materials and Methods

[0358] Freshly transformed E. coli BL21(DE3) were grown at 15.degree. C. in PowerBroth medium for 18 h. At an optical density of OD.sub.600=1.0 protein expression was induced by addition of 0.1 mM IPTG and allowed to proceed for a period of approximately 20 h. Pellets from 125 mL expression culture were pelleted by centrifugation (6,000.times.g, 10 min, 4.degree. C.). After a washing step with PBS, cells were re-suspended in 7.5 ml binding buffer (50 mM Tris pH 8.0, 300 mM NaCl) complemented with 40 .mu.g/ml Bestatin (Sigma), 1 .mu.g/ml Pepstatin (Applichem), 100 .mu.M PMSF (Stratagene) and sonified (Branson Digital Sonifier, 50% amplitude, 8.times.30 s, interrupted by cooling on ice). After centrifugation at 27,000.times.g for 30 min, the soluble fractions were directly loaded onto a HisTrap column (GE Healthcare) to enrich the recombinant proteins by immobilized metal ion affinity chromatography (IMAC). Columns were washed with binding buffer (50 mM Tris pH 8.0, 300 mM NaCl) and proteins eluted in step gradients using 10%, 30%, 50% and 100% elution buffer (binding buffer containing 500 mM imidazol). Fractions containing recombinant protein were pooled and the buffer exchanged to storage buffer (50 mM Tris pH 8.0, 50 mM NaCl for CsaA/CsaB; 50 mM Hepes pH 7.05, 100 mM NaCl, 5 mM MgCl.sub.2 and 1 mM EDTA for CsaC) using the HiPrep 26/10 Desalting column (GE Healthcare). Isolated proteins were concentrated using Amicon Ultra centrifugal devices (Millipore 30 MWCO). After separation into aliquots, samples were snap frozen in liquid nitrogen and stored at -80.degree. C.

[0359] For activity testing a radioactive assay system, which is an adaptation of a protocol described by Weisgerber (1990; J. Biol. Chem. 265, 1578-1587) was used. Briefly, assays were carried out with 5 .mu.l of the soluble fractions of bacterial lysates expressing either recombinant CsaB and CsaA or CsxA or the respective purified and epitope-tagged proteins in a total volume of 25 .mu.l assay buffer (50 mM Tris pH 8.0 or various pH for determination of the pH optimum). Divalent cations were added from stock solutions. The reaction was primed with 5 ng of the respective priming oligosaccharides (hydrolysates of the respective capsule polysaccharides) and started by the addition of 0.05 .mu.mol UDP-GlcNAc (Calbiochem) containing 0.05 .mu.Ci UDP-[.sup.14C]-GlcNAc (American Radiolabeled Chemicals). Samples were incubated at 37.degree. C. and 5 .mu.l aliquots spotted onto Whatman 3MM CHR paper after 0, 5, 10 and 30 min. Following descending paper chromatography, the chromatographically immobile .sup.14C-labeled CPSA was quantified by scintillation counting.

[0360] Activity Testing of CsaB (Together with CsaA) and CsxA by Use of a Multi-Enzyme Spectrophotometric Assay--

[0361] 1.2 .mu.M of CsaA and 1 .mu.M CsaB were assayed in the presence of 0.25 mM UDP-GlcNAc (Calbiochem), 20 mM MgCl.sub.2 and 50 mM Tris pH 8.0 in a total volume of 100 .mu.L. The consumption of UDP-GlcNAc was coupled to nicotinamide adenine dinucleotide (NADH) consumption using the following enzymes/substrates: 0.25 mM adenosine triphosphate (ATP, Roche), 1 mM phosphoenolpyruvate (PEP, ABCR), 0.3 mM (NADH, Roche), 9-15 units/ml pyruvate kinase, 13.5-21 units/ml lactic dehydrogenase (PK/LDH mix Sigma), 0.05 mg/ml nucleoside monophosphate kinase (Roche). Absorption was measured at 340 nm every 30 min using a Biotek EL 808 96-well plate reader.

Results

The Donor:Acceptor Ratio Influences the Product Distribution of CsaB- and CsxA-Reactions in a Different Manner

[0362] Previous studies have suggested that conjugate vaccines made with intermediate chain-length oligosaccharides, as opposed to high-molecular-weight polysaccharides, are more immunogenic and better at eliciting T cell-dependent antibody responses" (Broker M, Fantoni S. Minerva Med. (2007) 98(5):575-89). Very effective glycoconjugate vaccines are made of polysaccharides of intermediate size like for example the quadrivalent glycoconjugate vaccine Menveo (Novartis).

[0363] In two recent studies we demonstrated that the capsule polymerases from NmA (CsaB) and NmX (CsxA) were able to synthesise CPS chains in vitro which makes them a very attractive target for in vitro vaccine development. However, the synthesised chains were very long and thus not ready for immediate coupling to the toxin.

[0364] With the aim of producing oligosaccharide chains that can be readily coupled to a carrier protein, we wanted to investigate in this study if the product distribution produced by these enzymes can be manipulated.

[0365] In a first experiment, we incubated catalytic amounts of the enzymes (50 nM) in the presence of different ratios of UDP-GlcNAc, the donor sugar, and a mixture of small oligosaccharides ranging in size from DP1-DP10, the acceptor sugars. The reaction was allowed to proceed over night and the resulting products were analysed using high percentage PAGE stained with a combination of Alcian blue/silver. The expected DPs, indicated above the gels (FIG. 1), were calculated from the donor:acceptor ratio.

[0366] As a molecular weight marker, we used hydrolysed CPSA and CPSX samples that were used in the manufacturing process of Menveo. This way, we could judge immediately if the produced product pool had the desired size.

[0367] Indeed, CsaB was able to produce the desired product pool (see FIG. 1A, marker) containing chains ranging from .about.DP20-DP60. Moreover, the observed chains lengths correlate very well with the expected DPs in the respective reaction. This finding emphasizes that the chain lengths produced by CsaB are controlled by the donor:acceptor ratio, indicating a distributive elongation mechanism. It is of note that the repetition of the experiment in the presence of an excess of the epimerase CsaA lead to the same results, emphasizing that the observed finding are not due to limited supply with UDP-ManNAc.

[0368] In contrast, we observed a completely different elongation behaviour from CsxA (FIG. 1D). At very low donor: acceptor ratios CsxA produced only very short chains. However, with increasing donor:acceptor ratio we observed a second product distribution containing chains with a DP much higher than expected. The fact that no intermediate chain lengths were observed indicates that chains with a certain DP are elongated preferably while short chain are disregarded. To test this hypothesis, we repeated the experiment with CPSX oligos ranging from DP10-DP70. Again, the intermediate chain lengths disappeared emphasizing the possibility that those chains are elongated preferably.

Determination of the Minimal Active Domain of CsaB and CsxA

[0369] Both CsxA and CsaB belong to the family of stealth proteins that are involved in protecting pathogens from to the host immune defense system (Sperisen, PLoS Comput Biol. (2005) 1(6):e63). All members of stealth harbour, on amino acid level, four conserved regions separated and/or extended by non-conserved regions of varying length. FIG. 1C, showing a schematic alignment of CsxA and CsaB, illustrates that the two enzymes differ mostly by the length of their terminal extensions. Thus, we hypothesised that the difference in elongation behaviour might be encoded in the enzymes termini. Example 4 demonstrates that an N-terminal .DELTA.69-truncation of CsaB was active and very well expressed. The experiment shown in FIG. 1A was repeated with this construct and led to the same results, indicating that the N-terminal 69 aa are not important for the product distribution.

Determination of the Minimal Active Domain of CsaB and CsxA

[0370] Both CsxA and CsaB belong to the family of stealth proteins (Sperisen et al., 2005). All members of stealth harbour four conserved regions on amino acid level separated and/or extended by non-conserved regions of varying length. Based on these sequence characteristics, a schematic alignment of CsaB and CsxA shows that the two enzymes differ mainly in their terminal extensions (FIG. 1C). Thus, we hypothesised that the difference in elongation behaviour might be encoded in the enzyme's termini and designed truncated constructs (FIG. 1C). Starting with CsxA, primers were designed annealing in regions that were predicted to be unstructured by the secondary structure prediction software PHYRE2 (Kelley and Sternberg, 2009) to make sure that no important structural elements were destroyed. Like the full-length enzyme, the CsxA truncations were expressed as N-terminally MBP- and C-terminally His.sub.6-tagged fusion constructs. The radioactive incorporation assay was used to monitor enzymatic activity from lysates (Fiebig et al., 2014a). While no activity could be detected from constructs lacking CR1 (.DELTA.N104-CsxA) and CR4 (.DELTA.C174-CsxA), truncations of the N- (.DELTA.N58-CsxA) and/or C-terminal (.DELTA.C99-CsxA) extensions were active. A combination of IMAC and SEC was used for the purification of the active constructs. Interestingly, the truncation of the N-terminus led to a nearly six fold increase of purification yield for .DELTA.N58-CsxA (70 mg/l culture) and a two fold increase for .DELTA.N58.DELTA.C99-CsxA (27 mg/l culture) compared to full-length-CsxA (12 mg/l culture) or .DELTA.C99-CsxA (13 mg/l culture) and also reduced the co-purification of degradation products (FIG. 1B). To compare the enzymatic activity of the purified constructs to full-length-CsxA, an adaption of the spectrophotometric assay established for CsaB was used (Fiebig et al., 2014b). Interestingly, the activity of both single truncations was dramatically increased while the activity of the double truncation was comparable to full-length-CsxA (FIG. 31D).

Analysis of the Oligomerisation State

[0371] We recently demonstrated that MBP-CsxA-His.sub.6 forms complexes >669 kDa in the presence of low amounts of NaCl (25 mM) and partly dissociates into tri- to tetrameric assemblies in high salt conditions (700 mM NaCl) (Fiebig et al., 2014a). The preparative SEC step included in the purification procedure of the truncated CsxA constructs indicated that the termini might influence the oligomerisation behavior of this enzyme. To further interrogate this finding, analytical size exclusion chromatography was performed. .DELTA.C99-CsxA showed salt-dependent oligomerisation behavior similar to full-length-CsxA, suggesting that the C-terminus is not involved in the formation of the correct oligomeric state (FIG. 5). However, the truncation of the N-terminus resulted in roughly dimeric assemblies that could be observed at both high and low NaCl concentrations for both .DELTA.N58-CsxA and .DELTA.N58.DELTA.C99-CsxA (FIG. 5).

[0372] In contrast to CsxA, no data is available about the oligomerisation state of CsaB. Since full-length-CsaB is almost exclusively expressed as .DELTA.N69-truncation (Fiebig et al., 2014b), we used .DELTA.N69-CsaB for SEC analysis. Like CsxA, this construct also forms high-molecular weight complexes >669 kDa in low-salt buffer (FIG. 5). However, at high-salt conditions, no defined oligomerisation state could be detected.

The Termini of CsxA Influence its Elongation Mechanism

[0373] Asking if the truncations .DELTA.N58-CsxA, .DELTA.C99-CsxA and .DELTA.N58.DELTA.C99-CsxA showed a difference in their d/a-dependent product profiles, we repeated the experiment shown in FIG. 1 with these constructs. The product profile obtained for .DELTA.N58-CsxA was comparable to the profile of full-length-CsxA (compare FIGS. 1D and 2A) and a repetition of the experiment at additional d/a confirmed the appearance of two distinct product populations (FIG. 2B). Remarkably, the product profile of .DELTA.C99-CsxA (FIG. 2C) is unlike that of the full-length-CsxA, but resembles that of the distributive enzyme CsaB. Combined with testing at further d/a (FIG. 2D), these results demonstrate the absence of a processive step in chain elongation, and suggest that processivity is mediated by the truncated C-terminus of CsxA. Interestingly, this construct shares both elongation behaviour and its C-terminus, ending directly behind CR4, with the CsaB enzyme (FIG. 1C). In comparison to the product profiles obtained for .DELTA.C99-CsxA, .DELTA.N58.DELTA.C99-CsxA requires a far greater d/a to produce products of a similar size (compare FIGS. 2C and E).

[0374] Full-length-CsaB is mostly expressed as .DELTA.N69 truncation with only residual amounts of the original construct present in the purified sample (Fiebig et al., 2014b). Consequently, so far, only the product profile of .DELTA.N69-CsaB has been analysed (FIG. 1A). However, to investigate if the minor amounts of full-length-CsaB can influence the product profile, we also repeated the experiment shown in FIG. 1A with purified full-length-CsaB. No influence on the product profile could be observed (FIG. 2F) indicating that the first 69 amino acids are not important for the elongation behaviour of this polymerase.

Determination of the Minimal Acceptor Used by CsaB- and CsxA-Constructs

[0375] Protocols in glycoconjugate vaccine manufacturing often require free reducing ends for the conjugation of saccharides to the carrier protein (Costantino et al., 2011). Thus, we wanted to determine the minimal length of CPS acceptors with a free reducing end needed for chain elongation for each of the active constructs. We were especially interested if the d/a-dependent length control of .DELTA.N69-CsaB, .DELTA.C99-CsxA and .DELTA.N58.DELTA.C99-CsxA would be influenced by the DP of the acceptor. Acceptors of a single DP were purified from a mixture of hydrolysed, dephosphorylated CPSX and CPSA using anion exchange chromatography (FIG. 3A). The DP of purified material was confirmed by HPAEC-PAD and .sup.1H NMR using established protocols (Costantino et al., 1992; Berti et al., 2012). ManNAc-1P could already be excluded as acceptor for CsaB in an earlier study (Fiebig et al., 2014b). Consequently, the acceptor quality of monomeric compounds (GlcNAc, GlcNAc-1P) was only tested for CsxA. Since both CsaB and CsxA exhibit a de novo polymerisation activity which is considerably less efficient than elongation of an acceptor, we considered acceptors stimulating an increased amount of product synthesis compared to the de novo reaction to be acceptors for the enzymes.

[0376] Full-length-CsxA and the single truncations .DELTA.N58-CsxA and .DELTA.C99-CsxA were able to elongate acceptors with a DP.gtoreq.2 while the presence of the monomers GlcNAc-1P and GlcNAc did not lead to product signals beyond de novo background (FIG. 3 C, D, F). Interestingly, a repetition of the experiment with an acceptor concentration increased by 10-fold showed that acceptors of a DP.gtoreq.4 are particularly useful for the d/a-dependent length-control of .DELTA.C99-CsxA (FIG. 3E). Repeatedly no CPSX was synthesised by .DELTA.N58.DELTA.C99-CsxA in the presence of DP2 and activity seemed to be impaired also in reactions complemented with DP3 (FIG. 3G). It is of note that the products of .DELTA.N58.DELTA.C99-CsxA were considerably smaller than those of .DELTA.C99-CsxA under the same reaction conditions (compare FIGS. 3D and G).

[0377] For .DELTA.N69-CsaB, a first screening of short CPSA acceptors of a single DP showed elongation for chains .gtoreq.DP2 (FIG. 3I). In agreement with an earlier study suggesting that a phosphate, i.e. a phosphodiester (instead of a methyl-group) at the reducing end is helpful for DP2-uptake (Fiebig et al., 2014b), DP2 with a free reducing end was elongated less efficiently even though HPLC-AEC analysis of DP2 and DP3 at 214 nm indicated that the concentration used in the experiment was comparable for both acceptors. Interestingly, an increase of the acceptor concentration by 25-fold led to an increase in enzymatic activity in the presence of DP2 (FIG. 3J). However, in agreement with the observation for .DELTA.C99-CsxA, the best length-control of the product pool was achieved with acceptors of DP.gtoreq.4 (FIG. 3J) and even after a further 10-fold increase (compared to the concentrations used in FIG. 3J), the size of the product population in the presence of DP2 was not significantly reduced (FIG. 3H).

Analysis of the Time-Resolved Elongation Behavior of CsaB and CsxA

[0378] To provide further insight into the elongation mechanism used by the different constructs, we analysed the time-course of the polymerase reactions. For this analysis the donor and acceptor concentrations were increased to allow detection by HPLC-AEC at 214 nm while maintaining a d/a sufficient for the production of long chains. Samples of each reaction were heat-inactivated after the indicated time-points and analyzed via PAGE followed by Alcian-blue/silver staining or HPLC-AEC, which was calibrated using the purified DPs (FIG. 3A).

[0379] During the first 60 minutes, full-length-CsxA mainly synthesized short chains (FIG. 4A) that were at later time-point converted into a broad production population consisting of high-molecular weight material. Similar to the full-length construct, .DELTA.N58-CsxA showed the characteristics of a processive enzyme and a preference for intermediate chain lengths (FIG. 4B).

[0380] The fact that the product profile of .DELTA.C99-CsxA is perfectly controllable via the d/a suggests distributive elongation (FIG. 2C). However, this construct produces broad product profiles with high dispersity over time, which is an indication for a certain degree of processivity (FIG. 4E). Nevertheless, compared to full-length- and .DELTA.N58-CsxA (FIGS. 4A and B), no gap indicating biased acceptor binding could be detected (FIG. 4E and FIG. 32 A).

[0381] .DELTA.N58.DELTA.C99-CsxA appears to be fully distributive over time and again, produces much shorter chains and narrower distributions compared to .DELTA.C99-CsxA (compare FIGS. 4E/F and 32 A/B). However, a repeat of the experiment with lower acceptor concentrations indicated that the synthesis of longer chains is possible (FIG. 4D).

Experimental Procedures

[0382] General Cloning--

[0383] The generation of MBP-csxA-His.sub.6, csaB.sub.co-His.sub.6 and d69-csaB.sub.co-His.sub.6 has been reported elsewhere (Fiebig et al., 2014b; Fiebig et al., 2014a). All truncated constructs described herein were amplified by polymerase chain reaction (PCR) using the primers shown in Table 1 and genomic DNA of Nm strain Z2491, csaB.sub.co (Fiebig et al., 2014b) or pHC19 (for CsxA sequences)(Fiebig et al., 2014a) as template. After restriction digest using BamHI/XhoI csaB truncations were cloned into pET22b-Strep (Schwarzer et al., 2009) and csxA truncations were cloned into MBP-csxA-His.sub.6 (T7) or MBP-csxA-His.sub.6 (tac) (Fiebig et al., 2014a).

TABLE-US-00001 TABLE 1 Primers used in this study. Restriction sites are highlighted in bold. Primer pair Resulting construct GCAGATCTATTGATTTAGT StrepII-.DELTA.N235-CsaB-His.sub.6 ATTTACTTGG CCGCTCGAGTTTCTCAAAT GATGATGGTAATG GCAGATCTACTTTAAGTTC StrepII-.DELTA.N167-CsaB-His.sub.6 ATCTATATCT CCGCTCGAGTTTCTCAAAT GATGATGGTAATG GCAGATCTCCTTCTAATCT StrepII-.DELTA.N97-CsaB-His.sub.6 TACTCTTAAGC CCGCTCGAGTTTCTCAAAT GATGATGGTAATG GCAGATCTTTTATACTTAA StrepII-.DELTA.C41-CsaB-His.sub.6 TAACAGAAAATGGC CGCCTCGAGAAAATTCCTT TCTTTAGTTAAGG GCAGATCTTTTATACTTAA StrepII-.DELTA.C25-CsaB-His.sub.6 TAACAGAAAATGGC CGCCTCGAGGTGACTATCA GCACCATCG CGGGATCCCCAATTGAAGA MBP-.DELTA.N58-CsxA-His.sub.6 TCCATACCCAGTA CCGCTCGAGTTGTCCACTA GGCTGTGATG GCGGATCCATTATGAGCAA MBP-.DELTA.C99-CsxA-His.sub.6* AATTAGCAAATTG CCGCTCGAGGAGAATTTCT GCTTCTGATACATC CGGGATCCCCAATTGAAGA MBP-.DELTA.N58.DELTA.C99-CsxA-His.sub.6 TCCATACCCAGTA CCGCTCGAGGAGAATTTCT GCTTCTGATACATC CGGGATCCGAAAGCACCGA MBP-.DELTA.N104-CsxA-His.sub.6 TATTGCAAGATTCC CCGCTCGAGGAGAATTTCT GCTTCTGATACATC GCGGATCCATTATGAGCAA MBP-.DELTA.C174-CsxA-His.sub.6 AATTAGCAAATTG CCGCTCGAGTTGGCCTGTC AAAATGGCAAAATGGTGG *Leucin 388 of the CsxA sequence, normally encoded by ctt, is encoded by ctc from the XhoI site used for cloning.

[0384] Expression and Purification of Recombinant CsaB- and CsxA-Constructs--

[0385] Expression and purification of all CsaB- and CsxA-based sequences was performed as described in (Fiebig et al., 2014b) and (Fiebig et al., 2014a), respectively. For activity testing from lysates and subsequent analysis using the radioactive incorporation assay, csxA and its truncations were expressed from MBP-csxA-His.sub.6 (T7)-based constructs using BL21(DE3) as expression host (Fiebig et al., 2014a). Purification was performed using MBP-csxA-His.sub.6 (tac)-based constructs and M15(pREP4) as expression host.

[0386] Activity testing from lysates was performed using the radioactive incorporation assay as described before (Fiebig et al., 2014b; Fiebig et al., 2014a). The spectrophotometric assay published for CsaB (Fiebig et al., 2014b) was adapted for CsxA resulting in the alterations as follows; (i) no CsaA (epimerase) was used in the reaction mixture, (ii) the reaction was performed using 54 nM of the respective CsxA construct in the presence of (iii) 37 ng CPSX oligosaccharides of avDP18.6.

[0387] SDS-PAGE and Immunoblotting--

[0388] SDS-PAGE and immunoblotting was performed as described before (Fiebig et al., 2014b; Fiebig et al., 2014a).

[0389] Purification of CPSA and CPSX Oligosaccharides--

[0390] CPSA and CPSX oligosaccharide mixtures (Fiebig et al., 2014b; Fiebig et al., 2014a) were dephosphorylated using either acid phosphatase (Sigma) or alkaline phosphatase (CIP, NEB) according to the manufacturer's guidelines. Anion exchange chromatography (AEC) was performed on an AKTA.sub.FPLC (GE Healthcare) equipped with a MonoQ HR 5/5 column (Pharmacia Biotech) at a flow-rate of 1 ml/min. H.sub.2O and 1 M NaCl were used as mobile phases M.sub.1 and M.sub.2, respectively. Samples were separated using a combination of linear gradients (0% to 5% over 1 ml, 5% to 20% over 10 ml, 20% to 30% over 20 ml). The amount of oligosaccharide in each fraction was estimated from the peak area obtained at 214 nm under the assumption that each residue contributes equally to the absorbance of the respective oligomer. Fractions were dialysed against water (ZelluTrans, Roth, 1 kDa MWCO), freeze-dried and equal concentrations were adjusted with H.sub.2O. The identity and DP of the oligosaccharide fractions was confirmed by HPLC-PAD and .sup.1H NMR using established protocols (Berti et al., 2012; Ravenscroft et al., 1999; Costantino et al., 1992).

[0391] Analysis of CsaB and CsxA Reaction Products--

[0392] For the d/a dependent analysis, 100 nM CsaB and 50 nM CsxA constructs were incubated in 25-100 uL reaction buffer (50 mM Tris pH 8.0, 20 mM MgCl.sub.2, (2 mM DTT, only for CsxA)) in the presence of 2 mM (CsaB) or 5 mM (CsxA) UDP-GlcNAc and acceptors in a concentration resulting in the d/a ratios indicated in FIGS. 1 and 4. CsaB reactions were supplemented with 0.1 .mu.M (FIG. 1A) or 1 .mu.M (FIG. 4A) CsaA for in situ UDP-ManNAc production.

[0393] For the time-dependent analysis, the reaction volume was upscaled to 700 uL. The UDP-GlcNAc concentration was increased to 10 mM and the CsaB concentration was decreased to 50 nM. Aliquots were snap-frozen after the indicated time-points and heat-inactivated for 3-5 min at 98.degree. C. HPLC-AEC and high percentage PAGE followed by Alcian blue/silver staining was performed as described before (Fiebig et al., 2014b).

[0394] Analytical Size-Exclusion Chromatography--

[0395] Roughly 300 ug of recombinant protein was analysed at 280 nm on an AKTA.sub.FPLC (GE Healthcare) equipped with a Superdex 10/300 GL column (GE Healthcare) using 50 mM Tris pH 8.0 elution buffer supplemented with NaCl as indicated in FIG. 3. For the analysis of CsxA constructs, 1 mM of DTT was added. The column was calibrated using the Gel Filtration Markers Kit for Protein Molecular Weights 29,000-700,000 Da (Sigma) according to the manufacturer's guidelines.

REFERENCE LIST

[0396] Berti F, Romano M, Micoli F, Pinto V, Cappelletti E, Gavini M, Proietti D, Pluschke G, MacLennan C, Costantino P (2012) Relative stability of meningococcal serogroup A and X polysaccharides. Vaccine 30: 6409-6415

[0397] Costantino P, Rappuoli R, Berti F (2011) The design of semi-synthetic and synthetic glycoconjugate vaccines. Expert Opin Drug Discov 6: 1045-1066

[0398] Costantino P, Viti S, Podda A, Velmonte M, Nencioni L, Rappuoli R (1992) Development and phase 1 clinical testing of a conjugate vaccine against meningococcus A and C. Vaccine 10: 691-698

[0399] Fiebig T, Berti F, Freiberger F, Pinto V, Claus H, Romano M R, Proietti D, Brogioni B, Stummeyer K, Berger M, Vogel U, Costantino P, Gerardy-Schahn R (2014a) Functional expression of the capsule polymerase of Neisseria meningitidis serogroup X: a new perspective for vaccine development. Glycobiology 24: 150-158

[0400] Fiebig T, Freiberger F, Pinto V, Romano M R, Black A, Litschko C, Bethe A, Yashunsky D, Adamo R, Nikolaev A, Berti F, Gerardy-Schahn R (2014b) Molecular cloning and functional characterisation of components of the capsule biosynthesis complex of Neisseria meningitidis serogroup A: towards in vitro vaccine production. J Biol Chem

[0401] Kelley L A, Sternberg M J (2009) Protein structure prediction on the Web: a case study using the Phyre server. Nat Protoc 4: 363-371

[0402] Ravenscroft N, Averani G, Bartoloni A, Berti S, Bigio M, Carinci V, Costantino P, D'Ascenzi S, Giannozzi A, Norelli F, Pennatini C, Proietti D, Ceccarini C, Cescutti P (1999) Size determination of bacterial capsular oligosaccharides used to prepare conjugate vaccines. Vaccine 17: 2802-2816

[0403] Schwarzer D, Stummeyer K, Haselhorst T, Freiberger F, Rode B, Grove M, Scheper T, von Itzstein M, Muhlenhoff M, Gerardy-Schahn R (2009) Proteolytic release of the intramolecular chaperone domain confers processivity to endosialidase F. J Biol Chem 284: 9465-9474

[0404] Sperisen P, Schmid C D, Bucher P, Zilian O (2005) Stealth proteins: in silico identification of a novel protein family rendering bacterial pathogens invisible to host immune defense. PLoS Comput Biol 1: e63

EXAMPLE 2

Dissection of Hexosyl- and Sialyltransferase-Domain in the Bi-Functional Capsule Polymerases from Neisseria meningitidis W-135 and Y Provides Evidence for the Existence of a New Sialyltransferase Family

Abstract

[0405] Crucial virulence determinants of disease causing Neisseria meningitidis (Nm) species are their extracellular polysaccharide capsules (CPSs). In the serogroups W-135 and Y these are heteropolymers of the repeating units [.fwdarw.6)-.alpha.-D-Gal-(1-4)-.alpha.-Neu5Ac-(2.fwdarw.].sub.n in NmW-135 and [.fwdarw.6)-.alpha.-D-Glc-(1.fwdarw.4)-.alpha.-Neu5Ac-(2.fwdarw.].sub.n in NmY. We recently showed that the capsule polymerases, SiaD.sub.W-135 and SiaD.sub.Y, which synthesise these highly unusual polymers, are composed of two GT-B folded glycosyltransferase domains (an N-terminal hexosyl- and a C-terminal sialyltransferase domain) and a linker region (amino acids 399-762) of unknown function. Here we use a mutational approach and synthetic fluorescent substrates to define the boundaries of the hexosyl- and sialyltransferase domains. Our results reveal that the active sialyltransferase domain encompasses a large portion of the linker region and indeed may define a new family in the CAZy classification.

Experimental Procedures

[0406] Generation of Truncation Mutants and Purification of Recombinant Proteins.

[0407] To separate hexosyl- and sialyltransferase the clones pHC4 (SiaD.sub.W-135) and pHC5 (SiaD.sub.Y) (Claus (1997) Mol. Gen. Genet. 257, 28-34) were used as templates and truncation mutants were generated by PCR as shown in FIG. 12 and FIG. 16. Hot start Phusion-DNA-Polymerase (Thermo Scientific; Fermentas) was used in these experiments primers containing NdeI or XhoI sites and PCR conditions were: 1 cycle of 98.degree. C./120 s; 30 cycles 98.degree. C./15 s, 65.degree. C./30 s, 72.degree. C./30 s, and 1 cycle 72.degree. C./300 s. Used primers together with the obtained truncation variants are listed in Table 2. PCR-products after digestion with NdeI and XhoI (New England BioLabs.RTM..sub.ICn.) were purified and ligated into the respective sites of the expression vector pET22-b (Novagen). After transformation into E. coli XL-1 Blue (Stratagene), transformed colonies where selected on ampicillin and constructs controlled by restriction analysis and sequencing. Expressed proteins carried a C-terminal His.sub.6-epitope. Purification of recombinant proteins was carried out as described (Romanow, J Biol Chem. (2013) 26; 288(17):11718-30).

TABLE-US-00002 TABLE 1 Bacterial strains, plasmids and primers used in this study. Strains/ plasmids/ primers Description or sequence Reference E. coli strains BL21(DE3) B; F.sup.-ompT hsdS.sub.B(r.sub.B.sup.-m.sub.B.sup.-) gal dcm (DE3) Novagen XL1-Blue RecA1 ebdA1 gyrA96 thi-1 hsdR17 supE44 Stratagene Plasmids pET-22b(+) Novagen Primers KS422/KS273 5'-GCATCTCATATGGCTGTTATTATATTTGTTAACG-3' wt NmW-135 5'-CCGCTCGAGTTTTTCTTGGCCAAAAAACTG-3' KS422/KS273 5'-GCATCTCATATGGCTGTTATTATATTTGTTAACG-3' wt NmY 5'-CCGCTCGAGTTTTTCTTGGCCAAAAAACTG-3' Point mutants SiaD.sub.W-135 KS350/KS351 5'-CTGATCATGACATCAGAAAGTGCGGGATTTCCATATATATTTATG-3' E307A 5'-CATAAATATATATGGAAATCCCGCACTTTCTGATGTCATGATCAG-3' KS370/KS371 5'-ATCTCGCGTTGCTGTAGGTGTTTATGCAACTAGCTTATTTG-3' S972A 5'-CAAATAAGCTAGTTGCATAAACACCTACAGCAACGCGAGAT-3' Point mutants SiaD.gamma. AR11/AR12 5'-ATACAGATATCCTAATCATGACATCTCAAAGCGCAGGCTTTGGTTATATAT-3' E307A 5'-ATATATAACCAAAGCCTGCGCTTTGAGATGTCATGATTAGGATATCTGTAT-3' Truncations SiaD.sub.W-135 KS422/KS421 5'-GCATCTCATATGGCTGTTATTATATTTGTTAACG-3' C-.DELTA.639 5'-CCGCTCGAGGCTGCGCGGAAGAATAGTG-3' AR2/KS273 5'-GCATCTCATATGTTTAATAACGTATCATTATCGTC-3' N-.DELTA.398 5-'CCGCTCGAGTTTTTCTTGGCCAAAAAACTG-3' AR1/KS273 5'-GCATCTCATATGACTGATGATAATTTAATACCTAT-3' N-.DELTA.562 5-'CCGCTCGAGTTTTTCTTGGCCAAAAAACTG-3' AR9/AR273 5'-GCATCTCATATGAAATATTCTTATAAATATATCTA-3' N-.DELTA.609 5-'CCGCTCGAGTTTTTCTTGGCCAAAAAACTG-3' AR10/KS273 5'-GCATCTCATATGTCTTGGGAACTTATTCGTGCCTC-3' N-.DELTA.639 5-'CCGCTCGAGTTTTTCTTGGCCAAAAAACTG-3' KS433/KS273 5'-GCATCTCATATGGGTAAGCGTTCGATGGATG-3' N-.DELTA.676 5-'CCGCTCGAGTTTTTCTTGGCCAAAAAACTG-3' KS434/KS273 5'-GCATCTCATATGTCACTGAAAAGTAATGTAGTTG-3' N-.DELTA.729 5-'CCGCTCGAGTTTTTCTTGGCCAAAAAACTG-3' KS435/KS273 5'-GCATCTCATATGAATATCGAAGCATTTCTAAAACC-3' N-.DELTA.777 5-'CCGCTCGAGTTTTTCTTGGCCAAAAAACTG-3' Truncations SiaD.gamma. KS422/KS421 5'-GCATCTCATATGGCTGTTATTATATTTGTTAACG-3' C-.DELTA.639 5'-CCGCTCGAGGCTGCGCGGAAGAATAGTG-3' KS422/KS374 5'-GCATCTCATATGGCTGTTATTATATTTGTTAACG-3' C-.DELTA.479 5'-CCGCTCGAGCGTTTGCATGTTGGGTAAAG-3' AR2/KS273 5'-GCATCTCATATGTTTAATAACGTATCATTATCGTC-3' N-.DELTA.398 5-'CCGCTCGAGTTTTTCTTGGCCAAAAAACTG-3' AR1/KS273 5'-GCATCTCATATGACTGATGATAATTTAATACCTAT-3' N-.DELTA.562 5-'CCGCTCGAGTTTTTCTTGGCCAAAAAACTG-3' KS433/KS273 5'-GCATCTCATATGGGTAAGCGTTCGATGGATG-3' N-.DELTA.676 5-'CCGCTCGAGTTTTTCTTGGCCAAAAAACTG-3' KS434/KS273 5'-GCATCTCATATGTCACTGAAAAGTAATGTAGTTG-3' N-.DELTA.729 5-'CCGCTCGAGTTTTTCTTGGCCAAAAAACTG-3' Recloning 5'-GCATCTCATATGGGTAAGCGTTCGATGGATG-3' primer 5'-CCGCTCGAGTTTTTCTTGGCCAAAAAACTG-3'

[0408] Synthesis and Purification of Fluorescently Labelled Acceptors to Prime SiaD.sub.W-135 and SiaD.sub.Y Reactions.

[0409] Because activity testing in the classical radioactive incorporation assay depends on polymer formation (short oligosaccharide primers are washed out in the paper chromatography step (Weisgerber and Troy (1990) J. Biol. Chem. 265, 1578-1587), the reliable testing of monovalent mutants needed a test system in which single sugar transfers can be identified. We exploited the capability of SiaD.sub.W-135 and SiaD.sub.Y to extend sialic acid derivatives carrying the fluorescent label 2-(4-methylumbelliferyl) (4-MU) at the reducing end. The recombinant C-terminally His.sub.6-tagged monovalent full length enzymes NmW-135-(S972A)-His.sub.6 and NmY-(S972A)-His.sub.6 were used as hexosyltransferases and NmW-135-(E307A)-His.sub.6 and NmY-(E307A)-His.sub.6 as sialyltransferases. 4-MU-Sia-Gal/Glc were obtained by mixing 4 mM 4-MU-Sia*Na (Sigma or Iris Biotech GmbH) with 4 mM donor sugar UDP-Gal/UDP-Glc (Sigma) and 20 .mu.g ml.sup.-1 of the respective hexosyltransferase (NmW-135-(S972A)-His.sub.6 or NmY-(S972A)-His.sub.6) in reaction buffer (20 mM Tris/HCl pH 8.0, 20 mM MgCl.sub.2, and 2 mM DTT). After 24 h at 25.degree. C. enzymes were removed by ultra-filtration (Amicon.RTM.Ultra 10 Molecular Weight Cut-Off (MWCO) Millipore) and filtrates containing the reaction products (4-MU-Sia-Gal or 4-MU-Sia-Glc) were lyophilised (Christ; Alpha 1-2 LD plus). After dissolution in water, samples were desalted on P2-gel filtration columns (Bio-Rad). Reaction products were identified by high-performance liquid chromatography using an UFLC-RX system (Shimadzu) coupled to a fluorescence detector (RF-10A XL). Samples were excited at 315 nm and monitored at 375 nm. Although, under the conditions used, this reaction did not give product yields >90% (see FIG. 9) further product purification was omitted because the acceptor quality increased exponential from 4-MU-Sia to 4-MU-Sia-Hex-Sia, making 4-MU-Sia an irrelevant contaminant, which was however useful as an internal standard in consecutive HPLC runs. Obtained 4-MU-Sia-Gal/Glc were then the starting material for Sia transfer to obtain 4-MU-Sia-Gal/Glc-Sia. This reaction was carried out with either NmW-135-(E307A)-His.sub.6 or NmY-(E307A)-His.sub.6 in the presence of 2 mM CMP-Neu5Ac (Nacalai tesque). Reaction conditions were identical to those described in the first reaction step. However, due to significantly improved acceptor, the sialylation of the starting material was complete after 1 h incubation, after removal of enzyme and desalting, obtained compounds were used in iterative rounds to synthesise primers of the needed size.

[0410] Enzyme Testing.

[0411] Radioactive incorporation assays were performed as described (Romanow, J Biol Chem. (2013) 26; 288(17):11718-30). For activity testing with fluorescent compounds, reactions were carried out in a total volume of 25 .mu.l. Mixtures contained 50 mM Tris/HCl pH 8.0, 20 mM MgCl.sub.2, 2 mM DTT, 1 mM acceptor (4-MU-Sia-Gal/Glc, 4-MU-Sia-Gal/Glc-Sia, or 4-MU-Sia-Gal/Glc-Sia-Gal/Glc) plus 2 mM of the nucleotide sugar (UDP-Gal/Glc; Sigma and/or 2 mM CMP-Neu5Ac; Nacalai tesque) depending on the tested enzyme. Reactions were started by addition of 20 .mu.g ml.sup.-1 purified enzyme or, if the soluble fraction of bacterial lysates was used as enzyme source, with 72-100 .mu.g ml.sup.-1 total protein. After appropriate incubation times, reactions were stopped by shock freezing in liquid nitrogen.

[0412] Synthesised polymers were analysed and quantified via the fluorescent tag. Separation of 4-MU-labelled oligo- and polysaccharides was achieved by anion exchange chromatography (CarboPac.RTM. PA-100 column, Dionex) using an ultrafast HPLC system (UFLC-RX, Shimadzu) with coupled fluorescence detection (FD; detector RF-10A XL). Before loading onto the CarboPac.RTM. PA-100 column, samples were 500-fold diluted in water. For the elution the buffer components A (20 mM NaNO.sub.3) and B (1 M NaNO.sub.3) were used to establish a curved gradient, reaching 21.65% buffer B over 35 min. The flow rate was set to 0.6 ml min.sup.-1 and the column temperature to 50.degree. C. The curved portion of the gradient is described by the following formula in with the index -1.425 describes the slope (LS Solution; Shimadzu; Keys, Glycobiology (2013); 23(5):613-8).

B % = 21.65 * ( - 1.425 t / 25 - 1 ) ( - 1.425 - 1 ) ##EQU00001##

[0413] Elution profiles were monitored via fluorescence emission at 375 nm with 315 nm as extinction wave length. Under these conditions the separation of polysaccharides up to a degree of polymerisation (DP)>18 was easily achieved (see results).

[0414] SDS-PAGE and Immunoblotting.

[0415] SDS-PAGE was performed under reducing conditions using 2.5% (v/v) .beta.-mercaptoethanol. Western blot analysis was done on a PVDF membrane (Millipore). For detection of the hexahistidine tag, penta-His antibody (Qiagen) was used as first antibody at a concentration of 1 .mu.g ml.sup.-1 and detected with 0.05 .mu.g ml.sup.-1 IgG.sub.1 anti mouse IR Dye800 antibody (Odyssey Infrared Imaging.RTM.). Protein bands were visualised and quantified with the infrared fluorescence detection system (LI-COR.RTM.) according to the manufacturer's instructions.

Results

[0416] Synthesis of Fluorescently Labelled Oligosaccharides.

[0417] With the intention to physically separate hexosyl (Hex)- and sialyl (Sia)-transferase and functionally express the individual enzyme domains, it was prerequisite to have specific acceptors available that would allow the unequivocal detection of single sugar transfers. As detailed in Experimental Procedures the monovalent mutants NmW-135-(S972A)-His.sub.6 and NmY-(S972A)-His.sub.6, bearing only HexTF activity were used to generate 4-MU-Sia-Hex (4-MU-DP2), which then was substrate for the monovalent enzymes bearing only SiaTF activity (NmW-135-(E307A)-His.sub.6 and NmY-(E307A)-His.sub.6). 4-MU-Sia-Hex-Sia (4-MU-DP3) resulting from this reaction, was an efficient acceptor for the HexTFs. The structures of the generated acceptors (exemplarily shown for oligosaccharides with Gal as hexose) are given in FIG. 8.

[0418] Based on previous experiences with the separation and purification of Sia-containing oligo- and polymers (Glycobiology (2013) 23(5):613-8) a CarboPac.RTM. PA-100 column and a curved NaNO.sub.3 gradient were used to determine the elution positions of 4-MU-Sia and of the elongated products 4-MU-DP2-4-MU-DP4. Shown in FIGS. 9A and 9B, respectively, are the products formed with NmW-135-(S972A)-His.sub.6 and NmY-(S972A)-His.sub.6 as HexTFs and NmW-135-(E307A)-His.sub.6 or NmY-(E307A)-His.sub.6 as SiaTFs. Baseline separation was obtained for all tested compounds. Of note, transfer of the neutral sugar (Gal and Glc in the case of NmW-135 and NmY, respectively) shifted the product peaks left in relation to their precursors (compare 4-MU-DP2 and 4-MU-DP4 to 4-MU-DP1 and 4-MU-DP3, respectively), indicating that the surface charge density is decisive for the elution behaviour of the oligosaccharides. The chromatograms also show that the first hexosyl-transfer onto 4-MU-DP1 remained incomplete under the conditions used, while subsequent transfer reactions proceeded to completion.

[0419] It is important at this point to mention that trials to start the reaction with 4-MU-Gal and 4-MU-Glc as potential substrates for the SiaTF domains were unsuccessful with both, the wild-type enzymes and the monovalent point mutants NmW-135-(E307A)-His.sub.6 and NmY-(E307A)-His.sub.6. This finding strongly suggested that the SiaTF domain needs at least a disaccharide to be active or, alternatively, recognition of the acceptor may need the presence of at least one sialic acid residue.

[0420] The new assay system was then used to record the wild-type SiaD.sub.W-135/Y reactions over time. To visualize the initial reaction steps, the enzymes were primed with 1 mM 4-MU-DP3 in the presence of limited substrate concentrations (CMP-Sia and UDP-Gal/Glc, 2 mM each) and reactions run up to 40 min. Samples were taken between 0.25 min and 40 min as indicated (FIG. 10) and products displayed by HPLC-FD. Exemplarily shown are the results obtained with SiaD.sub.W-135. Under the conditions used the successive elongation of the 4-MU-DP3 primer up to DP19 (in the case of SiaD.sub.Y DP16) could be recorded. The well separated peaks migrated at precisely identical positions in three independent replicates and thus allowed the assignation of retention times to the individual DP (see also Table 2).

[0421] To quantify product formation over time, the relative abundance of each synthesized DP at a given time point was calculated and with the help of the LC Solution software (Shimadzu) normed and weighted to the total curve area giving the normed and weighted values according to the formula:

normed and weighted area = An n = 1 .infin. An * ( n - 1 ) ##EQU00002##

with A being the area underneath a single peak and n the number of transfers needed to form this product. Values were plotted against the reaction time. Data points as shown in (FIG. 10C) are from a single determination. The congruence of the curves obtained in three independent experiments confirms the reliability of the assay system.

TABLE-US-00003 TABLE 2 Degree of synthesised polysaccharides and their corresponding retention time in minutes. CP NmW-135 CP NmY Retention time Degree of Retention time Degree of [min] Polymerisation [min] Polymerisation 2.308 DP.sub.2 2.381 DP.sub.2 3.54 DP.sub.1 3.542 DP.sub.1 4.487 DP.sub.4 4.469 DP.sub.4 5.223 DP.sub.3 5.184 DP.sub.3 6.516 DP.sub.6 6.4 DP.sub.6 7.174 DP.sub.5 7.074 DP.sub.5 8.197 DP.sub.8 7.990 DP.sub.8 8.85 DP.sub.7 8.633 DP.sub.7 9.623 DP.sub.10 9.315 DP.sub.10 10.257 DP.sub.9 9.927 DP.sub.9 10.864 DP.sub.12 10.452 DP.sub.12 11.47 DP.sub.11 11.047 DP.sub.11 11.965 DP.sub.14 11.461 DP.sub.14 12.544 DP.sub.13 12.026 DP.sub.13 12.957 DP.sub.16 12.369 DP.sub.16 13.508 DP.sub.15 12.836 DP.sub.15 13.857 DP.sub.18 14.385 DP.sub.17 14.665 DP.sub.20 15.188 DP.sub.19 15.989 DP.sub.21

[0422] The illustrated table represents the retention time of appropriate produced capsule polysaccharides of NmW-135 and NmY during the elution with 1 M NaNO.sub.3 using an adapted curve gradient on the CarboPac PA-100 column. The elution is based on the charge of oligosaccharides and therefore all polysaccharides with sialic acid on the reducing end are represented by odd numbers and such with hexose on the reducing end are represented by even numbers.

[0423] Of note, while carbohydrate polymerases normally show a kinetic lag phase (Breyer, Protein Science 10, 1699-1711 (2001)), the progress curves obtained with SiaD.sub.W-135/Y represent typical enzyme reaction kinetic with an initial maximum velocity, indicating that products of progressively longer DP are not better acceptors than the starter compound 4-MU-DP3.

[0424] As separation of the two active glycosyltranferase domains was the goal of this study, it was important to reconfirm monovalence (Romanow, J Biol Chem. (2013) 26; 288(17):11718-30) of the single point mutant capsule polymerases NmW-135-(S972A)-His.sub.6 and NmW-135-(E307A)-His.sub.6, used as positive controls in subsequent experiments. The reaction profiles monitored in the presence of both donor sugars are shown in FIG. 11. Single transfers onto the acceptors 4-MU-DP2 and 4-MU-DP3 by NmW-135-(E307A)-His.sub.6 and NmW-135-(S972A)-His.sub.6, respectively, (FIG. 11A,D) could be shown. Instead, the absence of any visible transfer could be shown for reactions started with primers 4-MU-DP3 and 4-MU-DP2 with NmW-135-(E307A)-His.sub.6 and NmW-135-(S972A)-His.sub.6, respectively, (FIG. 11B,C) by providing unequivocal proof for the monovalent nature of these enzymes. Remarkably these pilot experiments in addition showed that Sia transfer onto 4-MU-DP2 occurred very fast. After only 15 sec >70% of the primer substrate (compare 4-MU-DP2 peak in FIG. 11C) were converted into 4-MU-DP3. However, the reaction did not proceed to completion, indicating that and equilibrium between forward and reverse reaction was reached.

[0425] Compared to the sialyltransferase reaction, transfer of Gal onto 4-MU-DP3 was slower, with only about 10% of the primer converted to 4-MU-DP4 after 15 sec reaction time. However, in contrast to the sialyl-transferase reaction this hexosyltransferase reaction proceeded to completeness within 30 min (FIG. 11D). Again, the absence of additional reaction products in FIG. 11D and the zero-activity of the control reaction (FIG. 11C) confirmed monovalence of the point mutant enzyme.

Truncation Mutagenesis to Separate Hexosyl- and Sialyltransferase in SiaD.sub.W-135 and SiaD.sub.Y.

[0426] In FIG. 12A a schematic representation of the linear sequence of SiaD.sub.W-135 and SiaD.sub.Y is shown. The GT-B folds comprising hexosyl- and sialyltransferase domain are shown separated by a stretch of 379 amino acids (dark bar, called linker hereafter). Since in bioinformatics analyses neither sequence homologies nor structural folds could be identified for the linker, we hypothesised that this region is not part of the catalytic domains, but maybe needed to give the GT-B folds the freedom to tertiary organize. This hypothesis was challenged when the GT-B folds (constructs C.DELTA.639 and N.DELTA.777 see FIG. 12A) were separately expressed and tested in the classical radioactive assay system in comparison to the monovalent enzymes. Though well expressed as soluble proteins (see FIG. 12B) activity was only detectable with the hexosyltransferase domains (NmW-135-C.DELTA.639-His.sub.6 and NmY-C.DELTA.639-His.sub.6) and not with construct NmW-135-N.DELTA.777-His.sub.6 (FIG. 12A), harboring the putative sialyltransferase of SiaD.sub.W-135 (data not shown). Because the radioactive assay gave no activity values also with the control enzymes (as outlined in Experimental Procedures, the radioactive assay is not well suited to analyse the single transfer reactions), the assays were also carried out with the newly synthesized acceptors 4-MU-DP2 and 4-MU-DP3. Now, product profiles obtained with the hexosyltransferase domains NmW-135-C.DELTA.639-His.sub.6 and NmY-C.DELTA.639-His.sub.6 were similar to the controls (see FIG. 11), but again, no activity could be detected with the isolated sialyltransferase domains (FIG. 13). These result confirmed that the N-terminal GT-B folds, but not the C-terminal GT-B folds comprise catalytically active enzymes.

[0427] Next, we asked, if the linker (parts of the linker) may be part of the active sialyltransferase. To test this assumption, mutant N.DELTA.398-His.sub.6, which comprises the entire linker sequence (see FIG. 12A) was constructed and was tested directly from bacterial lysates in parallel to the monovalent enzyme (NmW-135-(E307A)-His.sub.6) and the isolated GT-B fold (NmW-135-C.DELTA.639-His.sub.6). Addition of the linker in construct N.DELTA.398-His.sub.6 restored activity (FIG. 13).

[0428] To more precisely define the active sialyltransferase domain additional truncations as shown in FIG. 12A were carried out. In this step, attention was given to not hit predicted secondary structure elements (see FIG. 16). Constructs were expressed in E. coli BL21(DE3) bacterial cell line, protein expression monitored by western blotting and activity determined with the fluorescently labelled acceptor 4-MU-DP2 in the soluble fractions of the lysates of transformed bacteria. While the constructs N.DELTA.562 and N.DELTA.609 showed product profiles similar to the controls (FIG. 14) further truncation of 30 amino acids (N.DELTA.639) completely inactivated the sialyltransferase activity. As shown in FIG. 16, the cut introduced in N.DELTA.609 is likely to separate a .beta.-strand from a predicted helical stretch, which is missing in N.DELTA.639. The presence of this helical element seems of utmost importance not only for the activity but also for the stability of the truncated protein. While N.DELTA.609 is well expressed as a soluble protein, the mutant N.DELTA.639 was foremost found in the insoluble fraction (FIG. 12B). Further truncation as shown for construct N.DELTA.676, N.DELTA.729, and N.DELTA.777 generated recombinant proteins that were well expressed in the soluble fraction, but had not functional activity. This effect on protein stability was confirmed with SiaD.sub.Y (see truncations N.DELTA.676 and N.DELTA.729 in FIG. 12B).

[0429] Together the mutational studies demonstrated that hexosyl- and sialyltransferase can be separated in SiaD.sub.W-135 and SiaD.sub.Y and can be expressed as functionally active proteins. Different to the N-terminal GT-B folds, which encode classical hexosyltransferases belonging to CAZy family GT-4), the C-terminal GT-B folds comprise only parts of the functionally active sialyltransferases. The active sialyltransferases start with a predicted helical segment starting at F609 (see also FIG. 16). The sequence stretch connecting the two GT-B folds is thus not just a linking element, but part of the functional SiaTF domains in SiaD.sub.W-135/Y. Importantly, our data indicate that the capsule polymerases of NmW-135 and Y cannot be easily regulated via the donor-acceptor ratio; see FIG. 33.

[0430] The Sialyltransferase Domains in SiaD.sub.W-135/Y Define a New Sialyltransferase Family.

[0431] As discussed previously (Romanow, J Biol Chem. (2013) 26; 288(17):11718-30) Blast search analyses carried out with the C-terminal GT-B folds of SiaD.sub.W-135 and SiaD.sub.Y were unable to assign the sequences to one of the sialyltransferase families existing in the CAZy classification system (Coutinho (2003) J. Mol. Biol. 328, 307-317). Nevertheless, these searchers revealed weak homology to open reading frames in some bacterial proteins of unknown function. With the current knowledge that the functional sialyltransferase domains in SiaD.sub.W-135/Y encompass the C-terminal part of the linker region (at least F609-I778), the bioinformatics analyses was repeated and in parallel carried out with the programs BLAST and HMMER. Importantly, HMMER identified more than 30 sequences all comprising bacterial proteins of unknown function. After elimination of identical sequences further analyses by multi-sequence alignments clearly showed high conserved motifs (FIG. 15). The D/E-D/E-G motif, which is part of the catalytic centre in bacterial sialyltransferases of CAZy families GT-38, GT-52, GT-80 and in pfam 05855 (Freiberger (2007) Molecular Microbiology 65, 1258-1275), was found to be replaced by QHG (QYA in SiaD.sub.W-135/Y and a sequence encoding a putative sialyltransferase in Listeria). The known bacterial Sia-motifs HP and SS/T, that are involved in the binding of the nucleotide (sugar) (Freiberger (2007) Molecular Microbiology 65, 1258-1275; Yamamoto (2007) Biochem. Biophys. Res. Commun 365, 340-343), are highly conserved also in the newly identified sequences but seem to be part of more extended motifs. Moreover, the new alignment revealed a number of conserved positions many only present in the SiaD.sub.W-135/Y homologues. In summary this alignment suggests the existence of a new sialyltransferase family in which the sialyltransferase domains of the chimeric enzymes SiaD.sub.W-135/Y are the only functionally characterized members. No doubt, based on these data, the allocation of SiaD.sub.W-135/Y into CAZy family GT-4 needs new consideration.

EXAMPLE 3

Neutral Drift of a Polysialyltransferase Yields Enzymes with Processive and Distributive Mechanisms

[0432] The polysialyltransferase (polyST) from Neisseria meningitidis serogroup B (NmB) synthesises a homopolymer of .alpha.2,8-linked sialic acid residues known as polysialic acid. The chemical and immunological identity of the bacterial polymer with polysialic acid expressed in the human brain constitutes a highly effective molecular mimicry which contributes to virulence of NmB (Muhlenhoff, Curr. Opin. Struct. Biol. 8, 558-564 (1998); Roberts, Annu. Rev. Microbiol. 50, 285-315 (1996)). Recent studies have highlighted the potential of the NmB-polyST for polysialylation of therapeutic proteins (Lindhout, Proceedings of the National Academy of Sciences (2011), doi:10.1073/pnas.1019266108), and for direct therapeutic application of polysialic acid to tissues in vivo (Maarouf, J. Biol. Chem. 287, 32770-32779 (2012)). Despite the importance of this enzyme, little is known of its structure and function.

[0433] We have recently developed two new assay systems which enable high throughput screening and detailed characterisation of polyST activity (Keys, Analytical Biochemistry 427, 107-115 (2012); Keys, Analytical Biochemistry 427, 60-68 (2012)). In this study we have used these methods to conduct a neutral drift experiment--subjecting the NmB-polyST to high mutation rates followed by purifying selection to remove inactive variants--to explore functional regions of the polyST sequence space. Detailed analysis of over 50 drifted variants (with 7.3.+-.3.0 amino acid exchanges per sequence) revealed that some sequences had acquired new modes of chain elongation which correspond to either a processive or distributive mechanism of polymerisation. The study identifies sequence elements which control the mechanism of elongation and the dispersity of polymeric products.

[0434] The synthesis of uniform glycan structures is a necessary precursor to their evaluation as therapeutic reagents. We have explored the functional sequence space of a bacterial polysialyltransferase and identified sequence elements controlling the processive or distributive mechanism of chain elongation. The results demonstrate that these properties are independent of enzymatic activity and illuminate a pathway for the synthesis of uniform oligo- and polysaccharide structures for research and therapeutic applications.

[0435] The extracellular polysaccharides of diverse bacterial species, which have emerged from symbiotic or pathogenic coevolution with their human hosts, are a rich source of structures with important biological functions and applications as therapeutic reagents (DeAngelis, J. Glycobiology. 23, 764-777 (2013); Pollard Nat. Rev. Immunol. 9, 213-220 (2009) and Boltje, Nat. Chem. 1, 611-622 (2009)) The application of oligo- and polysaccharides will be greatly advanced by strategies to fine tune the functional properties of the polymerizing glycosyltransferases which synthesize these polysaccharides (Boltje, Nat. Chem. 1, 611-622 (2009) and Schmaltz, Chem. Rev. 111, 4259-4307 (2011)). Here we demonstrate that these enzymes can readily be engineered to produce uniform Poissonian product distributions.

[0436] Our study focused on the polysialyltransferase from Neisseria meningitidis serogroup B (polySTNmB), which synthesizes .alpha.2,8-linked polysialic acid (polySia), a low abundance polymer with unique biological functions and importance as a reagent for research and several emerging therapeutic applications. In mammals, polySia is a dynamically regulated posttranslational modification predominantly found on the neural cell adhesion molecule, NCAM (Muhlenhoff, Neurochem. Res. 38:1134-1143 (2013)). Due to its polyanionic nature, large size, and water binding capacity, polySia acts as a powerful anti-adhesive, globally down regulating cellular interactions and increasing cellular motility (Johnson, J. Biol. Chem. 280, 137-145 (2005)). These unique anti-adhesive properties provide plasticity in the developing and adult nervous system (Rutishauser, Nat. Rev. Neurosci. 9, 26-35 (2008)) and play a role in immune development (Drake, Proc. Natl. Acad. Sci. U.S.A. 106, 11995-12000 (2009)). PolySia is re-expressed at the cell surface by various tumors to promote invasion and metastatic potential (Hildebrandt, Adv. Exp. Med. Biol. 663, 95-109 (2010)). Moreover, some neuroinvasive bacteria produce an extracellular capsule of polySia which aids in evasion of the immune system (Corbett, Adv. Appl. Microbiol. 65, 1-26 (2008)). PolySia is currently being investigated for diverse therapeutic applications, primarily based on the ability to promote plastic processes involved in regeneration in neural tissues (El Maarouf, Adv. Exp. Med. Biol. 663, 137-147 (2010) and El Maarouf, Proc. Natl. Acad. Sci. U.S.A. 103, 16989-16994 (2006)) and its ability to improve the pharmacological profile of therapeutic proteins decorated with polySia (Constantinou, Bioconjugate Chem. 19, 643-650 (2008) and Lindhout Proc. Natl. Acad. Sci. U.S.A. 108, 7397-7402 (2011)). A major step towards these goals is the demonstration that gene therapy and chemical coupling can be avoided in these applications by using the polySTNmB to synthesize polySia directly onto therapeutic proteins (Lindhout, Proc. Natl. Acad. Sci. U.S.A. 108, 7397-7402 (2011)), cultured cells, and neural tissues in vivo (Maarouf, J. Biol. Chem. 287, 32770-32779 (2012)).

[0437] A critical feature of enzymes for use in glycoengineering applications is the ability to synthesize uniform products of defined length. The primary feature determining product length and dispersity for polymerases is the extent of their interaction with the growing polysaccharide acceptor (FIG. 17). An extended binding site confers increased affinity and an increased rate of transfer to longer chains which occupy more of the binding subsites, and may result in processive elongation (Levengood, J. Am. Chem. Soc. 133, 12758-12766 (2011) and Forsee, J. Biol. Chem. 281, 6283-6289 (2006)). This elongation-bias causes broad product distributions which are skewed towards longer chain lengths (FIG. 17a), and underlies the kinetic lag phase commonly observed for polymerizing glycosyltransferases Levengood, M. R., Splain, R. A. & Kiessling, L. L. J. Am. Chem. Soc. 133, 12758-12766 (2011) and Keys Anal. Biochem. 427, 107-115 (2012)). In contrast, the absence of extended interactions with the polysaccharide acceptor confers a distributive mechanism of elongation, characterized by the absence of length-bias during elongation, no kinetic lag phase, and uniform product distributions conforming to a Poisson distribution16 (FIG. 17b).

[0438] With very few exceptions (Jing, J. Biol. Chem. 279, 42345-42349 (2004)), the polymerizing glycosyltransferases which have been characterized to date exhibit an extended acceptor binding site and broad product distributions--properties which are not amenable to biotechnological applications. The polyST.sub.NmB was recently used for enzymatic polysialylation of therapeutic proteins, however, proteins were modified with a range of chain lengths from 4 to >70 residues at each site (Lindhout, Proc. Natl. Acad. Sci. U.S.A. 108, 7397-7402 (2011)). In this case, the assessment of optimal chain length is impossible, and the assessment of the desired modification is weakened due to averaging over many products. In the ideal case, enzymatic modification would be carried out by perfectly distributive enzymes, resulting in uniform chain elongation and a narrow distribution of product lengths (FIG. 17b). For successful application of polymerizing glycosyltransferases, strategies to engineer the product distribution are urgently needed.

[0439] Pioneering studies on ribonuclease A by delCardayre and Raines (delCardayre, Biochemistry 33, 6031-6037 (1994)) demonstrated that substrate binding could be modified independently of catalytic activity, enabling the introduction of processivity without altering activity. We proposed that this should hold true for polymerizing glycosyltransferases. As no structural data is available to identify residues interacting with the acceptor substrate, we conducted a small neutral drift to explore the polyST.sub.NmB sequence space. In a neutral drift, the target protein is subjected to high mutational loads under selection for the native level of catalytic activity, enabling a broad exploration of functional sequence space and facilitating the emergence of new properties (Gupta, Nat. Methods 5, 939-942 (2008)).

[0440] Our previous experiments have shown that truncation of the N-terminus of the polyST.sub.NmB increases soluble expression with little cost to enzymatic activity (Keys, Anal. Biochem. 427, 60-68 (2012)). The .sub..DELTA.25polyST.sub.NmB was therefore used as the starting point for the neutral drift. In total, three rounds of diversity generation and screening were conducted. An initial error prone PCR library was screened using an in vivo activity assay (Keys, Anal. Biochem. 427, 60-68 (2012)) and sequencing of 122 clones revealed 163 `neutral` amino acid exchanges (FIG. 17). In two subsequent rounds these mutations were shuffled through the sequence with a high frequency of recombination within short sequence elements and high overall mutation rates (see Methods). A second tier of in vitro activity screening of a small number of clones (94 in the second round and 188 in the final screen), using a HPLC based assay (Keys, Anal. Biochem. 427, 107-115 (2012)), ensured that the polySTs maintained a level of activity comparable to the .sub..DELTA.25polyST.sub.NmB reference sequence. The in vitro activity screen also proved to be a bottleneck for genetic diversity with only 21 clones passing into the third round of diversity generation. However, due to high mutagenesis rates, the diversity was sufficient to produce a broad range of elongation phenotypes. Final screening yielded a pool of 51 polymorphic sequences with 7.3.+-.3.0 exchanges per sequence (Supplementary Data Set 1) and a range of activity between approximately 25% and 300% of the reference sequences.

[0441] To examine the functional diversity present in the final pool, the reaction time course and pattern of chain elongation was determined for all 51 enzymes. This was achieved using a recently developed fluorescent acceptor, a 1,2-diamino-4,5-methylenedioxybenzene labeled trimer of sialic acid (DMBDP3), with HPLC separation of reaction products (Keys, Anal. Biochem. 427, 107-115 (2012)) adapted to 96-well format (see Methods). In order to be able to distinguish different modes of elongation and make inferences about substrate binding, it is important that reactions are carried out with a large substrate to enzyme ratio (Levengood, J. Am. Chem. Soc. 133, 12758-12766 (2011)), therefore an approximately 100 fold molar excess of the DMB-DP3 acceptor and a 4000 fold molar excess of the donor sugar, CMP-Neu5Ac, were used. The enzymes exhibited diverse reaction kinetics and product profiles suggesting that sequences had indeed drifted in the direction of both processive and distributive mechanisms of elongation. In order to quantify the tendency of each enzyme to produce uniform or disperse product profiles, the product dispersity (Xw/Xn; where Xw is massaverage DP, and Xn is number-average DP) (Stepto, Polym. Int. 59, 23-24 (2010)) was calculated, and the mean dispersity of products over the reaction course was determined for each of the enzymes. The enzymes exhibited a range of mean dispersity values from 1.06-1.33. As a guide, analysis of the .sub..DELTA.25polyST.sub.NmB in the same assay gave a mean dispersity of 1.16, and the dispersity of a uniform polymer sample with Poisson distribution of lengths approaches 1. The sequences were sorted into three categories according to high (>1.2), medium (1.1-1.2), and low (<1.1) dispersity. FIG. 18 shows examples of each category which demonstrate the diverse modes of chain elongation within the pool (results for all analyzed clones are given in Table 4).

[0442] The enzymes also exhibited different reaction kinetics which correlated broadly with the measured product dispersity. Enzymes in the high and medium dispersity categories displayed skewed product profiles and kinetic lag phases typical of polymerases with extended substrate binding sites (FIG. 18a and b). In stark contrast are the product profiles and reaction kinetics of enzymes in the low dispersity category. The majority of these enzymes have no lag phase (i.e. initial reaction rate is the maximum observed rate) and uniformly elongate acceptors giving narrow product distributions (FIG. 18c). For the low dispersity clones, the product distributions throughout the reaction closely fit a Poisson distribution, indicating that these enzymes use a distributive mechanism of elongation.

[0443] Remarkably, the Lys69Gln mutation is exclusively associated with enzymes in the low dispersity category, which display a distributive mechanism of elongation and Poissonian product distributions. The presence of numerous additional mutations in these enzymes indicates that the Lys69Gln exchange plays a dominant role in determining the mechanism of chain elongation. The molecular mechanism by which a single amino acid exchange potently affects chain elongation is of high interest for the rational engineering of related polymerases. We speculate that the mutation either alters interaction with the growing end of the polysaccharide or blocks entry of the polysaccharide to the extended binding site.

[0444] A number of single amino acid exchanges were observed to produce an elongation phenotype. The mutations Arg111His, Lys114Met, Lys143Thr and Lys412Ile increase the strength of interaction with chains of DP>10, increasing the observed lag phase and the dispersity of reaction products compared to .sub..DELTA.25polyST.sub.NmB (FIG. 20). The Lys69Arg, His78Leu and Asn100Ile mutations reduce interaction with the polymer to give a more distributive elongation mechanism (FIG. 20). Interestingly, almost all of the exchanges which were observed to alter the elongation mechanism involve the replacement of large basic amino acids. Together the results indicate that basic residues in the polyST sequence contribute to an extended interaction interface for binding of the polyanionic substrate, and that mutations within this site determine the mechanism of chain elongation.

[0445] Previous neutral drift experiments demonstrated that target enzymes were able to maintain native levels of activity under high mutational loads due to the accumulation of "global suppressor mutations", which stabilized the overall protein structure (Gupta, Nat. Methods 5, 939-942 (2008)). To test if this held true in our neutral drift, despite the considerably smaller screening effort, the most highly accumulated exchanges within the pool were cloned and examined. Impressively, the majority of exchanges which were highly accumulated during the neutral drift were observed to increase the yield of activity when present alone in the .sub..DELTA.25polyST.sub.NmB sequence (Table 3), and had little or no effect on product distribution. Notably, the Phe460Ile exchange was the most common mutation, present in 51% of the analyzed clones, and more than tripled the yield of activity when present alone in the .sub..DELTA.25polyST.sub.NmB sequence. The results are consistent with previous neutral drift experiments (Gupta, Nat. Methods 5, 939-942 (2008)), and suggest that the accumulated mutations may stabilize the polyST structure.

[0446] For the biological function of polymerizing glycosyltransferases it is advantageous to have extended interaction interfaces which bind and increase the rate of synthesis of their polysaccharide products. However, for biotechnological applications, the same interface represents a distinct disadvantage, constituting a barrier to the synthesis of uniform oligo- and polysaccharide structures. The results of this study demonstrate that these interaction interfaces can be readily engineered to yield enzymes with a distributive mechanism and product profiles favorable for research and therapeutic applications.

TABLE-US-00004 TABLE 3 Single mutations Frequency in Elongation Mean Initial reaction rate* Maximum reaction Mutation drifted clones Phenotype dispersity (pmoles/min) rate* (pmoles/min) K69R n.p. reduced lag 1.143 60 .+-. 11.2 96.2 .+-. 5.4 H78L 9% reduced lag 1.146 121.5 .+-. 8.5 179.7 .+-. 11.2 N100I 42% reduced lag 1.168 120 .+-. 22.2 184.1 .+-. 14.5 R111H 32% exacerbated lag 1.23 93.2 .+-. 3.5 274.1 .+-. 10.5 K114M n.p. exacerbated lag 1.214 92 .+-. 4.3 256.8 .+-. 2 K143T n.p. exacerbated lag 1.242 56.1 .+-. 8.9 220.6 .+-. 10.2 I151V n.p. none 1.153 182.2 .+-. 11.2 329.7 .+-. 8 K171T 19% none 1.238 37.2 .+-. 5.7 150.5 .+-. 10.9 N180I 13% none 1.21 86.6 .+-. 6.7 209.8 .+-. 1.7 I192V 21% none 1.18 135.4 .+-. 21.7 274.9 .+-. 18.5 I222V 25% none 1.151 162.8 .+-. 2.3 278 .+-. 15.1 I297T 36% none 1.132 197.5 .+-. 3.3 317.9 .+-. 14.1 I323V 25% none 1.184 102.6 .+-. 7.2 222.8 .+-. 4.6 E368V 32% none 1.213 50.9 .+-. 3.4 140.4 .+-. 3 T402S 21% none 1.16 133.7 .+-. 11.2 249.8 .+-. 11.9 K412I 42% exacerbated lag 1.211 64.4 .+-. 2.6 237.7 .+-. 3.9 F460I 51% none 1.194 273.8 .+-. 20.6 541.1 .+-. 16 .DELTA.25polyST.sub.NmB 1.181 79.8 .+-. 7 195.7 .+-. 1.9 n.p Not present. *Reaction rates are the average .+-. the standard deviation of 3 separate enzyme preparations (from different colonies) in the same experiment.

TABLE-US-00005 TABLE 4 PolyST clones of the neutral pool Mean Initial reaction Maximum reaction Lag phase Expression Level (% Category Clone dispersity rate (pmoles/min) rate (pmoles/min) (Rate.sub.max/Rate.sub.init) of soluble protein) high F154 1.201 57.74 217.11 3.8 1.34 dispersity F117 1.202 50.74 136.81 2.7 1.33 F139 1.205 65.38 248.35 3.8 1.71 F123 1.211 50.40 168.64 3.3 1.44 F162 1.212 36.57 187.99 5.1 1.33 F164 1.215 28.08 188.62 6.7 1.30 F072 1.216 131.50 650.61 4.9 1.65 F015 1.219 71.27 366.88 5.1 1.50 F161 1.221 49.65 186.94 3.8 1.45 F183 1.225 40.65 166.65 4.1 1.41 F126 1.236 89.80 233.34 2.6 1.25 F014 1.238 47.55 228.54 4.8 1.79 F105 1.239 37.50 198.49 5.3 1.18 F024 1.243 78.18 237.61 3.0 1.84 F180 1.248 36.75 191.93 5.2 1.27 F133 1.248 37.04 180.77 4.9 1.09 F102 1.280 69.54 449.11 6.5 1.65 F065 1.286 105.99 358.96 3.4 1.61 F093 1.311 61.08 261.75 4.3 2.70 F074 1.326 31.06 333.17 10.7 1.40 medium F119 1.128 91.88 142.32 1.5 1.13 dispersity F060 1.138 182.61 320.63 1.8 1.72 F069 1.148 105.44 220.62 2.1 1.48 F089 1.157 231.48 526.94 2.3 1.51 F146 1.168 130.02 258.55 2.0 1.55 F179 1.169 362.79 619.09 1.7 1.75 F182 1.170 37.98 105.10 2.8 1.21 F013 1.172 62.31 198.81 3.2 1.40 F043 1.175 265.16 419.78 1.6 2.29 F153 1.179 49.34 209.26 4.2 1.23 F103 1.182 119.99 324.98 2.7 1.31 F032 1.185 80.91 337.12 4.2 1.76 F175 1.187 119.34 348.12 2.9 1.35 F113 1.188 52.10 190.49 3.7 1.26 F036 1.188 53.16 193.45 3.6 1.26 F010 1.193 255.71 506.33 2.0 1.79 F159 1.195 94.80 214.46 2.3 1.26 F087 1.198 232.60 535.45 2.3 1.84 F185 1.198 113.24 278.22 2.5 1.37 low F184 1.060 173.76 173.76 1.0 1.68 dispersity F078 1.065 122.02 122.02 1.0 1.79 F079 1.067 205.99 205.99 1.0 1.53 F116 1.073 362.27 362.27 1.0 1.47 F034 1.077 79.31 79.31 1.0 1.34 F008 1.078 69.62 88.66 1.3 1.50 F129 1.080 111.50 111.50 1.0 1.39 F006 1.085 38.88 49.30 1.3 1.66 F138 1.087 152.25 172.60 1.1 1.35 F038 1.089 249.22 249.22 1.0 2.16 F028 1.092 76.46 134.30 1.8 1.33 F051 1.095 145.70 145.70 1.0 2.40 reference 3775_A 1.160 33.80 134.23 4.0 1.26 sequence 3775_B 1.159 40.65 143.34 3.5 1.29 (repeats) 3775_C 1.160 41.42 147.61 3.6 1.32 3775_D 1.159 35.09 132.17 3.8 1.31 indicates data missing or illegible when filed

Discussion

[0447] Aiming to identify sequence elements contributing to the extended polysialic acid binding site in the polyST.sub.NmB, the in vivo and the in vitro assays were combined into a throughput for exploring the polyST sequence space and identifying relationships with the mode of chain elongation. The sequence space was explored using a neutral drift experiment, where the target sequence is subjected to repeated rounds of mutagenesis and selection for the native level of activity (Bershtein, J. Mol. Biol. 379, 1029-1044 (2008) and Gupta, Nature Methods 5, 939-942 (2008)). Starting from the .sub..DELTA.25polyST.sub.NmB, three rounds of mutagenesis and screening were used to generate a pool of 51 polymorphic sequences (7.3.+-.3.0 amino acid exchanges per sequence) with a range of activity between approximately 25% and 300% of the original sequence. Each of these clones was then characterised in detail using the DMB-DP3 acceptor and HPLC assay. The mutated enzymes exhibited diverse modes of chain elongation. At one end of the spectrum were clones with product distributions suggestive of processive elongation. These clones were characterised by an exacerbated kinetic lag phase, a strong preference for elongated acceptor structures, and highly disperse reaction products. At the other end of the spectrum were clones with a highly distributive mechanism of chain elongation. These were characterised by the absence of a lag phase (i.e. initial reaction rate was the maximum reaction rate), no preference for elongated acceptors, and uniform product distributions.

[0448] The diverse elongation phenotypes were associated with specific amino acid exchanges. Remarkably, the Lys69Gln mutation was exclusively associated with the twelve clones exhibiting a distributive mechanism of elongation and the most uniform product distributions, indicating a dominant role for this residue in determining the mechanism of chain elongation. The Lys69Arg, His78Leu and Asn100Ile mutations also resulted in a narrowing of the product distribution, albeit less striking than that of Lys69Gln. In contrast, the mutations Arg111His, Lys114Met, Lys412Ile and Lys143Thr were demonstrated to increase activity specifically with acceptors of >10 residues in length, causing broadening of the product distributions.

[0449] The fact that most of the exchanges causing an elongation phenotype involve the replacement of basic amino acids suggests that the polyST's extended binding site is lined with basic residues which interact with the negative carboxyl groups of the polysialic acid chain. It is typical of enzymes which act on polymeric substrates to form many weak interactions with the polymer's repeating structural motifs, which prevents the formation of strong specific interactions and is thought to facilitate movement along the polymer (Breyer, Protein Science 10, 1699-1711 (2001)). However, during the initial stages of an elongation reaction, such an extended interaction interface confers an increasing affinity and increasing rate of transfer to chains which occupy more of the binding subsites (Levengood, J. Am. Chem. Soc. 133, 12758-12766 (2011)). The exact strength and extent of this interaction will sensitively affect the observed product distribution by altering the reaction rate in a chain-length dependent manner. This would explain the impressive diversity of product distributions in the neutral drift pool.

[0450] In contrast, a distributive mechanism of elongation indicates the absence of extended interactions with the polysaccharide chain and strictly nonprocessive elongation. Thus, distributive enzymes display no chain-length bias, but uniformly elongate the pool of acceptors resulting in narrow product distributions which approach a Poisson distribution (Chang, Journal of Molecular Biology 93, 219-235 (1975)), as observed for polySTs containing the Lys69Gln exchange. These considerations suggest that the Lys69Gln mutation blocks entry of the polysaccharide to the extended binding site. The proposition that residue 69 is involved in acceptor binding is supported by homology models of the polyST.sub.NmB structure which place this residue on a surface exposed loop in the vicinity of the proposed acceptor binding site (F. Freiberger and H. Fuchs, unpublished data).

[0451] Importantly, the rapid emergence of different mechanisms of elongation, as observed in this study, indicates that polymerising glycosyltransferases can be readily engineered to provide desired product distributions. Most promising in this respect is the introduction of a distributive mechanism with a single amino acid exchange. Distributive enzymes provide uniform (narrow) product distributions and product length is controlled simply by the ratio of donor to acceptor substrates. These are desirable properties for biotechnological applications where control of polysaccharide length is critical for the synthesis of defined structures and for the testing of optimal chain length for specific applications.

Methods

[0452] Molecular Biology.

[0453] Plasmids were isolated using NucleoSpin Plasmid.TM. or Extract.TM. kits from Machery-Nagel (Steinheim, Germany). Enzymes were purchased from New England Biolabs (Frankfurt, Germany) and all steps carried out according to the manufacturer's instructions. The DNA sequence of the .DELTA.25polySTNmB was optimized for expression in E. coli. according to guidelines set out by Welch and colleagues24 and synthesized by Eurofins MWG Operon (Ebersberg, Germany). The gene was cloned into pET32a-Strep22 via BamHI/NotI restriction sites giving the plasmid p3775.

[0454] The first library was created by two rounds of error-prone PCR (epPCR), starting on the p3775 template. Buffer condition 3 (320 .mu.M MnSO4; and an additional 40 .mu.M dGTP) of the Diversify.RTM. PCR random mutagenesis kit (Clontec, California, USA) was used with 100 ng of template plasmid in a 50 .mu.l reaction using and the T7/T7-term primer pair (Supplementary Table 2). PCR amplification was carried out with the following program:

TABLE-US-00006 ##STR00001##

[0455] Reaction products were separated on an agarose gel, the amplified gene purified with the NucleoSpin Extract.TM. kit and eluted in 50 .mu.l. Of the eluted product, 3 .mu.l, was used as template in a second epPCR reaction with the same buffer conditions and thermocycler program. The product of the second reaction was cloned via BamHI/NotI restriction sites into the pET32a-Strep vector22.

[0456] The protocol for the second library was designed to facilitate a high level of recombination within and between the clusters of high mutability identified from sequencing the first round clones (FIG. 19). Degenerate oligonucleotides encoding all of the diversity observed within short sequence elements were designed and ordered from Sigma-Aldrich (Steinheim, Germany) with the highest available purity (HPLC grade). The degenerate oligonucleotides were used to spike standard gene shuffling reactions with fragments (50-600 bp) of the sequenced first-round clones as template. As the optimal level of degenerate oligonucleotide incorporation could not be known, four reactions with different ratios of [oligonucleotides]:[gene fragments] were used and the reaction products pooled in the final library.

[0457] First, 122 plasmids from the first-round clones were pooled and used as template for amplification of the coding sequences using the T7/T7-term primer pair. The amplified product was purified via agarose gel electrophoresis and 1.6 .mu.g was digested with 0.5 units of DNaseI (New England Biolabs, Frankfurt am Main, Germany) in 40 .mu.l volume for 1 min 50 sec at 25.degree. C. The reaction was stopped by addition of 30 mM EDTA and incubation at 75.degree. C. for 10 min. DNaseI products were separated by agarose gel electrophoresis and fragments of 50-600 bp were extracted and purified. Four 30 .mu.l assembly reactions contained either 130 ng, 50 ng, 25 ng or 0 ng of the 38 pooled equimolar oligonucleotides (TK229-TK266; Supplementary Table 2), 130 ng of gene fragments, 3% DMSO, 200 .mu.M dNTPs, 0.5 units Phusion.RTM. DNA polymerase (New England Biolabs, Frankfurt am Main, Germany) and Phusion.RTM. HF buffer. The four assembly reactions were carried out with the following program:

TABLE-US-00007 ##STR00002##

[0458] Of each assembly reaction, 5 .mu.l was used as template to amplify full length genes in separate PCRs. Four 50 .mu.l amplification PCRs were carried out with 0.5 units Phusion.RTM. DNA polymerase, 6% DMSO, and the TK156/TK157 primer pair (Supplementary Table 2). Reactions were thermocycled according to the following program:

TABLE-US-00008 ##STR00003##

[0459] Analysis of 5 .mu.l samples confirmed amplification of similar amounts of the full gene in all reactions. Finally all four reactions were pooled and the products cloned as described for the first library. Sequencing of the library pool ensured incorporation of the desired diversity, and sequencing of 12 random clones indicated a mutation rate of 6.+-.5.9 amino acid exchanges per sequence.

[0460] The protocol for the third library was designed to enable fine-grade shuffling of sequences from the second round of screening with no further introduction of mutations. The 22 plasmids from the second-round clones were pooled, coding sequences were amplified and DNaseI digested as above, and gene fragments of 50-200 bp were extracted and purified. A single PCR assembly and amplification reaction was carried out as described above without the addition of synthetic oligonucleotides. The shuffled product was cloned into the expression vector as described above.

[0461] In vivo activity screening was carried out as previously described22. Briefly, library DNA was transformed into the screening strain, MB3109. Transformants were plated on Luria-Bertani (LB) agar at .about.2000 colonies per 140 mm plate, and grown for 10-12 h at 37.degree. C. until colonies had reached a diameter of 0.5-1 mm. A nitrocellulose membrane was used to copy colonies, then placed colonyside-up onto inducing plates. After incubation for 3 h at 37.degree. C., colonies were lysed and fixed on the membrane and polySia was detected as previously described22. Positive clones were picked from the master plates and the amount of polySia synthesised by each clone was quantified with the same assay in microtiter plate format22. In each round of screening 10,000-20,000 clones were screened and those producing at least 80% of the wild type level of polySia were considered to be neutrally drifting.

[0462] Preparation of Recombinant Protein.

[0463] Clones for analysis were transformed into BL21-gold(DE3). Colonies were picked into 150 .mu.l LB media with appropriate antibiotics in 96-well flat bottom plates to allow for observation of optical density (OD) on a plate reader (PowerWave 340, BioTek). Dense overnight cultures were diluted 1:20 into 200 .mu.l PowerBroth (AthenaES, Baltimore, Md., USA) and adjusted to uniform OD600. Cultures were grown at 37.degree. C. until OD600.apprxeq.1.8, then cooled on ice and supplemented with isopropyl .beta.-D-1-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM. Protein expression was carried out at 15.degree. C. for 20 h with shaking at 1000 rpm. Cultures typically reached an OD600 of 8.0-9.0. Expression cultures were harvested in two 100 .mu.l aliquots, pellets were washed with PBS, and stored at -80.degree. C. until analysis.

[0464] For cell lysis, pellets were resuspended in 100 .mu.l lysis buffer (50 mM Tris pH 8.0, 5% glycerol, 200 .mu.g/ml lysozyme, 10 .mu.g/ml Dnase I, 1 mM PMSF) then subjected to two cycles of freeze-sonication (15 min on ice, snap freezing in liquid nitrogen, and thawing in sonication bath at 23.degree. C.). Unlysed cells and debris were removed by centrifugation at 4000 g for 10 min, and cleared lysates were supplemented with 1 mM EDTA. The final cell free extracts were used directly in activity assays and protein determinations.

[0465] Polysialyltransferase Assays.

[0466] The in vitro activity of polySTs was determined as previously described18 except that all steps were carried out in 96-well microtiter plates using a 96-channel pipette for accuracy. The recombinant enzymes were assayed at 25.degree. C. in 100 .mu.l volumes containing 50 mM Tris-HCl pH 8.0, 25 mM KCl, 20 mM MgCl2, 5% glycerol, 200-1000 .mu.M CMP-Neu5Ac, and 5.32 .mu.M DMBDP3. Reactions were started by the addition of 25 .mu.l of cell free extract. Reaction samples were quenched by 10-fold dilution in 100 mM Tris-HCl pH 8.0, 20 mM EDTA, followed by 10 min at 50.degree. C. Stopped samples were centrifuged at 4000 g for 1 h prior to HPLC analysis. Separation of 10 .mu.l samples was carried out on a CarboPac PA-100 column as previously described18. During the neutral drift experiments each plate contained two copies of the .DELTA.25polySTNmB reference sequence and clones with at least 25% of the wild type level of activity were considered to be neutrally drifting.

[0467] Protein Determinations.

[0468] Total protein concentrations were determined with the BCA assay (Thermo Scientific, Rockford, Ill., USA) according to the manufacturer's instructions.

[0469] Determination of polyST expression level was carried out as previously described22. Briefly, samples of cell free extract were separated by 12% SDS-PAGE. For Western blot analysis, proteins were transferred onto PVDF (polyvinylidene fluoride) membranes (LI-COR Biosciences) and detected with 1 .mu.g/ml mouse anti-penta-his antibody (Qiagen) and anti-mouse IR800 (1:20,000, LI-COR Biosciences) and quantified by comparison with protein standards according to the recommendations of the Odyssey infrared imaging system (LI-COR Biosciences).

EXAMPLE 4

Exploring the Capsule Biosynthesis Machinery of NmA with Regard to its Suitability for In Vitro Vaccine Production

Introduction

[0470] Neisseria meningitidis serogroup A (NmA) is the major cause of meningococcal disease in the African meningitis belt. Besides of seasonal epidemics that occur with almost annual frequency, NmA has been the cause of severe pandemics in the last century (Stephens (2009) Vaccine, 27 Suppl 2, B71-B77). A major virulence factor of Nm is the negatively charged capsular polysaccharide (CPS). The NmA CPS (CPSA) consists of N-acetyl-mannosamine 1-phosphate units, linked by phosphodiester bonds to give the polymer [.fwdarw.6)-.alpha.-D-ManpNAc-(1.fwdarw.OPO.sub.3.fwdarw.].sub.n (Liu (1971) JBC 246 (9), 2849-2858). Of note, the six most virulent Nm serogroups (NmA, -B, -C, -W, -Y and -X) wear negative CPSs. Negative charge in CPSA and CPSX is due to the phosphodiester group, while negative charge in CPSB, -C, -W, and -Y results from the incorporation of the sialic acid (Stephens (2009) Vaccine 27 Suppl 2, B71-B77).

[0471] Besides of CPSB, which is identical with polysialic acid in the human host, all CPS are immunogenic and cause the production of antibodies that are bacteriotoxic in the presence of complement (Gotschlich (1969) The Journal of experimental medicine 129 (6), 1367-1384; Stephens (2007) Lancet 369 (9580), 2196-2210). In fact, this early observation has made the use of polysaccharide-protein conjugates the gold standard in the development of vaccines against Nm strains. A number of mono- and tetravalent (the latter comprising serogroups A, C, W and Y) conjugate vaccines against Nm have been licensed (Costantino (2011) Expert opinion on drug discovery 6 (10), 1045-1066).

[0472] Crucial to the success of vaccination programs in the sub-Saharan meningitis belt is the provision of a safe and high-quality vaccine. MenAfriVac.RTM., a conjugate vaccine with CPSA coupled to tetanus toxoid as carrier protein, has been specifically designed to address these needs (Frasch (2012) Human vaccines & immunotherapeutics 8 (6), 715-724). With under 50 cent per dose (Roberts (2010) Science 330 (6010), 1466-1467), mass vaccination campaigns were possible in Burkina Faso, Mali and Niger and installed herd immunity (Djingarey (2012) Vaccine 30, B40-B45; Caini (2013) Vaccine 31 (12), 1597-1603; LaForce (2011) Eliminating epidemic Group A meningococcal meningitis in Africa through a new vaccine. Health affairs (Project Hope) 30 (6), 1049-1057), protecting not only vaccinated but also non-vaccinated individuals and young children (Kristiansen (2013) Clinical infectious diseases 56 (3), 354-363). Recent progress made with the cloning and functional expression of capsule polymerases (CPs) (Freiberger (2007) Molecular microbiology 65 (5), 1258-1275; Romanow (2013) The Journal of biological chemistry 288 (17), 11718-11730; Peterson (2011) Journal of bacteriology 193 (7), 1576-1582) and the pioneering studies that demonstrate the suitability of the respective recombinant enzymes for the in vitro production of CPSs (McCarthy (2013) Glycoconjugate journal 30 (9), 857-870; Fiebig (2014) Glycobiology 24:150-8), have opened a new perspective for the economic and save production of conjugate vaccines. As NmA is a thread foremost in underdeveloped countries, the development of robust and economic regimes for vaccine production is of utmost relevance. Consequently, the goal of the current study was to isolate the capsular polymerase from NmA and to analyse the capability of the recombinant protein to produce CPSA in vitro. Because the sugar building block UDP-ManNAc is commercially not available, it was clear from the start of this project that a successful production chain depends on the in situ synthesis of UDP-ManNAc from cheap UDP-GlcNAc. Moreover, as CPSA is immunogenic only if O-acetylated (Berry (2002) Infection and immunity 70 (7), 3707-3713), an effective production chain needs, in addition, the O-acetyltransferase, which is able to perform this modification in a nature identical form. Based on these considerations we decided to isolate the three relevant enzymes from NmA and to explore the capacity of the recombinant proteins to work together in vitro to produce bio-identical CPSA.

[0473] The chromosomal locus (cps for capsular polysaccharide synthesis) contains the genetic information for CPS synthesis, modification and surface transport. The locus is sub-structured into six regions: A-D, D' and E (FIG. 21). The sequences encoded in region A are serogroup-specific and encode inter alia the polymerases responsible for CPS synthesis (Harrison (2013) Emerging infectious diseases 19 (4), 566-573). Regions B and C are highly conserved and encode the proteins necessary for export and assembly of the polysaccharide on the cell surface. In NmA, region A comprises four open reading frames (ORF) csaA, -B, -C and -D (previously designated sacA-D or mynA-D). Using insertion-mutagenesis Swartley et al. (1998, Journal of bacteriology 180 (6), 1533-1539) demonstrated that each of these genes is involved in the production of the NmA capsule. In a later study the gene product encoded in csaC was shown to be an acetyltransferase with specificity for the O-3 and O-4 positions in ManNAc (Gudlavalleti (2004) The Journal of biological chemistry 279 (41), 42765-42773). Based on their nucleotide and predicted amino acid sequence, csaA was presumed to encode an UDP-N-acetyl-.sub.D-glucosamine 2-epimerase and csaB a capsule polymerase (Swartley (1998) Journal of bacteriology 180 (6), 1533-1539). Additional evidence that CsaB is in fact the NmA specific capsule polymerase arose from the demonstration that it clusters together with CsxA, the proven CP from NmX (Muindi (2014) Glycobiology 24 (2), 139-149; Fiebig Glycobiology. (2014) 24:150-8) in the so called stealth protein family, which comprises exclusively D-hexose-1-phosphoryl transferases (Sperisen (2005) PLoS computational biology 1 (6)).

[0474] Here we describe the molecular cloning of the genes csaA, csaB and csaC from NmA, the production of functional recombinant proteins and provide proof for their functional properties. Using a series of synthetic primer compounds the ManNAc-dimer linked together by phosphodiester bonds and carrying a phosphodiester at the reducing end was identified to be the minimal acceptor structure, which was preferred by CsaB if presented in non-acetylated form. Using a two-step production protocol, O-acetylated CPSA was prepared in highly pure form and was recognized by the monoclonal antibody (mAb) 935, suggesting that the in vitro produced polymer represents the bioidentical CPSA.

Results

[0475] Cloning and Expression of csaA, csaB and csaC; Production of Recombinant Proteins.

[0476] Because previous analyses carried out on these genes provided strong evidence that csaA, csaB and csaC encode the UDP-GlcNAc epimerase, the poly-ManNAc-1-phosphoryl transferase (Swartley (1998) Journal of bacteriology 180 (6), 1533-1539) and the O-acetyl transferase (Gudlavalleti (2004) The Journal of biological chemistry 279 (41), 42765-42773), respectively, primers were constructed (see Material and Methods) to amplify these ORFs. The genomic DNA isolated from Nm strain Z2491 was used as a template. Obtained PCR products were cloned into the pET22b-Strep vector (Schwarzer (2009) The Journal of biological chemistry 284 (14), 9465-9474), allowing the expression of recombinant proteins with N-terminal StrepII- and/or C-terminal His.sub.6-tag. After transformation into BL21(DE3) and induction of protein expression (see Material and Methods) the distribution of recombinant proteins between the soluble (s) and insoluble (i) fractions of bacterial lysates was analyzed by western blotting against the affinity tags. The recombinant epitope tagged forms of CsaA and CsaC appeared mostly in the soluble fraction (data not shown) and could be purified directly from the bacterial lysates. CsaA was purified by IMAC (immobilized metal ion affinity chromatography) followed by a desalting step and yielded 40 mg protein/L expression culture. Though some additional faint bands were visible in Coosmassie stained SDS-PAGE (FIG. 22A), a protein fraction highly enriched in CsaA was obtained. CsaC was purified following the protocol described by Gudlavalleti (2004, The Journal of biological chemistry 279 (41), 42765-42773) and yielded 96 mg homogenously pure protein/L culture (FIG. 22A).

[0477] Similarly, the StrepII-CsaB-His.sub.6, encoding the putative poly-ManNAc-1-phosphoryl transferase, was well expressed and appeared with >60% in the soluble fraction. However, the major product revealed with the anti-penta-His antibody in western blot migrated with an apparent molecular mass of 50 kDa, strongly deviating from the calculated molecular mass of 67 kDa (FIG. 22B, left lanes). Because faint signals with molecular masses >50 kDa were additionally displayed with the anti-penta-His antibody, we concluded that StrepII-CsaB-His.sub.6 is either prone to N-terminal degradation or translated from an alternative start codon. Consequently, we reinvestigated the NmA genome with bioinformatics techniques. Indeed, two of the used gene prediction softwares (GeNmark and GeNmarkS) retrieved an additional ATG (starting with position 183528 of the NmA genome (NC_003116.1)). In PRODIGAL (Hyatt (2010) BMC bioinformatics 11, 119), the prediction for this second start codon was comparable to the published start codon (base No. 183321; Parkhill (2000) Nature 404 (6777), 502-506).

[0478] To investigate if translation from the alternative ATG leads to a stable protein, the corresponding truncation .DELTA.69CsaB was cloned with (StrepII-.DELTA.69-CsaB-His.sub.6) and without (.DELTA.69-CsaB-His.sub.6) the N-terminal StrepII-tag. Test expressions in BL21(DE3) demonstrated the occurrence of proteins of the expected molecular masses, but in repeated experiments the level of expressed protein was significantly lower than for the full length construct. Moreover, to our surprise, the construct cloned with free N-terminus (.DELTA.69-CsaB-His.sub.6) was routinely higher expressed than the StrepII-tagged construct (FIG. 22B). Since rare codons that exist in the CsaB sequence may negatively impact protein expression, csaB was codon optimized (using the DNA 2.0 software and the published codon frequency tables; Welch et al. (2009)) and expression tested with the constructs StrepII-CsaB.sub.co-His.sub.6 and .DELTA.69-CsaB.sub.co-His.sub.6. While increased degradation and concomitantly reduced expression was seen for StrepII-CsaB.sub.co-His.sub.6, .DELTA.69-CsaB.sub.co-His.sub.6 was well expressed and no degradation was detectable in western blot with the anti-penta-His antibody (FIG. 22B).

[0479] Consecutively, enzymatic activity within the soluble fractions of the bacterial lysates was determined, with a radioactive incorporation assay previously developed for the poly-GlcNAc-1-phosphoryl transferase from NmX (Fiebig, Glycobiology (2014) 24:150-8). The fractions containing the recombinant CsaB variants were tested in the presence of CsaA and UDP-[.sup.14C]GlcNAc. In accordance with the levels of expressed protein (FIG. 22B), StrepII-CsaB-His.sub.6 and .DELTA.69-CsaB.sub.co-His.sub.6 showed identical activity profiles (FIG. 22C). Based on these results the protein variant .DELTA.69-CsaB.sub.co-His.sub.6 was chosen for further experiments. The protein was purified from the soluble fraction of transformed BL21(DE3) by IMAC and SEC, yielding 60 mg of highly pure protein from 1 L bacterial culture (FIG. 22E).

[0480] Optimization of Test Conditions and Determination of CsaB Acceptors.

[0481] As the donor sugar (UDP-ManNAc) used by CsaB must be produced in situ in the epimerase reaction catalyzed by CsaA, the optimization of test conditions needed the presence of both enzymes. As for CPs of other Nm strains, a hydrolysate of CPSA (CPSA.sub.hyd) was used to prime the reaction in the presence of the CsaA substrate UDP-GlcNAc. Initial studies carried out to evaluate pH and salt conditions showed best activity values in the presence of 10-20 mM MgCl.sub.2 and a pH between 8.0-8.5 (data not shown). Replacement of Mg.sup.2+ by Ca.sup.2+ or Mn.sup.2+ inactivated the enzyme. While these results were similar to what we had seen with CsxA (CP of NmX), the CsaA/CsaB reaction, in contrast to the CsxA reaction, did not show sensitivity against DTT (up to 2 mM were tested).

[0482] The natural CPSA is O-acetylated in positions 3 and 4 of ManNAc (Liu (1971) The Journal of biological chemistry 246 (9), 2849-2858; Gudlavalleti et al., Carbohydr Res. 2006), we therefore interrogated if .DELTA.69-CsaB.sub.co-His.sub.6 recognizes and elongates acetylated and non-acetylated CPSA.sub.hyd with the same efficiency. Therefore, the hydrolysis of CPSA was carried out before and after base treatment to obtain O-acetylated (CPSA.sub.hyd(OAc)) and de-O-acetylated (CPSA.sub.hyd(deOAc)) shorter saccharide chains. Knowing that CPSA hydrolysis results in a large distribution of saccharide chain lengths (ranging in size between degree of polymerization, DP, 1 and 70), anion exchange chromatography was used to separate two fractions, the averaged DP (avDP) 6 (comprising DP1-DP10) and avDP15 (comprising DP10-DP70). Both fractions were used to prime the enzyme reactions as indicated in the subsequent experimental steps.

[0483] To quantitatively assess the enzyme reactions, we adapted a spectrophotometric assay previously designed to analyse the CP from NmB (Freiberger (2007) Molecular microbiology 65 (5), 1258-1275). In the multi-enzyme assay shown in FIG. 23, the .DELTA.69-CsaB.sub.co-His.sub.6 catalysed product formation is coupled to NADH consumption, a step, which can be continuously followed at 340 nm.

[0484] Using this assay the activity of CsaB was determined with CPSA.sub.hyd fractions (CPSA.sub.hyd(OAc) CPSA.sub.hyd(deOAc)) of avDP6 and avDP15 (FIG. 24). Moreover, because we recently demonstrated that the hydroxyl groups at position 6 (C.sub.6--OH) on the non-reducing end sugar in CPSA.sub.hyd is blocked by phophomonoesters (Ravenscroft (1999) Vaccine 17 (22), 2802-2816), both fractions were additionally tested after treatment with acid phosphatase (de-P) to remove this group. Independent of the size of the primers used to start the reaction, the native acetylated oligomers (CPSA.sub.hyd(OAc) of avDP6 and avDP15 CPSA.sub.hyd(OAc)) were found to be poor acceptors, but activity increased steep after removal of acetyl-groups and even steeper after release of the capping phosphate residue, making CPSA.sub.hyd(deOAc)-deP the most efficient acceptors. The size of the priming polymers was not of significance for enzymatic activity (FIG. 24; compare avDP6 and avDP15). The obtained results allowed the conclusion that the chain elongation by CsaB proceeds via the non-reducing end and by transfer of ManNAc-1P onto C.sub.6--OH groups. Furthermore, since CPSA.sub.hyd(deOAc) was a better acceptor than CPSA.sub.hyd(OAc), it is likely that O-acetylation takes place on the build polymer.

[0485] Because no information on the minimal length of the priming acceptor for CsaB could be derived from the CPSA.sub.hyd fractions, we used well characterized synthetic compounds to interrogate this question. The compounds synthesized are shown in FIG. 25 and varied not only in length, but also with respect to O-acetylation (compounds 2, 4, 6 were 3-O-acetylated) and reducing end modifications. In compounds 1, 2, 5, 6 the reducing ends were occupied by a decyl-phosphate-ester, while a methyl group (OMe) was present in the compounds 3 and 4. The .DELTA.69-CsaB.sub.co-His.sub.6 activity did not go beyond background (no acceptor) with compounds 1-4, but, intriguingly, steeply increased with disaccharides carrying a decyl-phosphate-ester at the reducing end (compounds 5, 6) (FIG. 25). With the non-acetylated compound 5, activity values similar to those obtained with the optimized acceptor CPSA.sub.hyd(deOAc)-deP were measured. In line with the above data (FIG. 24), O-acetylation of compound 5 (resulting in compound 6) reduced the quality of the acceptor. Based on these data, the minimal acceptor recognized by .DELTA.69-CsaB.sub.co-His.sub.6 could be defined as the dimer of ManNAc-1P units linked together by phosphodiester bonds. The presence of a phosphodiester at the reducing end seems obligatory, because compounds ending with OMe groups are not used.

[0486] In Vitro Synthesis of CPSA-Chains.

[0487] To analyze if long CPSA-chains can be produced with the recombinant enzymes, test reactions were carried out having the enzymes .DELTA.69-CsaB.sub.co-His.sub.6 and StrepII-CsaA-His.sub.6 at equal concentration (50 nM) and the priming oligosaccharide (CPSA.sub.hyd(deOAc)-deP of avDP6) in 100-fold molar excess.

[0488] Reactions were started by addition of 5 mM UDP-GlcNAc. Control reactions (FIG. 26A control-1-4), in which components were omitted as indicated, were carried out in parallel. After overnight incubation, samples were loaded onto high percentage PAGE and developed by alcian blue/silver staining. CPSA.sub.hyd(deOAc)-deP of avDP15 was loaded as size marker (FIG. 26A). In the presence of CsaA and CsaB the added oligosaccharide primers were efficiently elongated to long polymer chains (CPSA.sub.iv, for in vitro produced CPSA). However also in control-1, in the absence of priming compounds, a faint signal indicating long CPSA was seen. In .sup.31P NMR (FIG. 26B) the products obtained in the main reaction showed the phosphodiester signal, which is characteristic for CPSA as well as the signals that indicate the second reaction product UMP. Unexpected were the signals observed at -5.8 ppm and -9.5 ppm, which indicated the formation of UDP. As these latter signals were most prominent in control-4 (FIG. 26 B), with only StrepII-CsaA-His.sub.6 and UDP-GlcNAc present, we speculated that UDP is a side product of the epimerase reaction. Support for this assumption was obtained from literature, were Sala et al (1996; Journal of the American Chemical Society 118, 3033-3034) had shown that UDP-N-acetylglucosamine 2-epimerase from E. coli, if present at high concentration, accumulates UDP and 2-acetamidoglucal, the two intermediates of the epimerization reaction.

[0489] Since all relevant .sup.31P NMR signals were identified also in the product formed in control-1, this faint CPSA signal demonstrated that .DELTA.69-CsaB.sub.co-His.sub.6 is capable to induce product formation de novo in the absence of a priming oligosaccharide. Importantly, this same type of de novo synthesis was shown for the CP of NmX (Fiebig, Glycobiology (2014) 24:150-8). Still, the self-priming capacity came as a surprise in the case of CsaB, were de novo formation of product was not seen if the synthetic compounds 1 or 2 where tested as primers (see FIG. 25).

[0490] To reduce UDP-ManNAc hydrolysis and simultaneously complete the incorporation of ManNAc-1P into the product, the ratio between CsaA and CsaB was varied as indicated in FIG. 26C. The reactions carried out as described above with CPSA.sub.hyd(deOAc)-deP of avDP6 as primer. After overnight incubation, products were separated by HPLC and recorded at 214 nm (CPSA.sub.iv) and 280 nm (UDP and UMP). As long as the concentration of CsaB was equal or higher than the concentration of CsaA, no hydrolysis of UDP-ManNAc was detectable (FIG. 26C), even not, if the enzymes were present in only 50 nM concentration and the donor sugar was not completely consumed. These data convincingly show that UDP production is a side reaction of the CsaA.

[0491] With the intention to produce purified, bio-identical CPSA in milligram amounts, the CsaA/CsaB reaction was up-scaled. The long polymers that were obtained in this reaction were purified by anion exchange chromatography (AEC) using a protocol similar to the one described (Fiebig, Glycobiology (2014) 24:150-8). CPSA.sub.iv eluted at NaCl clearly separated from all other reaction components (FIG. 27A). 1 mg of the purified CPSA.sub.iv was then used for acetylation with the recombinant CsaC. In a second AEC-step CPSA.sub.iv/OAc eluted as a single peak (FIG. 27B) and the obtained material was recognized by mAB 932 in dot blot analysis (FIG. 27C). .sup.1H NMR spectra were recorded for CPSA.sub.iv and CPSA.sub.iv/OAc and were compared to CPSA from natural source (CPSA.sub.n) treated (CPSA.sub.n/deOAc) or not (CPSA.sub.n) with alkaline. The congruence of obtained spectra confirmed the bioidentity of the synthetic products (FIG. 27D).

[0492] Finally we explored at analytical scale, if bio-identically O-acetylated CPSA can also be produced in the one pot reaction. Therefore, as described above the reaction mixture was supplemented with recombinant CsaC-His.sub.6 and acetyl-CoA. Moreover, control reactions, with single compounds missing (see scheme added to FIG. 28) were carried out in parallel. After overnight incubation, products were analyzed by alcian-blue/silver stained high percentage PAGE (FIG. 28A) and immunoblotting with mAb 932 (FIG. 28B). In the presence of all components, a product recognized by mAb 932 was produced (FIG. 28B), indicating that CsaC-His.sub.6 can acetylate the produced CPSA in situ. The control reactions carried out in this experiment provided clear evidence for the functional nature of CsaA and CsaC being UDP-GlcNAc/UDP-ManNAc epimerase and O-acetyl-transferase, respectively. Lastly, the similarity (size and concentration) of reaction products identified in lanes 1, 4, and 6 (FIG. 28A) strongly argue for that suitable conditions were installed for all enzymes in the one-pot reaction scheme.

Discussion

[0493] Of all pathogenic Nm serogroups, NmA has caused the most disastrous epidemics in sub-saharan Africa. The prevalence of this pathogen caused an unprecedented attempt in terms of developing a highly effective and economic vaccine, MenAfriVac.RTM. (Roberts L. Science (2010) 330:1466-7). With coasts of less than 50 cent per dose, MenAfriVac.RTM. enabled mass vaccination campaigns in Burkina Faso, Mali and Niger (LaForce (2011) Eliminating epidemic Group A meningococcal meningitis in Africa through a new vaccine. Health affairs (Project Hope) 30 (6), 1049-1057; Djingarey (2012) Vaccine 30, B40-B45; Caini (2013) From Agadez to Zinder: estimating coverage of the MenAfriVac.GAMMA.ao conjugate vaccine against meningococcal serogroup A in Niger, September 2010-January 2012. Vaccine 31 (12), 1597-1603), which installed herd immunity, leading to the protection not only for vaccinated but also for non-vaccinated individuals and in particular for young children (Kristiansen (2013) Clinical infectious diseases 56 (3), 354-363).

[0494] All NmA vaccines licensed today are glycoconjugate vaccines, with CPSA oligosaccharides coupled to carrier proteins. CPSA oligosaccharides are thereby hydrolysis products of CPSA isolated from large scale NmA cultures (Bardotti (2008) Vaccine 26 (18), 2284-2296). To avoid the significant coast and biohazard in association with large scale NmA cultures and the pyrogen-free production of polysaccharides, the enzyme catalyzed in vitro synthesis of CPSA would provide an attractive alternative. Towards this goal, we describe in this study, the molecular cloning and functional expression of the three enzymes (UDP-GlcNAc 2-epimerase, CsaA; NmA capsule polymerase, CsaB; O-acetyltransferase, CsaC) that are part of the capsular biosynthesis complex in NmA and represent the minimal number of enzymes needed to produce immunologically active CPSA.sub.OAc in vitro starting from economic precursors. Using the well characterized BL21(DE3) strain as expression host, C-terminally His.sub.6-tagged versions of CsaA and CsaC could be purified in high quality and remarkable quantity (CsaA 40 mg and CsaC 96 mg per liter bacterial culture).

[0495] In CsaB a second start codon was identified starting with methionine-69 bp upstream of the first. Use of this second start codon generated a stable C-terminally His.sub.6-tagged protein. However, the level of expressed protein could be significantly increased (to 60 mg/L bacterial culture) after the cDNA sequence was optimized for codon usage in BL21(DE3). If the second start codon is actually used in the natural environment cannot be answered based on the current data. However, because a multi-sequence alignment demonstrated significant similarity between the N-termini of CsaB and other capsule polymerases starting with the first methionine (data not shown), it is likely that the first and not the second start codon is used in the natural environment.

[0496] Using a two-step protocol (in test reactions even a one pot reaction; see FIG. 28) 0-acetylated CPSA (CPSA.sub.iv/oAc) could be produced in vitro in high purity and at medium scale. CPSA.sub.iv/OAc was of same length as CPSA isolated from natural source (CPSA.sub.n), showed .sup.31PNMR and .sup.1HNMR profiles identical with the natural polymer and was recognized by mAb 932, a standard reagent in the characterization of immunologically active CPSA. Yet it is not proven that acetylation patters exactly mirror the natural product. However, it likely that also NmA glycoconjugate vaccines in which the glycan parts (CPSA.sub.hyd of avDP15) are derived from natural polymers by acidic hydrolysis, show some variation in the acetylation patterns. Even though this hydrolysis is carried out under optimized conditions, the probability that acetyl-groups are released is very high.

[0497] When the CsaA/CsaB reaction was carried out under suboptimal conditions (meaning that CsaA concentrations were higher than CsaB concentrations) UDP was formed as a side product. Based on control experiments and on earlier literature data the occurrence of UDP indicates that CsaA similar to UDP-N-acetylglucosamine 2-epimerase from E. coli, epimerizes UDP-GlcNAc to UDP-ManNAc by forming UDP and 2-acetamidoglucal as intermediate products (Sala (1996) Journal of the American Chemical Society 118, 3033-3034). If present in access, the epimerase releases these intermediates into solution (Sala Journal of the American Chemical Society 118, 3033-3034). Logically, the side reaction was avoided by adjusting the ratio in enzyme concentrations [CsaA]<[CsaB].

[0498] O-acetyltransferases from encapsulated bacterial strains have been cloned and functionally characterized (Bergfeld (2007) The Journal of biological chemistry 282 (30), 22217-22227). In these studies addressing also structure-function-relationships the enzymes could be grouped into two families. While O-acetyltransferases of NmW-135 and NmY group into the superfamily of left-handed .beta.-helix (L.beta.H) proteins the O-acetyltransferases isolated from NmA (CsaA) has sequence homology to members of the .alpha./.beta.-hydrolase fold family, harboring a catalytic serine-tried which is part of `nucleophilic elbow` motif (Bergfeld (2007) The Journal of biological chemistry 282 (30), 22217-22227; Bergfeld (2009) The Journal of biological chemistry 284 (1), 6-16). The .alpha./.beta.-hydrolase fold positions one of the catalytic serine-residues to the tip of a narrow turn. This structural motif may impact substrate recognition by the enzyme.

[0499] In the current study, we provide clear evidence that the acceptor quality of CPSA.sub.hyd increases significantly after release of O-acetyl-groups. Similarly, the synthetic disaccharide carrying 3-O-Ac-groups on ManNAc (compound 6) was a significantly less efficient primer than the respective compound (5) without O-acetyl-groups. These data argue for that O-acetylation takes place during or after the polymer is formed. This type of size-dependent substrate recognition has already been identified for other O-acetyltransferase that act on sugar polymers. With CPSA.sub.hyd a steep increase in activity was seen after dephosphorylation of the non-reducing ends. We previously demonstrated that acidic hydrolysis results (up to 70%) in fragments that are carrying phosphate at the non-reducing end. Since removal of phosphate improved the acceptor quality, it can be concluded that chain elongation proceeds by transfer of monosaccharide-phosphates to the non-reducing end.

[0500] The use of the synthetic compounds identified two other important features of CsaB: (i) the minimal efficient acceptor is the dimer and (ii) the reducing end phosphate group can be extended with rather large chemical groups (decyl-ester in the compounds tested in this study). This latter finding bears the perspective that chain elongation can be primed with reagents of very high purity and functional groups that facilitate conjugation of glycans to carrier proteins in the vaccine production chain.

[0501] Of note is the fact that CsaB is capable of self-priming. The mechanism underlying the de novo start of chain polymerisation is not understood, but was similarly observed with the CP of NmX (Fiebig, Glycobiology (2014) 24:150-8). In the case of CsaB the interpretation of the phenomenon is however further complicated by the fact that no such reaction was detectable in the presence of the artificial compounds 1-4. A possible explanation may be that these compounds compete against UDP-ManNAc in the active site pocket and thus block the initiation of the chain. However, more experimental work is needed to understand and explain this reaction.

[0502] Taken together, our study lays a new basis for the development of efficient and economic protocols with which immunologically active and highly pure CPSA.sub.OAc can be synthesized in vitro and used for glycoconjugate vaccine production. We provide initial evidence that CPSA.sub.OAc can be produced in a one pot reaction and purified to homogeneity in a single step. The used enzymes are without biohazard and provide the perspective that production chains can be established also in areas that are lacking advanced infrastructural conditions.

Material and Methods

[0503] General Cloning--

[0504] The genomic DNA isolated from Nm strain Z2491 was a kind gift from Dr. Heike Claus (Institute for Hygiene and Microbiology, University of Wurzburg). The csaB sequence was codon optimized for use in E. coli BL21(DE3) using the Gene Designer software package (DNA 2.0) (Villalobos (2006) BMC bioinformatics 7, 285) and the codon frequency tables published by Welch (2009; PloS ONE 4 (9)). The mean codon frequency for each amino acid was calculated from the codon frequency tables FreqA and FreqB (Welch (2009) PloS ONE 4 (9)) and the resulting codon frequency table used as template for the in silico generation of csaB.sub.co. csaB.sub.co flanked 5' by a BamHI site and 3' by a XhoI site was synthesized from Eurofins MWG Operon. All other csaA-C sequences described herein were amplified by polymerase chain reaction (PCR) using the primers shown in Table 4 and genomic DNA from Nm strain Z2491 or csaB.sub.co as template. PCR products were cloned via the restriction sites shown in Table 4 into the corresponding sites of the vector pET22b-Strep (Schwarzer (2009) The Journal of biological chemistry 284 (14), 9465-9474) driving the expression of recombinant proteins under the control of the T7 promoter. PCR products digested with BglII were cloned into the BamHI site of pET22b-Strep.

TABLE-US-00009 TABLE 4 Primers used in this study. Restriction sites are highlighted in bold. Primer pair Resulting construct GCGGATCCAAAGTCTTAACC StrepII-CsaA-His.sub.6 GTCTTTGGC CCGCTCGAGTCTATTCTTTA ATAAAGTTTCTACA GCAGATCTTTTATACTTAAT StrepII-CsaB-His.sub.6 AACAGAAAATGGC CCGCTCGAGTTTCTCAAATG ATGATGGTAATG CCGCTCGAGTTTCTCAAATG StrepII-.DELTA.69-CsaB-His.sub.6 ATGATGGTAATG GCAGATCTATGTTAATTCCT ATTAATTTTTTTAA CCGCTCGAGTTTCTCAAATG .DELTA.69-CsaB-His.sub.6 ATGATGGTAATG GCATCTCATATGTTAATTCC TATTAATTTTTTTTAATTT GCATCTCATATGCTGATCCC .DELTA.69-CsaB.sub.Co-His.sub.6 GATCAATTTCTTT CCGCTCGAGTTTCTCGAAGG AGCTCGGC CCGCTCGAGTATATTTTGGA StrepII-CsaC-His.sub.6 TTATGGT GCGGATCCTTATCTAATTTA AAAACAGG

[0505] Expression and Purification of Recombinant CsaA, CsaB and CsaC--

[0506] Freshly transformed E. coli BL21(DE3) were grown at 15.degree. C. in PowerBroth medium for 18 h. At an optical density of OD.sub.600=1.0 protein expression was induced by addition of 0.1 mM IPTG and allowed to proceed for a period of 20 h. In test expressions, 0.2 ml of culture-volume were pelleted with 16,000.times.g for 1 min. Cell pellets were lysed with 0.1 ml lysis buffer (50 mM Tris pH 8.0, 2 mM EDTA, 0.1 mg/ml lysozyme). The lysis was intensified by 3 cycles of sonication (Branson sonifier 450, 100% amplitude) interrupted by 3 min of cooling on ice. Soluble and insoluble fractions were separated by centrifugation (16,000.times.g, 30 min, 4.degree. C.), the supernatant mixed (1:1) with Laemmli-buffer and used for PAGE as described below.

[0507] For protein purification, pellets from 125 mL expression culture were pelleted by centrifugation (6,000.times.g, 10 min, 4.degree. C.). After a washing step with PBS, cells were re-suspended in 7.5 ml binding buffer (50 mM Tris pH 8.0, 300 mM NaCl) complemented with 40 .mu.g/ml Bestatin (Sigma), 1 .mu.g/ml Pepstatin (Applichem), 100 .mu.M PMSF (Stratagene) and sonified (Branson Digital Sonifier, 50% amplitude, 8.times.30 s, interrupted by cooling on ice). After centrifugation at 27,000.times.g for 30 min, the soluble fractions were directly loaded onto a HisTrap columns (GE Healthcare) to enrich the recombinant proteins by immobilized metal ion affinity chromatography (IMAC). Columns were washed with binding buffer (50 mM Tris pH 8.0, 300 mM NaCl) and proteins eluted in step gradients using 10%, 30%, 50% and 100% elution buffer (binding buffer containing 500 mM imidazol). Fractions containing recombinant protein were pooled and the buffer exchanged to storage buffer (50 mM Tris pH 8.0, 50 mM NaCl for CsaA/CsaB; 50 mM Hepes pH 7.05, 100 mM NaCl, 5 mM MgCl2 and 1 mM EDTA for CsaC) using the HiPrep 26/10 Desalting column (GE Healthcare). Isolated proteins were concentrated using Amicon Ultra centrifugal devices (Millipore 30 MWCO). After separation into aliquots, samples were snap frozen in liquid nitrogen and stored at -80.degree. C.

[0508] SDS-PAGE and Immunoblotting--

[0509] SDS-PAGE was performed under reducing conditions using 2.5% (v/v) 1-mercaptoethanol and 1.5% (w/v) SDS. Proteins were stained using Roti-Blue (Carl Roth GmbH) according to the manufacturer's guidelines. For western blot analysis samples and standard proteins were blotted onto PVDF membranes (Millipore). His-tagged proteins were detected with 0.5 .mu.g/ml anti-penta-His antibody (Qiagen) and goat anti-mouse IR680 or goat anti-mouse IR800 antibody (LI-COR) as second antibody. Second antibodies were used in a 1:20,000 dilution.

[0510] Preparation of CPSA Oligosaccharides--

[0511] CPSA oligosaccharide samples with an averaged degree of polymerization (avDP) of 6 and 15, respectively, were generated by acidic hydrolysis of long CPSA chains isolated from bacterial cultures (CPSA.sub.n). Therefore, solutions containing 2.5 mg CPSA/mL sodium acetate buffer (50 mM sodium acetate, pH 4.8) were incubated at 73.degree. C. for 6 h and two pool fractions (avDP 6 and 15, respectively) were purified by anionic exchange chromatography (Q-Sepharose column, GE Healthcare) using a sodium acetate gradient. The avDP and the dispersion of saccharide chains was determined by .sup.31P NMR and High Performance Anionic Exchange Chromatography-Pulsed Amperometric Detection (HPAEC-PAD) analysis following an established protocol (Berti (2012) Vaccine 30 (45), 6409-6415). If used in enzymatic reactions, hydrolyzed CPSA (CPSA.sub.hyd) was dephosphorylated (CPSA.sub.hyd-deP) using acid phosphatase (Sigma) according to the manufacturer's guidelines.

[0512] Activity Testing of CsaA/CsaB by Use of a Radioactive Assay System--

[0513] CsxA/CsaB activity was analysed using an adaptation of a radioactive incorporation assay previously described for the N-acetylglucosamine-1-phosphate-transferase from NmX (Fiebig, Glycobiology (2014) 24:150-8). Briefly, assays were carried out with 5 .mu.l of the soluble fractions of bacterial lysates expressing either recombinant CsaB or CsaA (see FIG. 22C) or with purified and epitope-tagged proteins (112 pmol StrepII-CsaA-His.sub.6; 88 pmol .DELTA.69CsaB.sub.co-His.sub.6) in a total volume of 25 .mu.l assay buffer (50 mM Tris pH 8.0 or various pH for determination of the pH optimum). Divalent cations were added from stock solutions. The reaction was primed with 5 ng avDP15 and started by the addition of 0.05 .mu.mol UDP-GlcNAc (Calbiochem) containing 0.05 .rho.Ci UDP-[.sup.14C]-GlcNAc (American Radiolabeled Chemicals). Samples were incubated at 37.degree. C. and 5 .mu.l aliquots spotted onto Whatman 3MM CHR paper after 0, 5, 10 and 30 min. Following descending paper chromatography, the chromatographically immobile .sup.14C-labeled CPSA was quantified by scintillation counting.

[0514] Activity Testing of CsaA/CsaB by Use of a Multi-Enzyme Spectrophotometric Assay--

[0515] 1.2 .mu.M of CsaA and 1 .mu.M CsaB were assayed in the presence of 0.25 mM UDP-GlcNAc (Calbiochem), 20 mM MgCl.sub.2 and 50 mM Tris pH 8.0 in a total volume of 100 .mu.L. The consumption of UDP-GlcNAc was coupled to nicotinamide adenine dinucleotide (NADH) consumption using the following enzymes/substrates: 0.25 mM adenosine triphosphate (ATP, Roche), 1 mM phosphoenolpyruvate (PEP, ABCR), 0.3 mM (NADH, Roche), 9-15 units/ml pyruvate kinase, 13.5-21 units/ml lactic dehydrogenase (PK/LDH mix Sigma), 0.05 mg/ml nucleoside monophosphate kinase (Roche). Absorption was measured at 340 nm every 30 min using a Biotek EL 808 96-well plate reader.

[0516] Physicochemical Analysis of CPSA.sub.iv.

[0517] To produce sufficient CPSA (CPSA.sub.iv) for PAGE and NMR analyses, 0.84 nmol (1.2 .mu.M final) of StrepII-CsaA-His.sub.6 and 37.5 pmol (50 nM final) of .DELTA.69CsaB.sub.co-His.sub.6 in reaction buffer (50 mM Tris pH 8.0, 20 mM MgCl.sub.2) were incubated with 5 mM UDP-GlcNAc and 6.8 .mu.g of CPSA.sub.hyd of avDP6 after de-O-acetylation and dephosphorylation (CPSA.sub.hyd(deOAc)-deP). The total reaction volume was adjusted to 750 .mu.l. In control 1 also StrepII-CsaA-His.sub.6 was used at 50 nM. Reactions as well as control samples were incubated over night at 37.degree. C. 5 .mu.l of each sample were then used for separation on high percentage (25%) PAGE and visualized by a combined Alcian blue/silver staining procedure (Min (1986) Analytical biochemistry 155 (2), 275-285).

[0518] The residual sample was freeze-dried, solubilized in 0.75 ml deuterium oxide (D.sub.2O, 99.9% atom D; Aldrich) to give a concentration of 0.5-1 mg saccharide and used for product characterization by NMR. All the .sup.1H and .sup.31P NMR experiments were recorded as previously described (Fiebig, Glycobiology. (2014) 24:150-8).

[0519] HPLC-AEC was performed on a Prominence UFLC-XR (Shimadzu) equipped with a CarboPac PA-100 column (2.times.250 mm, Dionex). Samples were separated as described by (Keys (2012) Analytical biochemistry 427 (2), 107-115) with the minor adjustment that H.sub.2O and 1M NaCl were used as mobile phases M.sub.1 and M.sub.2, respectively. 5 .mu.l of the samples were loaded for the detection of nucleotides at 280 nm and 50 .mu.l for the detection of CPSA at 214 nm. Products were separated using an elution gradient consisting of a -2 curved gradient from 0 to 30% M.sub.2 over 4 min followed by a linear gradient from 30 to 84% M.sub.2 over 33 min. Enzyme concentrations were used as indicated in FIG. 6. All other reactants were used in the amounts described above.

[0520] Analysis of 2-acetamidoglucal--

[0521] Assignments of 1H NMR spectrum were in agreement with those reported in literature (29). .sup.1H NMR (D.sub.2O, 400 MHz): .delta.=6.68 (d, 1H, J.sub.1,2 1.0, H-1), 4.25 (dd, H 1, J.sub.3,4 6.5 Hz, H-3), 3.99 (dt, 1H, J.sub.4,5 8.4, J.sub.5,6a=J.sub.5,6b 4.2 Hz, 6.5 Hz, H-5), 3.86 (d, 2H, H-6), 3.77 (dd, 1H, H-4), 2.05 (s, 3H, CH.sub.3CO). Significant signals from .sup.13C NMR (D.sub.2O, 100 MHz): .delta.=141.47 (C-1), 78.70 (C-5), 68.70 (C-3), 68.36 (C-4), 59.84 (C-6), 21.84 (2.times.CH.sub.3CO).

[0522] In Vitro Synthesis, Purification and Immunological Analysis of CPSA.sub.iv and CPSA.sub.iv(OAc)--

[0523] To generate CPSA.sub.iv, 10 nmol CsaA and 16 nmol CsaB were incubated over night at 37.degree. C. in reaction buffer (50 mM Tris pH 8.0, 20 mM MgCl.sub.2) complemented with 10 mM UDP-GlcNAc in a total volume of 9 ml. The reaction was primed with 1 .mu.g of CPSA.sub.hyd(deOAc)-deP of avDP6. Acetylation of 1 mg CPSA.sub.iv was performed for 4 h at 37.degree. C. in the presence of 1.2 nmol CsaC and 14 mM acetyl-CoA (Sigma) in a total volume of 0.5 ml acetylation buffer (25 mM Tris pH 7.5, 50 mM NaCl). Both CPSA.sub.iv and CPSA.sub.iv(OAc) were purified via anion exchange chromatography (AEC) using a MonoQ HR5/5 column (Pharmacia biotech) at a flow rate of 1 ml/min and a linear sodium chloride gradient. CPS containing fractions eluting at 540 mM NaCl were pooled, dialysed (ZelluTrans, Roth, 1 kDa MWCO) against water and freeze dried for further analysis. For dot blot analyses, small aliquots of the purified CPSA.sub.iv and CPSA.sub.iv(OAc) was spotted onto nitro-cellulose (Whatman) and incubated with mAb 932 specifically directed against CPSA.sub.(OAc) (mAb 935 was generated in the laboratory of Prof. Dr. D. Bitter-Suermann, Hannover Medical School, Institute for Medical Microbiology, and was kindly provided for this study) in a 1:10,000 dilution. Dot blots were developed with goat anti-mouse IR800 antibody (LI-COR) in 1:20,000 dilution.

EXAMPLE 5

Design and Expression of N- and C-Terminally Truncated CsxA-Constructs Carrying an N-Terminal MBP- and a C-Terminal His.sub.6-Tag

[0524] The materials and methods for this Example are described herein above and also shown in Fiebig, Glycobiology (2014) 24:150-8.

[0525] The secondary structure prediction software PHYRE (Kelley et al., 2009, Nat Protoc. 4, 363-371) was used to predict secondary structure elements within the CsxA amino acid sequence. Amplification of csxA using the truncation primers FF52 and FF53, annealing upstream of the conserved region (CR) 1 and downstream of CR2 in unstructured regions where no secondary structure was predicted, resulted in a dN58- or dC99-truncated CsxA, respectively.

[0526] The PCR products were cloned into a modified pET43.1 vector enabling the expression of the truncated genes as N-terminally MBP- and C-terminally His.sub.6-tagged fusion constructs. After expression in BL21(DE3)Gold followed by lysis of the pelletized cells, soluble fractions were used in a radioactive incorporation assay. Following SDS-PAGE and subsequent immuno-blotting against the His.sub.6-tag, the Odyssey-Software could be used to determine the amount of the respective constructs within the soluble and the insoluble fraction. The former was used to normalize the enzymatic activity obtained in the radioactive incorporation assay.

[0527] Judging from the western blot (FIG. 30. B), both full-length and truncated CsxA were expressed in good yields. The truncation of the N-terminus increased the ratio of soluble:insoluble construct from 2.4 (full-length) to 4.4, while the truncation of the C-terminus decreased the ratio to 1.2. However, the absolute amount of the .DELTA.C99 truncation within the soluble fraction was comparable to the amount of full-length construct. The amount of .DELTA.N58CsxA was increased by 20%. Of note, the radioactive incorporation assay showed that the activity of both the N-terminal and the C-terminal truncation was increased by 38% and 61%, respectively, if compared to the activity of the full-length construct.

[0528] With the aim of analyzing the influence of the termini towards the solubility of CsxA and furthermore defining the minimal catalytic domain of the enzyme, the truncated constructs shown in FIG. 29 were generated. It could be shown that the truncation of the N-terminus by 58 aa resulted in an increase of the ratio of the amount of protein between soluble and insoluble fraction by 80%. Moreover, the enzymatic activity was increased by 38% if compared to the full-length construct.

[0529] Group II polymerases are supposed to be part of the capsule transport complex (Steenbergen (2008) Mol. Microbiol. 68, 1252-1267). The improved solubility could indicate that hydrophobic aa within the first 58 aa of the CsxA sequence are exposed on the enzyme's surface to undergo protein-protein interactions with other components of the capsule transport complex.

[0530] The truncation of the C-terminus led to an increase of activity by 61% (compared to full-length). However, solubility was reduced by 50%. Since the C-terminus might play a role in the formation of the correct oligomerisation state of CsxA, the loss of the correct oligomerisation of .DELTA.C99CsxA might explain the reduced solubility. However, further investigation is needed to understand these structure-function relationships.

[0531] The truncation studies showed that the minimal catalytic domain of CsxA can be reduced to a polypeptide of 329 aa, which corresponds to a reduction of 33% of the full-length sequence. Furthermore, the results indicate that the truncated termini do not contain aa of CsxA's active center.

Further Studies

[0532] In later studies, the .DELTA.N58.DELTA.C99 double truncation was generated and cloned, as well as all afore mentioned construct, into optimised expression vectors. The optimised expression vector is described in Fiebig (Glycobiology (2014) 24:150-8) as MBP-CsxA-His.sub.6 (tac).

[0533] In accordance with the results described above, the truncation of the N-terminus led to an increase in purification yield by more than 500%, whereas the purification yield of the C-terminal truncation was comparable to the full-length construct (FIG. 31C).

[0534] Activity of the purified constructs, measured using an adaption of the multi-enzyme assay described by Freiberger et al. (2007, Molecular Microbiology 65, 1258-1275), confirmed the increased activity of both single CsxA truncations (FIG. 31D). However, the activity of the double truncation was comparable to the activity measured for the full-length construct.

[0535] Analytical size-exclusion chromatography showed that the C-terminus is not involved in oligomerisation/aggregation, since the C-terminal truncation, as well as the full-length enzyme (see Fiebig, Glycobiology. (2014) 24:150-8) forms aggregates in low salt buffer (FIG. 31A) and tri- to tetrameric assemblies in high salt buffer (FIG. 31B).

EXAMPLE 6

Regulation of .DELTA.C99-CP-X and .DELTA.N58.DELTA.C99-CP-X by the Reaction Time

[0536] The materials and methods for this Example are described herein above and also shown in Fiebig, Glycobiology (2014) 24:150-8.

[0537] The C-terminally (e.g. .DELTA.C99-CP-X) as well as the C- and N-terminally (e.g. .DELTA.N58.DELTA.C99-CP-X) truncated version of CP-X show a distributive polymerisation (FIG. 32). In addition, for both polypeptides the length of the produced capsular polysaccharides can be regulated via the reaction time (FIG. 32). However, if the C- and N-terminally truncated CP-X is regulated by the reaction time, the polydispersity is lower as it the C-terminally truncated version is used. Therefore, the C- and N-terminally truncated CP-X is the polypeptide which is most suitable for a regulation via the reaction time.

EXAMPLE 7

Production of Capsular Polysaccharides Using a Capsule Polymerase Coupled to a Solid Phase

[0538] Preparation of the Acceptor Substrate and Determination of the Donor:Acceptor Ratio (d/a).

[0539] Oligosaccharide fractions were prepared as described in Example 1 and fractions containing DP2-10 were pooled to get a mixture of short oligosaccharides ranging from DP2-10. Using the .DELTA.N58.DELTA.C99 CsxA truncation, the donor to acceptor ratio was experimentally determined using 10 mM UDP-GlcNAc and variable amounts of acceptor substrate in over-night reactions to give a product distribution ranging from DP10-DP60 (FIG. 34 a). All obtained product pools were within the DP10-DP60 range and the lowest d/a was chosen for the subsequent experiment. In particular, the d/a was approximately between 100:1 and 200:1.

[0540] Expression of the Enzyme and Coupling of the Construct to the Column

[0541] .DELTA.N58.DELTA.C99 CsxA was expressed in a 50 mL culture volume and purified via His-Trap as described in Fiebig et al 2014a. Briefly, the cells were lysed by sonication in binding buffer (50 mM Tris pH 8.0, 1 mM DTT, 500 mM NaCl) and coupled to a 1 mL His-Trap column. The column was washed with binding buffer containing 37.5 mM imidazole to remove cell debris and non-specifically bound protein before it was equilibrated with a buffer only containing 50 mM Tris pH 8.0 and 1 mM DTT.

[0542] Production of CPSX Using .DELTA.N58.DELTA.C99 CsxA Coupled to a Solid Phase

[0543] The reaction mix containing 50 mM Tris pH 8.0, 10 mM UDP-GlcNAc, 20 mM MgCl2, 1 mM DTT and the amount of acceptor determined in FIG. 34A was up-scaled to 5 mL and loaded onto the His-Trap column containing the immobilized .DELTA.N58.DELTA.C99 CsxA. The mix was circulated through the column using a Peristaltic P1 pump (GE Healthcare) for 2 h and aliquots were taken every 20 min. Aliquots were snap-frozen in liquid nitrogen and .DELTA.N58.DELTA.C99 CsxA was subsequently inactivated for 2.5 min at 98.degree. C. The analysis was performed using the HPLC-assay described in Example 4.

[0544] Following the consumption of the donor substrate UDP-GlcNAc and the production of the second reaction product UMP at 280 nm revealed that the reaction was finished after 20 min and shows that .DELTA.N58.DELTA.C99 CsxA is active even if it is bound/coupled to a solid phase (His-Trap beads) (FIG. 34B). Moreover, the HPLC-AEC (214 nm) analysis of the synthesized CPSX revealed that the ability of the distributive .DELTA.N58.DELTA.C99 CsxA to synthesize narrow product distribution is maintained even if the construct is coupled to a solid phase. The enzymatically produced CPSX pool is well within the range of the avDP15 pool (i.e. between DP10 and DP60) which is commonly used for vaccine manufacture (FIG. 24C). .DELTA.N58.DELTA.C99 CsxA was eluted after the reaction had been finished to demonstrate that it remained coupled to the column during the period of the reaction (FIG. 34D). The above presented data demonstrates that CPSX production using CsxA can be performed on a solid phase, thus omitting further purification steps to remove the enzyme from the CPSX fraction.

[0545] The present invention refers to the following nucleotide and amino acid sequences:

TABLE-US-00010 SEQ ID NO: 1: DNA CP-A/CsaB wildtyp/full-length (1632) atgtttatacttaataacagaaaatggcgtaaacttaaaagagaccct agcgctttctttcgagatagtaaatttaactttttaagatatttttct gctaaaaaatttgcaaagaattttaaaaattcatcacatatccataaa actaatataagtaaagctcaatcaaatatttcttcaaccttaaaacaa aatcggaaacaagatatgttaattcctattaatttttttaattttgaa tatatagttaaaaaacttaacaatcaaaacgcaataggtgtatatatt cttccttctaatcttactcttaagcctgcattatgtattctagaatca cataaagaagactttttaaataaatttcttcttactatttcctctgaa aatttaaagcttcaatacaaatttaatggacaaataaaaaatcctaag tccgtaaatgaaatttggacagatttatttagcattgctcatgttgac atgaaactcagcacagatagaactttaagttcatctatatctcaattt tggttcagattagagttctgtaaagaagataaggattttatcttattt cctacagctaacagatattctagaaaactttggaagcactctattaaa aataatcaattatttaaagaaggcatacgaaactattcagaaatatct tcattaccctatgaagaagatcataattttgatattgatttagtattt acttgggtcaactcagaagataagaattggcaagagttatataaaaaa tataagcccgactttaatagcgatgcaaccagtacatcaagattcctt agtagagatgaattaaaattcgcattacgctcttgggaaatgaatgga tccttcattcgaaaaatttttattgtctctaattgtgctcccccagca tggctagatttaaataaccctaaaattcaatgggtatatcacgaagaa attatgccacaaagtgcccttcctacttttagctcacatgctattgaa accagcttgcaccatataccaggaattagtaactattttatttacagc aatgacgacttcctattaactaaaccattgaataaagacaatttcttc tattcgaatggtattgcaaagttaagattagaagcatggggaaatgtt aatggtgaatgtactgaaggagaacctgactacttaaatggtgctcgc aatgcgaacactctcttagaaaaggaatttaaaaaatttactactaaa ctacatactcactcccctcaatccatgagaactgatattttatttgag atggaaaaaaaatatccagaagagtttaatagaacactacataataaa ttccgatctttagatgatattgcagtaacgggctatctctatcatcat tatgccctactctctggacgagcactacaaagttctgacaagacggaa cttgtacagcaaaatcatgatttcaaaaagaaactaaataatgtagtg accttaactaaagaaaggaattttgacaaacttcctttgagcgtatgt atcaacgatggtgctgatagtcacttgaatgaagaatggaatgttcaa gttattaagttcttagaaactcttttcccattaccatcatcatttgag aaa SEQ ID NO: 2: DNA CP-A/CsaB codon optimized (without) ATGTTCATCTTGAACAACCGCAAATGGCGCAAATTGAAGCGTGACCCA AGCGCGTTTTTTCGTGACAGCAAATTCAACTTTCTGCGCTATTTCTCC GCGAAAAAGTTTGCGAAGAATTTCAAAAACAGCTCGCATATCCATAAA ACCAACATTAGCAAAGCGCAGTCCAATATTTCCAGCACCTTGAAGCAG AACCGTAAGCAGGATATGCTGATCCCGATCAATTTCTTTAATTTTGAG TACATCGTGAAGAAACTGAATAACCAAAACGCAATCGGCGTGTACATT CTGCCGTCTAATCTGACCCTGAAACCAGCATTGTGCATCTTGGAGTCG CACAAAGAGGACTTCCTGAACAAATTTTTGTTGACCATTAGCAGCGAG AACCTGAAACTGCAGTATAAGTTCAATGGTCAGATCAAAAATCCGAAA AGCGTGAACGAAATCTGGACCGACCTGTTTAGCATTGCTCACGTCGAC ATGAAGCTGAGCACCGACCGTACGCTGTCCTCGTCCATCAGCCAATTT TGGTTTCGCCTGGAGTTCTGTAAAGAGGACAAGGACTTCATCCTGTTT CCGACGGCAAATCGTTACAGCCGCAAGCTGTGGAAGCACAGCATCAAA AATAATCAGCTGTTTAAGGAAGGTATCCGTAACTACAGCGAGATTAGC TCGCTGCCGTACGAGGAAGACCATAACTTCGACATCGATCTGGTCTTT ACCTGGGTCAATTCGGAAGACAAAAACTGGCAGGAACTGTACAAGAAA TATAAGCCGGATTTTAATAGCGATGCCACCTCGACGAGCCGTTTTCTG AGCCGTGACGAGCTGAAGTTTGCGCTGCGCTCGTGGGAAATGAACGGT AGCTTCATCCGTAAAATCTTTATCGTCAGCAACTGCGCGCCGCCGGCC TGGCTGGATCTGAACAATCCGAAGATCCAATGGGTGTATCACGAGGAG ATCATGCCACAGAGCGCCCTGCCAACCTTCAGCAGCCATGCTATTGAG ACTAGCTTGCATCACATTCCGGGCATCTCCAACTACTTCATCTACTCT AATGACGATTTTCTGTTGACCAAACCGCTGAACAAAGACAACTTCTTT TACTCCAACGGTATTGCTAAACTGCGTCTGGAAGCCTGGGGTAACGTT AACGGTGAATGTACCGAAGGCGAGCCGGATTACCTGAACGGCGCGCGT AACGCAAATACGCTGCTGGAGAAAGAGTTTAAAAAGTTTACCACCAAG CTGCACACCCACAGCCCGCAGAGCATGCGTACCGACATCCTGTTCGAG ATGGAGAAAAAATACCCAGAAGAGTTCAATCGCACGCTGCACAACAAG TTCCGCAGCCTGGATGACATCGCGGTTACCGGCTACCTGTACCATCAC TACGCATTGCTGTCTGGCCGCGCTCTGCAATCCAGCGATAAGACCGAA CTGGTCCAGCAGAATCACGACTTTAAAAAGAAGCTGAATAATGTTGTC ACCCTGACCAAAGAGCGTAACTTTGATAAGCTGCCGCTGAGCGTTTGT ATTAATGACGGTGCAGACAGCCACCTGAATGAGGAGTGGAATGTGCAA GTTATCAAATTCTTGGAGACCTTGTTCCCGTTGCCGAGCTCCTTCGAG AAA SEQ ID NO: 3: DNA .DELTA.N69-CP-A (codon optimized) (1425) CTGATCCCGATCAATTTCTTTAATTTTGAGTACATCGTGAAGAAACTG AATAACCAAAACGCAATCGGCGTGTACATTCTGCCGTCTAATCTGACC CTGAAACCAGCATTGTGCATCTTGGAGTCGCACAAAGAGGACTTCCTG AACAAATTTTTGTTGACCATTAGCAGCGAGAACCTGAAACTGCAGTAT AAGTTCAATGGTCAGATCAAAAATCCGAAAAGCGTGAACGAAATCTGG ACCGACCTGTTTAGCATTGCTCACGTCGACATGAAGCTGAGCACCGAC CGTACGCTGTCCTCGTCCATCAGCCAATTTTGGTTTCGCCTGGAGTTC TGTAAAGAGGACAAGGACTTCATCCTGTTTCCGACGGCAAATCGTTAC AGCCGCAAGCTGTGGAAGCACAGCATCAAAAATAATCAGCTGTTTAAG GAAGGTATCCGTAACTACAGCGAGATTAGCTCGCTGCCGTACGAGGAA GACCATAACTTCGACATCGATCTGGTCTTTACCTGGGTCAATTCGGAA GACAAAAACTGGCAGGAACTGTACAAGAAATATAAGCCGGATTTTAAT AGCGATGCCACCTCGACGAGCCGTTTTCTGAGCCGTGACGAGCTGAAG TTTGCGCTGCGCTCGTGGGAAATGAACGGTAGCTTCATCCGTAAAATC TTTATCGTCAGCAACTGCGCGCCGCCGGCCTGGCTGGATCTGAACAAT CCGAAGATCCAATGGGTGTATCACGAGGAGATCATGCCACAGAGCGCC CTGCCAACCTTCAGCAGCCATGCTATTGAGACTAGCTTGCATCACATT CCGGGCATCTCCAACTACTTCATCTACTCTAATGACGATTTTCTGTTG ACCAAACCGCTGAACAAAGACAACTTCTTTTACTCCAACGGTATTGCT AAACTGCGTCTGGAAGCCTGGGGTAACGTTAACGGTGAATGTACCGAA GGCGAGCCGGATTACCTGAACGGCGCGCGTAACGCAAATACGCTGCTG GAGAAAGAGTTTAAAAAGTTTACCACCAAGCTGCACACCCACAGCCCG CAGAGCATGCGTACCGACATCCTGTTCGAGATGGAGAAAAAATACCCA GAAGAGTTCAATCGCACGCTGCACAACAAGTTCCGCAGCCTGGATGAC ATCGCGGTTACCGGCTACCTGTACCATCACTACGCATTGCTGTCTGGC CGCGCTCTGCAATCCAGCGATAAGACCGAACTGGTCCAGCAGAATCAC GACTTTAAAAAGAAGCTGAATAATGTTGTCACCCTGACCAAAGAGCGT AACTTTGATAAGCTGCCGCTGAGCGTTTGTATTAATGACGGTGCAGAC AGCCACCTGAATGAGGAGTGGAATGTGCAAGTTATCAAATTCTTGGAG ACCTTGTTCCCGTTGCCGAGCTCCTTCGAGAAA SEQ ID NO: 4: DNA .DELTA.N97-CP-A (1344) ccttctaatcttactcttaagcctgcattatgtattctagaatcacat aaagaagactttttaaataaatttcttcttactatttcctctgaaaat ttaaagcttcaatacaaatttaatggacaaataaaaaatcctaagtcc gtaaatgaaatttggacagatttatttagcattgctcatgttgacatg aaactcagcacagatagaactttaagttcatctatatctcaattttgg ttcagattagagttctgtaaagaagataaggattttatcttatttcct acagctaacagatattctagaaaactttggaagcactctattaaaaat aatcaattatttaaagaaggcatacgaaactattcagaaatatcttca ttaccctatgaagaagatcataattttgatattgatttagtatttact tgggtcaactcagaagataagaattggcaagagttatataaaaaatat aagcccgactttaatagcgatgcaaccagtacatcaagattccttagt agagatgaattaaaattcgcattacgctcttgggaaatgaatggatcc ttcattcgaaaaatttttattgtctctaattgtgctcccccagcatgg ctagatttaaataaccctaaaattcaatgggtatatcacgaagaaatt atgccacaaagtgcccttcctacttttagctcacatgctattgaaacc agcttgcaccatataccaggaattagtaactattttatttacagcaat gacgacttcctattaactaaaccattgaataaagacaatttcttctat tcgaatggtattgcaaagttaagattagaagcatggggaaatgttaat ggtgaatgtactgaaggagaacctgactacttaaatggtgctcgcaat gcgaacactctcttagaaaaggaatttaaaaaatttactactaaacta catactcactcccctcaatccatgagaactgatattttatttgagatg

gaaaaaaaatatccagaagagtttaatagaacactacataataaattc cgatctttagatgatattgcagtaacgggctatctctatcatcattat gccctactctctggacgagcactacaaagttctgacaagacggaactt gtacagcaaaatcatgatttcaaaaagaaactaaataatgtagtgacc ttaactaaagaaaggaattttgacaaacttcctttgagcgtatgtatc aacgatggtgctgatagtcacttgaatgaagaatggaatgttcaagtt attaagttcttagaaactcttttcccattaccatcatcatttgagaaa SEQ ID NO: 5: DNA .DELTA.N167-CP-A (1134) Actttaagttcatctatatctcaattttggttcagattagagttctgt aaagaagataaggattttatcttatttcctacagctaacagatattct agaaaactttggaagcactctattaaaaataatcaattatttaaagaa ggcatacgaaactattcagaaatatcttcattaccctatgaagaagat cataattttgatattgatttagtatttacttgggtcaactcagaagat aagaattggcaagagttatataaaaaatataagcccgactttaatagc gatgcaaccagtacatcaagattccttagtagagatgaattaaaattc gcattacgctcttgggaaatgaatggatccttcattcgaaaaattttt attgtctctaattgtgctcccccagcatggctagatttaaataaccct aaaattcaatgggtatatcacgaagaaattatgccacaaagtgccctt cctacttttagctcacatgctattgaaaccagcttgcaccatatacca ggaattagtaactattttatttacagcaatgacgacttcctattaact aaaccattgaataaagacaatttcttctattcgaatggtattgcaaag ttaagattagaagcatggggaaatgttaatggtgaatgtactgaagga gaacctgactacttaaatggtgctcgcaatgcgaacactctcttagaa aaggaatttaaaaaatttactactaaactacatactcactcccctcaa tccatgagaactgatattttatttgagatggaaaaaaaatatccagaa gagtttaatagaacactacataataaattccgatctttagatgatatt gcagtaacgggctatctctatcatcattatgccctactctctggacga gcactacaaagttctgacaagacggaacttgtacagcaaaatcatgat ttcaaaaagaaactaaataatgtagtgaccttaactaaagaaaggaat tttgacaaacttcctttgagcgtatgtatcaacgatggtgctgatagt cacttgaatgaagaatggaatgttcaagttattaagttcttagaaact cttttcccattaccatcatcatttgagaaa SEQ ID NO: 6: DNA .DELTA.N235-CP-A (933) gatattgatttagtatttacttgggtcaactcagaagataagaattgg caagagttatataaaaaatataagcccgactttaatagcgatgcaacc agtacatcaagattccttagtagagatgaattaaaattcgcattacgc tcttgggaaatgaatggatccttcattcgaaaaatttttattgtctct aattgtgctcccccagcatggctagatttaaataaccctaaaattcaa tgggtatatcacgaagaaattatgccacaaagtgcccttcctactttt agctcacatgctattgaaaccagcttgcaccatataccaggaattagt aactattttatttacagcaatgacgacttcctattaactaaaccattg aataaagacaatttcttctattcgaatggtattgcaaagttaagatta gaagcatggggaaatgttaatggtgaatgtactgaaggagaacctgac tacttaaatggtgctcgcaatgcgaacactctcttagaaaaggaattt aaaaaatttactactaaactacatactcactcccctcaatccatgaga actgatattttatttgagatggaaaaaaaatatccagaagagtttaat agaacactacataataaattccgatctttagatgatattgcagtaacg ggctatctctatcatcattatgccctactctctggacgagcactacaa agttctgacaagacggaacttgtacagcaaaatcatgatttcaaaaag aaactaaataatgtagtgaccttaactaaagaaaggaattttgacaaa cttcctttgagcgtatgtatcaacgatggtgctgatagtcacttgaat gaagaatggaatgttcaagttattaagttcttagaaactcttttccca ttaccatcatcatttgagaaa SEQ ID NO: 7: DNA .DELTA.C45-CP-A (1497) tttatacttaataacagaaaatggcgtaaacttaaaagagaccctagc gctttctttcgagatagtaaatttaactttttaagatatttttctgct aaaaaatttgcaaagaattttaaaaattcatcacatatccataaaact aatataagtaaagctcaatcaaatatttcttcaaccttaaaacaaaat cggaaacaagatatgttaattcctattaatttttttaattttgaatat atagttaaaaaacttaacaatcaaaacgcaataggtgtatatattctt ccttctaatcttactcttaagcctgcattatgtattctagaatcacat aaagaagactttttaaataaatttcttcttactatttcctctgaaaat ttaaagcttcaatacaaatttaatggacaaataaaaaatcctaagtcc gtaaatgaaatttggacagatttatttagcattgctcatgttgacatg aaactcagcacagatagaactttaagttcatctatatctcaattttgg ttcagattagagttctgtaaagaagataaggattttatcttatttcct acagctaacagatattctagaaaactttggaagcactctattaaaaat aatcaattatttaaagaaggcatacgaaactattcagaaatatcttca ttaccctatgaagaagatcataattttgatattgatttagtatttact tgggtcaactcagaagataagaattggcaagagttatataaaaaatat aagcccgactttaatagcgatgcaaccagtacatcaagattccttagt agagatgaattaaaattcgcattacgctcttgggaaatgaatggatcc ttcattcgaaaaatttttattgtctctaattgtgctcccccagcatgg ctagatttaaataaccctaaaattcaatgggtatatcacgaagaaatt atgccacaaagtgcccttcctacttttagctcacatgctattgaaacc agcttgcaccatataccaggaattagtaactattttatttacagcaat gacgacttcctattaactaaaccattgaataaagacaatttcttctat tcgaatggtattgcaaagttaagattagaagcatggggaaatgttaat ggtgaatgtactgaaggagaacctgactacttaaatggtgctcgcaat gcgaacactctcttagaaaaggaatttaaaaaatttactactaaacta catactcactcccctcaatccatgagaactgatattttatttgagatg gaaaaaaaatatccagaagagtttaatagaacactacataataaattc cgatctttagatgatattgcagtaacgggctatctctatcatcattat gccctactctctggacgagcactacaaagttctgacaagacggaactt gtacagcaaaatcatgatttcaaaaagaaactaaataatgtagtgacc ttaactaaa SEQ ID NO: 8: DNA .DELTA.C25-CP-A (1557) tttatacttaataacagaaaatggcgtaaacttaaaagagaccctagc gctttctttcgagatagtaaatttaactttttaagatatttttctgct aaaaaatttgcaaagaattttaaaaattcatcacatatccataaaact aatataagtaaagctcaatcaaatatttcttcaaccttaaaacaaaat cggaaacaagatatgttaattcctattaatttttttaattttgaatat atagttaaaaaacttaacaatcaaaacgcaataggtgtatatattctt ccttctaatcttactcttaagcctgcattatgtattctagaatcacat aaagaagactttttaaataaatttcttcttactatttcctctgaaaat ttaaagcttcaatacaaatttaatggacaaataaaaaatcctaagtcc gtaaatgaaatttggacagatttatttagcattgctcatgttgacatg aaactcagcacagatagaactttaagttcatctatatctcaattttgg ttcagattagagttctgtaaagaagataaggattttatcttatttcct acagctaacagatattctagaaaactttggaagcactctattaaaaat aatcaattatttaaagaaggcatacgaaactattcagaaatatcttca ttaccctatgaagaagatcataattttgatattgatttagtatttact tgggtcaactcagaagataagaattggcaagagttatataaaaaatat aagcccgactttaatagcgatgcaaccagtacatcaagattccttagt agagatgaattaaaattcgcattacgctcttgggaaatgaatggatcc ttcattcgaaaaatttttattgtctctaattgtgctcccccagcatgg ctagatttaaataaccctaaaattcaatgggtatatcacgaagaaatt atgccacaaagtgcccttcctacttttagctcacatgctattgaaacc agcttgcaccatataccaggaattagtaactattttatttacagcaat gacgacttcctattaactaaaccattgaataaagacaatttcttctat tcgaatggtattgcaaagttaagattagaagcatggggaaatgttaat ggtgaatgtactgaaggagaacctgactacttaaatggtgctcgcaat gcgaacactctcttagaaaaggaatttaaaaaatttactactaaacta catactcactcccctcaatccatgagaactgatattttatttgagatg gaaaaaaaatatccagaagagtttaatagaacactacataataaattc cgatctttagatgatattgcagtaacgggctatctctatcatcattat gccctactctctggacgagcactacaaagttctgacaagacggaactt gtacagcaaaatcatgatttcaaaaagaaactaaataatgtagtgacc ttaactaaagaaaggaattttgacaaacttcctttgagcgtatgtatc aacgatggtgctgatagtcac SEQ ID NO: 9: AS CP-A/CsaB wildtyp/full-length (544) MFILNNRKWRKLKRDPSAFFRDSKFNFLRYFSAKKFAKNFKNSSHIHK TNISKAQSNISSTLKQNRKQDMLIPINFFNFEYIVKKLNNQNAIGVYI LPSNLTLKPALCILESHKEDFLNKFLLTISSENLKLQYKFNGQIKNPK SVNEIWTDLFSIAHVDMKLSTDRTLSSSISQFWFRLEFCKEDKDFILF PTANRYSRKLWKHSIKNNQLFKEGIRNYSEISSLPYEEDHNFDIDLVF

TWVNSEDKNWQELYKKYKPDFNSDATSTSRFLSRDELKFALRSWEMNG SFIRKIFIVSNCAPPAWLDLNNPKIQWVYHEEIMPQSALPTFSSHAIE TSLHHIPGISNYFIYSNDDFLLTKPLNKDNFFYSNGIAKLRLEAWGNV NGECTEGEPDYLNGARNANTLLEKEFKKFTTKLHTHSPQSMRTDILFE MEKKYPEEFNRTLHNKFRSLDDIAVTGYLYHHYALLSGRALQSSDKTE LVQQNHDFKKKLNNVVTLTKERNFDKLPLSVCINDGADSHLNEEWNVQ VIKFLETLFPLPSSFEK SEQ ID NO: 10: AS .DELTA.N69-CP-A (codon optimized) (475) LIPINFFNFEYIVKKLNNQNAIGVYILPSNLTLKPALCILESHKEDFL NKFLLTISSENLKLQYKFNGQIKNPKSVNEIWTDLFSIAHVDMKLSTD RTLSSSISQFWFRLEFCKEDKDFILFPTANRYSRKLWKHSIKNNQLFK EGIRNYSEISSLPYEEDHNFDIDLVFTWVNSEDKNWQELYKKYKPDFN SDATSTSRFLSRDELKFALRSWEMNGSFIRKIFIVSNCAPPAWLDLNN PKIQWVYHEEIMPQSALPTFSSHAIETSLHHIPGISNYFIYSNDDFLL TKPLNKDNFFYSNGIAKLRLEAWGNVNGECTEGEPDYLNGARNANTLL EKEFKKFTTKLHTHSPQSMRTDILFEMEKKYPEEFNRTLHNKFRSLDD IAVTGYLYHHYALLSGRALQSSDKTELVQQNHDFKKKLNNVVTLTKER NFDKLPLSVCINDGADSHLNEEWNVQVIKFLETLFPLPSSFEK SEQ ID NO: 11: AS .DELTA.N97-CP-A (448) PSNLTLKPALCILESHKEDFLNKFLLTISSENLKLQYKFNGQIKNPKS VNEIWTDLFSIAHVDMKLSTDRTLSSSISQFWFRLEFCKEDKDFILFP TANRYSRKLWKHSIKNNQLFKEGIRNYSEISSLPYEEDHNFDIDLVFT WVNSEDKNWQELYKKYKPDFNSDATSTSRFLSRDELKFALRSWEMNGS FIRKIFIVSNCAPPAWLDLNNPKIQWVYHEEIMPQSALPTFSSHAIET SLHHIPGISNYFIYSNDDFLLTKPLNKDNFFYSNGIAKLRLEAWGNVN GECTEGEPDYLNGARNANTLLEKEFKKFTTKLHTHSPQSMRTDILFEM EKKYPEEFNRTLHNKFRSLDDIAVTGYLYHHYALLSGRALQSSDKTEL VQQNHDFKKKLNNVVTLTKERNFDKLPLSVCINDGADSHLNEEWNVQV IKFLETLFPLPSSFEK SEQ ID NO: 12: AS .DELTA.N167-CP-A (378) TLSSSISQFWFRLEFCKEDKDFILFPTANRYSRKLWKHSIKNNQLFKE GIRNYSEISSLPYEEDHNFDIDLVFTWVNSEDKNWQELYKKYKPDFNS DATSTSRFLSRDELKFALRSWEMNGSFIRKIFIVSNCAPPAWLDLNNP KIQWVYHEEIMPQSALPTFSSHAIETSLHHIPGISNYFIYSNDDFLLT KPLNKDNFFYSNGIAKLRLEAWGNVNGECTEGEPDYLNGARNANTLLE KEFKKFTTKLHTHSPQSMRTDILFEMEKKYPEEFNRTLHNKFRSLDDI AVTGYLYHHYALLSGRALQSSDKTELVQQNHDFKKKLNNVVTLTKERN FDKLPLSVCINDGADSHLNEEWNVQVIKFLETLFPLPSSFEK SEQ ID NO: 13: AS .DELTA.N235-CP-A (311) DIDLVFTWVNSEDKNWQELYKKYKPDFNSDATSTSRFLSRDELKFALR SWEMNGSFIRKIFIVSNCAPPAWLDLNNPKIQWVYHEEIMPQSALPTF SSHAIETSLHHIPGISNYFIYSNDDFLLTKPLNKDNFFYSNGIAKLRL EAWGNVNGECTEGEPDYLNGARNANTLLEKEFKKFTTKLHTHSPQSMR TDILFEMEKKYPEEFNRTLHNKFRSLDDIAVTGYLYHHYALLSGRALQ SSDKTELVQQNHDFKKKLNNVVTLTKERNFDKLPLSVCINDGADSHLN EEWNVQVIKFLETLFPLPSSFEK SEQ ID NO: 14: AS .DELTA.C45-CP-A (499) FILNNRKWRKLKRDPSAFFRDSKFNFLRYFSAKKFAKNFKNSSHIHKT NISKAQSNISSTLKQNRKQDMLIPINFFNFEYIVKKLNNQNAIGVYIL PSNLTLKPALCILESHKEDFLNKFLLTISSENLKLQYKFNGQIKNPKS VNEIWTDLFSIAHVDMKLSTDRTLSSSISQFWFRLEFCKEDKDFILFP TANRYSRKLWKHSIKNNQLFKEGIRNYSEISSLPYEEDHNFDIDLVFT WVNSEDKNWQELYKKYKPDFNSDATSTSRFLSRDELKFALRSWEMNGS FIRKIFIVSNCAPPAWLDLNNPKIQWVYHEEIMPQSALPTFSSHAIET SLHHIPGISNYFIYSNDDFLLTKPLNKDNFFYSNGIAKLRLEAWGNVN GECTEGEPDYLNGARNANTLLEKEFKKFTTKLHTHSPQSMRTDILFEM EKKYPEEFNRTLHNKFRSLDDIAVTGYLYHHYALLSGRALQSSDKTEL VQQNHDFKKKLNNVVTLTK SEQ ID NO: 15: AS .DELTA.C25-CP-A (519) FILNNRKWRKLKRDPSAFFRDSKFNFLRYFSAKKFAKNFKNSSHIHKT NISKAQSNISSTLKQNRKQDMLIPINFFNFEYIVKKLNNQNAIGVYIL PSNLTLKPALCILESHKEDFLNKFLLTISSENLKLQYKFNGQIKNPKS VNEIWTDLFSIAHVDMKLSTDRTLSSSISQFWFRLEFCKEDKDFILFP TANRYSRKLWKHSIKNNQLFKEGIRNYSEISSLPYEEDHNFDIDLVFT WVNSEDKNWQELYKKYKPDFNSDATSTSRFLSRDELKFALRSWEMNGS FIRKIFIVSNCAPPAWLDLNNPKIQWVYHEEIMPQSALPTFSSHAIET SLHHIPGISNYFIYSNDDFLLTKPLNKDNFFYSNGIAKLRLEAWGNVN GECTEGEPDYLNGARNANTLLEKEFKKFTTKLHTHSPQSMRTDILFEM EKKYPEEFNRTLHNKFRSLDDIAVTGYLYHHYALLSGRALQSSDKTEL VQQNHDFKKKLNNVVTLTKERNFDKLPLSVCINDGADSH SEQ ID NO: 16: DNA CP-X/CsxA full-length (1455) ATTATGAGCAAAATTAGCAAATTGGTAACCCACCCAAACCTTTTCTTT CGAGATTATTTCTTAAAAAAAGCACCGTTAAATTATGGCGAAAATATT AAACCTTTACCAGTCGAAACCTCTTCTCATAGCAAAAAAAATACAGCC CATAAAACACCCGTATCATCCGACCAACCAATTGAAGATCCATACCCA GTAACATTTCCAATTGATGTAGTTTATACTTGGGTAGATTCAGATGAT GAAAAATTCAATGAAGAACGCCTAAAGTTTCAAAATTCAAGCACATCT GAGACTCTACAAGGCAAAGCAGAAAGCACCGATATTGCAAGATTCCAA TCACGCGACGAATTAAAATATTCGATTCGAAGCCTGATGAAGTATGCC CCATGGGTAAATCATATTTACATTGTAACAAATGGTCAAATACCAAAA TGGTTAGATACCAACAATACAAAGGTAACGATTATCCCTCACTCAACT ATTATCGACAGTCAATTTCTCCCTACTTTTAATTCTCACGTCATTGAA TCCTCTCTATATAAAATCCCAGGATTATCAGAGCATTACATTTATTTC AATGATGATGTCATGCTAGCTAGAGATTTAAGCCCATCTTATTTCTTT ACAAGCAGCGGATTAGCAAAACTGTTTATTACCAACTCTCGTCTACCA AATGGCTATAAGAATGTGAAAGACACACCAACCCAATGGGCCTCAAAA AATTCCCGTGAGCTTTTACATGCAGAAACAGGATTTTGGGCTGAAGCC ATGTTTGCACATACTTTTCATCCACAACGTAAAAGTGTACATGAATCT ATTGAACACCTATGGCATGAACAATTAAATGTTTGTCGTCAAAACCGT TTCCGTGATATTTCAGATATTAACATGGCGACATTCCTGCACCACCAT TTTGCCATTTTGACAGGCCAAGCTCTTGCTACACGCACTAAATGTATT TACTTTAACATTCGCTCTCCTCAAGCAGCTCAGCATTACAAAACATTA TTAGCTCGAAAAGGAAGCGAATACAGCCCACATTCTATCTGCTTAAAT GATCATACATCGAGCAATAAAAATATTTTATCTAATTACGAAGCCAAA TTACAAAGCTTTTTAGAAACATACTATCCAGATGTATCAGAAGCAGAA ATTCTCCTTCCTACTAAATCTGAAGTAGCTGAATTAGTTAAACATAAA GATTATTTAACTGTATATACTAAATTATTACCTATTATCAATAAGCAG CTGGTCAATAAATATAATAAACCTTATTCATATCTTTTCTATTATTTA GGTTTATCTGCCCGGTTTTTATTTGAAGAAACGCAACAAGAACACTAC CGGGAAACTGCTGAAGAAAATTTACAAATCTTTTGTGGCCTAAACCCA AAACATACACTAGCCCTCAAATACTTAGCGGATGTCACCCTCACATCA CAGCCTAGTGGACAA SEQ ID NO: 17: DNA .DELTA.N58-CP-X (1284) CCAATTGAAGATCCATACCCAGTAACATTTCCAATTGATGTAGTTTAT ACTTGGGTAGATTCAGATGATGAAAAATTCAATGAAGAACGCCTAAAG TTTCAAAATTCAAGCACATCTGAGACTCTACAAGGCAAAGCAGAAAGC ACCGATATTGCAAGATTCCAATCACGCGACGAATTAAAATATTCGATT CGAAGCCTGATGAAGTATGCCCCATGGGTAAATCATATTTACATTGTA ACAAATGGTCAAATACCAAAATGGTTAGATACCAACAATACAAAGGTA ACGATTATCCCTCACTCAACTATTATCGACAGTCAATTTCTCCCTACT TTTAATTCTCACGTCATTGAATCCTCTCTATATAAAATCCCAGGATTA TCAGAGCATTACATTTATTTCAATGATGATGTCATGCTAGCTAGAGAT TTAAGCCCATCTTATTTCTTTACAAGCAGCGGATTAGCAAAACTGTTT ATTACCAACTCTCGTCTACCAAATGGCTATAAGAATGTGAAAGACACA CCAACCCAATGGGCCTCAAAAAATTCCCGTGAGCTTTTACATGCAGAA ACAGGATTTTGGGCTGAAGCCATGTTTGCACATACTTTTCATCCACAA CGTAAAAGTGTACATGAATCTATTGAACACCTATGGCATGAACAATTA AATGTTTGTCGTCAAAACCGTTTCCGTGATATTTCAGATATTAACATG GCGACATTCCTGCACCACCATTTTGCCATTTTGACAGGCCAAGCTCTT GCTACACGCACTAAATGTATTTACTTTAACATTCGCTCTCCTCAAGCA GCTCAGCATTACAAAACATTATTAGCTCGAAAAGGAAGCGAATACAGC CCACATTCTATCTGCTTAAATGATCATACATCGAGCAATAAAAATATT TTATCTAATTACGAAGCCAAATTACAAAGCTTTTTAGAAACATACTAT CCAGATGTATCAGAAGCAGAAATTCTCCTTCCTACTAAATCTGAAGTA GCTGAATTAGTTAAACATAAAGATTATTTAACTGTATATACTAAATTA

TTACCTATTATCAATAAGCAGCTGGTCAATAAATATAATAAACCTTAT TCATATCTTTTCTATTATTTAGGTTTATCTGCCCGGTTTTTATTTGAA GAAACGCAACAAGAACACTACCGGGAAACTGCTGAAGAAAATTTACAA ATCTTTTGTGGCCTAAACCCAAAACATACACTAGCCCTCAAATACTTA GCGGATGTCACCCTCACATCACAGCCTAGTGGACAA SEQ ID NO: 18: DNA .DELTA.N104-CP-X (1146) GAAAGCACCGATATTGCAAGATTCCAATCACGCGACGAATTAAAATAT TCGATTCGAAGCCTGATGAAGTATGCCCCATGGGTAAATCATATTTAC ATTGTAACAAATGGTCAAATACCAAAATGGTTAGATACCAACAATACA AAGGTAACGATTATCCCTCACTCAACTATTATCGACAGTCAATTTCTC CCTACTTTTAATTCTCACGTCATTGAATCCTCTCTATATAAAATCCCA GGATTATCAGAGCATTACATTTATTTCAATGATGATGTCATGCTAGCT AGAGATTTAAGCCCATCTTATTTCTTTACAAGCAGCGGATTAGCAAAA CTGTTTATTACCAACTCTCGTCTACCAAATGGCTATAAGAATGTGAAA GACACACCAACCCAATGGGCCTCAAAAAATTCCCGTGAGCTTTTACAT GCAGAAACAGGATTTTGGGCTGAAGCCATGTTTGCACATACTTTTCAT CCACAACGTAAAAGTGTACATGAATCTATTGAACACCTATGGCATGAA CAATTAAATGTTTGTCGTCAAAACCGTTTCCGTGATATTTCAGATATT AACATGGCGACATTCCTGCACCACCATTTTGCCATTTTGACAGGCCAA GCTCTTGCTACACGCACTAAATGTATTTACTTTAACATTCGCTCTCCT CAAGCAGCTCAGCATTACAAAACATTATTAGCTCGAAAAGGAAGCGAA TACAGCCCACATTCTATCTGCTTAAATGATCATACATCGAGCAATAAA AATATTTTATCTAATTACGAAGCCAAATTACAAAGCTTTTTAGAAACA TACTATCCAGATGTATCAGAAGCAGAAATTCTCCTTCCTACTAAATCT GAAGTAGCTGAATTAGTTAAACATAAAGATTATTTAACTGTATATACT AAATTATTACCTATTATCAATAAGCAGCTGGTCAATAAATATAATAAA CCTTATTCATATCTTTTCTATTATTTAGGTTTATCTGCCCGGTTTTTA TTTGAAGAAACGCAACAAGAACACTACCGGGAAACTGCTGAAGAAAAT TTACAAATCTTTTGTGGCCTAAACCCAAAACATACACTAGCCCTCAAA TACTTAGCGGATGTCACCCTCACATCACAGCCTAGTGGACAA SEQ ID NO: 19: DNA .DELTA.C174-CP-X (933) ATTATGAGCAAAATTAGCAAATTGGTAACCCACCCAAACCTTTTCTTT CGAGATTATTTCTTAAAAAAAGCACCGTTAAATTATGGCGAAAATATT AAACCTTTACCAGTCGAAACCTCTTCTCATAGCAAAAAAAATACAGCC CATAAAACACCCGTATCATCCGACCAACCAATTGAAGATCCATACCCA GTAACATTTCCAATTGATGTAGTTTATACTTGGGTAGATTCAGATGAT GAAAAATTCAATGAAGAACGCCTAAAGTTTCAAAATTCAAGCACATCT GAGACTCTACAAGGCAAAGCAGAAAGCACCGATATTGCAAGATTCCAA TCACGCGACGAATTAAAATATTCGATTCGAAGCCTGATGAAGTATGCC CCATGGGTAAATCATATTTACATTGTAACAAATGGTCAAATACCAAAA TGGTTAGATACCAACAATACAAAGGTAACGATTATCCCTCACTCAACT ATTATCGACAGTCAATTTCTCCCTACTTTTAATTCTCACGTCATTGAA TCCTCTCTATATAAAATCCCAGGATTATCAGAGCATTACATTTATTTC AATGATGATGTCATGCTAGCTAGAGATTTAAGCCCATCTTATTTCTTT ACAAGCAGCGGATTAGCAAAACTGTTTATTACCAACTCTCGTCTACCA AATGGCTATAAGAATGTGAAAGACACACCAACCCAATGGGCCTCAAAA AATTCCCGTGAGCTTTTACATGCAGAAACAGGATTTTGGGCTGAAGCC ATGTTTGCACATACTTTTCATCCACAACGTAAAAGTGTACATGAATCT ATTGAACACCTATGGCATGAACAATTAAATGTTTGTCGTCAAAACCGT TTCCGTGATATTTCAGATATTAACATGGCGACATTCCTGCACCACCAT TTTGCCATTTTGACAGGCCAA SEQ ID NO: 20: DNA .DELTA.C99-CP-X (1158) ATTATGAGCAAAATTAGCAAATTGGTAACCCACCCAAACCTTTTCTTT CGAGATTATTTCTTAAAAAAAGCACCGTTAAATTATGGCGAAAATATT AAACCTTTACCAGTCGAAACCTCTTCTCATAGCAAAAAAAATACAGCC CATAAAACACCCGTATCATCCGACCAACCAATTGAAGATCCATACCCA GTAACATTTCCAATTGATGTAGTTTATACTTGGGTAGATTCAGATGAT GAAAAATTCAATGAAGAACGCCTAAAGTTTCAAAATTCAAGCACATCT GAGACTCTACAAGGCAAAGCAGAAAGCACCGATATTGCAAGATTCCAA TCACGCGACGAATTAAAATATTCGATTCGAAGCCTGATGAAGTATGCC CCATGGGTAAATCATATTTACATTGTAACAAATGGTCAAATACCAAAA TGGTTAGATACCAACAATACAAAGGTAACGATTATCCCTCACTCAACT ATTATCGACAGTCAATTTCTCCCTACTTTTAATTCTCACGTCATTGAA TCCTCTCTATATAAAATCCCAGGATTATCAGAGCATTACATTTATTTC AATGATGATGTCATGCTAGCTAGAGATTTAAGCCCATCTTATTTCTTT ACAAGCAGCGGATTAGCAAAACTGTTTATTACCAACTCTCGTCTACCA AATGGCTATAAGAATGTGAAAGACACACCAACCCAATGGGCCTCAAAA AATTCCCGTGAGCTTTTACATGCAGAAACAGGATTTTGGGCTGAAGCC ATGTTTGCACATACTTTTCATCCACAACGTAAAAGTGTACATGAATCT ATTGAACACCTATGGCATGAACAATTAAATGTTTGTCGTCAAAACCGT TTCCGTGATATTTCAGATATTAACATGGCGACATTCCTGCACCACCAT TTTGCCATTTTGACAGGCCAAGCTCTTGCTACACGCACTAAATGTATT TACTTTAACATTCGCTCTCCTCAAGCAGCTCAGCATTACAAAACATTA TTAGCTCGAAAAGGAAGCGAATACAGCCCACATTCTATCTGCTTAAAT GATCATACATCGAGCAATAAAAATATTTTATCTAATTACGAAGCCAAA TTACAAAGCTTTTTAGAAACATACTATCCAGATGTATCAGAAGCAGAA ATTCTC SEQ ID NO: 21: DNA .DELTA.N58.DELTA.C99-CP-X (987) CCAATTGAAGATCCATACCCAGTAACATTTCCAATTGATGTAGTTTAT ACTTGGGTAGATTCAGATGATGAAAAATTCAATGAAGAACGCCTAAAG TTTCAAAATTCAAGCACATCTGAGACTCTACAAGGCAAAGCAGAAAGC ACCGATATTGCAAGATTCCAATCACGCGACGAATTAAAATATTCGATT CGAAGCCTGATGAAGTATGCCCCATGGGTAAATCATATTTACATTGTA ACAAATGGTCAAATACCAAAATGGTTAGATACCAACAATACAAAGGTA ACGATTATCCCTCACTCAACTATTATCGACAGTCAATTTCTCCCTACT TTTAATTCTCACGTCATTGAATCCTCTCTATATAAAATCCCAGGATTA TCAGAGCATTACATTTATTTCAATGATGATGTCATGCTAGCTAGAGAT TTAAGCCCATCTTATTTCTTTACAAGCAGCGGATTAGCAAAACTGTTT ATTACCAACTCTCGTCTACCAAATGGCTATAAGAATGTGAAAGACACA CCAACCCAATGGGCCTCAAAAAATTCCCGTGAGCTTTTACATGCAGAA ACAGGATTTTGGGCTGAAGCCATGTTTGCACATACTTTTCATCCACAA CGTAAAAGTGTACATGAATCTATTGAACACCTATGGCATGAACAATTA AATGTTTGTCGTCAAAACCGTTTCCGTGATATTTCAGATATTAACATG GCGACATTCCTGCACCACCATTTTGCCATTTTGACAGGCCAAGCTCTT GCTACACGCACTAAATGTATTTACTTTAACATTCGCTCTCCTCAAGCA GCTCAGCATTACAAAACATTATTAGCTCGAAAAGGAAGCGAATACAGC CCACATTCTATCTGCTTAAATGATCATACATCGAGCAATAAAAATATT TTATCTAATTACGAAGCCAAATTACAAAGCTTTTTAGAAACATACTAT CCAGATGTATCAGAAGCAGAAATTCTC SEQ ID NO: 22: DNA .DELTA.N65.DELTA.C10-CP-X (1233) GTAACATTTCCAATTGATGTAGTTTATACTTGGGTAGATTCAGATGAT GAAAAATTCAATGAAGAACGCCTAAAGTTTCAAAATTCAAGCACATCT GAGACTCTACAAGGCAAAGCAGAAAGCACCGATATTGCAAGATTCCAA TCACGCGACGAATTAAAATATTCGATTCGAAGCCTGATGAAGTATGCC CCATGGGTAAATCATATTTACATTGTAACAAATGGTCAAATACCAAAA TGGTTAGATACCAACAATACAAAGGTAACGATTATCCCTCACTCAACT ATTATCGACAGTCAATTTCTCCCTACTTTTAATTCTCACGTCATTGAA TCCTCTCTATATAAAATCCCAGGATTATCAGAGCATTACATTTATTTC AATGATGATGTCATGCTAGCTAGAGATTTAAGCCCATCTTATTTCTTT ACAAGCAGCGGATTAGCAAAACTGTTTATTACCAACTCTCGTCTACCA AATGGCTATAAGAATGTGAAAGACACACCAACCCAATGGGCCTCAAAA AATTCCCGTGAGCTTTTACATGCAGAAACAGGATTTTGGGCTGAAGCC ATGTTTGCACATACTTTTCATCCACAACGTAAAAGTGTACATGAATCT ATTGAACACCTATGGCATGAACAATTAAATGTTTGTCGTCAAAACCGT TTCCGTGATATTTCAGATATTAACATGGCGACATTCCTGCACCACCAT TTTGCCATTTTGACAGGCCAAGCTCTTGCTACACGCACTAAATGTATT TACTTTAACATTCGCTCTCCTCAAGCAGCTCAGCATTACAAAACATTA TTAGCTCGAAAAGGAAGCGAATACAGCCCACATTCTATCTGCTTAAAT GATCATACATCGAGCAATAAAAATATTTTATCTAATTACGAAGCCAAA TTACAAAGCTTTTTAGAAACATACTATCCAGATGTATCAGAAGCAGAA ATTCTCCTTCCTACTAAATCTGAAGTAGCTGAATTAGTTAAACATAAA GATTATTTAACTGTATATACTAAATTATTACCTATTATCAATAAGCAG CTGGTCAATAAATATAATAAACCTTATTCATATCTTTTCTATTATTTA GGTTTATCTGCCCGGTTTTTATTTGAAGAAACGCAACAAGAACACTAC CGGGAAACTGCTGAAGAAAATTTACAAATCTTTTGTGGCCTAAACCCA AAACATACACTAGCCCTCAAATACTTAGCGGAT

SEQ ID NO: 23: DNA .DELTA.N67.DELTA.C99-CP-X (960) TTTCCAATTGATGTAGTTTATACTTGGGTAGATTCAGATGATGAAAAA TTCAATGAAGAACGCCTAAAGTTTCAAAATTCAAGCACATCTGAGACT CTACAAGGCAAAGCAGAAAGCACCGATATTGCAAGATTCCAATCACGC GACGAATTAAAATATTCGATTCGAAGCCTGATGAAGTATGCCCCATGG GTAAATCATATTTACATTGTAACAAATGGTCAAATACCAAAATGGTTA GATACCAACAATACAAAGGTAACGATTATCCCTCACTCAACTATTATC GACAGTCAATTTCTCCCTACTTTTAATTCTCACGTCATTGAATCCTCT CTATATAAAATCCCAGGATTATCAGAGCATTACATTTATTTCAATGAT GATGTCATGCTAGCTAGAGATTTAAGCCCATCTTATTTCTTTACAAGC AGCGGATTAGCAAAACTGTTTATTACCAACTCTCGTCTACCAAATGGC TATAAGAATGTGAAAGACACACCAACCCAATGGGCCTCAAAAAATTCC CGTGAGCTTTTACATGCAGAAACAGGATTTTGGGCTGAAGCCATGTTT GCACATACTTTTCATCCACAACGTAAAAGTGTACATGAATCTATTGAA CACCTATGGCATGAACAATTAAATGTTTGTCGTCAAAACCGTTTCCGT GATATTTCAGATATTAACATGGCGACATTCCTGCACCACCATTTTGCC ATTTTGACAGGCCAAGCTCTTGCTACACGCACTAAATGTATTTACTTT AACATTCGCTCTCCTCAAGCAGCTCAGCATTACAAAACATTATTAGCT CGAAAAGGAAGCGAATACAGCCCACATTCTATCTGCTTAAATGATCAT ACATCGAGCAATAAAAATATTTTATCTAATTACGAAGCCAAATTACAA AGCTTTTTAGAAACATACTATCCAGATGTATCAGAAGCAGAAATTCTC SEQ ID NO: 24: AS CP-X/CsxA full-length (485) IMSKISKLVTHPNLFFRDYFLKKAPLNYGENIKPLPVETSSHSKKNTA HKTPVSSDQPIEDPYPVTFPIDVVYTWVDSDDEKFNEERLKFQNSSTS ETLQGKAESTDIARFQSRDELKYSIRSLMKYAPWVNHIYIVTNGQIPK WLDTNNTKVTIIPHSTIIDSQFLPTFNSHVIESSLYKIPGLSEHYIYF NDDVMLARDLSPSYFFTSSGLAKLFITNSRLPNGYKNVKDTPTQWASK NSRELLHAETGFWAEAMFAHTFHPQRKSVHESIEHLWHEQLNVCRQNR FRDISDINMATFLHHHFAILTGQALATRTKCIYFNIRSPQAAQHYKTL LARKGSEYSPHSICLNDHTSSNKNILSNYEAKLQSFLETYYPDVSEAE ILLPTKSEVAELVKHKDYLTVYTKLLPIINKQLVNKYNKPYSYLFYYL GLSARFLFEETQQEHYRETAEENLQIFCGLNPKHTLALKYLADVTLTS QPSGQ SEQ ID NO: 25: AS .DELTA.N58-CP-X (428) PIEDPYPVTFPIDVVYTWVDSDDEKFNEERLKFQNSSTSETLQGKAES TDIARFQSRDELKYSIRSLMKYAPWVNHIYIVTNGQIPKWLDTNNTKV TIIPHSTIIDSQFLPTFNSHVIESSLYKIPGLSEHYIYFNDDVMLARD LSPSYFFTSSGLAKLFITNSRLPNGYKNVKDTPTQWASKNSRELLHAE TGFWAEAMFAHTFHPQRKSVHESIEHLWHEQLNVCRQNRFRDISDINM ATFLHHHFAILTGQALATRTKCIYFNIRSPQAAQHYKTLLARKGSEYS PHSICLNDHTSSNKNILSNYEAKLQSFLETYYPDVSEAEILLPTKSEV AELVKHKDYLTVYTKLLPIINKQLVNKYNKPYSYLFYYLGLSARFLFE ETQQEHYRETAEENLQIFCGLNPKHTLALKYLADVTLTSQPSGQ SEQ ID NO: 26: AS .DELTA.N104-CP-X (382) ESTDIARFQSRDELKYSIRSLMKYAPWVNHIYIVTNGQIPKWLDTNNT KVTIIPHSTIIDSQFLPTFNSHVIESSLYKIPGLSEHYIYFNDDVMLA RDLSPSYFFTSSGLAKLFITNSRLPNGYKNVKDTPTQWASKNSRELLH AETGFWAEAMFAHTFHPQRKSVHESIEHLWHEQLNVCRQNRFRDISDI NMATFLHHHFAILTGQALATRTKCIYFNIRSPQAAQHYKTLLARKGSE YSPHSICLNDHTSSNKNILSNYEAKLQSFLETYYPDVSEAEILLPTKS EVAELVKHKDYLTVYTKLLPIINKQLVNKYNKPYSYLFYYLGLSARFL FEETQQEHYRETAEENLQIFCGLNPKHTLALKYLADVTLTSQPSGQ SEQ ID NO: 27: AS .DELTA.C174-CP-X (311) IMSKISKLVTHPNLFFRDYFLKKAPLNYGENIKPLPVETSSHSKKNTA HKTPVSSDQPIEDPYPVTFPIDVVYTWVDSDDEKFNEERLKFQNSSTS ETLQGKAESTDIARFQSRDELKYSIRSLMKYAPWVNHIYIVTNGQIPK WLDTNNTKVTIIPHSTIIDSQFLPTFNSHVIESSLYKIPGLSEHYIYF NDDVMLARDLSPSYFFTSSGLAKLFITNSRLPNGYKNVKDTPTQWASK NSRELLHAETGFWAEAMFAHTFHPQRKSVHESIEHLWHEQLNVCRQNR FRDISDINMATFLHHHFAILTGQ SEQ ID NO: 28: AS .DELTA.C99-CP-X (386) IMSKISKLVTHPNLFFRDYFLKKAPLNYGENIKPLPVETSSHSKKNTA HKTPVSSDQPIEDPYPVTFPIDVVYTWVDSDDEKFNEERLKFQNSSTS ETLQGKAESTDIARFQSRDELKYSIRSLMKYAPWVNHIYIVTNGQIPK WLDTNNTKVTIIPHSTIIDSQFLPTFNSHVIESSLYKIPGLSEHYIYF NDDVMLARDLSPSYFFTSSGLAKLFITNSRLPNGYKNVKDTPTQWASK NSRELLHAETGFWAEAMFAHTFHPQRKSVHESIEHLWHEQLNVCRQNR FRDISDINMATFLHHHFAILTGQALATRTKCIYFNIRSPQAAQHYKTL LARKGSEYSPHSICLNDHTSSNKNILSNYEAKLQSFLETYYPDVSEAE IL SEQ ID NO: 29: AS .DELTA.N58.DELTA.C99-CP-X (329) PIEDPYPVTFPIDVVYTWVDSDDEKFNEERLKFQNSSTSETLQGKAES TDIARFQSRDELKYSIRSLMKYAPWVNHIYIVTNGQIPKWLDTNNTKV TIIPHSTIIDSQFLPTFNSHVIESSLYKIPGLSEHYIYFNDDVMLARD LSPSYFFTSSGLAKLFITNSRLPNGYKNVKDTPTQWASKNSRELLHAE TGFWAEAMFAHTFHPQRKSVHESIEHLWHEQLNVCRQNRFRDISDINM ATFLHHHFAILTGQALATRTKCIYFNIRSPQAAQHYKTLLARKGSEYS PHSICLNDHTSSNKNILSNYEAKLQSFLETYYPDVSEAEIL SEQ ID NO: 30: AS .DELTA.N65.DELTA.C10-CP-X (411) VTFPIDVVYTWVDSDDEKFNEERLKFQNSSTSETLQGKAESTDIARFQ SRDELKYSIRSLMKYAPWVNHIYIVTNGQIPKWLDTNNTKVTIIPHST IIDSQFLPTFNSHVIESSLYKIPGLSEHYIYFNDDVMLARDLSPSYFF TSSGLAKLFITNSRLPNGYKNVKDTPTQWASKNSRELLHAETGFWAEA MFAHTFHPQRKSVHESIEHLWHEQLNVCRQNRFRDISDINMATFLHHH FAILTGQALATRTKCIYFNIRSPQAAQHYKTLLARKGSEYSPHSICLN DHTSSNKNILSNYEAKLQSFLETYYPDVSEAEILLPTKSEVAELVKHK DYLTVYTKLLPIINKQLVNKYNKPYSYLFYYLGLSARFLFEETQQEHY RETAEENLQIFCGLNPKHTLALKYLAD SEQ ID NO: 31: AS .DELTA.N67.DELTA.C99-CP-X (320) FPIDVVYTWVDSDDEKFNEERLKFQNSSTSETLQGKAESTDIARFQSR DELKYSIRSLMKYAPWVNHIYIVTNGQIPKWLDTNNTKVTIIPHSTII DSQFLPTFNSHVIESSLYKIPGLSEHYIYFNDDVMLARDLSPSYFFTS SGLAKLFITNSRLPNGYKNVKDTPTQWASKNSRELLHAETGFWAEAMF AHTFHPQRKSVHESIEHLWHEQLNVCRQNRFRDISDINMATFLHHHFA ILTGQALATRTKCIYFNIRSPQAAQHYKTLLARKGSEYSPHSICLNDH TSSNKNILSNYEAKLQSFLETYYPDVSEAEIL SEQ ID NO: 32: UDP-GIcNAc-Epimerase (NmA) cloned from Neisseria meningitidis serogroup A, coding sequence >UDP-GIcNAc-Epimerase-NmA (AF019760 REGION: 479..1597) Atgaaagtcttaaccgtctttggcactcgccctgaagctattaaaatg gcgcctgtaattctagagttacaaaaacataacacaattacttcaaaa gtttgcattactgcacagcatcgtgaaatgctagatcaggttttgagc ctattcgaaatcaaagctgattatgatttaaatatcatgaaacccaac cagagcctacaagaaatcacaacaaatatcatctcaagccttaccgat gttcttgaagatttcaaacctgactgcgtccttgctcacggagacacc acaacaacttttgcagctagccttgctgcattctatcaaaaaatacct gttggccacattgaagcaggcctgagaacttataatttatactctcct tggccagaggaagcaaataggcgtttaacaagcgttctaagccagtgg cattttgcacctactgaagattctaaaaataacttactatctgaatca ataccttctgacaaagttattgttactggaaatactgtcatagatgca ctaatggtatctctagaaaaactaaaaataactacaattaaaaaacaa atggaacaagcttttccatttattcaggacaactctaaagtaatttta attaccgctcatagaagagaaaatcatggggaaggtattaaaaatatt ggactttctatcttagaattagctaaaaaatacccaacattctctttt gtgattccgctccatttaaatcctaacgttagaaaaccaattcaagat ttattatcctctgtgcacaatgttcatcttattgagccacaagaatac ttaccattcgtatatttaatgtctaaaagccatataatattaagtgat tcaggcggcatacaagaagaagctccatccctaggaaaaccagttctt gtattaagagatactacagaacgtcctgaagctgtagctgcaggaact gtaaaattagtaggttctgaaactcaaaatattattgagagctttaca caactaattgaataccctgaatattatgaaaaaatggctaatattgaa aacccttacgggataggtaatgcctcaaaaatcattgtagaaacttta ttaaagaatagataa SEQ ID NO: 33: UDP-GIcNAc-Epimerase (NmA) cloned from Neisseria meningitidis serogroup A amino acid sequence >UDP-GIcNAc-Epimerase-NmA (AAC38285).pro mkvltvfgtrpeaikmapvilelqkhntitskvcitaqhremldqvls

lfeikadydlnimkpnqslgeittniissltdvledfkpdcvlahgdt tttfaaslaafyqkipvghieaglrtynlyspwpeeanrrltsvlsqw hfaptedsknnllsesipsdkvivtgntvidalmvsleklkittikkq megafpfiqdnskvilitahrrenhgegikniglsilelakkyptfsf yiplhlnpnvrkpiqdllssvhnvhliepqeylpfvylmskshiilsd sggiqeeapslgkpvlvlrdtterpeavaagtvklygsetqniiesft qlieypeyyekmanienpygignaskiivetllknr

[0546] The sequences provided herein (e.g. the sequences corresponding to CP-A or fragments thereof) may be linked to (a) tag(s). For example, the sequences may have a StrepII-thrombin-tag at the N-terminus and/or a his-tag at the C-terminus. The constructs may be prepared by using appropriate restriction sites. For example, CP-A-constructs may be cloned by using BamHI/BglII and XhoI. It is mentioned that, if the N-terminus is extended (e.g. by tags), then ATG/Met1 can be omitted. Accordingly, a construct comprising the full length CP-A may comprise not 545 but 544 CP-A-amino acids. SEQ ID NOs: 34 and 37 show an example for a StrepII-thrombin-BamHI/BglII-CP-A-XhoI-his construct. In context of the invention, all herein disclosed sequences may be used to produce constructs as demonstrated for CP-A in SEQ ID NOs: 34 and 37.

TABLE-US-00011 SEQ ID NO: 34: DNA StrepII-Thrombin-BamHI/BgIII-CsaB-XhoI-His6 atggctagctggagccacccgcagttcgaaaaaggcgccctggttccg cgtgGATCTtttatacttaataacagaaaatggcgtaaacttaaaaga gaccctagcgctttctttcgagatagtaaatttaactttttaagatat ttttctgctaaaaaatttgcaaagaattttaaaaattcatcacatatc cataaaactaatataagtaaagctcaatcaaatatttcttcaacctta aaacaaaatcggaaacaagatatgttaattcctattaatttttttaat tttgaatatatagttaaaaaacttaacaatcaaaacgcaataggtgta tatattcttccttctaatcttactcttaagcctgcattatgtattcta gaatcacataaagaagactttttaaataaatttcttcttactatttcc tctgaaaatttaaagcttcaatacaaatttaatggacaaataaaaaat cctaagtccgtaaatgaaatttggacagatttatttagcattgctcat gttgacatgaaactcagcacagatagaactttaagttcatctatatct caattttggttcagattagagttctgtaaagaagataaggattttatc ttatttcctacagctaacagatattctagaaaactttggaagcactct attaaaaataatcaattatttaaagaaggcatacgaaactattcagaa atatcttcattaccctatgaagaagatcataattttgatattgattta gtatttacttgggtcaactcagaagataagaattggcaagagttatat aaaaaatataagcccgactttaatagcgatgcaaccagtacatcaaga ttccttagtagagatgaattaaaattcgcattacgctcttgggaaatg aatggatccttcattcgaaaaatttttattgtctctaattgtgctccc ccagcatggctagatttaaataaccctaaaattcaatgggtatatcac gaagaaattatgccacaaagtgcccttcctacttttagctcacatgct attgaaaccagcttgcaccatataccaggaattagtaactattttatt tacagcaatgacgacttcctattaactaaaccattgaataaagacaat ttcttctattcgaatggtattgcaaagttaagattagaagcatgggga aatgttaatggtgaatgtactgaaggagaacctgactacttaaatggt gctcgcaatgcgaacactctcttagaaaaggaatttaaaaaatttact actaaactacatactcactcccctcaatccatgagaactgatatttta tttgagatggaaaaaaaatatccagaagagtttaatagaacactacat aataaattccgatctttagatgatattgcagtaacgggctatctctat catcattatgccctactctctggacgagcactacaaagttctgacaag acggaacttgtacagcaaaatcatgatttcaaaaagaaactaaataat gtagtgaccttaactaaagaaaggaattttgacaaacttcctttgagc gtatgtatcaacgatggtgctgatagtcacttgaatgaagaatggaat gttcaagttattaagttcttagaaactcttttcccattaccatcatca tttgagaaacTCGAGcaccaccaccaccaccac SEQ ID NO: 35: DNA StrepII-Thrombin-BamHI/BgIII atggctagctggagccacccgcagttcgaaaaaggcgccctggttccg cgtgGATCT SEQ ID NO: 36: DNA XhoI-His6 cTCGAGcaccaccaccaccaccac SEQ ID NO: 37: AS StrepII-Thrombin-BamHI/BgIII-CsaB-XhoI-His6 MASWSHPQFEKGALVPRGSFILNNRKWRKLKRDPSAFFRDSKFNFLRY FSAKKFAKNFKNSSHIHKTNISKAQSNISSTLKQNRKQDMLIPINFFN FEYIVKKLNNQNAIGVYILPSNLTLKPALCILESHKEDFLNKFLLTIS SENLKLQYKFNGQIKNPKSVNEIWTDLFSIAHVDMKLSTDRTLSSSIS QFWFRLEFCKEDKDFILFPTANRYSRKLWKHSIKNNQLFKEGIRNYSE ISSLPYEEDHNFDIDLVFTWVNSEDKNWQELYKKYKPDFNSDATSTSR FLSRDELKFALRSWEMNGSFIRKIFIVSNCAPPAWLDLNNPKIQWVYH EEIMPQSALPTFSSHAIETSLHHIPGISNYFIYSNDDFLLTKPLNKDN FFYSNGIAKLRLEAWGNVNGECTEGEPDYLNGARNANTLLEKEFKKFT TKLHTHSPQSMRTDILFEMEKKYPEEFNRTLHNKFRSLDDIAVTGYLY HHYALLSGRALQSSDKTELVQQNHDFKKKLNNVVTLTKERNFDKLPLS VCINDGADSHLNEEWNVQVIKFLETLFPLPSSFEKLEHHHHHH SEQ ID NO: 38: AS StrepII-Thrombin-BamHI MASWSHPQFEKGALVPRGS SEQ ID NO: 39: AS XhoI-His6 LEHHHHHH

[0547] The sequences provided herein (e.g. the sequences corresponding to .DELTA.69-CP-A) may be cloned into the vector by using NdeI/XhoI. SEQ ID NOs: 40 and 43 show an example for a NdeI-.DELTA.N69-CP-A-XhoI-his construct. In context of the invention, all herein disclosed sequences may be used to produce constructs as demonstrated for the codon optimized .DELTA.N69-CP-A in SEQ ID NOs: 40 and 43.

TABLE-US-00012 SEQ ID NO: 40: DNA NdeI-dN69CsaB(co)-XhoI-His6 catatgCTGATCCCGATCAATTTCTTTAATTTTGAGTACATCGTGAAG AAACTGAATAACCAAAACGCAATCGGCGTGTACATTCTGCCGTCTAAT CTGACCCTGAAACCAGCATTGTGCATCTTGGAGTCGCACAAAGAGGAC TTCCTGAACAAATTTTTGTTGACCATTAGCAGCGAGAACCTGAAACTG CAGTATAAGTTCAATGGTCAGATCAAAAATCCGAAAAGCGTGAACGAA ATCTGGACCGACCTGTTTAGCATTGCTCACGTCGACATGAAGCTGAGC ACCGACCGTACGCTGTCCTCGTCCATCAGCCAATTTTGGTTTCGCCTG GAGTTCTGTAAAGAGGACAAGGACTTCATCCTGTTTCCGACGGCAAAT CGTTACAGCCGCAAGCTGTGGAAGCACAGCATCAAAAATAATCAGCTG TTTAAGGAAGGTATCCGTAACTACAGCGAGATTAGCTCGCTGCCGTAC GAGGAAGACCATAACTTCGACATCGATCTGGTCTTTACCTGGGTCAAT TCGGAAGACAAAAACTGGCAGGAACTGTACAAGAAATATAAGCCGGAT TTTAATAGCGATGCCACCTCGACGAGCCGTTTTCTGAGCCGTGACGAG CTGAAGTTTGCGCTGCGCTCGTGGGAAATGAACGGTAGCTTCATCCGT AAAATCTTTATCGTCAGCAACTGCGCGCCGCCGGCCTGGCTGGATCTG AACAATCCGAAGATCCAATGGGTGTATCACGAGGAGATCATGCCACAG AGCGCCCTGCCAACCTTCAGCAGCCATGCTATTGAGACTAGCTTGCAT CACATTCCGGGCATCTCCAACTACTTCATCTACTCTAATGACGATTTT CTGTTGACCAAACCGCTGAACAAAGACAACTTCTTTTACTCCAACGGT ATTGCTAAACTGCGTCTGGAAGCCTGGGGTAACGTTAACGGTGAATGT ACCGAAGGCGAGCCGGATTACCTGAACGGCGCGCGTAACGCAAATACG CTGCTGGAGAAAGAGTTTAAAAAGTTTACCACCAAGCTGCACACCCAC AGCCCGCAGAGCATGCGTACCGACATCCTGTTCGAGATGGAGAAAAAA TACCCAGAAGAGTTCAATCGCACGCTGCACAACAAGTTCCGCAGCCTG GATGACATCGCGGTTACCGGCTACCTGTACCATCACTACGCATTGCTG TCTGGCCGCGCTCTGCAATCCAGCGATAAGACCGAACTGGTCCAGCAG AATCACGACTTTAAAAAGAAGCTGAATAATGTTGTCACCCTGACCAAA GAGCGTAACTTTGATAAGCTGCCGCTGAGCGTTTGTATTAATGACGGT GCAGACAGCCACCTGAATGAGGAGTGGAATGTGCAAGTTATCAAATTC TTGGAGACCTTGTTCCCGTTGCCGAGCTCCTTCGAGAAAcTCGAGcac caccaccaccaccac SEQ ID NO: 41: DNA NdeI catatg SEQ ID NO: 42: DNA XhoI-His6 cTCGAGcaccaccaccaccaccac SEQ ID NO: 43: AS NdeI-dN69CsaB(co)-XhoI-His6 MLIPINFFNFEYIVKKLNNQNAIGVYILPSNLTLKPALCILESHKEDF LNKFLLTISSENLKLQYKFNGQIKNPKSVNEIWTDLFSIAHVDMKLST DRTLSSSISQFWFRLEFCKEDKDFILFPTANRYSRKLWKHSIKNNQLF KEGIRNYSEISSLPYEEDHNFDIDLVFTWVNSEDKNWQELYKKYKPDF NSDATSTSRFLSRDELKFALRSWEMNGSFIRKIFIVSNCAPPAWLDLN NPKIQWVYHEEIMPQSALPTFSSHAIETSLHHIPGISNYFIYSNDDFL LTKPLNKDNFFYSNGIAKLRLEAWGNVNGECTEGEPDYLNGARNANTL LEKEFKKFTTKLHTHSPQSMRTDILFEMEKKYPEEFNRTLHNKFRSLD DIAVTGYLYHHYALLSGRALQSSDKTELVQQNHDFKKKLNNVVTLTKE RNFDKLPLSVCINDGADSHLNEEWNVQVIKFLETLFPLPSSFEKLEHH HHHH SEQ ID NO: 44: AS NdeI M SEQ ID NO: 45: AS XhoI-His6 LEHHHHHH SEQ ID NO: 46: DNA .DELTA.N69-CP-A(wt)(1425) ttaattcctattaatttttttaattttgaatatatagttaaaaaactt aacaatcaaaacgcaataggtgtatatattcttccttctaatcttact cttaagcctgcattatgtattctagaatcacataaagaagacttttta aataaatttcttcttactatttcctctgaaaatttaaagcttcaatac aaatttaatggacaaataaaaaatcctaagtccgtaaatgaaatttgg acagatttatttagcattgctcatgttgacatgaaactcagcacagat agaactttaagttcatctatatctcaattttggttcagattagagttc tgtaaagaagataaggattttatcttatttcctacagctaacagatat tctagaaaactttggaagcactctattaaaaataatcaattatttaaa gaaggcatacgaaactattcagaaatatcttcattaccctatgaagaa gatcataattttgatattgatttagtatttacttgggtcaactcagaa gataagaattggcaagagttatataaaaaatataagcccgactttaat agcgatgcaaccagtacatcaagattccttagtagagatgaattaaaa ttcgcattacgctcttgggaaatgaatggatccttcattcgaaaaatt tttattgtctctaattgtgctcccccagcatggctagatttaaataac cctaaaattcaatgggtatatcacgaagaaattatgccacaaagtgcc cttcctacttttagctcacatgctattgaaaccagcttgcaccatata ccaggaattagtaactattttatttacagcaatgacgacttcctatta actaaaccattgaataaagacaatttcttctattcgaatggtattgca aagttaagattagaagcatggggaaatgttaatggtgaatgtactgaa ggagaacctgactacttaaatggtgctcgcaatgcgaacactctctta gaaaaggaatttaaaaaatttactactaaactacatactcactcccct caatccatgagaactgatattttatttgagatggaaaaaaaatatcca gaagagtttaatagaacactacataataaattccgatctttagatgat attgcagtaacgggctatctctatcatcattatgccctactctctgga cgagcactacaaagttctgacaagacggaacttgtacagcaaaatcat gatttcaaaaagaaactaaataatgtagtgaccttaactaaagaaagg aattttgacaaacttcctttgagcgtatgtatcaacgatggtgctgat agtcacttgaatgaagaatggaatgttcaagttattaagttcttagaa actcttttcccattaccatcatcatttgagaaa

[0548] The sequences provided herein (e.g. the sequences corresponding to CP-X or fragments thereof) may be linked to (a) tag(s). For example, the sequences may have an N-terminal MBP-tag followed by the protease resistant linker S3N10, and/or a C-terminal his-tag. The sequences provided herein (e.g. the sequences corresponding to CP-X or fragments thereof) may be cloned into the vector by using the restriction sites BamHI and/or XhoI. In particular in the constructs of CP-X (or fragments thereof), if the N-terminus is not truncated, then the ATG1/Met1 may not be in the construct. SEQ ID NOs: 47 and 49 show an example for a MBP-S3N10-BamHI-CP-X-XhoI-his construct. In context of the invention, all herein disclosed sequences may be used to produce constructs as demonstrated for CP-X in SEQ ID NOs: 47 and 49.

TABLE-US-00013 SEQ ID NO: 47: DNA MBP-S3N10-BamHI-CsxA-Xho1-His6 atgaaaactgaagaaggtaaactggtaatctggattaacggcgataa aggctataacggtctcgctgaagtcggtaagaaattcgagaaagata ccggaattaaagtcaccgttgagcatccggataaactggaagagaaa ttcccacaggttgcggcaactggcgatggccctgacattatcttctg ggcacacgaccgctttggtggctacgctcaatctggcctgttggctg aaatcaccccggacaaagcgttccaggacaagctgtatccgtttacc tgggatgccgtacgttacaacggcaagctgattgcttacccgatcgc tgttgaagcgttatcgctgatttataacaaagatctgctgccgaacc cgccaaaaacctgggaagagatcccggcgctggataaagaactgaaa gcgaaaggtaagagcgcgctgatgttcaacctgcaagaaccgtactt cacctggccgctgattgctgctgacgggggttatgcgttcaagtatg aaaacggcaagtacgacattaaagacgtgggcgtggataacgctggc gcgaaagcgggtctgaccttcctggttgacctgattaaaaacaaaca catgaatgcagacaccgattactccatcgcagaagctgcctttaata aaggcgaaacagcgatgaccatcaacggcccgtgggcatggtccaac atcgacaccagcaaagtgaattatggtgtaacggtactgccgacctt caagggtcaaccatccaaaccgttcgttggcgtgctgagcgcaggta ttaacgccgccagtccgaacaaagagctggcgaaagagttcctcgaa aactatctgctgactgatgaaggtctggaagcggttaataaagacaa accgctgggtgccgtagcgctgaagtcttacgaggaagagttggcga aagatccacgtattgccgccaccatggaaaacgcccagaaaggtgaa atcatgccgaacatcccgcagatgtccgctttctggtatgccgtgcg tactgcggtgatcaacgccgccagcggtcgtcagactgtcgatgaag ccctgaaagacgcgcagactaatTCGAGCTCCAATAACAATAACAAC AACAATAAcAATAACGGATCCATTATGAGCAAAATTAGCAAATTGGT AACCCACCCAAACCTTTTCTTTCGAGATTATTTCTTAAAAAAAGCAC CGTTAAATTATGGCGAAAATATTAAACCTTTACCAGTCGAAACCTCT TCTCATAGCAAAAAAAATACAGCCCATAAAACACCCGTATCATCCGA CCAACCAATTGAAGATCCATACCCAGTAACATTTCCAATTGATGTAG TTTATACTTGGGTAGATTCAGATGATGAAAAATTCAATGAAGAACGC CTAAAGTTTCAAAATTCAAGCACATCTGAGACTCTACAAGGCAAAGC AGAAAGCACCGATATTGCAAGATTCCAATCACGCGACGAATTAAAAT ATTCGATTCGAAGCCTGATGAAGTATGCCCCATGGGTAAATCATATT TACATTGTAACAAATGGTCAAATACCAAAATGGTTAGATACCAACAA TACAAAGGTAACGATTATCCCTCACTCAACTATTATCGACAGTCAAT TTCTCCCTACTTTTAATTCTCACGTCATTGAATCCTCTCTATATAAA ATCCCAGGATTATCAGAGCATTACATTTATTTCAATGATGATGTCAT GCTAGCTAGAGATTTAAGCCCATCTTATTTCTTTACAAGCAGCGGAT TAGCAAAACTGTTTATTACCAACTCTCGTCTACCAAATGGCTATAAG AATGTGAAAGACACACCAACCCAATGGGCCTCAAAAAATTCCCGTGA GCTTTTACATGCAGAAACAGGATTTTGGGCTGAAGCCATGTTTGCAC ATACTTTTCATCCACAACGTAAAAGTGTACATGAATCTATTGAACAC CTATGGCATGAACAATTAAATGTTTGTCGTCAAAACCGTTTCCGTGA TATTTCAGATATTAACATGGCGACATTCCTGCACCACCATTTTGCCA TTTTGACAGGCCAAGCTCTTGCTACACGCACTAAATGTATTTACTTT AACATTCGCTCTCCTCAAGCAGCTCAGCATTACAAAACATTATTAGC TCGAAAAGGAAGCGAATACAGCCCACATTCTATCTGCTTAAATGATC ATACATCGAGCAATAAAAATATTTTATCTAATTACGAAGCCAAATTA CAAAGCTTTTTAGAAACATACTATCCAGATGTATCAGAAGCAGAAAT TCTCCTTCCTACTAAATCTGAAGTAGCTGAATTAGTTAAACATAAAG ATTATTTAACTGTATATACTAAATTATTACCTATTATCAATAAGCAG CTGGTCAATAAATATAATAAACCTTATTCATATCTTTTCTATTATTT AGGTTTATCTGCCCGGTTTTTATTTGAAGAAACGCAACAAGAACACT ACCGGGAAACTGCTGAAGAAAATTTACAAATCTTTTGTGGCCTAAAC CCAAAACATACACTAGCCCTCAAATACTTAGCGGATGTCACCCTCAC ATCACAGCCTAGTGGACAACtcgagcaccaccaccaccaccac SEQ ID NO: 48: DNA MBP-S3N10-BamHI atgaaaactgaagaaggtaaactggtaatctggattaacggcgataaa ggctataacggtctcgctgaagtcggtaagaaattcgagaaagatacc ggaattaaagtcaccgttgagcatccggataaactggaagagaaattc ccacaggttgcggcaactggcgatggccctgacattatcttctgggca cacgaccgctttggtggctacgctcaatctggcctgttggctgaaatc accccggacaaagcgttccaggacaagctgtatccgtttacctgggat gccgtacgttacaacggcaagctgattgcttacccgatcgctgttgaa gcgttatcgctgatttataacaaagatctgctgccgaacccgccaaaa acctgggaagagatcccggcgctggataaagaactgaaagcgaaaggt aagagcgcgctgatgttcaacctgcaagaaccgtacttcacctggccg ctgattgctgctgacgggggttatgcgttcaagtatgaaaacggcaag tacgacattaaagacgtgggcgtggataacgctggcgcgaaagcgggt ctgaccttcctggttgacctgattaaaaacaaacacatgaatgcagac accgattactccatcgcagaagctgcctttaataaaggcgaaacagcg atgaccatcaacggcccgtgggcatggtccaacatcgacaccagcaaa gtgaattatggtgtaacggtactgccgaccttcaagggtcaaccatcc aaaccgttcgttggcgtgctgagcgcaggtattaacgccgccagtccg aacaaagagctggcgaaagagttcctcgaaaactatctgctgactgat gaaggtctggaagcggttaataaagacaaaccgctgggtgccgtagcg ctgaagtcttacgaggaagagttggcgaaagatccacgtattgccgcc accatggaaaacgcccagaaaggtgaaatcatgccgaacatcccgcag atgtccgctttctggtatgccgtgcgtactgcggtgatcaacgccgcc agcggtcgtcagactgtcgatgaagccctgaaagacgcgcagactaat TCGAGCTCCAATAACAATAACAACAACAATAAcAATAACGGATCC SEQ ID NO: 49: AS MBP-S3N10-BamHI-CsxA-Xho1-His6 MKTEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKF PQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWD AVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKG KSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAG LTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSK VNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTD EGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQ MSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNNNNNGSI MSKISKLVTHPNLFFRDYFLKKAPLNYGENIKPLPVETSSHSKKNTAH KTPVSSDQPIEDPYPVTFPIDVVYTWVDSDDEKFNEERLKFQNSSTSE TLQGKAESTDIARFQSRDELKYSIRSLMKYAPWVNHIYIVTNGQIPKW LDTNNTKVTIIPHSTIIDSQFLPTFNSHVIESSLYKIPGLSEHYIYFN DDVMLARDLSPSYFFTSSGLAKLFITNSRLPNGYKNVKDTPTQWASKN SRELLHAETGFWAEAMFAHTFHPQRKSVHESIEHLWHEQLNVCRQNRF RDISDINMATFLHHHFAILTGQALATRTKCIYFNIRSPQAAQHYKTLL ARKGSEYSPHSICLNDHTSSNKNILSNYEAKLQSFLETYYPDVSEAEI LLPTKSEVAELVKHKDYLTVYTKLLPIINKQLVNKYNKPYSYLFYYLG LSARFLFEETQQEHYRETAEENLQIFCGLNPKHTLALKYLADVTLTSQ PSGQLEHHHHHH SEQ ID NO: 50: AS MBP-S3N10-BamHI MKTEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKF PQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWD AVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKG KSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAG LTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSK VNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTD EGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQ MSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNNNNNGS

[0549] The sequences provided herein (e.g. the sequences corresponding to .DELTA.N65.DELTA.C10-CP-X) may have a free N-terminus. These constructs may be used, inter alia for crystallization approaches. For example, the sequences may be cloned into a vector by using NdeI and/or XhoI. SEQ ID NOs: 51 and 52 show an example for an NdeI-.DELTA.N65.DELTA.C10-CP-X-XhoI-his construct. In context of the invention, all herein disclosed sequences may be used to produce constructs as demonstrated for .DELTA.N65.DELTA.C10-CP-X in SEQ ID NOs: 51 and 52.

TABLE-US-00014 SEQ ID NO: 51: DNA NdeI-dN65dC10-CsxA-XhoI-His6 caTATGGTAACATTTCCAATTGATGTAGTTTATACTTGGGTAGATTCA GATGATGAAAAATTCAATGAAGAACGCCTAAAGTTTCAAAATTCAAGC ACATCTGAGACTCTACAAGGCAAAGCAGAAAGCACCGATATTGCAAGA TTCCAATCACGCGACGAATTAAAATATTCGATTCGAAGCCTGATGAAG TATGCCCCATGGGTAAATCATATTTACATTGTAACAAATGGTCAAATA CCAAAATGGTTAGATACCAACAATACAAAGGTAACGATTATCCCTCAC TCAACTATTATCGACAGTCAATTTCTCCCTACTTTTAATTCTCACGTC ATTGAATCCTCTCTATATAAAATCCCAGGATTATCAGAGCATTACATT TATTTCAATGATGATGTCATGCTAGCTAGAGATTTAAGCCCATCTTAT TTCTTTACAAGCAGCGGATTAGCAAAACTGTTTATTACCAACTCTCGT CTACCAAATGGCTATAAGAATGTGAAAGACACACCAACCCAATGGGCC TCAAAAAATTCCCGTGAGCTTTTACATGCAGAAACAGGATTTTGGGCT GAAGCCATGTTTGCACATACTTTTCATCCACAACGTAAAAGTGTACAT GAATCTATTGAACACCTATGGCATGAACAATTAAATGTTTGTCGTCAA AACCGTTTCCGTGATATTTCAGATATTAACATGGCGACATTCCTGCAC CACCATTTTGCCATTTTGACAGGCCAAGCTCTTGCTACACGCACTAAA TGTATTTACTTTAACATTCGCTCTCCTCAAGCAGCTCAGCATTACAAA ACATTATTAGCTCGAAAAGGAAGCGAATACAGCCCACATTCTATCTGC TTAAATGATCATACATCGAGCAATAAAAATATTTTATCTAATTACGAA GCCAAATTACAAAGCTTTTTAGAAACATACTATCCAGATGTATCAGAA GCAGAAATTCTCCTTCCTACTAAATCTGAAGTAGCTGAATTAGTTAAA CATAAAGATTATTTAACTGTATATACTAAATTATTACCTATTATCAAT AAGCAGCTGGTCAATAAATATAATAAACCTTATTCATATCTTTTCTAT TATTTAGGTTTATCTGCCCGGTTTTTATTTGAAGAAACGCAACAAGAA CACTACCGGGAAACTGCTGAAGAAAATTTACAAATCTTTTGTGGCCTA AACCCAAAACATACACTAGCCCTCAAATACTTAGCGGATctcgagcac caccaccaccaccac SEQ ID NO: 52: AS NdeI-dN65dC10-CsxA-XhoI-His6 MVTFPIDVVYTWVDSDDEKFNEERLKFQNSSTSETLQGKAESTDIARF QSRDELKYSIRSLMKYAPWVNHIYIVTNGQIPKWLDTNNTKVTIIPHS TIIDSQFLPTFNSHVIESSLYKIPGLSEHYIYFNDDVMLARDLSPSYF FTSSGLAKLFITNSRLPNGYKNVKDTPTQWASKNSRELLHAETGFWAE AMFAHTFHPQRKSVHESIEHLWHEQLNVCRQNRFRDISDINMATFLHH HFAILTGQALATRTKCIYFNIRSPQAAQHYKTLLARKGSEYSPHSICL NDHTSSNKNILSNYEAKLQSFLETYYPDVSEAEILLPTKSEVAELVKH KDYLTVYTKLLPIINKQLVNKYNKPYSYLFYYLGLSARFLFEETQQEH YRETAEENLQIFCGLNPKHTLALKYLADLEHHHHHH SEQ ID NO: 53: DNA CsaC Atgttatctaatttaaaaacaggaaataatatcttaggattacctgaa tttgagttgaatggctgccgattcttatataaaaaaggtatagaaaaa acaattattactttttcagcatttcctcctaaagatattgctcaaaaa tataattatataaaagattttttaagttctaattatacttttttagca ttcttagataccaaatatccagaagatgatgctagaggcacttattac attactaatgagttagataatggatatttacaaaccatacattgtatt attcaattattatcgaatacaaatcaagaagatacctaccttttgggt tcaagtaaaggtggcgttggcgcacttctactcggtcttacatataat tatcctaatataattattaatgctcctcaagccaaattagcagattat atcaaaacacgctcgaaaaccattctttcatatatgcttggaacctct aaaagatttcaagatattaattacgattatatcaatgacttcttacta tctaaaattaagacttgcgactcctcacttaaatggaatattcatata acttgcggaaaagatgattcatatcatttaaatgaattagaaattcta aaaaatgaatttaatataaaagctattacgattaaaaccaaactaatt tctggcgggcatgataatgaagcaattgcccactatagagaatacttt aaaaccataatccaaaatata SEQ ID NO: 54: AS CsaC MLSNLKTGNNILGLPEFELNGCRFLYKKGIEKTIITFSAFPPKDIAQK YNYIKDFLSSNYTFLAFLDTKYPEDDARGTYYITNELDNGYLQTIHCI IQLLSNTNQEDTYLLGSSKGGVGALLLGLTYNYPNIIINAPQAKLADY IKTRSKTILSYMLGTSKRFQDINYDYINDFLLSKIKTCDSSLKWNIHI TCGKDDSYHLNELEILKNEFNIKAITIKTKLISGGHDNEAIAHYREYF KTIIQNI

Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 135 <210> SEQ ID NO 1 <211> LENGTH: 1635 <212> TYPE: DNA <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: DNA CP-A/CsaB wildtyp/full-length <400> SEQUENCE: 1 atgtttatac ttaataacag aaaatggcgt aaacttaaaa gagaccctag cgctttcttt 60 cgagatagta aatttaactt tttaagatat ttttctgcta aaaaatttgc aaagaatttt 120 aaaaattcat cacatatcca taaaactaat ataagtaaag ctcaatcaaa tatttcttca 180 accttaaaac aaaatcggaa acaagatatg ttaattccta ttaatttttt taattttgaa 240 tatatagtta aaaaacttaa caatcaaaac gcaataggtg tatatattct tccttctaat 300 cttactctta agcctgcatt atgtattcta gaatcacata aagaagactt tttaaataaa 360 tttcttctta ctatttcctc tgaaaattta aagcttcaat acaaatttaa tggacaaata 420 aaaaatccta agtccgtaaa tgaaatttgg acagatttat ttagcattgc tcatgttgac 480 atgaaactca gcacagatag aactttaagt tcatctatat ctcaattttg gttcagatta 540 gagttctgta aagaagataa ggattttatc ttatttccta cagctaacag atattctaga 600 aaactttgga agcactctat taaaaataat caattattta aagaaggcat acgaaactat 660 tcagaaatat cttcattacc ctatgaagaa gatcataatt ttgatattga tttagtattt 720 acttgggtca actcagaaga taagaattgg caagagttat ataaaaaata taagcccgac 780 tttaatagcg atgcaaccag tacatcaaga ttccttagta gagatgaatt aaaattcgca 840 ttacgctctt gggaaatgaa tggatccttc attcgaaaaa tttttattgt ctctaattgt 900 gctcccccag catggctaga tttaaataac cctaaaattc aatgggtata tcacgaagaa 960 attatgccac aaagtgccct tcctactttt agctcacatg ctattgaaac cagcttgcac 1020 catataccag gaattagtaa ctattttatt tacagcaatg acgacttcct attaactaaa 1080 ccattgaata aagacaattt cttctattcg aatggtattg caaagttaag attagaagca 1140 tggggaaatg ttaatggtga atgtactgaa ggagaacctg actacttaaa tggtgctcgc 1200 aatgcgaaca ctctcttaga aaaggaattt aaaaaattta ctactaaact acatactcac 1260 tcccctcaat ccatgagaac tgatatttta tttgagatgg aaaaaaaata tccagaagag 1320 tttaatagaa cactacataa taaattccga tctttagatg atattgcagt aacgggctat 1380 ctctatcatc attatgccct actctctgga cgagcactac aaagttctga caagacggaa 1440 cttgtacagc aaaatcatga tttcaaaaag aaactaaata atgtagtgac cttaactaaa 1500 gaaaggaatt ttgacaaact tcctttgagc gtatgtatca acgatggtgc tgatagtcac 1560 ttgaatgaag aatggaatgt tcaagttatt aagttcttag aaactctttt cccattacca 1620 tcatcatttg agaaa 1635 <210> SEQ ID NO 2 <211> LENGTH: 1635 <212> TYPE: DNA <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: DNA CP-A/CsaB codon optimized <400> SEQUENCE: 2 atgttcatct tgaacaaccg caaatggcgc aaattgaagc gtgacccaag cgcgtttttt 60 cgtgacagca aattcaactt tctgcgctat ttctccgcga aaaagtttgc gaagaatttc 120 aaaaacagct cgcatatcca taaaaccaac attagcaaag cgcagtccaa tatttccagc 180 accttgaagc agaaccgtaa gcaggatatg ctgatcccga tcaatttctt taattttgag 240 tacatcgtga agaaactgaa taaccaaaac gcaatcggcg tgtacattct gccgtctaat 300 ctgaccctga aaccagcatt gtgcatcttg gagtcgcaca aagaggactt cctgaacaaa 360 tttttgttga ccattagcag cgagaacctg aaactgcagt ataagttcaa tggtcagatc 420 aaaaatccga aaagcgtgaa cgaaatctgg accgacctgt ttagcattgc tcacgtcgac 480 atgaagctga gcaccgaccg tacgctgtcc tcgtccatca gccaattttg gtttcgcctg 540 gagttctgta aagaggacaa ggacttcatc ctgtttccga cggcaaatcg ttacagccgc 600 aagctgtgga agcacagcat caaaaataat cagctgttta aggaaggtat ccgtaactac 660 agcgagatta gctcgctgcc gtacgaggaa gaccataact tcgacatcga tctggtcttt 720 acctgggtca attcggaaga caaaaactgg caggaactgt acaagaaata taagccggat 780 tttaatagcg atgccacctc gacgagccgt tttctgagcc gtgacgagct gaagtttgcg 840 ctgcgctcgt gggaaatgaa cggtagcttc atccgtaaaa tctttatcgt cagcaactgc 900 gcgccgccgg cctggctgga tctgaacaat ccgaagatcc aatgggtgta tcacgaggag 960 atcatgccac agagcgccct gccaaccttc agcagccatg ctattgagac tagcttgcat 1020 cacattccgg gcatctccaa ctacttcatc tactctaatg acgattttct gttgaccaaa 1080 ccgctgaaca aagacaactt cttttactcc aacggtattg ctaaactgcg tctggaagcc 1140 tggggtaacg ttaacggtga atgtaccgaa ggcgagccgg attacctgaa cggcgcgcgt 1200 aacgcaaata cgctgctgga gaaagagttt aaaaagttta ccaccaagct gcacacccac 1260 agcccgcaga gcatgcgtac cgacatcctg ttcgagatgg agaaaaaata cccagaagag 1320 ttcaatcgca cgctgcacaa caagttccgc agcctggatg acatcgcggt taccggctac 1380 ctgtaccatc actacgcatt gctgtctggc cgcgctctgc aatccagcga taagaccgaa 1440 ctggtccagc agaatcacga ctttaaaaag aagctgaata atgttgtcac cctgaccaaa 1500 gagcgtaact ttgataagct gccgctgagc gtttgtatta atgacggtgc agacagccac 1560 ctgaatgagg agtggaatgt gcaagttatc aaattcttgg agaccttgtt cccgttgccg 1620 agctccttcg agaaa 1635 <210> SEQ ID NO 3 <211> LENGTH: 1425 <212> TYPE: DNA <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: DNA deltaN69-CP-A (codon optimized) (1425) <400> SEQUENCE: 3 ctgatcccga tcaatttctt taattttgag tacatcgtga agaaactgaa taaccaaaac 60 gcaatcggcg tgtacattct gccgtctaat ctgaccctga aaccagcatt gtgcatcttg 120 gagtcgcaca aagaggactt cctgaacaaa tttttgttga ccattagcag cgagaacctg 180 aaactgcagt ataagttcaa tggtcagatc aaaaatccga aaagcgtgaa cgaaatctgg 240 accgacctgt ttagcattgc tcacgtcgac atgaagctga gcaccgaccg tacgctgtcc 300 tcgtccatca gccaattttg gtttcgcctg gagttctgta aagaggacaa ggacttcatc 360 ctgtttccga cggcaaatcg ttacagccgc aagctgtgga agcacagcat caaaaataat 420 cagctgttta aggaaggtat ccgtaactac agcgagatta gctcgctgcc gtacgaggaa 480 gaccataact tcgacatcga tctggtcttt acctgggtca attcggaaga caaaaactgg 540 caggaactgt acaagaaata taagccggat tttaatagcg atgccacctc gacgagccgt 600 tttctgagcc gtgacgagct gaagtttgcg ctgcgctcgt gggaaatgaa cggtagcttc 660 atccgtaaaa tctttatcgt cagcaactgc gcgccgccgg cctggctgga tctgaacaat 720 ccgaagatcc aatgggtgta tcacgaggag atcatgccac agagcgccct gccaaccttc 780 agcagccatg ctattgagac tagcttgcat cacattccgg gcatctccaa ctacttcatc 840 tactctaatg acgattttct gttgaccaaa ccgctgaaca aagacaactt cttttactcc 900 aacggtattg ctaaactgcg tctggaagcc tggggtaacg ttaacggtga atgtaccgaa 960 ggcgagccgg attacctgaa cggcgcgcgt aacgcaaata cgctgctgga gaaagagttt 1020 aaaaagttta ccaccaagct gcacacccac agcccgcaga gcatgcgtac cgacatcctg 1080 ttcgagatgg agaaaaaata cccagaagag ttcaatcgca cgctgcacaa caagttccgc 1140 agcctggatg acatcgcggt taccggctac ctgtaccatc actacgcatt gctgtctggc 1200 cgcgctctgc aatccagcga taagaccgaa ctggtccagc agaatcacga ctttaaaaag 1260 aagctgaata atgttgtcac cctgaccaaa gagcgtaact ttgataagct gccgctgagc 1320 gtttgtatta atgacggtgc agacagccac ctgaatgagg agtggaatgt gcaagttatc 1380 aaattcttgg agaccttgtt cccgttgccg agctccttcg agaaa 1425 <210> SEQ ID NO 4 <211> LENGTH: 1344 <212> TYPE: DNA <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: DNA deltaN97-CP-A (1344) <400> SEQUENCE: 4 ccttctaatc ttactcttaa gcctgcatta tgtattctag aatcacataa agaagacttt 60 ttaaataaat ttcttcttac tatttcctct gaaaatttaa agcttcaata caaatttaat 120 ggacaaataa aaaatcctaa gtccgtaaat gaaatttgga cagatttatt tagcattgct 180 catgttgaca tgaaactcag cacagataga actttaagtt catctatatc tcaattttgg 240 ttcagattag agttctgtaa agaagataag gattttatct tatttcctac agctaacaga 300 tattctagaa aactttggaa gcactctatt aaaaataatc aattatttaa agaaggcata 360 cgaaactatt cagaaatatc ttcattaccc tatgaagaag atcataattt tgatattgat 420 ttagtattta cttgggtcaa ctcagaagat aagaattggc aagagttata taaaaaatat 480 aagcccgact ttaatagcga tgcaaccagt acatcaagat tccttagtag agatgaatta 540 aaattcgcat tacgctcttg ggaaatgaat ggatccttca ttcgaaaaat ttttattgtc 600 tctaattgtg ctcccccagc atggctagat ttaaataacc ctaaaattca atgggtatat 660 cacgaagaaa ttatgccaca aagtgccctt cctactttta gctcacatgc tattgaaacc 720 agcttgcacc atataccagg aattagtaac tattttattt acagcaatga cgacttccta 780 ttaactaaac cattgaataa agacaatttc ttctattcga atggtattgc aaagttaaga 840 ttagaagcat ggggaaatgt taatggtgaa tgtactgaag gagaacctga ctacttaaat 900 ggtgctcgca atgcgaacac tctcttagaa aaggaattta aaaaatttac tactaaacta 960 catactcact cccctcaatc catgagaact gatattttat ttgagatgga aaaaaaatat 1020 ccagaagagt ttaatagaac actacataat aaattccgat ctttagatga tattgcagta 1080 acgggctatc tctatcatca ttatgcccta ctctctggac gagcactaca aagttctgac 1140 aagacggaac ttgtacagca aaatcatgat ttcaaaaaga aactaaataa tgtagtgacc 1200 ttaactaaag aaaggaattt tgacaaactt cctttgagcg tatgtatcaa cgatggtgct 1260 gatagtcact tgaatgaaga atggaatgtt caagttatta agttcttaga aactcttttc 1320 ccattaccat catcatttga gaaa 1344 <210> SEQ ID NO 5 <211> LENGTH: 1134 <212> TYPE: DNA <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: DNA deltaN167-CP-A (1134) <400> SEQUENCE: 5 actttaagtt catctatatc tcaattttgg ttcagattag agttctgtaa agaagataag 60 gattttatct tatttcctac agctaacaga tattctagaa aactttggaa gcactctatt 120 aaaaataatc aattatttaa agaaggcata cgaaactatt cagaaatatc ttcattaccc 180 tatgaagaag atcataattt tgatattgat ttagtattta cttgggtcaa ctcagaagat 240 aagaattggc aagagttata taaaaaatat aagcccgact ttaatagcga tgcaaccagt 300 acatcaagat tccttagtag agatgaatta aaattcgcat tacgctcttg ggaaatgaat 360 ggatccttca ttcgaaaaat ttttattgtc tctaattgtg ctcccccagc atggctagat 420 ttaaataacc ctaaaattca atgggtatat cacgaagaaa ttatgccaca aagtgccctt 480 cctactttta gctcacatgc tattgaaacc agcttgcacc atataccagg aattagtaac 540 tattttattt acagcaatga cgacttccta ttaactaaac cattgaataa agacaatttc 600 ttctattcga atggtattgc aaagttaaga ttagaagcat ggggaaatgt taatggtgaa 660 tgtactgaag gagaacctga ctacttaaat ggtgctcgca atgcgaacac tctcttagaa 720 aaggaattta aaaaatttac tactaaacta catactcact cccctcaatc catgagaact 780 gatattttat ttgagatgga aaaaaaatat ccagaagagt ttaatagaac actacataat 840 aaattccgat ctttagatga tattgcagta acgggctatc tctatcatca ttatgcccta 900 ctctctggac gagcactaca aagttctgac aagacggaac ttgtacagca aaatcatgat 960 ttcaaaaaga aactaaataa tgtagtgacc ttaactaaag aaaggaattt tgacaaactt 1020 cctttgagcg tatgtatcaa cgatggtgct gatagtcact tgaatgaaga atggaatgtt 1080 caagttatta agttcttaga aactcttttc ccattaccat catcatttga gaaa 1134 <210> SEQ ID NO 6 <211> LENGTH: 933 <212> TYPE: DNA <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: DNA deltaN235-CP-A (933) <400> SEQUENCE: 6 gatattgatt tagtatttac ttgggtcaac tcagaagata agaattggca agagttatat 60 aaaaaatata agcccgactt taatagcgat gcaaccagta catcaagatt ccttagtaga 120 gatgaattaa aattcgcatt acgctcttgg gaaatgaatg gatccttcat tcgaaaaatt 180 tttattgtct ctaattgtgc tcccccagca tggctagatt taaataaccc taaaattcaa 240 tgggtatatc acgaagaaat tatgccacaa agtgcccttc ctacttttag ctcacatgct 300 attgaaacca gcttgcacca tataccagga attagtaact attttattta cagcaatgac 360 gacttcctat taactaaacc attgaataaa gacaatttct tctattcgaa tggtattgca 420 aagttaagat tagaagcatg gggaaatgtt aatggtgaat gtactgaagg agaacctgac 480 tacttaaatg gtgctcgcaa tgcgaacact ctcttagaaa aggaatttaa aaaatttact 540 actaaactac atactcactc ccctcaatcc atgagaactg atattttatt tgagatggaa 600 aaaaaatatc cagaagagtt taatagaaca ctacataata aattccgatc tttagatgat 660 attgcagtaa cgggctatct ctatcatcat tatgccctac tctctggacg agcactacaa 720 agttctgaca agacggaact tgtacagcaa aatcatgatt tcaaaaagaa actaaataat 780 gtagtgacct taactaaaga aaggaatttt gacaaacttc ctttgagcgt atgtatcaac 840 gatggtgctg atagtcactt gaatgaagaa tggaatgttc aagttattaa gttcttagaa 900 actcttttcc cattaccatc atcatttgag aaa 933 <210> SEQ ID NO 7 <211> LENGTH: 1497 <212> TYPE: DNA <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: DNA deltaC45-CP-A (1497) <400> SEQUENCE: 7 tttatactta ataacagaaa atggcgtaaa cttaaaagag accctagcgc tttctttcga 60 gatagtaaat ttaacttttt aagatatttt tctgctaaaa aatttgcaaa gaattttaaa 120 aattcatcac atatccataa aactaatata agtaaagctc aatcaaatat ttcttcaacc 180 ttaaaacaaa atcggaaaca agatatgtta attcctatta atttttttaa ttttgaatat 240 atagttaaaa aacttaacaa tcaaaacgca ataggtgtat atattcttcc ttctaatctt 300 actcttaagc ctgcattatg tattctagaa tcacataaag aagacttttt aaataaattt 360 cttcttacta tttcctctga aaatttaaag cttcaataca aatttaatgg acaaataaaa 420 aatcctaagt ccgtaaatga aatttggaca gatttattta gcattgctca tgttgacatg 480 aaactcagca cagatagaac tttaagttca tctatatctc aattttggtt cagattagag 540 ttctgtaaag aagataagga ttttatctta tttcctacag ctaacagata ttctagaaaa 600 ctttggaagc actctattaa aaataatcaa ttatttaaag aaggcatacg aaactattca 660 gaaatatctt cattacccta tgaagaagat cataattttg atattgattt agtatttact 720 tgggtcaact cagaagataa gaattggcaa gagttatata aaaaatataa gcccgacttt 780 aatagcgatg caaccagtac atcaagattc cttagtagag atgaattaaa attcgcatta 840 cgctcttggg aaatgaatgg atccttcatt cgaaaaattt ttattgtctc taattgtgct 900 cccccagcat ggctagattt aaataaccct aaaattcaat gggtatatca cgaagaaatt 960 atgccacaaa gtgcccttcc tacttttagc tcacatgcta ttgaaaccag cttgcaccat 1020 ataccaggaa ttagtaacta ttttatttac agcaatgacg acttcctatt aactaaacca 1080 ttgaataaag acaatttctt ctattcgaat ggtattgcaa agttaagatt agaagcatgg 1140 ggaaatgtta atggtgaatg tactgaagga gaacctgact acttaaatgg tgctcgcaat 1200 gcgaacactc tcttagaaaa ggaatttaaa aaatttacta ctaaactaca tactcactcc 1260 cctcaatcca tgagaactga tattttattt gagatggaaa aaaaatatcc agaagagttt 1320 aatagaacac tacataataa attccgatct ttagatgata ttgcagtaac gggctatctc 1380 tatcatcatt atgccctact ctctggacga gcactacaaa gttctgacaa gacggaactt 1440 gtacagcaaa atcatgattt caaaaagaaa ctaaataatg tagtgacctt aactaaa 1497 <210> SEQ ID NO 8 <211> LENGTH: 1557 <212> TYPE: DNA <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: DNA deltaC25-CP-A (1557) <400> SEQUENCE: 8 tttatactta ataacagaaa atggcgtaaa cttaaaagag accctagcgc tttctttcga 60 gatagtaaat ttaacttttt aagatatttt tctgctaaaa aatttgcaaa gaattttaaa 120 aattcatcac atatccataa aactaatata agtaaagctc aatcaaatat ttcttcaacc 180 ttaaaacaaa atcggaaaca agatatgtta attcctatta atttttttaa ttttgaatat 240 atagttaaaa aacttaacaa tcaaaacgca ataggtgtat atattcttcc ttctaatctt 300 actcttaagc ctgcattatg tattctagaa tcacataaag aagacttttt aaataaattt 360 cttcttacta tttcctctga aaatttaaag cttcaataca aatttaatgg acaaataaaa 420 aatcctaagt ccgtaaatga aatttggaca gatttattta gcattgctca tgttgacatg 480 aaactcagca cagatagaac tttaagttca tctatatctc aattttggtt cagattagag 540 ttctgtaaag aagataagga ttttatctta tttcctacag ctaacagata ttctagaaaa 600 ctttggaagc actctattaa aaataatcaa ttatttaaag aaggcatacg aaactattca 660 gaaatatctt cattacccta tgaagaagat cataattttg atattgattt agtatttact 720 tgggtcaact cagaagataa gaattggcaa gagttatata aaaaatataa gcccgacttt 780 aatagcgatg caaccagtac atcaagattc cttagtagag atgaattaaa attcgcatta 840 cgctcttggg aaatgaatgg atccttcatt cgaaaaattt ttattgtctc taattgtgct 900 cccccagcat ggctagattt aaataaccct aaaattcaat gggtatatca cgaagaaatt 960 atgccacaaa gtgcccttcc tacttttagc tcacatgcta ttgaaaccag cttgcaccat 1020 ataccaggaa ttagtaacta ttttatttac agcaatgacg acttcctatt aactaaacca 1080 ttgaataaag acaatttctt ctattcgaat ggtattgcaa agttaagatt agaagcatgg 1140 ggaaatgtta atggtgaatg tactgaagga gaacctgact acttaaatgg tgctcgcaat 1200 gcgaacactc tcttagaaaa ggaatttaaa aaatttacta ctaaactaca tactcactcc 1260 cctcaatcca tgagaactga tattttattt gagatggaaa aaaaatatcc agaagagttt 1320 aatagaacac tacataataa attccgatct ttagatgata ttgcagtaac gggctatctc 1380 tatcatcatt atgccctact ctctggacga gcactacaaa gttctgacaa gacggaactt 1440 gtacagcaaa atcatgattt caaaaagaaa ctaaataatg tagtgacctt aactaaagaa 1500 aggaattttg acaaacttcc tttgagcgta tgtatcaacg atggtgctga tagtcac 1557 <210> SEQ ID NO 9 <211> LENGTH: 545 <212> TYPE: PRT <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: AS CP-A/CsaB wildtyp/full-length (544 without M1) <400> SEQUENCE: 9 Met Phe Ile Leu Asn Asn Arg Lys Trp Arg Lys Leu Lys Arg Asp Pro 1 5 10 15 Ser Ala Phe Phe Arg Asp Ser Lys Phe Asn Phe Leu Arg Tyr Phe Ser 20 25 30 Ala Lys Lys Phe Ala Lys Asn Phe Lys Asn Ser Ser His Ile His Lys 35 40 45 Thr Asn Ile Ser Lys Ala Gln Ser Asn Ile Ser Ser Thr Leu Lys Gln 50 55 60 Asn Arg Lys Gln Asp Met Leu Ile Pro Ile Asn Phe Phe Asn Phe Glu 65 70 75 80 Tyr Ile Val Lys Lys Leu Asn Asn Gln Asn Ala Ile Gly Val Tyr Ile 85 90 95 Leu Pro Ser Asn Leu Thr Leu Lys Pro Ala Leu Cys Ile Leu Glu Ser 100 105 110 His Lys Glu Asp Phe Leu Asn Lys Phe Leu Leu Thr Ile Ser Ser Glu 115 120 125 Asn Leu Lys Leu Gln Tyr Lys Phe Asn Gly Gln Ile Lys Asn Pro Lys 130 135 140 Ser Val Asn Glu Ile Trp Thr Asp Leu Phe Ser Ile Ala His Val Asp 145 150 155 160 Met Lys Leu Ser Thr Asp Arg Thr Leu Ser Ser Ser Ile Ser Gln Phe 165 170 175 Trp Phe Arg Leu Glu Phe Cys Lys Glu Asp Lys Asp Phe Ile Leu Phe 180 185 190 Pro Thr Ala Asn Arg Tyr Ser Arg Lys Leu Trp Lys His Ser Ile Lys 195 200 205 Asn Asn Gln Leu Phe Lys Glu Gly Ile Arg Asn Tyr Ser Glu Ile Ser 210 215 220 Ser Leu Pro Tyr Glu Glu Asp His Asn Phe Asp Ile Asp Leu Val Phe 225 230 235 240 Thr Trp Val Asn Ser Glu Asp Lys Asn Trp Gln Glu Leu Tyr Lys Lys 245 250 255 Tyr Lys Pro Asp Phe Asn Ser Asp Ala Thr Ser Thr Ser Arg Phe Leu 260 265 270 Ser Arg Asp Glu Leu Lys Phe Ala Leu Arg Ser Trp Glu Met Asn Gly 275 280 285 Ser Phe Ile Arg Lys Ile Phe Ile Val Ser Asn Cys Ala Pro Pro Ala 290 295 300 Trp Leu Asp Leu Asn Asn Pro Lys Ile Gln Trp Val Tyr His Glu Glu 305 310 315 320 Ile Met Pro Gln Ser Ala Leu Pro Thr Phe Ser Ser His Ala Ile Glu 325 330 335 Thr Ser Leu His His Ile Pro Gly Ile Ser Asn Tyr Phe Ile Tyr Ser 340 345 350 Asn Asp Asp Phe Leu Leu Thr Lys Pro Leu Asn Lys Asp Asn Phe Phe 355 360 365 Tyr Ser Asn Gly Ile Ala Lys Leu Arg Leu Glu Ala Trp Gly Asn Val 370 375 380 Asn Gly Glu Cys Thr Glu Gly Glu Pro Asp Tyr Leu Asn Gly Ala Arg 385 390 395 400 Asn Ala Asn Thr Leu Leu Glu Lys Glu Phe Lys Lys Phe Thr Thr Lys 405 410 415 Leu His Thr His Ser Pro Gln Ser Met Arg Thr Asp Ile Leu Phe Glu 420 425 430 Met Glu Lys Lys Tyr Pro Glu Glu Phe Asn Arg Thr Leu His Asn Lys 435 440 445 Phe Arg Ser Leu Asp Asp Ile Ala Val Thr Gly Tyr Leu Tyr His His 450 455 460 Tyr Ala Leu Leu Ser Gly Arg Ala Leu Gln Ser Ser Asp Lys Thr Glu 465 470 475 480 Leu Val Gln Gln Asn His Asp Phe Lys Lys Lys Leu Asn Asn Val Val 485 490 495 Thr Leu Thr Lys Glu Arg Asn Phe Asp Lys Leu Pro Leu Ser Val Cys 500 505 510 Ile Asn Asp Gly Ala Asp Ser His Leu Asn Glu Glu Trp Asn Val Gln 515 520 525 Val Ile Lys Phe Leu Glu Thr Leu Phe Pro Leu Pro Ser Ser Phe Glu 530 535 540 Lys 545 <210> SEQ ID NO 10 <211> LENGTH: 475 <212> TYPE: PRT <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: AS deltaN69-CP-A (codon optimized) (475) <400> SEQUENCE: 10 Leu Ile Pro Ile Asn Phe Phe Asn Phe Glu Tyr Ile Val Lys Lys Leu 1 5 10 15 Asn Asn Gln Asn Ala Ile Gly Val Tyr Ile Leu Pro Ser Asn Leu Thr 20 25 30 Leu Lys Pro Ala Leu Cys Ile Leu Glu Ser His Lys Glu Asp Phe Leu 35 40 45 Asn Lys Phe Leu Leu Thr Ile Ser Ser Glu Asn Leu Lys Leu Gln Tyr 50 55 60 Lys Phe Asn Gly Gln Ile Lys Asn Pro Lys Ser Val Asn Glu Ile Trp 65 70 75 80 Thr Asp Leu Phe Ser Ile Ala His Val Asp Met Lys Leu Ser Thr Asp 85 90 95 Arg Thr Leu Ser Ser Ser Ile Ser Gln Phe Trp Phe Arg Leu Glu Phe 100 105 110 Cys Lys Glu Asp Lys Asp Phe Ile Leu Phe Pro Thr Ala Asn Arg Tyr 115 120 125 Ser Arg Lys Leu Trp Lys His Ser Ile Lys Asn Asn Gln Leu Phe Lys 130 135 140 Glu Gly Ile Arg Asn Tyr Ser Glu Ile Ser Ser Leu Pro Tyr Glu Glu 145 150 155 160 Asp His Asn Phe Asp Ile Asp Leu Val Phe Thr Trp Val Asn Ser Glu 165 170 175 Asp Lys Asn Trp Gln Glu Leu Tyr Lys Lys Tyr Lys Pro Asp Phe Asn 180 185 190 Ser Asp Ala Thr Ser Thr Ser Arg Phe Leu Ser Arg Asp Glu Leu Lys 195 200 205 Phe Ala Leu Arg Ser Trp Glu Met Asn Gly Ser Phe Ile Arg Lys Ile 210 215 220 Phe Ile Val Ser Asn Cys Ala Pro Pro Ala Trp Leu Asp Leu Asn Asn 225 230 235 240 Pro Lys Ile Gln Trp Val Tyr His Glu Glu Ile Met Pro Gln Ser Ala 245 250 255 Leu Pro Thr Phe Ser Ser His Ala Ile Glu Thr Ser Leu His His Ile 260 265 270 Pro Gly Ile Ser Asn Tyr Phe Ile Tyr Ser Asn Asp Asp Phe Leu Leu 275 280 285 Thr Lys Pro Leu Asn Lys Asp Asn Phe Phe Tyr Ser Asn Gly Ile Ala 290 295 300 Lys Leu Arg Leu Glu Ala Trp Gly Asn Val Asn Gly Glu Cys Thr Glu 305 310 315 320 Gly Glu Pro Asp Tyr Leu Asn Gly Ala Arg Asn Ala Asn Thr Leu Leu 325 330 335 Glu Lys Glu Phe Lys Lys Phe Thr Thr Lys Leu His Thr His Ser Pro 340 345 350 Gln Ser Met Arg Thr Asp Ile Leu Phe Glu Met Glu Lys Lys Tyr Pro 355 360 365 Glu Glu Phe Asn Arg Thr Leu His Asn Lys Phe Arg Ser Leu Asp Asp 370 375 380 Ile Ala Val Thr Gly Tyr Leu Tyr His His Tyr Ala Leu Leu Ser Gly 385 390 395 400 Arg Ala Leu Gln Ser Ser Asp Lys Thr Glu Leu Val Gln Gln Asn His 405 410 415 Asp Phe Lys Lys Lys Leu Asn Asn Val Val Thr Leu Thr Lys Glu Arg 420 425 430 Asn Phe Asp Lys Leu Pro Leu Ser Val Cys Ile Asn Asp Gly Ala Asp 435 440 445 Ser His Leu Asn Glu Glu Trp Asn Val Gln Val Ile Lys Phe Leu Glu 450 455 460 Thr Leu Phe Pro Leu Pro Ser Ser Phe Glu Lys 465 470 475 <210> SEQ ID NO 11 <211> LENGTH: 448 <212> TYPE: PRT <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: AS deltaN97-CP-A (448) <400> SEQUENCE: 11 Pro Ser Asn Leu Thr Leu Lys Pro Ala Leu Cys Ile Leu Glu Ser His 1 5 10 15 Lys Glu Asp Phe Leu Asn Lys Phe Leu Leu Thr Ile Ser Ser Glu Asn 20 25 30 Leu Lys Leu Gln Tyr Lys Phe Asn Gly Gln Ile Lys Asn Pro Lys Ser 35 40 45 Val Asn Glu Ile Trp Thr Asp Leu Phe Ser Ile Ala His Val Asp Met 50 55 60 Lys Leu Ser Thr Asp Arg Thr Leu Ser Ser Ser Ile Ser Gln Phe Trp 65 70 75 80 Phe Arg Leu Glu Phe Cys Lys Glu Asp Lys Asp Phe Ile Leu Phe Pro 85 90 95 Thr Ala Asn Arg Tyr Ser Arg Lys Leu Trp Lys His Ser Ile Lys Asn 100 105 110 Asn Gln Leu Phe Lys Glu Gly Ile Arg Asn Tyr Ser Glu Ile Ser Ser 115 120 125 Leu Pro Tyr Glu Glu Asp His Asn Phe Asp Ile Asp Leu Val Phe Thr 130 135 140 Trp Val Asn Ser Glu Asp Lys Asn Trp Gln Glu Leu Tyr Lys Lys Tyr 145 150 155 160 Lys Pro Asp Phe Asn Ser Asp Ala Thr Ser Thr Ser Arg Phe Leu Ser 165 170 175 Arg Asp Glu Leu Lys Phe Ala Leu Arg Ser Trp Glu Met Asn Gly Ser 180 185 190 Phe Ile Arg Lys Ile Phe Ile Val Ser Asn Cys Ala Pro Pro Ala Trp 195 200 205 Leu Asp Leu Asn Asn Pro Lys Ile Gln Trp Val Tyr His Glu Glu Ile 210 215 220 Met Pro Gln Ser Ala Leu Pro Thr Phe Ser Ser His Ala Ile Glu Thr 225 230 235 240 Ser Leu His His Ile Pro Gly Ile Ser Asn Tyr Phe Ile Tyr Ser Asn 245 250 255 Asp Asp Phe Leu Leu Thr Lys Pro Leu Asn Lys Asp Asn Phe Phe Tyr 260 265 270 Ser Asn Gly Ile Ala Lys Leu Arg Leu Glu Ala Trp Gly Asn Val Asn 275 280 285 Gly Glu Cys Thr Glu Gly Glu Pro Asp Tyr Leu Asn Gly Ala Arg Asn 290 295 300 Ala Asn Thr Leu Leu Glu Lys Glu Phe Lys Lys Phe Thr Thr Lys Leu 305 310 315 320 His Thr His Ser Pro Gln Ser Met Arg Thr Asp Ile Leu Phe Glu Met 325 330 335 Glu Lys Lys Tyr Pro Glu Glu Phe Asn Arg Thr Leu His Asn Lys Phe 340 345 350 Arg Ser Leu Asp Asp Ile Ala Val Thr Gly Tyr Leu Tyr His His Tyr 355 360 365 Ala Leu Leu Ser Gly Arg Ala Leu Gln Ser Ser Asp Lys Thr Glu Leu 370 375 380 Val Gln Gln Asn His Asp Phe Lys Lys Lys Leu Asn Asn Val Val Thr 385 390 395 400 Leu Thr Lys Glu Arg Asn Phe Asp Lys Leu Pro Leu Ser Val Cys Ile 405 410 415 Asn Asp Gly Ala Asp Ser His Leu Asn Glu Glu Trp Asn Val Gln Val 420 425 430 Ile Lys Phe Leu Glu Thr Leu Phe Pro Leu Pro Ser Ser Phe Glu Lys 435 440 445 <210> SEQ ID NO 12 <211> LENGTH: 378 <212> TYPE: PRT <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: AS deltaN167-CP-A (378) <400> SEQUENCE: 12 Thr Leu Ser Ser Ser Ile Ser Gln Phe Trp Phe Arg Leu Glu Phe Cys 1 5 10 15 Lys Glu Asp Lys Asp Phe Ile Leu Phe Pro Thr Ala Asn Arg Tyr Ser 20 25 30 Arg Lys Leu Trp Lys His Ser Ile Lys Asn Asn Gln Leu Phe Lys Glu 35 40 45 Gly Ile Arg Asn Tyr Ser Glu Ile Ser Ser Leu Pro Tyr Glu Glu Asp 50 55 60 His Asn Phe Asp Ile Asp Leu Val Phe Thr Trp Val Asn Ser Glu Asp 65 70 75 80 Lys Asn Trp Gln Glu Leu Tyr Lys Lys Tyr Lys Pro Asp Phe Asn Ser 85 90 95 Asp Ala Thr Ser Thr Ser Arg Phe Leu Ser Arg Asp Glu Leu Lys Phe 100 105 110 Ala Leu Arg Ser Trp Glu Met Asn Gly Ser Phe Ile Arg Lys Ile Phe 115 120 125 Ile Val Ser Asn Cys Ala Pro Pro Ala Trp Leu Asp Leu Asn Asn Pro 130 135 140 Lys Ile Gln Trp Val Tyr His Glu Glu Ile Met Pro Gln Ser Ala Leu 145 150 155 160 Pro Thr Phe Ser Ser His Ala Ile Glu Thr Ser Leu His His Ile Pro 165 170 175 Gly Ile Ser Asn Tyr Phe Ile Tyr Ser Asn Asp Asp Phe Leu Leu Thr 180 185 190 Lys Pro Leu Asn Lys Asp Asn Phe Phe Tyr Ser Asn Gly Ile Ala Lys 195 200 205 Leu Arg Leu Glu Ala Trp Gly Asn Val Asn Gly Glu Cys Thr Glu Gly 210 215 220 Glu Pro Asp Tyr Leu Asn Gly Ala Arg Asn Ala Asn Thr Leu Leu Glu 225 230 235 240 Lys Glu Phe Lys Lys Phe Thr Thr Lys Leu His Thr His Ser Pro Gln 245 250 255 Ser Met Arg Thr Asp Ile Leu Phe Glu Met Glu Lys Lys Tyr Pro Glu 260 265 270 Glu Phe Asn Arg Thr Leu His Asn Lys Phe Arg Ser Leu Asp Asp Ile 275 280 285 Ala Val Thr Gly Tyr Leu Tyr His His Tyr Ala Leu Leu Ser Gly Arg 290 295 300 Ala Leu Gln Ser Ser Asp Lys Thr Glu Leu Val Gln Gln Asn His Asp 305 310 315 320 Phe Lys Lys Lys Leu Asn Asn Val Val Thr Leu Thr Lys Glu Arg Asn 325 330 335 Phe Asp Lys Leu Pro Leu Ser Val Cys Ile Asn Asp Gly Ala Asp Ser 340 345 350 His Leu Asn Glu Glu Trp Asn Val Gln Val Ile Lys Phe Leu Glu Thr 355 360 365 Leu Phe Pro Leu Pro Ser Ser Phe Glu Lys 370 375 <210> SEQ ID NO 13 <211> LENGTH: 311 <212> TYPE: PRT <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: AS deltaN235-CP-A (311) <400> SEQUENCE: 13 Asp Ile Asp Leu Val Phe Thr Trp Val Asn Ser Glu Asp Lys Asn Trp 1 5 10 15 Gln Glu Leu Tyr Lys Lys Tyr Lys Pro Asp Phe Asn Ser Asp Ala Thr 20 25 30 Ser Thr Ser Arg Phe Leu Ser Arg Asp Glu Leu Lys Phe Ala Leu Arg 35 40 45 Ser Trp Glu Met Asn Gly Ser Phe Ile Arg Lys Ile Phe Ile Val Ser 50 55 60 Asn Cys Ala Pro Pro Ala Trp Leu Asp Leu Asn Asn Pro Lys Ile Gln 65 70 75 80 Trp Val Tyr His Glu Glu Ile Met Pro Gln Ser Ala Leu Pro Thr Phe 85 90 95 Ser Ser His Ala Ile Glu Thr Ser Leu His His Ile Pro Gly Ile Ser 100 105 110 Asn Tyr Phe Ile Tyr Ser Asn Asp Asp Phe Leu Leu Thr Lys Pro Leu 115 120 125 Asn Lys Asp Asn Phe Phe Tyr Ser Asn Gly Ile Ala Lys Leu Arg Leu 130 135 140 Glu Ala Trp Gly Asn Val Asn Gly Glu Cys Thr Glu Gly Glu Pro Asp 145 150 155 160 Tyr Leu Asn Gly Ala Arg Asn Ala Asn Thr Leu Leu Glu Lys Glu Phe 165 170 175 Lys Lys Phe Thr Thr Lys Leu His Thr His Ser Pro Gln Ser Met Arg 180 185 190 Thr Asp Ile Leu Phe Glu Met Glu Lys Lys Tyr Pro Glu Glu Phe Asn 195 200 205 Arg Thr Leu His Asn Lys Phe Arg Ser Leu Asp Asp Ile Ala Val Thr 210 215 220 Gly Tyr Leu Tyr His His Tyr Ala Leu Leu Ser Gly Arg Ala Leu Gln 225 230 235 240 Ser Ser Asp Lys Thr Glu Leu Val Gln Gln Asn His Asp Phe Lys Lys 245 250 255 Lys Leu Asn Asn Val Val Thr Leu Thr Lys Glu Arg Asn Phe Asp Lys 260 265 270 Leu Pro Leu Ser Val Cys Ile Asn Asp Gly Ala Asp Ser His Leu Asn 275 280 285 Glu Glu Trp Asn Val Gln Val Ile Lys Phe Leu Glu Thr Leu Phe Pro 290 295 300 Leu Pro Ser Ser Phe Glu Lys 305 310 <210> SEQ ID NO 14 <211> LENGTH: 499 <212> TYPE: PRT <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: AS deltaC45-CP-A (499) <400> SEQUENCE: 14 Phe Ile Leu Asn Asn Arg Lys Trp Arg Lys Leu Lys Arg Asp Pro Ser 1 5 10 15 Ala Phe Phe Arg Asp Ser Lys Phe Asn Phe Leu Arg Tyr Phe Ser Ala 20 25 30 Lys Lys Phe Ala Lys Asn Phe Lys Asn Ser Ser His Ile His Lys Thr 35 40 45 Asn Ile Ser Lys Ala Gln Ser Asn Ile Ser Ser Thr Leu Lys Gln Asn 50 55 60 Arg Lys Gln Asp Met Leu Ile Pro Ile Asn Phe Phe Asn Phe Glu Tyr 65 70 75 80 Ile Val Lys Lys Leu Asn Asn Gln Asn Ala Ile Gly Val Tyr Ile Leu 85 90 95 Pro Ser Asn Leu Thr Leu Lys Pro Ala Leu Cys Ile Leu Glu Ser His 100 105 110 Lys Glu Asp Phe Leu Asn Lys Phe Leu Leu Thr Ile Ser Ser Glu Asn 115 120 125 Leu Lys Leu Gln Tyr Lys Phe Asn Gly Gln Ile Lys Asn Pro Lys Ser 130 135 140 Val Asn Glu Ile Trp Thr Asp Leu Phe Ser Ile Ala His Val Asp Met 145 150 155 160 Lys Leu Ser Thr Asp Arg Thr Leu Ser Ser Ser Ile Ser Gln Phe Trp 165 170 175 Phe Arg Leu Glu Phe Cys Lys Glu Asp Lys Asp Phe Ile Leu Phe Pro 180 185 190 Thr Ala Asn Arg Tyr Ser Arg Lys Leu Trp Lys His Ser Ile Lys Asn 195 200 205 Asn Gln Leu Phe Lys Glu Gly Ile Arg Asn Tyr Ser Glu Ile Ser Ser 210 215 220 Leu Pro Tyr Glu Glu Asp His Asn Phe Asp Ile Asp Leu Val Phe Thr 225 230 235 240 Trp Val Asn Ser Glu Asp Lys Asn Trp Gln Glu Leu Tyr Lys Lys Tyr 245 250 255 Lys Pro Asp Phe Asn Ser Asp Ala Thr Ser Thr Ser Arg Phe Leu Ser 260 265 270 Arg Asp Glu Leu Lys Phe Ala Leu Arg Ser Trp Glu Met Asn Gly Ser 275 280 285 Phe Ile Arg Lys Ile Phe Ile Val Ser Asn Cys Ala Pro Pro Ala Trp 290 295 300 Leu Asp Leu Asn Asn Pro Lys Ile Gln Trp Val Tyr His Glu Glu Ile 305 310 315 320 Met Pro Gln Ser Ala Leu Pro Thr Phe Ser Ser His Ala Ile Glu Thr 325 330 335 Ser Leu His His Ile Pro Gly Ile Ser Asn Tyr Phe Ile Tyr Ser Asn 340 345 350 Asp Asp Phe Leu Leu Thr Lys Pro Leu Asn Lys Asp Asn Phe Phe Tyr 355 360 365 Ser Asn Gly Ile Ala Lys Leu Arg Leu Glu Ala Trp Gly Asn Val Asn 370 375 380 Gly Glu Cys Thr Glu Gly Glu Pro Asp Tyr Leu Asn Gly Ala Arg Asn 385 390 395 400 Ala Asn Thr Leu Leu Glu Lys Glu Phe Lys Lys Phe Thr Thr Lys Leu 405 410 415 His Thr His Ser Pro Gln Ser Met Arg Thr Asp Ile Leu Phe Glu Met 420 425 430 Glu Lys Lys Tyr Pro Glu Glu Phe Asn Arg Thr Leu His Asn Lys Phe 435 440 445 Arg Ser Leu Asp Asp Ile Ala Val Thr Gly Tyr Leu Tyr His His Tyr 450 455 460 Ala Leu Leu Ser Gly Arg Ala Leu Gln Ser Ser Asp Lys Thr Glu Leu 465 470 475 480 Val Gln Gln Asn His Asp Phe Lys Lys Lys Leu Asn Asn Val Val Thr 485 490 495 Leu Thr Lys <210> SEQ ID NO 15 <211> LENGTH: 519 <212> TYPE: PRT <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: AS deltaC25-CP-A (519) <400> SEQUENCE: 15 Phe Ile Leu Asn Asn Arg Lys Trp Arg Lys Leu Lys Arg Asp Pro Ser 1 5 10 15 Ala Phe Phe Arg Asp Ser Lys Phe Asn Phe Leu Arg Tyr Phe Ser Ala 20 25 30 Lys Lys Phe Ala Lys Asn Phe Lys Asn Ser Ser His Ile His Lys Thr 35 40 45 Asn Ile Ser Lys Ala Gln Ser Asn Ile Ser Ser Thr Leu Lys Gln Asn 50 55 60 Arg Lys Gln Asp Met Leu Ile Pro Ile Asn Phe Phe Asn Phe Glu Tyr 65 70 75 80 Ile Val Lys Lys Leu Asn Asn Gln Asn Ala Ile Gly Val Tyr Ile Leu 85 90 95 Pro Ser Asn Leu Thr Leu Lys Pro Ala Leu Cys Ile Leu Glu Ser His 100 105 110 Lys Glu Asp Phe Leu Asn Lys Phe Leu Leu Thr Ile Ser Ser Glu Asn 115 120 125 Leu Lys Leu Gln Tyr Lys Phe Asn Gly Gln Ile Lys Asn Pro Lys Ser 130 135 140 Val Asn Glu Ile Trp Thr Asp Leu Phe Ser Ile Ala His Val Asp Met 145 150 155 160 Lys Leu Ser Thr Asp Arg Thr Leu Ser Ser Ser Ile Ser Gln Phe Trp 165 170 175 Phe Arg Leu Glu Phe Cys Lys Glu Asp Lys Asp Phe Ile Leu Phe Pro 180 185 190 Thr Ala Asn Arg Tyr Ser Arg Lys Leu Trp Lys His Ser Ile Lys Asn 195 200 205 Asn Gln Leu Phe Lys Glu Gly Ile Arg Asn Tyr Ser Glu Ile Ser Ser 210 215 220 Leu Pro Tyr Glu Glu Asp His Asn Phe Asp Ile Asp Leu Val Phe Thr 225 230 235 240 Trp Val Asn Ser Glu Asp Lys Asn Trp Gln Glu Leu Tyr Lys Lys Tyr 245 250 255 Lys Pro Asp Phe Asn Ser Asp Ala Thr Ser Thr Ser Arg Phe Leu Ser 260 265 270 Arg Asp Glu Leu Lys Phe Ala Leu Arg Ser Trp Glu Met Asn Gly Ser 275 280 285 Phe Ile Arg Lys Ile Phe Ile Val Ser Asn Cys Ala Pro Pro Ala Trp 290 295 300 Leu Asp Leu Asn Asn Pro Lys Ile Gln Trp Val Tyr His Glu Glu Ile 305 310 315 320 Met Pro Gln Ser Ala Leu Pro Thr Phe Ser Ser His Ala Ile Glu Thr 325 330 335 Ser Leu His His Ile Pro Gly Ile Ser Asn Tyr Phe Ile Tyr Ser Asn 340 345 350 Asp Asp Phe Leu Leu Thr Lys Pro Leu Asn Lys Asp Asn Phe Phe Tyr 355 360 365 Ser Asn Gly Ile Ala Lys Leu Arg Leu Glu Ala Trp Gly Asn Val Asn 370 375 380 Gly Glu Cys Thr Glu Gly Glu Pro Asp Tyr Leu Asn Gly Ala Arg Asn 385 390 395 400 Ala Asn Thr Leu Leu Glu Lys Glu Phe Lys Lys Phe Thr Thr Lys Leu 405 410 415 His Thr His Ser Pro Gln Ser Met Arg Thr Asp Ile Leu Phe Glu Met 420 425 430 Glu Lys Lys Tyr Pro Glu Glu Phe Asn Arg Thr Leu His Asn Lys Phe 435 440 445 Arg Ser Leu Asp Asp Ile Ala Val Thr Gly Tyr Leu Tyr His His Tyr 450 455 460 Ala Leu Leu Ser Gly Arg Ala Leu Gln Ser Ser Asp Lys Thr Glu Leu 465 470 475 480 Val Gln Gln Asn His Asp Phe Lys Lys Lys Leu Asn Asn Val Val Thr 485 490 495 Leu Thr Lys Glu Arg Asn Phe Asp Lys Leu Pro Leu Ser Val Cys Ile 500 505 510 Asn Asp Gly Ala Asp Ser His 515 <210> SEQ ID NO 16 <211> LENGTH: 1455 <212> TYPE: DNA <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: DNA CP-X/CsxA full-length (1455) <400> SEQUENCE: 16 attatgagca aaattagcaa attggtaacc cacccaaacc ttttctttcg agattatttc 60 ttaaaaaaag caccgttaaa ttatggcgaa aatattaaac ctttaccagt cgaaacctct 120 tctcatagca aaaaaaatac agcccataaa acacccgtat catccgacca accaattgaa 180 gatccatacc cagtaacatt tccaattgat gtagtttata cttgggtaga ttcagatgat 240 gaaaaattca atgaagaacg cctaaagttt caaaattcaa gcacatctga gactctacaa 300 ggcaaagcag aaagcaccga tattgcaaga ttccaatcac gcgacgaatt aaaatattcg 360 attcgaagcc tgatgaagta tgccccatgg gtaaatcata tttacattgt aacaaatggt 420 caaataccaa aatggttaga taccaacaat acaaaggtaa cgattatccc tcactcaact 480 attatcgaca gtcaatttct ccctactttt aattctcacg tcattgaatc ctctctatat 540 aaaatcccag gattatcaga gcattacatt tatttcaatg atgatgtcat gctagctaga 600 gatttaagcc catcttattt ctttacaagc agcggattag caaaactgtt tattaccaac 660 tctcgtctac caaatggcta taagaatgtg aaagacacac caacccaatg ggcctcaaaa 720 aattcccgtg agcttttaca tgcagaaaca ggattttggg ctgaagccat gtttgcacat 780 acttttcatc cacaacgtaa aagtgtacat gaatctattg aacacctatg gcatgaacaa 840 ttaaatgttt gtcgtcaaaa ccgtttccgt gatatttcag atattaacat ggcgacattc 900 ctgcaccacc attttgccat tttgacaggc caagctcttg ctacacgcac taaatgtatt 960 tactttaaca ttcgctctcc tcaagcagct cagcattaca aaacattatt agctcgaaaa 1020 ggaagcgaat acagcccaca ttctatctgc ttaaatgatc atacatcgag caataaaaat 1080 attttatcta attacgaagc caaattacaa agctttttag aaacatacta tccagatgta 1140 tcagaagcag aaattctcct tcctactaaa tctgaagtag ctgaattagt taaacataaa 1200 gattatttaa ctgtatatac taaattatta cctattatca ataagcagct ggtcaataaa 1260 tataataaac cttattcata tcttttctat tatttaggtt tatctgcccg gtttttattt 1320 gaagaaacgc aacaagaaca ctaccgggaa actgctgaag aaaatttaca aatcttttgt 1380 ggcctaaacc caaaacatac actagccctc aaatacttag cggatgtcac cctcacatca 1440 cagcctagtg gacaa 1455 <210> SEQ ID NO 17 <211> LENGTH: 1284 <212> TYPE: DNA <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: DNA deltaN58-CP-X (1284) <400> SEQUENCE: 17 ccaattgaag atccataccc agtaacattt ccaattgatg tagtttatac ttgggtagat 60 tcagatgatg aaaaattcaa tgaagaacgc ctaaagtttc aaaattcaag cacatctgag 120 actctacaag gcaaagcaga aagcaccgat attgcaagat tccaatcacg cgacgaatta 180 aaatattcga ttcgaagcct gatgaagtat gccccatggg taaatcatat ttacattgta 240 acaaatggtc aaataccaaa atggttagat accaacaata caaaggtaac gattatccct 300 cactcaacta ttatcgacag tcaatttctc cctactttta attctcacgt cattgaatcc 360 tctctatata aaatcccagg attatcagag cattacattt atttcaatga tgatgtcatg 420 ctagctagag atttaagccc atcttatttc tttacaagca gcggattagc aaaactgttt 480 attaccaact ctcgtctacc aaatggctat aagaatgtga aagacacacc aacccaatgg 540 gcctcaaaaa attcccgtga gcttttacat gcagaaacag gattttgggc tgaagccatg 600 tttgcacata cttttcatcc acaacgtaaa agtgtacatg aatctattga acacctatgg 660 catgaacaat taaatgtttg tcgtcaaaac cgtttccgtg atatttcaga tattaacatg 720 gcgacattcc tgcaccacca ttttgccatt ttgacaggcc aagctcttgc tacacgcact 780 aaatgtattt actttaacat tcgctctcct caagcagctc agcattacaa aacattatta 840 gctcgaaaag gaagcgaata cagcccacat tctatctgct taaatgatca tacatcgagc 900 aataaaaata ttttatctaa ttacgaagcc aaattacaaa gctttttaga aacatactat 960 ccagatgtat cagaagcaga aattctcctt cctactaaat ctgaagtagc tgaattagtt 1020 aaacataaag attatttaac tgtatatact aaattattac ctattatcaa taagcagctg 1080 gtcaataaat ataataaacc ttattcatat cttttctatt atttaggttt atctgcccgg 1140 tttttatttg aagaaacgca acaagaacac taccgggaaa ctgctgaaga aaatttacaa 1200 atcttttgtg gcctaaaccc aaaacataca ctagccctca aatacttagc ggatgtcacc 1260 ctcacatcac agcctagtgg acaa 1284 <210> SEQ ID NO 18 <211> LENGTH: 1146 <212> TYPE: DNA <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: DNA deltaN104-CP-X (1146) <400> SEQUENCE: 18 gaaagcaccg atattgcaag attccaatca cgcgacgaat taaaatattc gattcgaagc 60 ctgatgaagt atgccccatg ggtaaatcat atttacattg taacaaatgg tcaaatacca 120 aaatggttag ataccaacaa tacaaaggta acgattatcc ctcactcaac tattatcgac 180 agtcaatttc tccctacttt taattctcac gtcattgaat cctctctata taaaatccca 240 ggattatcag agcattacat ttatttcaat gatgatgtca tgctagctag agatttaagc 300 ccatcttatt tctttacaag cagcggatta gcaaaactgt ttattaccaa ctctcgtcta 360 ccaaatggct ataagaatgt gaaagacaca ccaacccaat gggcctcaaa aaattcccgt 420 gagcttttac atgcagaaac aggattttgg gctgaagcca tgtttgcaca tacttttcat 480 ccacaacgta aaagtgtaca tgaatctatt gaacacctat ggcatgaaca attaaatgtt 540 tgtcgtcaaa accgtttccg tgatatttca gatattaaca tggcgacatt cctgcaccac 600 cattttgcca ttttgacagg ccaagctctt gctacacgca ctaaatgtat ttactttaac 660 attcgctctc ctcaagcagc tcagcattac aaaacattat tagctcgaaa aggaagcgaa 720 tacagcccac attctatctg cttaaatgat catacatcga gcaataaaaa tattttatct 780 aattacgaag ccaaattaca aagcttttta gaaacatact atccagatgt atcagaagca 840 gaaattctcc ttcctactaa atctgaagta gctgaattag ttaaacataa agattattta 900 actgtatata ctaaattatt acctattatc aataagcagc tggtcaataa atataataaa 960 ccttattcat atcttttcta ttatttaggt ttatctgccc ggtttttatt tgaagaaacg 1020 caacaagaac actaccggga aactgctgaa gaaaatttac aaatcttttg tggcctaaac 1080 ccaaaacata cactagccct caaatactta gcggatgtca ccctcacatc acagcctagt 1140 ggacaa 1146 <210> SEQ ID NO 19 <211> LENGTH: 933 <212> TYPE: DNA <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: DNA deltaC174-CP-X (933) <400> SEQUENCE: 19 attatgagca aaattagcaa attggtaacc cacccaaacc ttttctttcg agattatttc 60 ttaaaaaaag caccgttaaa ttatggcgaa aatattaaac ctttaccagt cgaaacctct 120 tctcatagca aaaaaaatac agcccataaa acacccgtat catccgacca accaattgaa 180 gatccatacc cagtaacatt tccaattgat gtagtttata cttgggtaga ttcagatgat 240 gaaaaattca atgaagaacg cctaaagttt caaaattcaa gcacatctga gactctacaa 300 ggcaaagcag aaagcaccga tattgcaaga ttccaatcac gcgacgaatt aaaatattcg 360 attcgaagcc tgatgaagta tgccccatgg gtaaatcata tttacattgt aacaaatggt 420 caaataccaa aatggttaga taccaacaat acaaaggtaa cgattatccc tcactcaact 480 attatcgaca gtcaatttct ccctactttt aattctcacg tcattgaatc ctctctatat 540 aaaatcccag gattatcaga gcattacatt tatttcaatg atgatgtcat gctagctaga 600 gatttaagcc catcttattt ctttacaagc agcggattag caaaactgtt tattaccaac 660 tctcgtctac caaatggcta taagaatgtg aaagacacac caacccaatg ggcctcaaaa 720 aattcccgtg agcttttaca tgcagaaaca ggattttggg ctgaagccat gtttgcacat 780 acttttcatc cacaacgtaa aagtgtacat gaatctattg aacacctatg gcatgaacaa 840 ttaaatgttt gtcgtcaaaa ccgtttccgt gatatttcag atattaacat ggcgacattc 900 ctgcaccacc attttgccat tttgacaggc caa 933 <210> SEQ ID NO 20 <211> LENGTH: 1158 <212> TYPE: DNA <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: DNA deltaC99-CP-X (1158) <400> SEQUENCE: 20 attatgagca aaattagcaa attggtaacc cacccaaacc ttttctttcg agattatttc 60 ttaaaaaaag caccgttaaa ttatggcgaa aatattaaac ctttaccagt cgaaacctct 120 tctcatagca aaaaaaatac agcccataaa acacccgtat catccgacca accaattgaa 180 gatccatacc cagtaacatt tccaattgat gtagtttata cttgggtaga ttcagatgat 240 gaaaaattca atgaagaacg cctaaagttt caaaattcaa gcacatctga gactctacaa 300 ggcaaagcag aaagcaccga tattgcaaga ttccaatcac gcgacgaatt aaaatattcg 360 attcgaagcc tgatgaagta tgccccatgg gtaaatcata tttacattgt aacaaatggt 420 caaataccaa aatggttaga taccaacaat acaaaggtaa cgattatccc tcactcaact 480 attatcgaca gtcaatttct ccctactttt aattctcacg tcattgaatc ctctctatat 540 aaaatcccag gattatcaga gcattacatt tatttcaatg atgatgtcat gctagctaga 600 gatttaagcc catcttattt ctttacaagc agcggattag caaaactgtt tattaccaac 660 tctcgtctac caaatggcta taagaatgtg aaagacacac caacccaatg ggcctcaaaa 720 aattcccgtg agcttttaca tgcagaaaca ggattttggg ctgaagccat gtttgcacat 780 acttttcatc cacaacgtaa aagtgtacat gaatctattg aacacctatg gcatgaacaa 840 ttaaatgttt gtcgtcaaaa ccgtttccgt gatatttcag atattaacat ggcgacattc 900 ctgcaccacc attttgccat tttgacaggc caagctcttg ctacacgcac taaatgtatt 960 tactttaaca ttcgctctcc tcaagcagct cagcattaca aaacattatt agctcgaaaa 1020 ggaagcgaat acagcccaca ttctatctgc ttaaatgatc atacatcgag caataaaaat 1080 attttatcta attacgaagc caaattacaa agctttttag aaacatacta tccagatgta 1140 tcagaagcag aaattctc 1158 <210> SEQ ID NO 21 <211> LENGTH: 987 <212> TYPE: DNA <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: DNA deltaN58 deltaC99-CP-X (987) <400> SEQUENCE: 21 ccaattgaag atccataccc agtaacattt ccaattgatg tagtttatac ttgggtagat 60 tcagatgatg aaaaattcaa tgaagaacgc ctaaagtttc aaaattcaag cacatctgag 120 actctacaag gcaaagcaga aagcaccgat attgcaagat tccaatcacg cgacgaatta 180 aaatattcga ttcgaagcct gatgaagtat gccccatggg taaatcatat ttacattgta 240 acaaatggtc aaataccaaa atggttagat accaacaata caaaggtaac gattatccct 300 cactcaacta ttatcgacag tcaatttctc cctactttta attctcacgt cattgaatcc 360 tctctatata aaatcccagg attatcagag cattacattt atttcaatga tgatgtcatg 420 ctagctagag atttaagccc atcttatttc tttacaagca gcggattagc aaaactgttt 480 attaccaact ctcgtctacc aaatggctat aagaatgtga aagacacacc aacccaatgg 540 gcctcaaaaa attcccgtga gcttttacat gcagaaacag gattttgggc tgaagccatg 600 tttgcacata cttttcatcc acaacgtaaa agtgtacatg aatctattga acacctatgg 660 catgaacaat taaatgtttg tcgtcaaaac cgtttccgtg atatttcaga tattaacatg 720 gcgacattcc tgcaccacca ttttgccatt ttgacaggcc aagctcttgc tacacgcact 780 aaatgtattt actttaacat tcgctctcct caagcagctc agcattacaa aacattatta 840 gctcgaaaag gaagcgaata cagcccacat tctatctgct taaatgatca tacatcgagc 900 aataaaaata ttttatctaa ttacgaagcc aaattacaaa gctttttaga aacatactat 960 ccagatgtat cagaagcaga aattctc 987 <210> SEQ ID NO 22 <211> LENGTH: 1233 <212> TYPE: DNA <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: DNA deltaN65deltaC10-CP-X (1233) <400> SEQUENCE: 22 gtaacatttc caattgatgt agtttatact tgggtagatt cagatgatga aaaattcaat 60 gaagaacgcc taaagtttca aaattcaagc acatctgaga ctctacaagg caaagcagaa 120 agcaccgata ttgcaagatt ccaatcacgc gacgaattaa aatattcgat tcgaagcctg 180 atgaagtatg ccccatgggt aaatcatatt tacattgtaa caaatggtca aataccaaaa 240 tggttagata ccaacaatac aaaggtaacg attatccctc actcaactat tatcgacagt 300 caatttctcc ctacttttaa ttctcacgtc attgaatcct ctctatataa aatcccagga 360 ttatcagagc attacattta tttcaatgat gatgtcatgc tagctagaga tttaagccca 420 tcttatttct ttacaagcag cggattagca aaactgttta ttaccaactc tcgtctacca 480 aatggctata agaatgtgaa agacacacca acccaatggg cctcaaaaaa ttcccgtgag 540 cttttacatg cagaaacagg attttgggct gaagccatgt ttgcacatac ttttcatcca 600 caacgtaaaa gtgtacatga atctattgaa cacctatggc atgaacaatt aaatgtttgt 660 cgtcaaaacc gtttccgtga tatttcagat attaacatgg cgacattcct gcaccaccat 720 tttgccattt tgacaggcca agctcttgct acacgcacta aatgtattta ctttaacatt 780 cgctctcctc aagcagctca gcattacaaa acattattag ctcgaaaagg aagcgaatac 840 agcccacatt ctatctgctt aaatgatcat acatcgagca ataaaaatat tttatctaat 900 tacgaagcca aattacaaag ctttttagaa acatactatc cagatgtatc agaagcagaa 960 attctccttc ctactaaatc tgaagtagct gaattagtta aacataaaga ttatttaact 1020 gtatatacta aattattacc tattatcaat aagcagctgg tcaataaata taataaacct 1080 tattcatatc ttttctatta tttaggttta tctgcccggt ttttatttga agaaacgcaa 1140 caagaacact accgggaaac tgctgaagaa aatttacaaa tcttttgtgg cctaaaccca 1200 aaacatacac tagccctcaa atacttagcg gat 1233 <210> SEQ ID NO 23 <211> LENGTH: 960 <212> TYPE: DNA <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: DNA deltaN67deltaC99-CP-X (960) <400> SEQUENCE: 23 tttccaattg atgtagttta tacttgggta gattcagatg atgaaaaatt caatgaagaa 60 cgcctaaagt ttcaaaattc aagcacatct gagactctac aaggcaaagc agaaagcacc 120 gatattgcaa gattccaatc acgcgacgaa ttaaaatatt cgattcgaag cctgatgaag 180 tatgccccat gggtaaatca tatttacatt gtaacaaatg gtcaaatacc aaaatggtta 240 gataccaaca atacaaaggt aacgattatc cctcactcaa ctattatcga cagtcaattt 300 ctccctactt ttaattctca cgtcattgaa tcctctctat ataaaatccc aggattatca 360 gagcattaca tttatttcaa tgatgatgtc atgctagcta gagatttaag cccatcttat 420 ttctttacaa gcagcggatt agcaaaactg tttattacca actctcgtct accaaatggc 480 tataagaatg tgaaagacac accaacccaa tgggcctcaa aaaattcccg tgagctttta 540 catgcagaaa caggattttg ggctgaagcc atgtttgcac atacttttca tccacaacgt 600 aaaagtgtac atgaatctat tgaacaccta tggcatgaac aattaaatgt ttgtcgtcaa 660 aaccgtttcc gtgatatttc agatattaac atggcgacat tcctgcacca ccattttgcc 720 attttgacag gccaagctct tgctacacgc actaaatgta tttactttaa cattcgctct 780 cctcaagcag ctcagcatta caaaacatta ttagctcgaa aaggaagcga atacagccca 840 cattctatct gcttaaatga tcatacatcg agcaataaaa atattttatc taattacgaa 900 gccaaattac aaagcttttt agaaacatac tatccagatg tatcagaagc agaaattctc 960 <210> SEQ ID NO 24 <211> LENGTH: 485 <212> TYPE: PRT <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: AS CP-X/CsxA full-length (485) <400> SEQUENCE: 24 Ile Met Ser Lys Ile Ser Lys Leu Val Thr His Pro Asn Leu Phe Phe 1 5 10 15 Arg Asp Tyr Phe Leu Lys Lys Ala Pro Leu Asn Tyr Gly Glu Asn Ile 20 25 30 Lys Pro Leu Pro Val Glu Thr Ser Ser His Ser Lys Lys Asn Thr Ala 35 40 45 His Lys Thr Pro Val Ser Ser Asp Gln Pro Ile Glu Asp Pro Tyr Pro 50 55 60 Val Thr Phe Pro Ile Asp Val Val Tyr Thr Trp Val Asp Ser Asp Asp 65 70 75 80 Glu Lys Phe Asn Glu Glu Arg Leu Lys Phe Gln Asn Ser Ser Thr Ser 85 90 95 Glu Thr Leu Gln Gly Lys Ala Glu Ser Thr Asp Ile Ala Arg Phe Gln 100 105 110 Ser Arg Asp Glu Leu Lys Tyr Ser Ile Arg Ser Leu Met Lys Tyr Ala 115 120 125 Pro Trp Val Asn His Ile Tyr Ile Val Thr Asn Gly Gln Ile Pro Lys 130 135 140 Trp Leu Asp Thr Asn Asn Thr Lys Val Thr Ile Ile Pro His Ser Thr 145 150 155 160 Ile Ile Asp Ser Gln Phe Leu Pro Thr Phe Asn Ser His Val Ile Glu 165 170 175 Ser Ser Leu Tyr Lys Ile Pro Gly Leu Ser Glu His Tyr Ile Tyr Phe 180 185 190 Asn Asp Asp Val Met Leu Ala Arg Asp Leu Ser Pro Ser Tyr Phe Phe 195 200 205 Thr Ser Ser Gly Leu Ala Lys Leu Phe Ile Thr Asn Ser Arg Leu Pro 210 215 220 Asn Gly Tyr Lys Asn Val Lys Asp Thr Pro Thr Gln Trp Ala Ser Lys 225 230 235 240 Asn Ser Arg Glu Leu Leu His Ala Glu Thr Gly Phe Trp Ala Glu Ala 245 250 255 Met Phe Ala His Thr Phe His Pro Gln Arg Lys Ser Val His Glu Ser 260 265 270 Ile Glu His Leu Trp His Glu Gln Leu Asn Val Cys Arg Gln Asn Arg 275 280 285 Phe Arg Asp Ile Ser Asp Ile Asn Met Ala Thr Phe Leu His His His 290 295 300 Phe Ala Ile Leu Thr Gly Gln Ala Leu Ala Thr Arg Thr Lys Cys Ile 305 310 315 320 Tyr Phe Asn Ile Arg Ser Pro Gln Ala Ala Gln His Tyr Lys Thr Leu 325 330 335 Leu Ala Arg Lys Gly Ser Glu Tyr Ser Pro His Ser Ile Cys Leu Asn 340 345 350 Asp His Thr Ser Ser Asn Lys Asn Ile Leu Ser Asn Tyr Glu Ala Lys 355 360 365 Leu Gln Ser Phe Leu Glu Thr Tyr Tyr Pro Asp Val Ser Glu Ala Glu 370 375 380 Ile Leu Leu Pro Thr Lys Ser Glu Val Ala Glu Leu Val Lys His Lys 385 390 395 400 Asp Tyr Leu Thr Val Tyr Thr Lys Leu Leu Pro Ile Ile Asn Lys Gln 405 410 415 Leu Val Asn Lys Tyr Asn Lys Pro Tyr Ser Tyr Leu Phe Tyr Tyr Leu 420 425 430 Gly Leu Ser Ala Arg Phe Leu Phe Glu Glu Thr Gln Gln Glu His Tyr 435 440 445 Arg Glu Thr Ala Glu Glu Asn Leu Gln Ile Phe Cys Gly Leu Asn Pro 450 455 460 Lys His Thr Leu Ala Leu Lys Tyr Leu Ala Asp Val Thr Leu Thr Ser 465 470 475 480 Gln Pro Ser Gly Gln 485 <210> SEQ ID NO 25 <211> LENGTH: 428 <212> TYPE: PRT <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: AS deltaN58-CP-X (428) <400> SEQUENCE: 25 Pro Ile Glu Asp Pro Tyr Pro Val Thr Phe Pro Ile Asp Val Val Tyr 1 5 10 15 Thr Trp Val Asp Ser Asp Asp Glu Lys Phe Asn Glu Glu Arg Leu Lys 20 25 30 Phe Gln Asn Ser Ser Thr Ser Glu Thr Leu Gln Gly Lys Ala Glu Ser 35 40 45 Thr Asp Ile Ala Arg Phe Gln Ser Arg Asp Glu Leu Lys Tyr Ser Ile 50 55 60 Arg Ser Leu Met Lys Tyr Ala Pro Trp Val Asn His Ile Tyr Ile Val 65 70 75 80 Thr Asn Gly Gln Ile Pro Lys Trp Leu Asp Thr Asn Asn Thr Lys Val 85 90 95 Thr Ile Ile Pro His Ser Thr Ile Ile Asp Ser Gln Phe Leu Pro Thr 100 105 110 Phe Asn Ser His Val Ile Glu Ser Ser Leu Tyr Lys Ile Pro Gly Leu 115 120 125 Ser Glu His Tyr Ile Tyr Phe Asn Asp Asp Val Met Leu Ala Arg Asp 130 135 140 Leu Ser Pro Ser Tyr Phe Phe Thr Ser Ser Gly Leu Ala Lys Leu Phe 145 150 155 160 Ile Thr Asn Ser Arg Leu Pro Asn Gly Tyr Lys Asn Val Lys Asp Thr 165 170 175 Pro Thr Gln Trp Ala Ser Lys Asn Ser Arg Glu Leu Leu His Ala Glu 180 185 190 Thr Gly Phe Trp Ala Glu Ala Met Phe Ala His Thr Phe His Pro Gln 195 200 205 Arg Lys Ser Val His Glu Ser Ile Glu His Leu Trp His Glu Gln Leu 210 215 220 Asn Val Cys Arg Gln Asn Arg Phe Arg Asp Ile Ser Asp Ile Asn Met 225 230 235 240 Ala Thr Phe Leu His His His Phe Ala Ile Leu Thr Gly Gln Ala Leu 245 250 255 Ala Thr Arg Thr Lys Cys Ile Tyr Phe Asn Ile Arg Ser Pro Gln Ala 260 265 270 Ala Gln His Tyr Lys Thr Leu Leu Ala Arg Lys Gly Ser Glu Tyr Ser 275 280 285 Pro His Ser Ile Cys Leu Asn Asp His Thr Ser Ser Asn Lys Asn Ile 290 295 300 Leu Ser Asn Tyr Glu Ala Lys Leu Gln Ser Phe Leu Glu Thr Tyr Tyr 305 310 315 320 Pro Asp Val Ser Glu Ala Glu Ile Leu Leu Pro Thr Lys Ser Glu Val 325 330 335 Ala Glu Leu Val Lys His Lys Asp Tyr Leu Thr Val Tyr Thr Lys Leu 340 345 350 Leu Pro Ile Ile Asn Lys Gln Leu Val Asn Lys Tyr Asn Lys Pro Tyr 355 360 365 Ser Tyr Leu Phe Tyr Tyr Leu Gly Leu Ser Ala Arg Phe Leu Phe Glu 370 375 380 Glu Thr Gln Gln Glu His Tyr Arg Glu Thr Ala Glu Glu Asn Leu Gln 385 390 395 400 Ile Phe Cys Gly Leu Asn Pro Lys His Thr Leu Ala Leu Lys Tyr Leu 405 410 415 Ala Asp Val Thr Leu Thr Ser Gln Pro Ser Gly Gln 420 425 <210> SEQ ID NO 26 <211> LENGTH: 382 <212> TYPE: PRT <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: AS deltaN104-CP-X (382) <400> SEQUENCE: 26 Glu Ser Thr Asp Ile Ala Arg Phe Gln Ser Arg Asp Glu Leu Lys Tyr 1 5 10 15 Ser Ile Arg Ser Leu Met Lys Tyr Ala Pro Trp Val Asn His Ile Tyr 20 25 30 Ile Val Thr Asn Gly Gln Ile Pro Lys Trp Leu Asp Thr Asn Asn Thr 35 40 45 Lys Val Thr Ile Ile Pro His Ser Thr Ile Ile Asp Ser Gln Phe Leu 50 55 60 Pro Thr Phe Asn Ser His Val Ile Glu Ser Ser Leu Tyr Lys Ile Pro 65 70 75 80 Gly Leu Ser Glu His Tyr Ile Tyr Phe Asn Asp Asp Val Met Leu Ala 85 90 95 Arg Asp Leu Ser Pro Ser Tyr Phe Phe Thr Ser Ser Gly Leu Ala Lys 100 105 110 Leu Phe Ile Thr Asn Ser Arg Leu Pro Asn Gly Tyr Lys Asn Val Lys 115 120 125 Asp Thr Pro Thr Gln Trp Ala Ser Lys Asn Ser Arg Glu Leu Leu His 130 135 140 Ala Glu Thr Gly Phe Trp Ala Glu Ala Met Phe Ala His Thr Phe His 145 150 155 160 Pro Gln Arg Lys Ser Val His Glu Ser Ile Glu His Leu Trp His Glu 165 170 175 Gln Leu Asn Val Cys Arg Gln Asn Arg Phe Arg Asp Ile Ser Asp Ile 180 185 190 Asn Met Ala Thr Phe Leu His His His Phe Ala Ile Leu Thr Gly Gln 195 200 205 Ala Leu Ala Thr Arg Thr Lys Cys Ile Tyr Phe Asn Ile Arg Ser Pro 210 215 220 Gln Ala Ala Gln His Tyr Lys Thr Leu Leu Ala Arg Lys Gly Ser Glu 225 230 235 240 Tyr Ser Pro His Ser Ile Cys Leu Asn Asp His Thr Ser Ser Asn Lys 245 250 255 Asn Ile Leu Ser Asn Tyr Glu Ala Lys Leu Gln Ser Phe Leu Glu Thr 260 265 270 Tyr Tyr Pro Asp Val Ser Glu Ala Glu Ile Leu Leu Pro Thr Lys Ser 275 280 285 Glu Val Ala Glu Leu Val Lys His Lys Asp Tyr Leu Thr Val Tyr Thr 290 295 300 Lys Leu Leu Pro Ile Ile Asn Lys Gln Leu Val Asn Lys Tyr Asn Lys 305 310 315 320 Pro Tyr Ser Tyr Leu Phe Tyr Tyr Leu Gly Leu Ser Ala Arg Phe Leu 325 330 335 Phe Glu Glu Thr Gln Gln Glu His Tyr Arg Glu Thr Ala Glu Glu Asn 340 345 350 Leu Gln Ile Phe Cys Gly Leu Asn Pro Lys His Thr Leu Ala Leu Lys 355 360 365 Tyr Leu Ala Asp Val Thr Leu Thr Ser Gln Pro Ser Gly Gln 370 375 380 <210> SEQ ID NO 27 <211> LENGTH: 311 <212> TYPE: PRT <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: AS deltaC174-CP-X (311) <400> SEQUENCE: 27 Ile Met Ser Lys Ile Ser Lys Leu Val Thr His Pro Asn Leu Phe Phe 1 5 10 15 Arg Asp Tyr Phe Leu Lys Lys Ala Pro Leu Asn Tyr Gly Glu Asn Ile 20 25 30 Lys Pro Leu Pro Val Glu Thr Ser Ser His Ser Lys Lys Asn Thr Ala 35 40 45 His Lys Thr Pro Val Ser Ser Asp Gln Pro Ile Glu Asp Pro Tyr Pro 50 55 60 Val Thr Phe Pro Ile Asp Val Val Tyr Thr Trp Val Asp Ser Asp Asp 65 70 75 80 Glu Lys Phe Asn Glu Glu Arg Leu Lys Phe Gln Asn Ser Ser Thr Ser 85 90 95 Glu Thr Leu Gln Gly Lys Ala Glu Ser Thr Asp Ile Ala Arg Phe Gln 100 105 110 Ser Arg Asp Glu Leu Lys Tyr Ser Ile Arg Ser Leu Met Lys Tyr Ala 115 120 125 Pro Trp Val Asn His Ile Tyr Ile Val Thr Asn Gly Gln Ile Pro Lys 130 135 140 Trp Leu Asp Thr Asn Asn Thr Lys Val Thr Ile Ile Pro His Ser Thr 145 150 155 160 Ile Ile Asp Ser Gln Phe Leu Pro Thr Phe Asn Ser His Val Ile Glu 165 170 175 Ser Ser Leu Tyr Lys Ile Pro Gly Leu Ser Glu His Tyr Ile Tyr Phe 180 185 190 Asn Asp Asp Val Met Leu Ala Arg Asp Leu Ser Pro Ser Tyr Phe Phe 195 200 205 Thr Ser Ser Gly Leu Ala Lys Leu Phe Ile Thr Asn Ser Arg Leu Pro 210 215 220 Asn Gly Tyr Lys Asn Val Lys Asp Thr Pro Thr Gln Trp Ala Ser Lys 225 230 235 240 Asn Ser Arg Glu Leu Leu His Ala Glu Thr Gly Phe Trp Ala Glu Ala 245 250 255 Met Phe Ala His Thr Phe His Pro Gln Arg Lys Ser Val His Glu Ser 260 265 270 Ile Glu His Leu Trp His Glu Gln Leu Asn Val Cys Arg Gln Asn Arg 275 280 285 Phe Arg Asp Ile Ser Asp Ile Asn Met Ala Thr Phe Leu His His His 290 295 300 Phe Ala Ile Leu Thr Gly Gln 305 310 <210> SEQ ID NO 28 <211> LENGTH: 386 <212> TYPE: PRT <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: AS deltaC99-CP-X (386) <400> SEQUENCE: 28 Ile Met Ser Lys Ile Ser Lys Leu Val Thr His Pro Asn Leu Phe Phe 1 5 10 15 Arg Asp Tyr Phe Leu Lys Lys Ala Pro Leu Asn Tyr Gly Glu Asn Ile 20 25 30 Lys Pro Leu Pro Val Glu Thr Ser Ser His Ser Lys Lys Asn Thr Ala 35 40 45 His Lys Thr Pro Val Ser Ser Asp Gln Pro Ile Glu Asp Pro Tyr Pro 50 55 60 Val Thr Phe Pro Ile Asp Val Val Tyr Thr Trp Val Asp Ser Asp Asp 65 70 75 80 Glu Lys Phe Asn Glu Glu Arg Leu Lys Phe Gln Asn Ser Ser Thr Ser 85 90 95 Glu Thr Leu Gln Gly Lys Ala Glu Ser Thr Asp Ile Ala Arg Phe Gln 100 105 110 Ser Arg Asp Glu Leu Lys Tyr Ser Ile Arg Ser Leu Met Lys Tyr Ala 115 120 125 Pro Trp Val Asn His Ile Tyr Ile Val Thr Asn Gly Gln Ile Pro Lys 130 135 140 Trp Leu Asp Thr Asn Asn Thr Lys Val Thr Ile Ile Pro His Ser Thr 145 150 155 160 Ile Ile Asp Ser Gln Phe Leu Pro Thr Phe Asn Ser His Val Ile Glu 165 170 175 Ser Ser Leu Tyr Lys Ile Pro Gly Leu Ser Glu His Tyr Ile Tyr Phe 180 185 190 Asn Asp Asp Val Met Leu Ala Arg Asp Leu Ser Pro Ser Tyr Phe Phe 195 200 205 Thr Ser Ser Gly Leu Ala Lys Leu Phe Ile Thr Asn Ser Arg Leu Pro 210 215 220 Asn Gly Tyr Lys Asn Val Lys Asp Thr Pro Thr Gln Trp Ala Ser Lys 225 230 235 240 Asn Ser Arg Glu Leu Leu His Ala Glu Thr Gly Phe Trp Ala Glu Ala 245 250 255 Met Phe Ala His Thr Phe His Pro Gln Arg Lys Ser Val His Glu Ser 260 265 270 Ile Glu His Leu Trp His Glu Gln Leu Asn Val Cys Arg Gln Asn Arg 275 280 285 Phe Arg Asp Ile Ser Asp Ile Asn Met Ala Thr Phe Leu His His His 290 295 300 Phe Ala Ile Leu Thr Gly Gln Ala Leu Ala Thr Arg Thr Lys Cys Ile 305 310 315 320 Tyr Phe Asn Ile Arg Ser Pro Gln Ala Ala Gln His Tyr Lys Thr Leu 325 330 335 Leu Ala Arg Lys Gly Ser Glu Tyr Ser Pro His Ser Ile Cys Leu Asn 340 345 350 Asp His Thr Ser Ser Asn Lys Asn Ile Leu Ser Asn Tyr Glu Ala Lys 355 360 365 Leu Gln Ser Phe Leu Glu Thr Tyr Tyr Pro Asp Val Ser Glu Ala Glu 370 375 380 Ile Leu 385 <210> SEQ ID NO 29 <211> LENGTH: 329 <212> TYPE: PRT <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: AS deltaN58deltaC99-CP-X (329) <400> SEQUENCE: 29 Pro Ile Glu Asp Pro Tyr Pro Val Thr Phe Pro Ile Asp Val Val Tyr 1 5 10 15 Thr Trp Val Asp Ser Asp Asp Glu Lys Phe Asn Glu Glu Arg Leu Lys 20 25 30 Phe Gln Asn Ser Ser Thr Ser Glu Thr Leu Gln Gly Lys Ala Glu Ser 35 40 45 Thr Asp Ile Ala Arg Phe Gln Ser Arg Asp Glu Leu Lys Tyr Ser Ile 50 55 60 Arg Ser Leu Met Lys Tyr Ala Pro Trp Val Asn His Ile Tyr Ile Val 65 70 75 80 Thr Asn Gly Gln Ile Pro Lys Trp Leu Asp Thr Asn Asn Thr Lys Val 85 90 95 Thr Ile Ile Pro His Ser Thr Ile Ile Asp Ser Gln Phe Leu Pro Thr 100 105 110 Phe Asn Ser His Val Ile Glu Ser Ser Leu Tyr Lys Ile Pro Gly Leu 115 120 125 Ser Glu His Tyr Ile Tyr Phe Asn Asp Asp Val Met Leu Ala Arg Asp 130 135 140 Leu Ser Pro Ser Tyr Phe Phe Thr Ser Ser Gly Leu Ala Lys Leu Phe 145 150 155 160 Ile Thr Asn Ser Arg Leu Pro Asn Gly Tyr Lys Asn Val Lys Asp Thr 165 170 175 Pro Thr Gln Trp Ala Ser Lys Asn Ser Arg Glu Leu Leu His Ala Glu 180 185 190 Thr Gly Phe Trp Ala Glu Ala Met Phe Ala His Thr Phe His Pro Gln 195 200 205 Arg Lys Ser Val His Glu Ser Ile Glu His Leu Trp His Glu Gln Leu 210 215 220 Asn Val Cys Arg Gln Asn Arg Phe Arg Asp Ile Ser Asp Ile Asn Met 225 230 235 240 Ala Thr Phe Leu His His His Phe Ala Ile Leu Thr Gly Gln Ala Leu 245 250 255 Ala Thr Arg Thr Lys Cys Ile Tyr Phe Asn Ile Arg Ser Pro Gln Ala 260 265 270 Ala Gln His Tyr Lys Thr Leu Leu Ala Arg Lys Gly Ser Glu Tyr Ser 275 280 285 Pro His Ser Ile Cys Leu Asn Asp His Thr Ser Ser Asn Lys Asn Ile 290 295 300 Leu Ser Asn Tyr Glu Ala Lys Leu Gln Ser Phe Leu Glu Thr Tyr Tyr 305 310 315 320 Pro Asp Val Ser Glu Ala Glu Ile Leu 325 <210> SEQ ID NO 30 <211> LENGTH: 411 <212> TYPE: PRT <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: AS deltaN65deltaC10-CP-X (411) <400> SEQUENCE: 30 Val Thr Phe Pro Ile Asp Val Val Tyr Thr Trp Val Asp Ser Asp Asp 1 5 10 15 Glu Lys Phe Asn Glu Glu Arg Leu Lys Phe Gln Asn Ser Ser Thr Ser 20 25 30 Glu Thr Leu Gln Gly Lys Ala Glu Ser Thr Asp Ile Ala Arg Phe Gln 35 40 45 Ser Arg Asp Glu Leu Lys Tyr Ser Ile Arg Ser Leu Met Lys Tyr Ala 50 55 60 Pro Trp Val Asn His Ile Tyr Ile Val Thr Asn Gly Gln Ile Pro Lys 65 70 75 80 Trp Leu Asp Thr Asn Asn Thr Lys Val Thr Ile Ile Pro His Ser Thr 85 90 95 Ile Ile Asp Ser Gln Phe Leu Pro Thr Phe Asn Ser His Val Ile Glu 100 105 110 Ser Ser Leu Tyr Lys Ile Pro Gly Leu Ser Glu His Tyr Ile Tyr Phe 115 120 125 Asn Asp Asp Val Met Leu Ala Arg Asp Leu Ser Pro Ser Tyr Phe Phe 130 135 140 Thr Ser Ser Gly Leu Ala Lys Leu Phe Ile Thr Asn Ser Arg Leu Pro 145 150 155 160 Asn Gly Tyr Lys Asn Val Lys Asp Thr Pro Thr Gln Trp Ala Ser Lys 165 170 175 Asn Ser Arg Glu Leu Leu His Ala Glu Thr Gly Phe Trp Ala Glu Ala 180 185 190 Met Phe Ala His Thr Phe His Pro Gln Arg Lys Ser Val His Glu Ser 195 200 205 Ile Glu His Leu Trp His Glu Gln Leu Asn Val Cys Arg Gln Asn Arg 210 215 220 Phe Arg Asp Ile Ser Asp Ile Asn Met Ala Thr Phe Leu His His His 225 230 235 240 Phe Ala Ile Leu Thr Gly Gln Ala Leu Ala Thr Arg Thr Lys Cys Ile 245 250 255 Tyr Phe Asn Ile Arg Ser Pro Gln Ala Ala Gln His Tyr Lys Thr Leu 260 265 270 Leu Ala Arg Lys Gly Ser Glu Tyr Ser Pro His Ser Ile Cys Leu Asn 275 280 285 Asp His Thr Ser Ser Asn Lys Asn Ile Leu Ser Asn Tyr Glu Ala Lys 290 295 300 Leu Gln Ser Phe Leu Glu Thr Tyr Tyr Pro Asp Val Ser Glu Ala Glu 305 310 315 320 Ile Leu Leu Pro Thr Lys Ser Glu Val Ala Glu Leu Val Lys His Lys 325 330 335 Asp Tyr Leu Thr Val Tyr Thr Lys Leu Leu Pro Ile Ile Asn Lys Gln 340 345 350 Leu Val Asn Lys Tyr Asn Lys Pro Tyr Ser Tyr Leu Phe Tyr Tyr Leu 355 360 365 Gly Leu Ser Ala Arg Phe Leu Phe Glu Glu Thr Gln Gln Glu His Tyr 370 375 380 Arg Glu Thr Ala Glu Glu Asn Leu Gln Ile Phe Cys Gly Leu Asn Pro 385 390 395 400 Lys His Thr Leu Ala Leu Lys Tyr Leu Ala Asp 405 410 <210> SEQ ID NO 31 <211> LENGTH: 320 <212> TYPE: PRT <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: AS deltaN67deltaC99-CP-X (320) <400> SEQUENCE: 31 Phe Pro Ile Asp Val Val Tyr Thr Trp Val Asp Ser Asp Asp Glu Lys 1 5 10 15 Phe Asn Glu Glu Arg Leu Lys Phe Gln Asn Ser Ser Thr Ser Glu Thr 20 25 30 Leu Gln Gly Lys Ala Glu Ser Thr Asp Ile Ala Arg Phe Gln Ser Arg 35 40 45 Asp Glu Leu Lys Tyr Ser Ile Arg Ser Leu Met Lys Tyr Ala Pro Trp 50 55 60 Val Asn His Ile Tyr Ile Val Thr Asn Gly Gln Ile Pro Lys Trp Leu 65 70 75 80 Asp Thr Asn Asn Thr Lys Val Thr Ile Ile Pro His Ser Thr Ile Ile 85 90 95 Asp Ser Gln Phe Leu Pro Thr Phe Asn Ser His Val Ile Glu Ser Ser 100 105 110 Leu Tyr Lys Ile Pro Gly Leu Ser Glu His Tyr Ile Tyr Phe Asn Asp 115 120 125 Asp Val Met Leu Ala Arg Asp Leu Ser Pro Ser Tyr Phe Phe Thr Ser 130 135 140 Ser Gly Leu Ala Lys Leu Phe Ile Thr Asn Ser Arg Leu Pro Asn Gly 145 150 155 160 Tyr Lys Asn Val Lys Asp Thr Pro Thr Gln Trp Ala Ser Lys Asn Ser 165 170 175 Arg Glu Leu Leu His Ala Glu Thr Gly Phe Trp Ala Glu Ala Met Phe 180 185 190 Ala His Thr Phe His Pro Gln Arg Lys Ser Val His Glu Ser Ile Glu 195 200 205 His Leu Trp His Glu Gln Leu Asn Val Cys Arg Gln Asn Arg Phe Arg 210 215 220 Asp Ile Ser Asp Ile Asn Met Ala Thr Phe Leu His His His Phe Ala 225 230 235 240 Ile Leu Thr Gly Gln Ala Leu Ala Thr Arg Thr Lys Cys Ile Tyr Phe 245 250 255 Asn Ile Arg Ser Pro Gln Ala Ala Gln His Tyr Lys Thr Leu Leu Ala 260 265 270 Arg Lys Gly Ser Glu Tyr Ser Pro His Ser Ile Cys Leu Asn Asp His 275 280 285 Thr Ser Ser Asn Lys Asn Ile Leu Ser Asn Tyr Glu Ala Lys Leu Gln 290 295 300 Ser Phe Leu Glu Thr Tyr Tyr Pro Asp Val Ser Glu Ala Glu Ile Leu 305 310 315 320 <210> SEQ ID NO 32 <211> LENGTH: 1119 <212> TYPE: DNA <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: UDP-GlcNAc-Epimerase (NmA) cloned from Neisseria meningitidis serogroup A, coding sequence >UDP-GlcNAc-Epimerase-NmA (AF019760 REGION: 479..1597) <400> SEQUENCE: 32 atgaaagtct taaccgtctt tggcactcgc cctgaagcta ttaaaatggc gcctgtaatt 60 ctagagttac aaaaacataa cacaattact tcaaaagttt gcattactgc acagcatcgt 120 gaaatgctag atcaggtttt gagcctattc gaaatcaaag ctgattatga tttaaatatc 180 atgaaaccca accagagcct acaagaaatc acaacaaata tcatctcaag ccttaccgat 240 gttcttgaag atttcaaacc tgactgcgtc cttgctcacg gagacaccac aacaactttt 300 gcagctagcc ttgctgcatt ctatcaaaaa atacctgttg gccacattga agcaggcctg 360 agaacttata atttatactc tccttggcca gaggaagcaa ataggcgttt aacaagcgtt 420 ctaagccagt ggcattttgc acctactgaa gattctaaaa ataacttact atctgaatca 480 ataccttctg acaaagttat tgttactgga aatactgtca tagatgcact aatggtatct 540 ctagaaaaac taaaaataac tacaattaaa aaacaaatgg aacaagcttt tccatttatt 600 caggacaact ctaaagtaat tttaattacc gctcatagaa gagaaaatca tggggaaggt 660 attaaaaata ttggactttc tatcttagaa ttagctaaaa aatacccaac attctctttt 720 gtgattccgc tccatttaaa tcctaacgtt agaaaaccaa ttcaagattt attatcctct 780 gtgcacaatg ttcatcttat tgagccacaa gaatacttac cattcgtata tttaatgtct 840 aaaagccata taatattaag tgattcaggc ggcatacaag aagaagctcc atccctagga 900 aaaccagttc ttgtattaag agatactaca gaacgtcctg aagctgtagc tgcaggaact 960 gtaaaattag taggttctga aactcaaaat attattgaga gctttacaca actaattgaa 1020 taccctgaat attatgaaaa aatggctaat attgaaaacc cttacgggat aggtaatgcc 1080 tcaaaaatca ttgtagaaac tttattaaag aatagataa 1119 <210> SEQ ID NO 33 <211> LENGTH: 372 <212> TYPE: PRT <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: UDP-GlcNAc-Epimerase (NmA) cloned from Neisseria meningitidis serogroup A amino acid sequence >UDP-GlcNAc-Epimerase-NmA (AAC38285).pro <400> SEQUENCE: 33 Met Lys Val Leu Thr Val Phe Gly Thr Arg Pro Glu Ala Ile Lys Met 1 5 10 15 Ala Pro Val Ile Leu Glu Leu Gln Lys His Asn Thr Ile Thr Ser Lys 20 25 30 Val Cys Ile Thr Ala Gln His Arg Glu Met Leu Asp Gln Val Leu Ser 35 40 45 Leu Phe Glu Ile Lys Ala Asp Tyr Asp Leu Asn Ile Met Lys Pro Asn 50 55 60 Gln Ser Leu Gln Glu Ile Thr Thr Asn Ile Ile Ser Ser Leu Thr Asp 65 70 75 80 Val Leu Glu Asp Phe Lys Pro Asp Cys Val Leu Ala His Gly Asp Thr 85 90 95 Thr Thr Thr Phe Ala Ala Ser Leu Ala Ala Phe Tyr Gln Lys Ile Pro 100 105 110 Val Gly His Ile Glu Ala Gly Leu Arg Thr Tyr Asn Leu Tyr Ser Pro 115 120 125 Trp Pro Glu Glu Ala Asn Arg Arg Leu Thr Ser Val Leu Ser Gln Trp 130 135 140 His Phe Ala Pro Thr Glu Asp Ser Lys Asn Asn Leu Leu Ser Glu Ser 145 150 155 160 Ile Pro Ser Asp Lys Val Ile Val Thr Gly Asn Thr Val Ile Asp Ala 165 170 175 Leu Met Val Ser Leu Glu Lys Leu Lys Ile Thr Thr Ile Lys Lys Gln 180 185 190 Met Glu Gln Ala Phe Pro Phe Ile Gln Asp Asn Ser Lys Val Ile Leu 195 200 205 Ile Thr Ala His Arg Arg Glu Asn His Gly Glu Gly Ile Lys Asn Ile 210 215 220 Gly Leu Ser Ile Leu Glu Leu Ala Lys Lys Tyr Pro Thr Phe Ser Phe 225 230 235 240 Val Ile Pro Leu His Leu Asn Pro Asn Val Arg Lys Pro Ile Gln Asp 245 250 255 Leu Leu Ser Ser Val His Asn Val His Leu Ile Glu Pro Gln Glu Tyr 260 265 270 Leu Pro Phe Val Tyr Leu Met Ser Lys Ser His Ile Ile Leu Ser Asp 275 280 285 Ser Gly Gly Ile Gln Glu Glu Ala Pro Ser Leu Gly Lys Pro Val Leu 290 295 300 Val Leu Arg Asp Thr Thr Glu Arg Pro Glu Ala Val Ala Ala Gly Thr 305 310 315 320 Val Lys Leu Val Gly Ser Glu Thr Gln Asn Ile Ile Glu Ser Phe Thr 325 330 335 Gln Leu Ile Glu Tyr Pro Glu Tyr Tyr Glu Lys Met Ala Asn Ile Glu 340 345 350 Asn Pro Tyr Gly Ile Gly Asn Ala Ser Lys Ile Ile Val Glu Thr Leu 355 360 365 Leu Lys Asn Arg 370 <210> SEQ ID NO 34 <211> LENGTH: 1713 <212> TYPE: DNA <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: DNA StrepII-Thrombin-BamHI/BglII-CsaB-XhoI-His6 <400> SEQUENCE: 34 atggctagct ggagccaccc gcagttcgaa aaaggcgccc tggttccgcg tggatctttt 60 atacttaata acagaaaatg gcgtaaactt aaaagagacc ctagcgcttt ctttcgagat 120 agtaaattta actttttaag atatttttct gctaaaaaat ttgcaaagaa ttttaaaaat 180 tcatcacata tccataaaac taatataagt aaagctcaat caaatatttc ttcaacctta 240 aaacaaaatc ggaaacaaga tatgttaatt cctattaatt tttttaattt tgaatatata 300 gttaaaaaac ttaacaatca aaacgcaata ggtgtatata ttcttccttc taatcttact 360 cttaagcctg cattatgtat tctagaatca cataaagaag actttttaaa taaatttctt 420 cttactattt cctctgaaaa tttaaagctt caatacaaat ttaatggaca aataaaaaat 480 cctaagtccg taaatgaaat ttggacagat ttatttagca ttgctcatgt tgacatgaaa 540 ctcagcacag atagaacttt aagttcatct atatctcaat tttggttcag attagagttc 600 tgtaaagaag ataaggattt tatcttattt cctacagcta acagatattc tagaaaactt 660 tggaagcact ctattaaaaa taatcaatta tttaaagaag gcatacgaaa ctattcagaa 720 atatcttcat taccctatga agaagatcat aattttgata ttgatttagt atttacttgg 780 gtcaactcag aagataagaa ttggcaagag ttatataaaa aatataagcc cgactttaat 840 agcgatgcaa ccagtacatc aagattcctt agtagagatg aattaaaatt cgcattacgc 900 tcttgggaaa tgaatggatc cttcattcga aaaattttta ttgtctctaa ttgtgctccc 960 ccagcatggc tagatttaaa taaccctaaa attcaatggg tatatcacga agaaattatg 1020 ccacaaagtg cccttcctac ttttagctca catgctattg aaaccagctt gcaccatata 1080 ccaggaatta gtaactattt tatttacagc aatgacgact tcctattaac taaaccattg 1140 aataaagaca atttcttcta ttcgaatggt attgcaaagt taagattaga agcatgggga 1200 aatgttaatg gtgaatgtac tgaaggagaa cctgactact taaatggtgc tcgcaatgcg 1260 aacactctct tagaaaagga atttaaaaaa tttactacta aactacatac tcactcccct 1320 caatccatga gaactgatat tttatttgag atggaaaaaa aatatccaga agagtttaat 1380 agaacactac ataataaatt ccgatcttta gatgatattg cagtaacggg ctatctctat 1440 catcattatg ccctactctc tggacgagca ctacaaagtt ctgacaagac ggaacttgta 1500 cagcaaaatc atgatttcaa aaagaaacta aataatgtag tgaccttaac taaagaaagg 1560 aattttgaca aacttccttt gagcgtatgt atcaacgatg gtgctgatag tcacttgaat 1620 gaagaatgga atgttcaagt tattaagttc ttagaaactc ttttcccatt accatcatca 1680 tttgagaaac tcgagcacca ccaccaccac cac 1713 <210> SEQ ID NO 35 <211> LENGTH: 57 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: DNA StrepII-Thrombin-BamHI/BglII <400> SEQUENCE: 35 atggctagct ggagccaccc gcagttcgaa aaaggcgccc tggttccgcg tggatct 57 <210> SEQ ID NO 36 <211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: DNA XhoI-His6 <400> SEQUENCE: 36 ctcgagcacc accaccacca ccac 24 <210> SEQ ID NO 37 <211> LENGTH: 571 <212> TYPE: PRT <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: AS StrepII-Thrombin-BamHI/BglII-CsaB-XhoI-His6 <400> SEQUENCE: 37 Met Ala Ser Trp Ser His Pro Gln Phe Glu Lys Gly Ala Leu Val Pro 1 5 10 15 Arg Gly Ser Phe Ile Leu Asn Asn Arg Lys Trp Arg Lys Leu Lys Arg 20 25 30 Asp Pro Ser Ala Phe Phe Arg Asp Ser Lys Phe Asn Phe Leu Arg Tyr 35 40 45 Phe Ser Ala Lys Lys Phe Ala Lys Asn Phe Lys Asn Ser Ser His Ile 50 55 60 His Lys Thr Asn Ile Ser Lys Ala Gln Ser Asn Ile Ser Ser Thr Leu 65 70 75 80 Lys Gln Asn Arg Lys Gln Asp Met Leu Ile Pro Ile Asn Phe Phe Asn 85 90 95 Phe Glu Tyr Ile Val Lys Lys Leu Asn Asn Gln Asn Ala Ile Gly Val 100 105 110 Tyr Ile Leu Pro Ser Asn Leu Thr Leu Lys Pro Ala Leu Cys Ile Leu 115 120 125 Glu Ser His Lys Glu Asp Phe Leu Asn Lys Phe Leu Leu Thr Ile Ser 130 135 140 Ser Glu Asn Leu Lys Leu Gln Tyr Lys Phe Asn Gly Gln Ile Lys Asn 145 150 155 160 Pro Lys Ser Val Asn Glu Ile Trp Thr Asp Leu Phe Ser Ile Ala His 165 170 175 Val Asp Met Lys Leu Ser Thr Asp Arg Thr Leu Ser Ser Ser Ile Ser 180 185 190 Gln Phe Trp Phe Arg Leu Glu Phe Cys Lys Glu Asp Lys Asp Phe Ile 195 200 205 Leu Phe Pro Thr Ala Asn Arg Tyr Ser Arg Lys Leu Trp Lys His Ser 210 215 220 Ile Lys Asn Asn Gln Leu Phe Lys Glu Gly Ile Arg Asn Tyr Ser Glu 225 230 235 240 Ile Ser Ser Leu Pro Tyr Glu Glu Asp His Asn Phe Asp Ile Asp Leu 245 250 255 Val Phe Thr Trp Val Asn Ser Glu Asp Lys Asn Trp Gln Glu Leu Tyr 260 265 270 Lys Lys Tyr Lys Pro Asp Phe Asn Ser Asp Ala Thr Ser Thr Ser Arg 275 280 285 Phe Leu Ser Arg Asp Glu Leu Lys Phe Ala Leu Arg Ser Trp Glu Met 290 295 300 Asn Gly Ser Phe Ile Arg Lys Ile Phe Ile Val Ser Asn Cys Ala Pro 305 310 315 320 Pro Ala Trp Leu Asp Leu Asn Asn Pro Lys Ile Gln Trp Val Tyr His 325 330 335 Glu Glu Ile Met Pro Gln Ser Ala Leu Pro Thr Phe Ser Ser His Ala 340 345 350 Ile Glu Thr Ser Leu His His Ile Pro Gly Ile Ser Asn Tyr Phe Ile 355 360 365 Tyr Ser Asn Asp Asp Phe Leu Leu Thr Lys Pro Leu Asn Lys Asp Asn 370 375 380 Phe Phe Tyr Ser Asn Gly Ile Ala Lys Leu Arg Leu Glu Ala Trp Gly 385 390 395 400 Asn Val Asn Gly Glu Cys Thr Glu Gly Glu Pro Asp Tyr Leu Asn Gly 405 410 415 Ala Arg Asn Ala Asn Thr Leu Leu Glu Lys Glu Phe Lys Lys Phe Thr 420 425 430 Thr Lys Leu His Thr His Ser Pro Gln Ser Met Arg Thr Asp Ile Leu 435 440 445 Phe Glu Met Glu Lys Lys Tyr Pro Glu Glu Phe Asn Arg Thr Leu His 450 455 460 Asn Lys Phe Arg Ser Leu Asp Asp Ile Ala Val Thr Gly Tyr Leu Tyr 465 470 475 480 His His Tyr Ala Leu Leu Ser Gly Arg Ala Leu Gln Ser Ser Asp Lys 485 490 495 Thr Glu Leu Val Gln Gln Asn His Asp Phe Lys Lys Lys Leu Asn Asn 500 505 510 Val Val Thr Leu Thr Lys Glu Arg Asn Phe Asp Lys Leu Pro Leu Ser 515 520 525 Val Cys Ile Asn Asp Gly Ala Asp Ser His Leu Asn Glu Glu Trp Asn 530 535 540 Val Gln Val Ile Lys Phe Leu Glu Thr Leu Phe Pro Leu Pro Ser Ser 545 550 555 560 Phe Glu Lys Leu Glu His His His His His His 565 570 <210> SEQ ID NO 38 <211> LENGTH: 19 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: AS StrepII-Thrombin-BamHI <400> SEQUENCE: 38 Met Ala Ser Trp Ser His Pro Gln Phe Glu Lys Gly Ala Leu Val Pro 1 5 10 15 Arg Gly Ser <210> SEQ ID NO 39 <211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: AS XhoI-His6 <400> SEQUENCE: 39 Leu Glu His His His His His His 1 5 <210> SEQ ID NO 40 <211> LENGTH: 1455 <212> TYPE: DNA <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: DNA NdeI-dN69CsaB(co)-XhoI-His6 <400> SEQUENCE: 40 catatgctga tcccgatcaa tttctttaat tttgagtaca tcgtgaagaa actgaataac 60 caaaacgcaa tcggcgtgta cattctgccg tctaatctga ccctgaaacc agcattgtgc 120 atcttggagt cgcacaaaga ggacttcctg aacaaatttt tgttgaccat tagcagcgag 180 aacctgaaac tgcagtataa gttcaatggt cagatcaaaa atccgaaaag cgtgaacgaa 240 atctggaccg acctgtttag cattgctcac gtcgacatga agctgagcac cgaccgtacg 300 ctgtcctcgt ccatcagcca attttggttt cgcctggagt tctgtaaaga ggacaaggac 360 ttcatcctgt ttccgacggc aaatcgttac agccgcaagc tgtggaagca cagcatcaaa 420 aataatcagc tgtttaagga aggtatccgt aactacagcg agattagctc gctgccgtac 480 gaggaagacc ataacttcga catcgatctg gtctttacct gggtcaattc ggaagacaaa 540 aactggcagg aactgtacaa gaaatataag ccggatttta atagcgatgc cacctcgacg 600 agccgttttc tgagccgtga cgagctgaag tttgcgctgc gctcgtggga aatgaacggt 660 agcttcatcc gtaaaatctt tatcgtcagc aactgcgcgc cgccggcctg gctggatctg 720 aacaatccga agatccaatg ggtgtatcac gaggagatca tgccacagag cgccctgcca 780 accttcagca gccatgctat tgagactagc ttgcatcaca ttccgggcat ctccaactac 840 ttcatctact ctaatgacga ttttctgttg accaaaccgc tgaacaaaga caacttcttt 900 tactccaacg gtattgctaa actgcgtctg gaagcctggg gtaacgttaa cggtgaatgt 960 accgaaggcg agccggatta cctgaacggc gcgcgtaacg caaatacgct gctggagaaa 1020 gagtttaaaa agtttaccac caagctgcac acccacagcc cgcagagcat gcgtaccgac 1080 atcctgttcg agatggagaa aaaataccca gaagagttca atcgcacgct gcacaacaag 1140 ttccgcagcc tggatgacat cgcggttacc ggctacctgt accatcacta cgcattgctg 1200 tctggccgcg ctctgcaatc cagcgataag accgaactgg tccagcagaa tcacgacttt 1260 aaaaagaagc tgaataatgt tgtcaccctg accaaagagc gtaactttga taagctgccg 1320 ctgagcgttt gtattaatga cggtgcagac agccacctga atgaggagtg gaatgtgcaa 1380 gttatcaaat tcttggagac cttgttcccg ttgccgagct ccttcgagaa actcgagcac 1440 caccaccacc accac 1455 <210> SEQ ID NO 41 <400> SEQUENCE: 41 000 <210> SEQ ID NO 42 <211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: DNA XhoI-His6 <400> SEQUENCE: 42 ctcgagcacc accaccacca ccac 24 <210> SEQ ID NO 43 <211> LENGTH: 484 <212> TYPE: PRT <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: AS NdeI-dN69CsaB(co)-XhoI-His6 <400> SEQUENCE: 43 Met Leu Ile Pro Ile Asn Phe Phe Asn Phe Glu Tyr Ile Val Lys Lys 1 5 10 15 Leu Asn Asn Gln Asn Ala Ile Gly Val Tyr Ile Leu Pro Ser Asn Leu 20 25 30 Thr Leu Lys Pro Ala Leu Cys Ile Leu Glu Ser His Lys Glu Asp Phe 35 40 45 Leu Asn Lys Phe Leu Leu Thr Ile Ser Ser Glu Asn Leu Lys Leu Gln 50 55 60 Tyr Lys Phe Asn Gly Gln Ile Lys Asn Pro Lys Ser Val Asn Glu Ile 65 70 75 80 Trp Thr Asp Leu Phe Ser Ile Ala His Val Asp Met Lys Leu Ser Thr 85 90 95 Asp Arg Thr Leu Ser Ser Ser Ile Ser Gln Phe Trp Phe Arg Leu Glu 100 105 110 Phe Cys Lys Glu Asp Lys Asp Phe Ile Leu Phe Pro Thr Ala Asn Arg 115 120 125 Tyr Ser Arg Lys Leu Trp Lys His Ser Ile Lys Asn Asn Gln Leu Phe 130 135 140 Lys Glu Gly Ile Arg Asn Tyr Ser Glu Ile Ser Ser Leu Pro Tyr Glu 145 150 155 160 Glu Asp His Asn Phe Asp Ile Asp Leu Val Phe Thr Trp Val Asn Ser 165 170 175 Glu Asp Lys Asn Trp Gln Glu Leu Tyr Lys Lys Tyr Lys Pro Asp Phe 180 185 190 Asn Ser Asp Ala Thr Ser Thr Ser Arg Phe Leu Ser Arg Asp Glu Leu 195 200 205 Lys Phe Ala Leu Arg Ser Trp Glu Met Asn Gly Ser Phe Ile Arg Lys 210 215 220 Ile Phe Ile Val Ser Asn Cys Ala Pro Pro Ala Trp Leu Asp Leu Asn 225 230 235 240 Asn Pro Lys Ile Gln Trp Val Tyr His Glu Glu Ile Met Pro Gln Ser 245 250 255 Ala Leu Pro Thr Phe Ser Ser His Ala Ile Glu Thr Ser Leu His His 260 265 270 Ile Pro Gly Ile Ser Asn Tyr Phe Ile Tyr Ser Asn Asp Asp Phe Leu 275 280 285 Leu Thr Lys Pro Leu Asn Lys Asp Asn Phe Phe Tyr Ser Asn Gly Ile 290 295 300 Ala Lys Leu Arg Leu Glu Ala Trp Gly Asn Val Asn Gly Glu Cys Thr 305 310 315 320 Glu Gly Glu Pro Asp Tyr Leu Asn Gly Ala Arg Asn Ala Asn Thr Leu 325 330 335 Leu Glu Lys Glu Phe Lys Lys Phe Thr Thr Lys Leu His Thr His Ser 340 345 350 Pro Gln Ser Met Arg Thr Asp Ile Leu Phe Glu Met Glu Lys Lys Tyr 355 360 365 Pro Glu Glu Phe Asn Arg Thr Leu His Asn Lys Phe Arg Ser Leu Asp 370 375 380 Asp Ile Ala Val Thr Gly Tyr Leu Tyr His His Tyr Ala Leu Leu Ser 385 390 395 400 Gly Arg Ala Leu Gln Ser Ser Asp Lys Thr Glu Leu Val Gln Gln Asn 405 410 415 His Asp Phe Lys Lys Lys Leu Asn Asn Val Val Thr Leu Thr Lys Glu 420 425 430 Arg Asn Phe Asp Lys Leu Pro Leu Ser Val Cys Ile Asn Asp Gly Ala 435 440 445 Asp Ser His Leu Asn Glu Glu Trp Asn Val Gln Val Ile Lys Phe Leu 450 455 460 Glu Thr Leu Phe Pro Leu Pro Ser Ser Phe Glu Lys Leu Glu His His 465 470 475 480 His His His His <210> SEQ ID NO 44 <400> SEQUENCE: 44 000 <210> SEQ ID NO 45 <211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: AS XhoI-His6 <400> SEQUENCE: 45 Leu Glu His His His His His His 1 5 <210> SEQ ID NO 46 <211> LENGTH: 1425 <212> TYPE: DNA <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: DNA deltaN69-CP-A (wt) (1425) <400> SEQUENCE: 46 ttaattccta ttaatttttt taattttgaa tatatagtta aaaaacttaa caatcaaaac 60 gcaataggtg tatatattct tccttctaat cttactctta agcctgcatt atgtattcta 120 gaatcacata aagaagactt tttaaataaa tttcttctta ctatttcctc tgaaaattta 180 aagcttcaat acaaatttaa tggacaaata aaaaatccta agtccgtaaa tgaaatttgg 240 acagatttat ttagcattgc tcatgttgac atgaaactca gcacagatag aactttaagt 300 tcatctatat ctcaattttg gttcagatta gagttctgta aagaagataa ggattttatc 360 ttatttccta cagctaacag atattctaga aaactttgga agcactctat taaaaataat 420 caattattta aagaaggcat acgaaactat tcagaaatat cttcattacc ctatgaagaa 480 gatcataatt ttgatattga tttagtattt acttgggtca actcagaaga taagaattgg 540 caagagttat ataaaaaata taagcccgac tttaatagcg atgcaaccag tacatcaaga 600 ttccttagta gagatgaatt aaaattcgca ttacgctctt gggaaatgaa tggatccttc 660 attcgaaaaa tttttattgt ctctaattgt gctcccccag catggctaga tttaaataac 720 cctaaaattc aatgggtata tcacgaagaa attatgccac aaagtgccct tcctactttt 780 agctcacatg ctattgaaac cagcttgcac catataccag gaattagtaa ctattttatt 840 tacagcaatg acgacttcct attaactaaa ccattgaata aagacaattt cttctattcg 900 aatggtattg caaagttaag attagaagca tggggaaatg ttaatggtga atgtactgaa 960 ggagaacctg actacttaaa tggtgctcgc aatgcgaaca ctctcttaga aaaggaattt 1020 aaaaaattta ctactaaact acatactcac tcccctcaat ccatgagaac tgatatttta 1080 tttgagatgg aaaaaaaata tccagaagag tttaatagaa cactacataa taaattccga 1140 tctttagatg atattgcagt aacgggctat ctctatcatc attatgccct actctctgga 1200 cgagcactac aaagttctga caagacggaa cttgtacagc aaaatcatga tttcaaaaag 1260 aaactaaata atgtagtgac cttaactaaa gaaaggaatt ttgacaaact tcctttgagc 1320 gtatgtatca acgatggtgc tgatagtcac ttgaatgaag aatggaatgt tcaagttatt 1380 aagttcttag aaactctttt cccattacca tcatcatttg agaaa 1425 <210> SEQ ID NO 47 <211> LENGTH: 2628 <212> TYPE: DNA <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: DNA MBP-S3N10-BamHI-CsxA-Xho1-His6 <400> SEQUENCE: 47 atgaaaactg aagaaggtaa actggtaatc tggattaacg gcgataaagg ctataacggt 60 ctcgctgaag tcggtaagaa attcgagaaa gataccggaa ttaaagtcac cgttgagcat 120 ccggataaac tggaagagaa attcccacag gttgcggcaa ctggcgatgg ccctgacatt 180 atcttctggg cacacgaccg ctttggtggc tacgctcaat ctggcctgtt ggctgaaatc 240 accccggaca aagcgttcca ggacaagctg tatccgttta cctgggatgc cgtacgttac 300 aacggcaagc tgattgctta cccgatcgct gttgaagcgt tatcgctgat ttataacaaa 360 gatctgctgc cgaacccgcc aaaaacctgg gaagagatcc cggcgctgga taaagaactg 420 aaagcgaaag gtaagagcgc gctgatgttc aacctgcaag aaccgtactt cacctggccg 480 ctgattgctg ctgacggggg ttatgcgttc aagtatgaaa acggcaagta cgacattaaa 540 gacgtgggcg tggataacgc tggcgcgaaa gcgggtctga ccttcctggt tgacctgatt 600 aaaaacaaac acatgaatgc agacaccgat tactccatcg cagaagctgc ctttaataaa 660 ggcgaaacag cgatgaccat caacggcccg tgggcatggt ccaacatcga caccagcaaa 720 gtgaattatg gtgtaacggt actgccgacc ttcaagggtc aaccatccaa accgttcgtt 780 ggcgtgctga gcgcaggtat taacgccgcc agtccgaaca aagagctggc gaaagagttc 840 ctcgaaaact atctgctgac tgatgaaggt ctggaagcgg ttaataaaga caaaccgctg 900 ggtgccgtag cgctgaagtc ttacgaggaa gagttggcga aagatccacg tattgccgcc 960 accatggaaa acgcccagaa aggtgaaatc atgccgaaca tcccgcagat gtccgctttc 1020 tggtatgccg tgcgtactgc ggtgatcaac gccgccagcg gtcgtcagac tgtcgatgaa 1080 gccctgaaag acgcgcagac taattcgagc tccaataaca ataacaacaa caataacaat 1140 aacggatcca ttatgagcaa aattagcaaa ttggtaaccc acccaaacct tttctttcga 1200 gattatttct taaaaaaagc accgttaaat tatggcgaaa atattaaacc tttaccagtc 1260 gaaacctctt ctcatagcaa aaaaaataca gcccataaaa cacccgtatc atccgaccaa 1320 ccaattgaag atccataccc agtaacattt ccaattgatg tagtttatac ttgggtagat 1380 tcagatgatg aaaaattcaa tgaagaacgc ctaaagtttc aaaattcaag cacatctgag 1440 actctacaag gcaaagcaga aagcaccgat attgcaagat tccaatcacg cgacgaatta 1500 aaatattcga ttcgaagcct gatgaagtat gccccatggg taaatcatat ttacattgta 1560 acaaatggtc aaataccaaa atggttagat accaacaata caaaggtaac gattatccct 1620 cactcaacta ttatcgacag tcaatttctc cctactttta attctcacgt cattgaatcc 1680 tctctatata aaatcccagg attatcagag cattacattt atttcaatga tgatgtcatg 1740 ctagctagag atttaagccc atcttatttc tttacaagca gcggattagc aaaactgttt 1800 attaccaact ctcgtctacc aaatggctat aagaatgtga aagacacacc aacccaatgg 1860 gcctcaaaaa attcccgtga gcttttacat gcagaaacag gattttgggc tgaagccatg 1920 tttgcacata cttttcatcc acaacgtaaa agtgtacatg aatctattga acacctatgg 1980 catgaacaat taaatgtttg tcgtcaaaac cgtttccgtg atatttcaga tattaacatg 2040 gcgacattcc tgcaccacca ttttgccatt ttgacaggcc aagctcttgc tacacgcact 2100 aaatgtattt actttaacat tcgctctcct caagcagctc agcattacaa aacattatta 2160 gctcgaaaag gaagcgaata cagcccacat tctatctgct taaatgatca tacatcgagc 2220 aataaaaata ttttatctaa ttacgaagcc aaattacaaa gctttttaga aacatactat 2280 ccagatgtat cagaagcaga aattctcctt cctactaaat ctgaagtagc tgaattagtt 2340 aaacataaag attatttaac tgtatatact aaattattac ctattatcaa taagcagctg 2400 gtcaataaat ataataaacc ttattcatat cttttctatt atttaggttt atctgcccgg 2460 tttttatttg aagaaacgca acaagaacac taccgggaaa ctgctgaaga aaatttacaa 2520 atcttttgtg gcctaaaccc aaaacataca ctagccctca aatacttagc ggatgtcacc 2580 ctcacatcac agcctagtgg acaactcgag caccaccacc accaccac 2628 <210> SEQ ID NO 48 <211> LENGTH: 1149 <212> TYPE: DNA <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: DNA MBP-S3N10-BamHI <400> SEQUENCE: 48 atgaaaactg aagaaggtaa actggtaatc tggattaacg gcgataaagg ctataacggt 60 ctcgctgaag tcggtaagaa attcgagaaa gataccggaa ttaaagtcac cgttgagcat 120 ccggataaac tggaagagaa attcccacag gttgcggcaa ctggcgatgg ccctgacatt 180 atcttctggg cacacgaccg ctttggtggc tacgctcaat ctggcctgtt ggctgaaatc 240 accccggaca aagcgttcca ggacaagctg tatccgttta cctgggatgc cgtacgttac 300 aacggcaagc tgattgctta cccgatcgct gttgaagcgt tatcgctgat ttataacaaa 360 gatctgctgc cgaacccgcc aaaaacctgg gaagagatcc cggcgctgga taaagaactg 420 aaagcgaaag gtaagagcgc gctgatgttc aacctgcaag aaccgtactt cacctggccg 480 ctgattgctg ctgacggggg ttatgcgttc aagtatgaaa acggcaagta cgacattaaa 540 gacgtgggcg tggataacgc tggcgcgaaa gcgggtctga ccttcctggt tgacctgatt 600 aaaaacaaac acatgaatgc agacaccgat tactccatcg cagaagctgc ctttaataaa 660 ggcgaaacag cgatgaccat caacggcccg tgggcatggt ccaacatcga caccagcaaa 720 gtgaattatg gtgtaacggt actgccgacc ttcaagggtc aaccatccaa accgttcgtt 780 ggcgtgctga gcgcaggtat taacgccgcc agtccgaaca aagagctggc gaaagagttc 840 ctcgaaaact atctgctgac tgatgaaggt ctggaagcgg ttaataaaga caaaccgctg 900 ggtgccgtag cgctgaagtc ttacgaggaa gagttggcga aagatccacg tattgccgcc 960 accatggaaa acgcccagaa aggtgaaatc atgccgaaca tcccgcagat gtccgctttc 1020 tggtatgccg tgcgtactgc ggtgatcaac gccgccagcg gtcgtcagac tgtcgatgaa 1080 gccctgaaag acgcgcagac taattcgagc tccaataaca ataacaacaa caataacaat 1140 aacggatcc 1149 <210> SEQ ID NO 49 <211> LENGTH: 876 <212> TYPE: PRT <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: AS MBP-S3N10-BamHI-CsxA-Xho1-His6 <400> SEQUENCE: 49 Met Lys Thr Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp Lys 1 5 10 15 Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp Thr 20 25 30 Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys Phe 35 40 45 Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp Ala 50 55 60 His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu Ile 65 70 75 80 Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp Asp 85 90 95 Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val Glu 100 105 110 Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro Lys 115 120 125 Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys Gly 130 135 140 Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp Pro 145 150 155 160 Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly Lys 165 170 175 Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala Gly 180 185 190 Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala Asp 195 200 205 Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala 210 215 220 Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser Lys 225 230 235 240 Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro Ser 245 250 255 Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser Pro 260 265 270 Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr Asp 275 280 285 Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val Ala 290 295 300 Leu Lys Ser Tyr Glu Glu Glu Leu Ala Lys Asp Pro Arg Ile Ala Ala 305 310 315 320 Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro Gln 325 330 335 Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala Ala 340 345 350 Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr Asn 355 360 365 Ser Ser Ser Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Gly Ser Ile 370 375 380 Met Ser Lys Ile Ser Lys Leu Val Thr His Pro Asn Leu Phe Phe Arg 385 390 395 400 Asp Tyr Phe Leu Lys Lys Ala Pro Leu Asn Tyr Gly Glu Asn Ile Lys 405 410 415 Pro Leu Pro Val Glu Thr Ser Ser His Ser Lys Lys Asn Thr Ala His 420 425 430 Lys Thr Pro Val Ser Ser Asp Gln Pro Ile Glu Asp Pro Tyr Pro Val 435 440 445 Thr Phe Pro Ile Asp Val Val Tyr Thr Trp Val Asp Ser Asp Asp Glu 450 455 460 Lys Phe Asn Glu Glu Arg Leu Lys Phe Gln Asn Ser Ser Thr Ser Glu 465 470 475 480 Thr Leu Gln Gly Lys Ala Glu Ser Thr Asp Ile Ala Arg Phe Gln Ser 485 490 495 Arg Asp Glu Leu Lys Tyr Ser Ile Arg Ser Leu Met Lys Tyr Ala Pro 500 505 510 Trp Val Asn His Ile Tyr Ile Val Thr Asn Gly Gln Ile Pro Lys Trp 515 520 525 Leu Asp Thr Asn Asn Thr Lys Val Thr Ile Ile Pro His Ser Thr Ile 530 535 540 Ile Asp Ser Gln Phe Leu Pro Thr Phe Asn Ser His Val Ile Glu Ser 545 550 555 560 Ser Leu Tyr Lys Ile Pro Gly Leu Ser Glu His Tyr Ile Tyr Phe Asn 565 570 575 Asp Asp Val Met Leu Ala Arg Asp Leu Ser Pro Ser Tyr Phe Phe Thr 580 585 590 Ser Ser Gly Leu Ala Lys Leu Phe Ile Thr Asn Ser Arg Leu Pro Asn 595 600 605 Gly Tyr Lys Asn Val Lys Asp Thr Pro Thr Gln Trp Ala Ser Lys Asn 610 615 620 Ser Arg Glu Leu Leu His Ala Glu Thr Gly Phe Trp Ala Glu Ala Met 625 630 635 640 Phe Ala His Thr Phe His Pro Gln Arg Lys Ser Val His Glu Ser Ile 645 650 655 Glu His Leu Trp His Glu Gln Leu Asn Val Cys Arg Gln Asn Arg Phe 660 665 670 Arg Asp Ile Ser Asp Ile Asn Met Ala Thr Phe Leu His His His Phe 675 680 685 Ala Ile Leu Thr Gly Gln Ala Leu Ala Thr Arg Thr Lys Cys Ile Tyr 690 695 700 Phe Asn Ile Arg Ser Pro Gln Ala Ala Gln His Tyr Lys Thr Leu Leu 705 710 715 720 Ala Arg Lys Gly Ser Glu Tyr Ser Pro His Ser Ile Cys Leu Asn Asp 725 730 735 His Thr Ser Ser Asn Lys Asn Ile Leu Ser Asn Tyr Glu Ala Lys Leu 740 745 750 Gln Ser Phe Leu Glu Thr Tyr Tyr Pro Asp Val Ser Glu Ala Glu Ile 755 760 765 Leu Leu Pro Thr Lys Ser Glu Val Ala Glu Leu Val Lys His Lys Asp 770 775 780 Tyr Leu Thr Val Tyr Thr Lys Leu Leu Pro Ile Ile Asn Lys Gln Leu 785 790 795 800 Val Asn Lys Tyr Asn Lys Pro Tyr Ser Tyr Leu Phe Tyr Tyr Leu Gly 805 810 815 Leu Ser Ala Arg Phe Leu Phe Glu Glu Thr Gln Gln Glu His Tyr Arg 820 825 830 Glu Thr Ala Glu Glu Asn Leu Gln Ile Phe Cys Gly Leu Asn Pro Lys 835 840 845 His Thr Leu Ala Leu Lys Tyr Leu Ala Asp Val Thr Leu Thr Ser Gln 850 855 860 Pro Ser Gly Gln Leu Glu His His His His His His 865 870 875 <210> SEQ ID NO 50 <211> LENGTH: 383 <212> TYPE: PRT <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: AS MBP-S3N10-BamHI <400> SEQUENCE: 50 Met Lys Thr Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp Lys 1 5 10 15 Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp Thr 20 25 30 Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys Phe 35 40 45 Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp Ala 50 55 60 His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu Ile 65 70 75 80 Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp Asp 85 90 95 Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val Glu 100 105 110 Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro Lys 115 120 125 Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys Gly 130 135 140 Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp Pro 145 150 155 160 Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly Lys 165 170 175 Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala Gly 180 185 190 Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala Asp 195 200 205 Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala 210 215 220 Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser Lys 225 230 235 240 Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro Ser 245 250 255 Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser Pro 260 265 270 Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr Asp 275 280 285 Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val Ala 290 295 300 Leu Lys Ser Tyr Glu Glu Glu Leu Ala Lys Asp Pro Arg Ile Ala Ala 305 310 315 320 Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro Gln 325 330 335 Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala Ala 340 345 350 Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr Asn 355 360 365 Ser Ser Ser Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Gly Ser 370 375 380 <210> SEQ ID NO 51 <211> LENGTH: 1263 <212> TYPE: DNA <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: DNA NdeI-dN65dC10-CsxA-XhoI-His6 <400> SEQUENCE: 51 catatggtaa catttccaat tgatgtagtt tatacttggg tagattcaga tgatgaaaaa 60 ttcaatgaag aacgcctaaa gtttcaaaat tcaagcacat ctgagactct acaaggcaaa 120 gcagaaagca ccgatattgc aagattccaa tcacgcgacg aattaaaata ttcgattcga 180 agcctgatga agtatgcccc atgggtaaat catatttaca ttgtaacaaa tggtcaaata 240 ccaaaatggt tagataccaa caatacaaag gtaacgatta tccctcactc aactattatc 300 gacagtcaat ttctccctac ttttaattct cacgtcattg aatcctctct atataaaatc 360 ccaggattat cagagcatta catttatttc aatgatgatg tcatgctagc tagagattta 420 agcccatctt atttctttac aagcagcgga ttagcaaaac tgtttattac caactctcgt 480 ctaccaaatg gctataagaa tgtgaaagac acaccaaccc aatgggcctc aaaaaattcc 540 cgtgagcttt tacatgcaga aacaggattt tgggctgaag ccatgtttgc acatactttt 600 catccacaac gtaaaagtgt acatgaatct attgaacacc tatggcatga acaattaaat 660 gtttgtcgtc aaaaccgttt ccgtgatatt tcagatatta acatggcgac attcctgcac 720 caccattttg ccattttgac aggccaagct cttgctacac gcactaaatg tatttacttt 780 aacattcgct ctcctcaagc agctcagcat tacaaaacat tattagctcg aaaaggaagc 840 gaatacagcc cacattctat ctgcttaaat gatcatacat cgagcaataa aaatatttta 900 tctaattacg aagccaaatt acaaagcttt ttagaaacat actatccaga tgtatcagaa 960 gcagaaattc tccttcctac taaatctgaa gtagctgaat tagttaaaca taaagattat 1020 ttaactgtat atactaaatt attacctatt atcaataagc agctggtcaa taaatataat 1080 aaaccttatt catatctttt ctattattta ggtttatctg cccggttttt atttgaagaa 1140 acgcaacaag aacactaccg ggaaactgct gaagaaaatt tacaaatctt ttgtggccta 1200 aacccaaaac atacactagc cctcaaatac ttagcggatc tcgagcacca ccaccaccac 1260 cac 1263 <210> SEQ ID NO 52 <211> LENGTH: 420 <212> TYPE: PRT <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: AS NdeI-dN65dC10-CsxA-XhoI-His6 <400> SEQUENCE: 52 Met Val Thr Phe Pro Ile Asp Val Val Tyr Thr Trp Val Asp Ser Asp 1 5 10 15 Asp Glu Lys Phe Asn Glu Glu Arg Leu Lys Phe Gln Asn Ser Ser Thr 20 25 30 Ser Glu Thr Leu Gln Gly Lys Ala Glu Ser Thr Asp Ile Ala Arg Phe 35 40 45 Gln Ser Arg Asp Glu Leu Lys Tyr Ser Ile Arg Ser Leu Met Lys Tyr 50 55 60 Ala Pro Trp Val Asn His Ile Tyr Ile Val Thr Asn Gly Gln Ile Pro 65 70 75 80 Lys Trp Leu Asp Thr Asn Asn Thr Lys Val Thr Ile Ile Pro His Ser 85 90 95 Thr Ile Ile Asp Ser Gln Phe Leu Pro Thr Phe Asn Ser His Val Ile 100 105 110 Glu Ser Ser Leu Tyr Lys Ile Pro Gly Leu Ser Glu His Tyr Ile Tyr 115 120 125 Phe Asn Asp Asp Val Met Leu Ala Arg Asp Leu Ser Pro Ser Tyr Phe 130 135 140 Phe Thr Ser Ser Gly Leu Ala Lys Leu Phe Ile Thr Asn Ser Arg Leu 145 150 155 160 Pro Asn Gly Tyr Lys Asn Val Lys Asp Thr Pro Thr Gln Trp Ala Ser 165 170 175 Lys Asn Ser Arg Glu Leu Leu His Ala Glu Thr Gly Phe Trp Ala Glu 180 185 190 Ala Met Phe Ala His Thr Phe His Pro Gln Arg Lys Ser Val His Glu 195 200 205 Ser Ile Glu His Leu Trp His Glu Gln Leu Asn Val Cys Arg Gln Asn 210 215 220 Arg Phe Arg Asp Ile Ser Asp Ile Asn Met Ala Thr Phe Leu His His 225 230 235 240 His Phe Ala Ile Leu Thr Gly Gln Ala Leu Ala Thr Arg Thr Lys Cys 245 250 255 Ile Tyr Phe Asn Ile Arg Ser Pro Gln Ala Ala Gln His Tyr Lys Thr 260 265 270 Leu Leu Ala Arg Lys Gly Ser Glu Tyr Ser Pro His Ser Ile Cys Leu 275 280 285 Asn Asp His Thr Ser Ser Asn Lys Asn Ile Leu Ser Asn Tyr Glu Ala 290 295 300 Lys Leu Gln Ser Phe Leu Glu Thr Tyr Tyr Pro Asp Val Ser Glu Ala 305 310 315 320 Glu Ile Leu Leu Pro Thr Lys Ser Glu Val Ala Glu Leu Val Lys His 325 330 335 Lys Asp Tyr Leu Thr Val Tyr Thr Lys Leu Leu Pro Ile Ile Asn Lys 340 345 350 Gln Leu Val Asn Lys Tyr Asn Lys Pro Tyr Ser Tyr Leu Phe Tyr Tyr 355 360 365 Leu Gly Leu Ser Ala Arg Phe Leu Phe Glu Glu Thr Gln Gln Glu His 370 375 380 Tyr Arg Glu Thr Ala Glu Glu Asn Leu Gln Ile Phe Cys Gly Leu Asn 385 390 395 400 Pro Lys His Thr Leu Ala Leu Lys Tyr Leu Ala Asp Leu Glu His His 405 410 415 His His His His 420 <210> SEQ ID NO 53 <211> LENGTH: 741 <212> TYPE: DNA <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: DNA CsaC <400> SEQUENCE: 53 atgttatcta atttaaaaac aggaaataat atcttaggat tacctgaatt tgagttgaat 60 ggctgccgat tcttatataa aaaaggtata gaaaaaacaa ttattacttt ttcagcattt 120 cctcctaaag atattgctca aaaatataat tatataaaag attttttaag ttctaattat 180 acttttttag cattcttaga taccaaatat ccagaagatg atgctagagg cacttattac 240 attactaatg agttagataa tggatattta caaaccatac attgtattat tcaattatta 300 tcgaatacaa atcaagaaga tacctacctt ttgggttcaa gtaaaggtgg cgttggcgca 360 cttctactcg gtcttacata taattatcct aatataatta ttaatgctcc tcaagccaaa 420 ttagcagatt atatcaaaac acgctcgaaa accattcttt catatatgct tggaacctct 480 aaaagatttc aagatattaa ttacgattat atcaatgact tcttactatc taaaattaag 540 acttgcgact cctcacttaa atggaatatt catataactt gcggaaaaga tgattcatat 600 catttaaatg aattagaaat tctaaaaaat gaatttaata taaaagctat tacgattaaa 660 accaaactaa tttctggcgg gcatgataat gaagcaattg cccactatag agaatacttt 720 aaaaccataa tccaaaatat a 741 <210> SEQ ID NO 54 <211> LENGTH: 247 <212> TYPE: PRT <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: AS CsaC <400> SEQUENCE: 54 Met Leu Ser Asn Leu Lys Thr Gly Asn Asn Ile Leu Gly Leu Pro Glu 1 5 10 15 Phe Glu Leu Asn Gly Cys Arg Phe Leu Tyr Lys Lys Gly Ile Glu Lys 20 25 30 Thr Ile Ile Thr Phe Ser Ala Phe Pro Pro Lys Asp Ile Ala Gln Lys 35 40 45 Tyr Asn Tyr Ile Lys Asp Phe Leu Ser Ser Asn Tyr Thr Phe Leu Ala 50 55 60 Phe Leu Asp Thr Lys Tyr Pro Glu Asp Asp Ala Arg Gly Thr Tyr Tyr 65 70 75 80 Ile Thr Asn Glu Leu Asp Asn Gly Tyr Leu Gln Thr Ile His Cys Ile 85 90 95 Ile Gln Leu Leu Ser Asn Thr Asn Gln Glu Asp Thr Tyr Leu Leu Gly 100 105 110 Ser Ser Lys Gly Gly Val Gly Ala Leu Leu Leu Gly Leu Thr Tyr Asn 115 120 125 Tyr Pro Asn Ile Ile Ile Asn Ala Pro Gln Ala Lys Leu Ala Asp Tyr 130 135 140 Ile Lys Thr Arg Ser Lys Thr Ile Leu Ser Tyr Met Leu Gly Thr Ser 145 150 155 160 Lys Arg Phe Gln Asp Ile Asn Tyr Asp Tyr Ile Asn Asp Phe Leu Leu 165 170 175 Ser Lys Ile Lys Thr Cys Asp Ser Ser Leu Lys Trp Asn Ile His Ile 180 185 190 Thr Cys Gly Lys Asp Asp Ser Tyr His Leu Asn Glu Leu Glu Ile Leu 195 200 205 Lys Asn Glu Phe Asn Ile Lys Ala Ile Thr Ile Lys Thr Lys Leu Ile 210 215 220 Ser Gly Gly His Asp Asn Glu Ala Ile Ala His Tyr Arg Glu Tyr Phe 225 230 235 240 Lys Thr Ile Ile Gln Asn Ile 245 <210> SEQ ID NO 55 <211> LENGTH: 34 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer KS422/KS273 <400> SEQUENCE: 55 gcatctcata tggctgttat tatatttgtt aacg 34 <210> SEQ ID NO 56 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer wt NmW-135 <400> SEQUENCE: 56 ccgctcgagt ttttcttggc caaaaaactg 30 <210> SEQ ID NO 57 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer wt NmY <400> SEQUENCE: 57 ccgctcgagt ttttcttggc caaaaaactg 30 <210> SEQ ID NO 58 <211> LENGTH: 45 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer KS350/KS351 <400> SEQUENCE: 58 ctgatcatga catcagaaag tgcgggattt ccatatatat ttatg 45 <210> SEQ ID NO 59 <211> LENGTH: 45 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer E307A <400> SEQUENCE: 59 cataaatata tatggaaatc ccgcactttc tgatgtcatg atcag 45 <210> SEQ ID NO 60 <211> LENGTH: 41 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer KS370/KS371 <400> SEQUENCE: 60 atctcgcgtt gctgtaggtg tttatgcaac tagcttattt g 41 <210> SEQ ID NO 61 <211> LENGTH: 41 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer S972A <400> SEQUENCE: 61 caaataagct agttgcataa acacctacag caacgcgaga t 41 <210> SEQ ID NO 62 <211> LENGTH: 51 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer AR11/AR12 <400> SEQUENCE: 62 atacagatat cctaatcatg acatctcaaa gcgcaggctt tggttatata t 51 <210> SEQ ID NO 63 <211> LENGTH: 51 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer E307A <400> SEQUENCE: 63 atatataacc aaagcctgcg ctttgagatg tcatgattag gatatctgta t 51 <210> SEQ ID NO 64 <211> LENGTH: 34 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer KS422/KS421 <400> SEQUENCE: 64 gcatctcata tggctgttat tatatttgtt aacg 34 <210> SEQ ID NO 65 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer C-delta639 <400> SEQUENCE: 65 ccgctcgagg ctgcgcggaa gaatagtg 28 <210> SEQ ID NO 66 <211> LENGTH: 35 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer AR2/KS273 <400> SEQUENCE: 66 gcatctcata tgtttaataa cgtatcatta tcgtc 35 <210> SEQ ID NO 67 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer N-delta398 <400> SEQUENCE: 67 ccgctcgagt ttttcttggc caaaaaactg 30 <210> SEQ ID NO 68 <211> LENGTH: 35 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer AR1/KS273 <400> SEQUENCE: 68 gcatctcata tgactgatga taatttaata cctat 35 <210> SEQ ID NO 69 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer N-delta562 <400> SEQUENCE: 69 ccgctcgagt ttttcttggc caaaaaactg 30 <210> SEQ ID NO 70 <211> LENGTH: 35 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer AR9/AR273 <400> SEQUENCE: 70 gcatctcata tgaaatattc ttataaatat atcta 35 <210> SEQ ID NO 71 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer N-delta609 <400> SEQUENCE: 71 ccgctcgagt ttttcttggc caaaaaactg 30 <210> SEQ ID NO 72 <211> LENGTH: 35 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer AR10/KS273 <400> SEQUENCE: 72 gcatctcata tgtcttggga acttattcgt gcctc 35 <210> SEQ ID NO 73 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer N-delta639 <400> SEQUENCE: 73 ccgctcgagt ttttcttggc caaaaaactg 30 <210> SEQ ID NO 74 <211> LENGTH: 31 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer KS433/KS273 <400> SEQUENCE: 74 gcatctcata tgggtaagcg ttcgatggat g 31 <210> SEQ ID NO 75 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer N-delta676 <400> SEQUENCE: 75 ccgctcgagt ttttcttggc caaaaaactg 30 <210> SEQ ID NO 76 <211> LENGTH: 34 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer KS434/KS273 <400> SEQUENCE: 76 gcatctcata tgtcactgaa aagtaatgta gttg 34 <210> SEQ ID NO 77 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer N-delta729 <400> SEQUENCE: 77 ccgctcgagt ttttcttggc caaaaaactg 30 <210> SEQ ID NO 78 <211> LENGTH: 35 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer KS435/KS273 <400> SEQUENCE: 78 gcatctcata tgaatatcga agcatttcta aaacc 35 <210> SEQ ID NO 79 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer N-delta777 <400> SEQUENCE: 79 ccgctcgagt ttttcttggc caaaaaactg 30 <210> SEQ ID NO 80 <211> LENGTH: 34 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer KS422/KS421 <400> SEQUENCE: 80 gcatctcata tggctgttat tatatttgtt aacg 34 <210> SEQ ID NO 81 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer C-delta639 <400> SEQUENCE: 81 ccgctcgagg ctgcgcggaa gaatagtg 28 <210> SEQ ID NO 82 <211> LENGTH: 34 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer KS422/KS374 <400> SEQUENCE: 82 gcatctcata tggctgttat tatatttgtt aacg 34 <210> SEQ ID NO 83 <211> LENGTH: 29 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer C-delta479 <400> SEQUENCE: 83 ccgctcgagc gtttgcatgt tgggtaaag 29 <210> SEQ ID NO 84 <211> LENGTH: 35 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer AR2/KS273 <400> SEQUENCE: 84 gcatctcata tgtttaataa cgtatcatta tcgtc 35 <210> SEQ ID NO 85 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer N-delta398 <400> SEQUENCE: 85 ccgctcgagt ttttcttggc caaaaaactg 30 <210> SEQ ID NO 86 <211> LENGTH: 35 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer AR1/KS273 <400> SEQUENCE: 86 gcatctcata tgactgatga taatttaata cctat 35 <210> SEQ ID NO 87 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer N-delta562 <400> SEQUENCE: 87 ccgctcgagt ttttcttggc caaaaaactg 30 <210> SEQ ID NO 88 <211> LENGTH: 31 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer KS433/KS273 <400> SEQUENCE: 88 gcatctcata tgggtaagcg ttcgatggat g 31 <210> SEQ ID NO 89 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer N-delta 676 <400> SEQUENCE: 89 ccgctcgagt ttttcttggc caaaaaactg 30 <210> SEQ ID NO 90 <211> LENGTH: 34 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer KS434/KS273 <400> SEQUENCE: 90 gcatctcata tgtcactgaa aagtaatgta gttg 34 <210> SEQ ID NO 91 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: N-delta 729 <400> SEQUENCE: 91 ccgctcgagt ttttcttggc caaaaaactg 30 <210> SEQ ID NO 92 <211> LENGTH: 31 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Recloning primer <400> SEQUENCE: 92 gcatctcata tgggtaagcg ttcgatggat g 31 <210> SEQ ID NO 93 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Recloning Primer <400> SEQUENCE: 93 ccgctcgagt ttttcttggc caaaaaactg 30 <210> SEQ ID NO 94 <211> LENGTH: 29 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer StrepII-CsaA-His6 <400> SEQUENCE: 94 gcggatccaa agtcttaacc gtctttggc 29 <210> SEQ ID NO 95 <211> LENGTH: 34 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer StrepII-CsaA-His6 <400> SEQUENCE: 95 ccgctcgagt ctattcttta ataaagtttc taca 34 <210> SEQ ID NO 96 <211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer StrepII-CsaB-His6 <400> SEQUENCE: 96 gcagatcttt tatacttaat aacagaaaat ggc 33 <210> SEQ ID NO 97 <211> LENGTH: 32 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer StrepII-CsaB-His6 <400> SEQUENCE: 97 ccgctcgagt ttctcaaatg atgatggtaa tg 32 <210> SEQ ID NO 98 <211> LENGTH: 32 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer StrepII-delta69-CsaB-His6 <400> SEQUENCE: 98 ccgctcgagt ttctcaaatg atgatggtaa tg 32 <210> SEQ ID NO 99 <211> LENGTH: 34 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer StrepII-delta69-CsaB-His6 <400> SEQUENCE: 99 gcagatctat gttaattcct attaattttt ttaa 34 <210> SEQ ID NO 100 <211> LENGTH: 32 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer delta69-CsaB-His6 <400> SEQUENCE: 100 ccgctcgagt ttctcaaatg atgatggtaa tg 32 <210> SEQ ID NO 101 <211> LENGTH: 39 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer delta69-CsaB-His6 <400> SEQUENCE: 101 gcatctcata tgttaattcc tattaatttt ttttaattt 39 <210> SEQ ID NO 102 <211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer delta69-CsaBCo-His6 <400> SEQUENCE: 102 gcatctcata tgctgatccc gatcaatttc ttt 33 <210> SEQ ID NO 103 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer delta69-CsaBCo-His6 <400> SEQUENCE: 103 ccgctcgagt ttctcgaagg agctcggc 28 <210> SEQ ID NO 104 <211> LENGTH: 27 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer StrepII-CsaC-His6 <400> SEQUENCE: 104 ccgctcgagt atattttgga ttatggt 27 <210> SEQ ID NO 105 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer StrepII-CsaC-His6 <400> SEQUENCE: 105 gcggatcctt atctaattta aaaacagg 28 <210> SEQ ID NO 106 <211> LENGTH: 366 <212> TYPE: PRT <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: NmL (CsIA); Homologue of CsxA/CsaB <400> SEQUENCE: 106 Met Lys Arg Leu Lys Lys Leu Thr Arg Glu Pro Gly Val Phe Phe Arg 1 5 10 15 Asp Tyr Phe Asn Lys Lys Tyr Pro Val Arg Asn Ile Glu Gln Arg Ile 20 25 30 Asn Glu Ile Glu Glu Pro Ala Ile Ile Ala Asn Ser Leu His Leu Ala 35 40 45 Ala Val Glu Ser Ala Ile His Leu Ser Pro Phe Lys Ile Asp Val Val 50 55 60 Phe Thr Trp Val Asp Asn Ser Asp Thr Gln Trp Gln Gln Arg His Gln 65 70 75 80 Gln Tyr Cys His Ala Ala Ser Pro Asn Asn Leu Tyr Ser Asn Asp Glu 85 90 95 Thr Arg Phe Ala Asn His Asn Glu Leu Tyr Tyr Ser Leu His Ser Val 100 105 110 Arg Ser Phe Leu Pro Trp Val Asn His Ile Tyr Ile Ile Thr Asp Ser 115 120 125 Gln Thr Pro Lys Trp Phe Lys Ser Ala Glu Tyr Pro Asn Val Ser Ile 130 135 140 Ile Asp His Ser Glu Ile Ile Asp Lys Gln Tyr Leu Pro Thr Phe Asn 145 150 155 160 Ser His Val Ile Glu Ala His Leu His Asn Ile Pro Asn Leu Ser Glu 165 170 175 His Phe Ile Tyr Phe Asn Asp Asp Val Phe Val Ala Arg Pro Leu His 180 185 190 Arg Glu His Phe Phe His Ala Asn Gly Ile Ala Ser Leu Phe Ile Ala 195 200 205 Asp Lys Ser Leu Gln Lys Met Ala Thr Lys Gly Thr Ile Thr Pro Thr 210 215 220 Leu Ser Ala Ser Gln Asn Cys Ile Arg Leu Leu Asn Gln Arg Tyr Asp 225 230 235 240 Cys Asn Leu Asp His Pro Leu Val His Thr Tyr Val Pro Leu Arg Lys 245 250 255 Ser Gly Phe Gln Thr Ala Trp Gln Tyr Tyr Arg Glu Glu Ile Lys Ala 260 265 270 Phe Leu Pro Asn Lys Phe Arg Thr Asn Gln Asp Leu Asn Leu Ala Thr 275 280 285 Phe Leu Val Pro Trp Leu Met Tyr Leu Asp Gly Lys Ser Ile Pro Asn 290 295 300 Asn Asp Ile Cys Tyr Tyr Phe Asn Ile Arg Ser Asn Lys Ala Pro Thr 305 310 315 320 Gln Tyr Leu Lys Leu Leu Gln Lys Asn Glu Asp Asn Gln Gln Pro His 325 330 335 Ser Phe Cys Ala Asn Asp Phe His Ser Glu Gln Gln Leu Tyr Asp Tyr 340 345 350 His Ala Lys Leu Ile Ala Met Leu Lys Asp Tyr Phe Lys Ile 355 360 365 <210> SEQ ID NO 107 <211> LENGTH: 1037 <212> TYPE: PRT <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: polymerase of serogroup Y <400> SEQUENCE: 107 Met Ala Val Ile Ile Phe Val Asn Gly Ile Arg Ala Val Asn Gly Leu 1 5 10 15 Val Lys Ser Ser Ile Asn Thr Ala Asn Ala Phe Ala Glu Glu Gly Leu 20 25 30 Asp Val His Leu Ile Asn Phe Val Gly Asn Ile Thr Gly Ala Glu His 35 40 45 Leu Ser Pro Pro Phe His Leu His Pro Asn Val Lys Thr Ser Ser Ile 50 55 60 Ile Asp Leu Phe Asn Asp Ile Pro Glu Asn Val Ser Cys Arg Asn Ile 65 70 75 80 Pro Phe Tyr Ser Ile His Gln Gln Phe Phe Lys Ala Glu Tyr Ser Ala 85 90 95 His Tyr Lys His Val Leu Met Lys Ile Glu Ser Leu Leu Ser Glu Glu 100 105 110 Asp Ser Ile Ile Phe Thr His Pro Leu Gln Leu Glu Met Tyr Arg Leu 115 120 125 Ala Asn Asn Asn Ile Lys Ser Lys Ala Lys Leu Ile Val Gln Ile His 130 135 140 Gly Asn Tyr Met Glu Glu Ile His Asn Tyr Glu Ile Trp Ala Arg Asn 145 150 155 160 Ile Asp Tyr Val Asp Tyr Leu Gln Thr Val Ser Asp Glu Met Leu Glu 165 170 175 Glu Met His Ser His Phe Lys Ile Lys Lys Asp Lys Leu Val Phe Ile 180 185 190 Pro Asn Ile Thr Tyr Pro Ile Ser Leu Glu Lys Lys Glu Ala Asp Phe 195 200 205 Phe Ile Lys Asp Asn Glu Asp Ile Asp Asn Ala Gln Lys Phe Lys Arg 210 215 220 Ile Ser Ile Val Gly Ser Ile Gln Pro Arg Lys Asn Gln Leu Asp Ala 225 230 235 240 Ile Lys Ile Ile Asn Lys Ile Lys Asn Glu Asn Tyr Ile Leu Gln Ile 245 250 255 Tyr Gly Lys Ser Ile Asn Lys Asp Tyr Phe Glu Leu Ile Lys Lys Tyr 260 265 270 Ile Lys Asp Asn Lys Leu Gln Asn Arg Ile Leu Phe Lys Gly Glu Ser 275 280 285 Ser Glu Gln Glu Ile Tyr Glu Asn Thr Asp Ile Leu Ile Met Thr Ser 290 295 300 Gln Ser Glu Gly Phe Gly Tyr Ile Phe Leu Glu Gly Met Val Tyr Asp 305 310 315 320 Ile Pro Ile Leu Ala Tyr Asn Phe Lys Tyr Gly Ala Asn Asp Phe Ser 325 330 335 Asn Tyr Asn Glu Asn Ala Ser Val Phe Lys Thr Gly Asp Ile Ser Gly 340 345 350 Met Ala Lys Lys Ile Ile Glu Leu Leu Asn Asn Pro Glu Lys Tyr Lys 355 360 365 Glu Leu Val Gln Tyr Asn His Asn Arg Phe Leu Lys Glu Tyr Ala Lys 370 375 380 Asp Val Val Met Ala Lys Tyr Phe Thr Ile Leu Pro Arg Ser Phe Asn 385 390 395 400 Asn Val Ser Leu Ser Ser Ala Phe Ser Arg Lys Glu Leu Asp Glu Phe 405 410 415 Gln Asn Ile Thr Phe Ser Ile Glu Asp Ser Asn Asp Leu Ala His Ile 420 425 430 Trp Asn Phe Glu Leu Thr Asn Pro Ala Gln Asn Met Asn Phe Phe Ala 435 440 445 Leu Val Gly Lys Arg Lys Phe Pro Met Asp Ala His Ile Gln Gly Thr 450 455 460 Gln Cys Thr Ile Lys Ile Ala His Lys Lys Thr Gly Asn Leu Leu Ser 465 470 475 480 Leu Leu Leu Lys Lys Arg Asn Gln Leu Asn Leu Ser Arg Gly Tyr Thr 485 490 495 Leu Ile Ala Glu Asp Asn Ser Tyr Glu Lys Tyr Ile Gly Ala Ile Ser 500 505 510 Asn Lys Gly Asn Phe Glu Ile Ile Ala Asn Lys Lys Asn Ser Leu Val 515 520 525 Thr Ile Asn Lys Ser Thr Leu Glu Leu His Glu Ile Pro His Glu Leu 530 535 540 His Gln Asn Lys Leu Leu Ile Ala Leu Pro Asn Met Gln Thr Pro Leu 545 550 555 560 Lys Ile Thr Asp Asp Asn Leu Ile Pro Ile Gln Ala Ser Ile Lys Leu 565 570 575 Glu Lys Ile Gly Asn Thr Tyr Tyr Pro Cys Phe Leu Pro Ser Gly Ile 580 585 590 Phe Asn Asn Ile Cys Leu Asp Tyr Gly Glu Glu Ser Lys Ile Ile Asn 595 600 605 Phe Ser Lys Tyr Ser Tyr Lys Tyr Ile Tyr Asp Ser Ile Arg His Ile 610 615 620 Glu Gln His Thr Asp Ile Ser Asp Ile Ile Val Cys Asn Val Tyr Ser 625 630 635 640 Trp Glu Leu Ile Arg Ala Ser Val Ile Glu Ser Leu Met Glu Phe Thr 645 650 655 Gly Lys Trp Glu Lys His Phe Gln Thr Ser Pro Lys Ile Asp Tyr Arg 660 665 670 Phe Asp His Glu Gly Lys Arg Ser Met Asp Asp Val Phe Ser Glu Glu 675 680 685 Thr Phe Ile Met Glu Phe Pro Arg Lys Asn Gly Ile Asp Lys Lys Thr 690 695 700 Ala Ala Phe Gln Asn Ile Pro Asn Ser Ile Val Met Glu Tyr Pro Gln 705 710 715 720 Thr Asn Gly Tyr Ser Met Arg Ser His Ser Leu Lys Ser Asn Val Val 725 730 735 Ala Ala Lys His Phe Leu Glu Lys Leu Asn Lys Ile Lys Val Asp Ile 740 745 750 Lys Phe Lys Lys His Asp Leu Ala Asn Ile Lys Lys Met Asn Arg Ile 755 760 765 Ile Tyr Glu His Leu Gly Ile Asn Ile Asn Ile Glu Ala Phe Leu Lys 770 775 780 Pro Arg Leu Glu Lys Phe Lys Arg Glu Glu Lys Tyr Phe His Asp Phe 785 790 795 800 Phe Lys Arg Asn Asn Phe Lys Glu Val Ile Phe Pro Ser Thr Tyr Trp 805 810 815 Asn Pro Gly Ile Ile Cys Ala Ala His Lys Gln Gly Ile Lys Val Ser 820 825 830 Asp Ile Gln Tyr Ala Ala Ile Thr Pro Tyr His Pro Ala Tyr Phe Lys 835 840 845 Ser Pro Lys Ser His Tyr Val Ala Asp Lys Leu Phe Leu Trp Ser Glu 850 855 860 Tyr Trp Asn His Glu Leu Leu Pro Asn Pro Thr Arg Glu Ile Gly Ser 865 870 875 880 Gly Ala Ala Tyr Trp Tyr Ala Leu Asp Asp Val Arg Phe Ser Glu Lys 885 890 895 Leu Asn Tyr Asp Tyr Ile Phe Leu Ser Gln Ser Arg Ile Ser Ser Arg 900 905 910 Leu Leu Ser Phe Ala Ile Glu Phe Ala Leu Lys Asn Pro Gln Leu Gln 915 920 925 Leu Leu Phe Ser Lys His Leu Asp Glu Asn Ile Asp Leu Lys Asn Arg 930 935 940 Ile Ile Pro Asp Asn Leu Ile Ile Ser Thr Glu Ser Ser Ile Gln Gly 945 950 955 960 Ile Asn Glu Ser Arg Val Ala Val Gly Val Tyr Ser Thr Ser Leu Phe 965 970 975 Glu Ala Leu Ala Cys Gly Lys Gln Thr Phe Val Val Lys Tyr Pro Gly 980 985 990 Tyr Glu Ile Met Ser Asn Glu Ile Asp Ser Gly Leu Phe Phe Ala Val 995 1000 1005 Glu Thr Pro Glu Glu Met Leu Glu Lys Thr Ser Pro Asn Trp Val Ala 1010 1015 1020 Val Ala Asp Ile Glu Asn Gln Phe Phe Gly Gln Glu Lys 1025 1030 1035 <210> SEQ ID NO 108 <211> LENGTH: 1037 <212> TYPE: PRT <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: polymerase of serogroup W-135 <400> SEQUENCE: 108 Met Ala Val Ile Ile Phe Val Asn Gly Ile Arg Ala Val Asn Gly Leu 1 5 10 15 Val Lys Ser Ser Ile Asn Thr Ala Asn Ala Phe Ala Glu Glu Gly Leu 20 25 30 Asp Val His Leu Ile Asn Phe Val Gly Asn Ile Thr Gly Ala Glu His 35 40 45 Leu Tyr Pro Pro Phe His Leu His Pro Asn Val Lys Thr Ser Ser Ile 50 55 60 Ile Asp Leu Phe Asn Asp Ile Pro Glu Asn Val Ser Cys Arg Asn Thr 65 70 75 80 Pro Phe Tyr Ser Ile His Gln Gln Phe Phe Lys Ala Glu Tyr Ser Ala 85 90 95 His Tyr Lys His Val Leu Met Lys Ile Glu Ser Leu Leu Ser Ala Glu 100 105 110 Asp Ser Ile Ile Phe Thr His Pro Leu Gln Leu Glu Met Tyr Arg Leu 115 120 125 Ala Asn Asn Asp Ile Lys Ser Lys Ala Lys Leu Ile Val Gln Ile His 130 135 140 Gly Asn Tyr Met Glu Glu Ile His Asn Tyr Glu Ile Leu Ala Arg Asn 145 150 155 160 Ile Asp Tyr Val Asp Tyr Leu Gln Thr Val Ser Asp Glu Met Leu Glu 165 170 175 Glu Met His Ser His Phe Lys Ile Lys Lys Asp Lys Leu Val Phe Ile 180 185 190 Pro Asn Ile Thr Tyr Pro Ile Ser Leu Glu Lys Lys Glu Ala Asp Phe 195 200 205 Phe Ile Lys Asp Asn Glu Asp Ile Asp Asn Ala Gln Lys Phe Lys Arg 210 215 220 Ile Ser Ile Val Gly Ser Ile Gln Pro Arg Lys Asn Gln Leu Asp Ala 225 230 235 240 Ile Lys Ile Ile Asn Lys Ile Lys Asn Glu Asn Tyr Ile Leu Gln Ile 245 250 255 Tyr Gly Lys Ser Ile Asn Lys Asp Tyr Phe Glu Leu Ile Lys Lys Tyr 260 265 270 Ile Lys Asp Asn Lys Leu Gln Asn Arg Ile Leu Phe Lys Gly Glu Ser 275 280 285 Ser Glu Gln Glu Ile Tyr Glu Asn Thr Asp Ile Leu Ile Met Thr Ser 290 295 300 Glu Ser Glu Gly Phe Pro Tyr Ile Phe Met Glu Gly Met Val Tyr Asp 305 310 315 320 Ile Pro Ile Val Val Tyr Asp Phe Lys Tyr Gly Ala Asn Asp Tyr Ser 325 330 335 Asn Tyr Asn Glu Asn Gly Cys Val Phe Lys Thr Gly Asp Ile Ser Gly 340 345 350 Met Ala Lys Lys Ile Ile Glu Leu Leu Asn Asn Pro Glu Lys Tyr Lys 355 360 365 Glu Leu Val Gln Tyr Asn His Asn Arg Phe Leu Lys Glu Tyr Ala Lys 370 375 380 Asp Val Val Met Ala Lys Tyr Phe Thr Ile Leu Pro Arg Ser Phe Asn 385 390 395 400 Asn Val Ser Leu Ser Ser Ala Phe Ser Arg Lys Glu Leu Asp Glu Phe 405 410 415 Gln Asn Ile Thr Phe Ser Ile Glu Asp Ser Asn Asp Leu Ala His Ile 420 425 430 Trp Asn Phe Glu Leu Thr Asn Pro Ala Gln Asn Met Asn Phe Phe Ala 435 440 445 Leu Val Gly Lys Arg Lys Phe Pro Met Asp Ala His Ile Gln Gly Thr 450 455 460 Gln Cys Thr Ile Lys Ile Ala His Lys Lys Thr Gly Asn Leu Leu Ser 465 470 475 480 Leu Leu Leu Lys Lys Arg Asn Gln Leu Asn Leu Ser Arg Gly Tyr Thr 485 490 495 Leu Ile Ala Glu Asp Asn Ser Tyr Glu Lys Tyr Ile Gly Ala Ile Ser 500 505 510 Asn Lys Gly Asn Phe Glu Ile Ile Ala Asn Lys Lys Ser Ser Leu Val 515 520 525 Thr Ile Asn Lys Ser Thr Leu Glu Leu His Glu Ile Pro His Glu Leu 530 535 540 His Gln Asn Lys Leu Leu Ile Ala Leu Pro Asn Met Gln Thr Pro Leu 545 550 555 560 Lys Ile Thr Asp Asp Asn Leu Ile Pro Ile Gln Ala Ser Ile Lys Leu 565 570 575 Glu Lys Ile Gly Asn Thr Tyr Tyr Pro Cys Phe Leu Pro Ser Gly Ile 580 585 590 Phe Asn Asn Ile Cys Leu Asp Tyr Gly Glu Glu Ser Lys Ile Ile Asn 595 600 605 Phe Ser Lys Tyr Ser Tyr Lys Tyr Ile Tyr Asp Ser Ile Arg His Ile 610 615 620 Glu Gln His Thr Asp Ile Ser Asp Ile Ile Val Cys Asn Val Tyr Ser 625 630 635 640 Trp Glu Leu Ile Arg Ala Ser Val Ile Glu Ser Leu Met Glu Phe Thr 645 650 655 Gly Lys Trp Glu Lys His Phe Gln Thr Ser Pro Lys Ile Asp Tyr Arg 660 665 670 Phe Asp His Glu Gly Lys Arg Ser Met Asp Asp Val Phe Ser Glu Glu 675 680 685 Thr Phe Ile Met Glu Phe Pro Arg Lys Asn Gly Ile Asp Lys Lys Thr 690 695 700 Ala Ala Phe Gln Asn Ile Pro Asn Ser Ile Val Met Glu Tyr Pro Gln 705 710 715 720 Thr Asn Gly Tyr Ser Met Arg Ser His Ser Leu Lys Ser Asn Val Val 725 730 735 Ala Ala Lys His Phe Leu Glu Lys Leu Asn Lys Ile Lys Val Asp Ile 740 745 750 Lys Phe Lys Lys His Asp Leu Ala Asn Ile Lys Lys Met Asn Arg Ile 755 760 765 Ile Tyr Glu His Leu Gly Ile Asn Ile Asn Ile Glu Ala Phe Leu Lys 770 775 780 Pro Arg Leu Glu Lys Phe Lys Arg Glu Glu Lys Tyr Phe His Asp Phe 785 790 795 800 Phe Lys Arg Asn Asn Phe Lys Glu Val Ile Phe Pro Ser Thr Tyr Trp 805 810 815 Asn Pro Gly Ile Ile Cys Ala Ala His Lys Gln Gly Ile Lys Val Ser 820 825 830 Asp Ile Gln Tyr Ala Ala Ile Thr Pro Tyr His Pro Ala Tyr Phe Lys 835 840 845 Ser Pro Lys Ser His Tyr Val Ala Asp Lys Leu Phe Leu Trp Ser Glu 850 855 860 Tyr Trp Asn His Glu Leu Leu Pro Asn Pro Thr Arg Glu Ile Gly Ser 865 870 875 880 Gly Ala Ala Tyr Trp Tyr Ala Leu Asp Asp Val Arg Phe Ser Glu Lys 885 890 895 Leu Asn Tyr Asp Tyr Ile Phe Leu Ser Gln Ser Arg Ile Ser Ser Arg 900 905 910 Leu Leu Ser Phe Ala Ile Glu Phe Ala Leu Lys Asn Pro Gln Leu Gln 915 920 925 Leu Leu Phe Ser Lys His Pro Asp Glu Asn Ile Asp Leu Lys Asn Arg 930 935 940 Ile Ile Pro Asp Asn Leu Ile Ile Ser Thr Glu Ser Ser Ile Gln Gly 945 950 955 960 Ile Asn Glu Ser Arg Val Ala Val Gly Val Tyr Ser Thr Ser Leu Phe 965 970 975 Glu Ala Leu Ala Cys Gly Lys Gln Thr Phe Val Val Lys Tyr Pro Gly 980 985 990 Tyr Glu Ile Met Ser Asn Glu Ile Asp Ser Gly Leu Phe Phe Ala Val 995 1000 1005 Glu Thr Pro Glu Glu Met Leu Glu Lys Thr Ser Pro Asn Trp Val Ala 1010 1015 1020 Val Ala Asp Ile Glu Asn Gln Phe Phe Gly Gln Glu Lys 1025 1030 1035 <210> SEQ ID NO 109 <211> LENGTH: 495 <212> TYPE: PRT <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: Polysialyltransferase of NmB <400> SEQUENCE: 109 Met Leu Lys Lys Ile Lys Lys Ala Leu Phe Gln Pro Lys Lys Phe Phe 1 5 10 15 Gln Asp Ser Met Trp Leu Thr Thr Ser Pro Phe Tyr Leu Thr Pro Pro 20 25 30 Arg Asn Asn Leu Phe Val Ile Ser Asn Leu Gly Gln Leu Asn Gln Val 35 40 45 Gln Ser Leu Ile Lys Ile Gln Lys Leu Thr Asn Asn Leu Leu Val Ile 50 55 60 Leu Tyr Thr Ser Lys Asn Leu Lys Met Pro Lys Leu Val His Gln Ser 65 70 75 80 Ala Asn Lys Asn Leu Phe Glu Ser Ile Tyr Leu Phe Glu Leu Pro Arg 85 90 95 Ser Pro Asn Asn Ile Thr Pro Lys Lys Leu Leu Tyr Ile Tyr Arg Ser 100 105 110 Tyr Lys Lys Ile Leu Asn Ile Ile Gln Pro Ala His Leu Tyr Met Leu 115 120 125 Ser Phe Thr Gly His Tyr Ser Tyr Leu Ile Ser Ile Ala Lys Lys Lys 130 135 140 Asn Ile Thr Thr His Leu Ile Asp Glu Gly Thr Gly Thr Tyr Ala Pro 145 150 155 160 Leu Leu Glu Ser Phe Ser Tyr His Pro Thr Lys Leu Glu Arg Tyr Leu 165 170 175 Ile Gly Asn Asn Leu Asn Ile Lys Gly Tyr Ile Asp His Phe Asp Ile 180 185 190 Leu His Val Pro Phe Pro Glu Tyr Ala Lys Lys Ile Phe Asn Ala Lys 195 200 205 Lys Tyr Asn Arg Phe Phe Ala His Ala Gly Gly Ile Ser Ile Asn Asn 210 215 220 Asn Ile Ala Asn Leu Gln Lys Lys Tyr Gln Ile Ser Lys Asn Asp Tyr 225 230 235 240 Ile Phe Val Ser Gln Arg Tyr Pro Ile Ser Asp Asp Leu Tyr Tyr Lys 245 250 255 Ser Ile Val Glu Ile Leu Asn Ser Ile Ser Leu Gln Ile Lys Gly Lys 260 265 270 Ile Phe Ile Lys Leu His Pro Lys Glu Met Gly Asn Asn Tyr Val Met 275 280 285 Ser Leu Phe Leu Asn Met Val Glu Ile Asn Pro Arg Leu Val Val Ile 290 295 300 Asn Glu Pro Pro Phe Leu Ile Glu Pro Leu Ile Tyr Leu Thr Asn Pro 305 310 315 320 Lys Gly Ile Ile Gly Leu Ala Ser Ser Ser Leu Ile Tyr Thr Pro Leu 325 330 335 Leu Ser Pro Ser Thr Gln Cys Leu Ser Ile Gly Glu Leu Ile Ile Asn 340 345 350 Leu Ile Gln Lys Tyr Ser Met Val Glu Asn Thr Glu Met Ile Gln Glu 355 360 365 His Leu Glu Ile Ile Lys Lys Phe Asn Phe Ile Asn Ile Leu Asn Asp 370 375 380 Leu Asn Gly Val Ile Ser Asn Pro Leu Phe Lys Thr Glu Glu Thr Phe 385 390 395 400 Glu Thr Leu Leu Lys Ser Ala Glu Phe Ala Tyr Lys Ser Lys Asn Tyr 405 410 415 Phe Gln Ala Ile Phe Tyr Trp Gln Leu Ala Ser Lys Asn Asn Ile Thr 420 425 430 Leu Leu Gly His Lys Ala Leu Trp Tyr Tyr Asn Ala Leu Tyr Asn Val 435 440 445 Lys Gln Ile Tyr Lys Met Glu Tyr Ser Asp Ile Phe Tyr Ile Asp Asn 450 455 460 Ile Ser Val Asp Phe His Ser Lys Asp Lys Leu Thr Trp Glu Lys Ile 465 470 475 480 Lys His Tyr Tyr Tyr Ser Ala Asp Asn Arg Ile Gly Arg Asp Arg 485 490 495 <210> SEQ ID NO 110 <211> LENGTH: 492 <212> TYPE: PRT <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: Polysialyltransferase of NmC <400> SEQUENCE: 110 Met Leu Gln Lys Ile Arg Lys Ala Leu Phe His Pro Lys Lys Phe Phe 1 5 10 15 Gln Asp Ser Gln Trp Phe Ala Thr Pro Leu Phe Ser Ser Phe Ala Pro 20 25 30 Lys Ser Asn Leu Phe Ile Ile Ser Thr Phe Ala Gln Leu Asn Gln Ala 35 40 45 His Ser Leu Thr Lys Met Gln Lys Leu Lys Asn Asn Leu Leu Val Ile 50 55 60 Leu Tyr Thr Thr Gln Asn Met Lys Met Pro Lys Leu Ile Gln Lys Ser 65 70 75 80 Val Asp Lys Glu Leu Phe Ser Val Thr Tyr Met Phe Glu Leu Pro Arg 85 90 95 Lys Pro Gly Ile Val Ser Pro Lys Lys Phe Leu Tyr Ile Gln Arg Gly 100 105 110 Tyr Lys Lys Leu Leu Lys Thr Ile Gln Pro Ala His Leu Tyr Val Met 115 120 125 Ser Phe Ala Gly His Tyr Ser Ser Leu Leu Ser Leu Ala Lys Lys Met 130 135 140 Asn Ile Thr Thr His Leu Val Glu Glu Gly Thr Ala Thr Tyr Ala Pro 145 150 155 160 Leu Leu Glu Ser Phe Thr Tyr Lys Pro Thr Lys Phe Glu Gln Arg Phe 165 170 175 Val Gly Asn Asn Leu His Gln Lys Gly Tyr Phe Asp Lys Phe Asp Ile 180 185 190 Leu His Val Ala Phe Pro Glu Tyr Ala Lys Lys Ile Phe Asn Ala Asn 195 200 205 Glu Tyr His Arg Phe Phe Ala His Ser Gly Gly Ile Ser Thr Ser Gln 210 215 220 Ser Ile Ala Lys Ile Gln Asp Lys Tyr Arg Ile Ser Gln Asn Asp Tyr 225 230 235 240 Ile Phe Val Ser Gln Arg Tyr Pro Val Ser Asp Glu Val Tyr Tyr Lys 245 250 255 Thr Ile Val Glu Thr Leu Asn Gln Met Ser Leu Arg Ile Glu Gly Lys 260 265 270 Ile Phe Ile Lys Leu His Pro Lys Glu Met Glu Asn Lys Asn Ile Met 275 280 285 Ser Leu Phe Leu Asn Met Val Thr Ile Asn Pro Arg Leu Val Val Ile 290 295 300 Asn Glu Pro Pro Phe Leu Ile Asp Pro Leu Ile Tyr Leu Thr Thr Pro 305 310 315 320 Lys Gly Ile Ile Gly Leu Thr Ser Thr Ser Ile Val Tyr Thr Pro Leu 325 330 335 Leu Ser Pro Thr Thr Gln Cys Leu Ser Ile Gly Gln Ile Val Ile Asp 340 345 350 Ser Ile His His Thr Ala Gln Gln Glu Asn Thr Ala Leu Ile Glu Glu 355 360 365 His Leu Glu Ile Val Lys Gln Phe Asp Phe Ile Lys Ile Leu Ser Ser 370 375 380 Ile Glu Asp Gly Ile Asp Thr Asn Ser Phe Lys Thr Glu Glu Thr Leu 385 390 395 400 Glu Met Leu Leu Lys Ser Ala Glu Tyr Ala Tyr Lys Asn Lys Asn Phe 405 410 415 Tyr Gln Ala Ile Phe Tyr Trp Gln Leu Ala Ser Asn Asn Asp Leu Ser 420 425 430 Val Leu Gly Tyr Lys Ser Leu Trp Tyr Tyr Asn Ala Leu Asn Lys Val 435 440 445 Lys Gln Asn Tyr Lys Met Lys Tyr Leu Glu Ile Asn Tyr Ile Glu Arg 450 455 460 Ile Ser Leu Tyr Phe Asn Asp Lys Asp Lys Met Ile Trp Gln Asn Ile 465 470 475 480 Lys Asn Asp Phe Phe Lys Tyr Ser Leu Cys Asn Gln 485 490 <210> SEQ ID NO 111 <211> LENGTH: 409 <212> TYPE: PRT <213> ORGANISM: Escherichia coli <220> FEATURE: <223> OTHER INFORMATION: Escherichia coli K1 <400> SEQUENCE: 111 Met Ile Phe Asp Ala Ser Leu Lys Lys Leu Arg Lys Leu Phe Val Asn 1 5 10 15 Pro Ile Gly Phe Phe Arg Asp Ser Trp Phe Phe Asn Ser Lys Asn Lys 20 25 30 Ala Glu Glu Leu Leu Ser Pro Leu Lys Ile Lys Ser Lys Asn Ile Phe 35 40 45 Ile Ile Ser Asn Leu Gly Gln Leu Lys Lys Ala Glu Ser Phe Val Gln 50 55 60 Lys Phe Ser Lys Arg Ser Asn Tyr Leu Ile Val Leu Ala Thr Glu Lys 65 70 75 80 Asn Thr Glu Met Pro Lys Ile Ile Val Glu Gln Ile Asn Asn Lys Leu 85 90 95 Phe Ser Ser Tyr Lys Val Leu Phe Ile Pro Thr Phe Pro Asn Val Phe 100 105 110 Ser Leu Lys Lys Val Ile Trp Phe Tyr Asn Val Tyr Asn Tyr Leu Val 115 120 125 Leu Asn Ser Lys Ala Lys Asp Ala Tyr Phe Met Ser Tyr Ala Gln His 130 135 140 Tyr Ala Ile Phe Val Tyr Leu Phe Lys Lys Asn Asn Ile Arg Cys Ser 145 150 155 160 Leu Ile Glu Glu Gly Thr Gly Thr Tyr Lys Thr Glu Lys Glu Asn Pro 165 170 175 Val Val Asn Ile Asn Phe Tyr Ser Glu Ile Ile Asn Ser Ile Ile Leu 180 185 190 Phe His Tyr Pro Asp Leu Lys Phe Glu Asn Val Tyr Gly Thr Tyr Pro 195 200 205 Ile Leu Leu Lys Lys Lys Phe Asn Ala Gln Lys Phe Val Glu Phe Lys 210 215 220 Gly Ala Pro Ser Val Lys Ser Ser Thr Arg Ile Asp Asn Val Ile His 225 230 235 240 Lys Tyr Ser Ile Thr Arg Asp Asp Ile Ile Tyr Ala Asn Gln Lys Tyr 245 250 255 Leu Ile Glu His Thr Leu Phe Ala Asp Ser Leu Ile Ser Ile Leu Leu 260 265 270 Arg Ile Asp Lys Pro Asp Asn Ala Arg Ile Phe Ile Lys Pro His Pro 275 280 285 Lys Glu Pro Lys Lys Asn Ile Asn Ala Ile Gln Lys Ala Ile Lys Lys 290 295 300 Ala Lys Cys Arg Asp Ile Ile Leu Ile Thr Glu Pro Asp Phe Leu Ile 305 310 315 320 Glu Pro Val Ile Lys Lys Ala Lys Ile Lys His Leu Ile Gly Leu Thr 325 330 335 Ser Ser Ser Leu Val Tyr Ala Pro Leu Val Ser Lys Arg Cys Gln Ser 340 345 350 Tyr Ser Ile Ala Pro Leu Met Ile Lys Leu Cys Asp Asn Asp Lys Ser 355 360 365 Gln Lys Gly Ile Asn Thr Leu Arg Leu His Phe Asp Ile Leu Lys Asn 370 375 380 Phe Asp Asn Val Lys Ile Leu Ser Asp Asp Ile Thr Ser Pro Ser Leu 385 390 395 400 His Asp Lys Arg Ile Phe Leu Gly Glu 405 <210> SEQ ID NO 112 <211> LENGTH: 409 <212> TYPE: PRT <213> ORGANISM: Escherichia coli <220> FEATURE: <223> OTHER INFORMATION: Escherichia coli K92 <400> SEQUENCE: 112 Met Ile Phe Asp Ala Ser Leu Lys Lys Leu Arg Lys Leu Phe Val Asn 1 5 10 15 Pro Ile Gly Phe Phe Arg Asp Ser Trp Phe Phe Asn Ser Lys Asn Lys 20 25 30 Ala Glu Glu Leu Leu Ser Pro Leu Lys Ile Lys Ser Lys Asn Ile Phe 35 40 45 Ile Val Ala His Leu Gly Gln Leu Lys Lys Ala Glu Leu Phe Ile Gln 50 55 60 Lys Phe Ser Arg Arg Ser Asn Phe Leu Ile Val Leu Ala Thr Lys Lys 65 70 75 80 Asn Thr Glu Met Pro Arg Leu Ile Leu Glu Gln Met Asn Lys Lys Leu 85 90 95 Phe Ser Ser Tyr Lys Leu Leu Phe Ile Pro Thr Glu Pro Asn Thr Phe 100 105 110 Ser Leu Lys Lys Val Ile Trp Phe Tyr Asn Val Tyr Lys Tyr Ile Val 115 120 125 Leu Asn Ser Lys Ala Lys Asp Ala Tyr Phe Met Ser Tyr Ala Gln His 130 135 140 Tyr Ala Ile Phe Ile Trp Leu Phe Lys Lys Asn Asn Ile Arg Cys Ser 145 150 155 160 Leu Ile Glu Glu Gly Thr Gly Thr Tyr Lys Thr Glu Lys Lys Lys Pro 165 170 175 Leu Val Asn Ile Asn Phe Tyr Ser Trp Ile Ile Asn Ser Ile Ile Leu 180 185 190 Phe His Tyr Pro Asp Leu Lys Phe Glu Asn Val Tyr Gly Thr Phe Pro 195 200 205 Asn Leu Leu Lys Glu Lys Phe Asp Ala Lys Lys Ile Phe Glu Phe Lys 210 215 220 Thr Ile Pro Leu Val Lys Ser Ser Thr Arg Met Asp Asn Leu Ile His 225 230 235 240 Lys Tyr Arg Ile Thr Arg Asp Asp Ile Ile Tyr Val Ser Gln Arg Tyr 245 250 255 Trp Ile Asp Asn Glu Leu Tyr Ala His Leu Leu Ile Ser Thr Leu Met 260 265 270 Arg Ile Asp Lys Ser Asp Asn Ala Arg Val Phe Ile Lys Pro His Pro 275 280 285 Lys Glu Thr Lys Lys Tyr Ile Asn Ala Ile Gln Gly Ala Ile Asn Lys 290 295 300 Ala Lys Arg Arg Asp Ile Ile Ile Ile Val Glu Lys Asp Phe Leu Ile 305 310 315 320 Glu Ser Ile Ile Lys Lys Cys Lys Ile Lys His Leu Ile Gly Leu Ala 325 330 335 Ser Ser Ser Leu Val Tyr Ala Pro Leu Val Tyr Lys Glu Cys Lys Thr 340 345 350 Tyr Ser Ile Ala Pro Ile Ile Ile Lys Leu Cys Asn Asn Glu Lys Ser 355 360 365 Gln Lys Gly Ile Asn Thr Leu Arg Leu His Phe Asp Ile Leu Lys Asn 370 375 380 Phe Asp Asn Val Lys Ile Leu Ser Asp Asp Ile Thr Ser Pro Ser Leu 385 390 395 400 His Asp Lys Arg Ile Phe Leu Gly Glu 405 <210> SEQ ID NO 113 <211> LENGTH: 29 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer StrepII-deltaN235-CsaB-His6 <400> SEQUENCE: 113 gcagatctat tgatttagta tttacttgg 29 <210> SEQ ID NO 114 <211> LENGTH: 32 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer StrepII-deltaN235-CsaB-His6 <400> SEQUENCE: 114 ccgctcgagt ttctcaaatg atgatggtaa tg 32 <210> SEQ ID NO 115 <211> LENGTH: 29 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer StrepII-deltaN167-CsaB-His6 <400> SEQUENCE: 115 gcagatctac tttaagttca tctatatct 29 <210> SEQ ID NO 116 <211> LENGTH: 32 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer StrepII-deltaN167-CsaB-His6 <400> SEQUENCE: 116 ccgctcgagt ttctcaaatg atgatggtaa tg 32 <210> SEQ ID NO 117 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer StrepII-deltaN97-CsaB-His6 <400> SEQUENCE: 117 gcagatctcc ttctaatctt actcttaagc 30 <210> SEQ ID NO 118 <211> LENGTH: 32 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer StrepII-deltaN97-CsaB-His6 <400> SEQUENCE: 118 ccgctcgagt ttctcaaatg atgatggtaa tg 32 <210> SEQ ID NO 119 <211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer StrepII-deltaC41-CsaB-His6 <400> SEQUENCE: 119 gcagatcttt tatacttaat aacagaaaat ggc 33 <210> SEQ ID NO 120 <211> LENGTH: 32 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer StrepII-deltaC41-CsaB-His6 <400> SEQUENCE: 120 cgcctcgaga aaattccttt ctttagttaa gg 32 <210> SEQ ID NO 121 <211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer StrepII-deltaC25-CsaB-His6 <400> SEQUENCE: 121 gcagatcttt tatacttaat aacagaaaat ggc 33 <210> SEQ ID NO 122 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer StrepII-deltaC25-CsaB-His6 <400> SEQUENCE: 122 cgcctcgagg tgactatcag caccatcg 28 <210> SEQ ID NO 123 <211> LENGTH: 32 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer MBP-deltaN58-CsxA-His6 <400> SEQUENCE: 123 cgggatcccc aattgaagat ccatacccag ta 32 <210> SEQ ID NO 124 <211> LENGTH: 29 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer MBP-deltaN58-CsxA-His6 <400> SEQUENCE: 124 ccgctcgagt tgtccactag gctgtgatg 29 <210> SEQ ID NO 125 <211> LENGTH: 32 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer MBP-deltaC99-CsxA-His6 <400> SEQUENCE: 125 gcggatccat tatgagcaaa attagcaaat tg 32 <210> SEQ ID NO 126 <211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer MBP-deltaC99-CsxA-His6 <400> SEQUENCE: 126 ccgctcgagg agaatttctg cttctgatac atc 33 <210> SEQ ID NO 127 <211> LENGTH: 32 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer MBP-deltaN58deltaC99-CsxA-His6 <400> SEQUENCE: 127 cgggatcccc aattgaagat ccatacccag ta 32 <210> SEQ ID NO 128 <211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer MBP-deltaN58deltaC99-CsxA-His6 <400> SEQUENCE: 128 ccgctcgagg agaatttctg cttctgatac atc 33 <210> SEQ ID NO 129 <211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer MBP-deltaN104-CsxA-His6 <400> SEQUENCE: 129 cgggatccga aagcaccgat attgcaagat tcc 33 <210> SEQ ID NO 130 <211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer MBP-deltaN104-CsxA-His6 <400> SEQUENCE: 130 ccgctcgagg agaatttctg cttctgatac atc 33 <210> SEQ ID NO 131 <211> LENGTH: 32 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer MBP-deltaC174-CsxA-His6 <400> SEQUENCE: 131 gcggatccat tatgagcaaa attagcaaat tg 32 <210> SEQ ID NO 132 <211> LENGTH: 37 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer MBP-deltaC174-CsxA-His6 <400> SEQUENCE: 132 ccgctcgagt tggcctgtca aaatggcaaa atggtgg 37 <210> SEQ ID NO 133 <211> LENGTH: 58 <212> TYPE: PRT <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: Fragment Figure 7 CslA <400> SEQUENCE: 133 Thr Phe Leu Val Pro Trp Leu Met Tyr Leu Asp Gly Lys Ser Ile Pro 1 5 10 15 Asn Asn Asp Ile Cys Tyr Tyr Phe Asn Ile Arg Ser Asn Lys Ala Pro 20 25 30 Thr Gln Tyr Leu Lys Leu Leu Gln Lys Asn Glu Asp Asn Gln Gln Pro 35 40 45 His Ser Phe Cys Ala Asn Asp Phe His Ser 50 55 <210> SEQ ID NO 134 <211> LENGTH: 60 <212> TYPE: PRT <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: Fragment Figure 7 CsaB <400> SEQUENCE: 134 Gly Tyr Leu Tyr His His Tyr Ala Leu Leu Ser Gly Arg Ala Leu Gln 1 5 10 15 Ser Ser Asp Lys Thr Glu Leu Val Gln Gln Asn His Asp Phe Lys Lys 20 25 30 Lys Leu Asn Asn Val Val Thr Leu Thr Lys Glu Arg Asn Phe Asp Lys 35 40 45 Leu Pro Leu Ser Val Cys Ile Asn Asp Gly Ala Asp 50 55 60 <210> SEQ ID NO 135 <211> LENGTH: 58 <212> TYPE: PRT <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: Fragment Figure 7 CsxA <400> SEQUENCE: 135 Thr Phe Leu His His His Phe Ala Ile Leu Thr Gly Gln Ala Leu Ala 1 5 10 15 Thr Arg Thr Lys Cys Ile Tyr Phe Asn Ile Arg Ser Pro Gln Ala Ala 20 25 30 Gln His Tyr Lys Thr Leu Leu Ala Arg Lys Gly Ser Glu Tyr Ser Pro 35 40 45 His Ser Ile Cys Leu Asn Asp His Thr Ser 50 55

1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 135 <210> SEQ ID NO 1 <211> LENGTH: 1635 <212> TYPE: DNA <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: DNA CP-A/CsaB wildtyp/full-length <400> SEQUENCE: 1 atgtttatac ttaataacag aaaatggcgt aaacttaaaa gagaccctag cgctttcttt 60 cgagatagta aatttaactt tttaagatat ttttctgcta aaaaatttgc aaagaatttt 120 aaaaattcat cacatatcca taaaactaat ataagtaaag ctcaatcaaa tatttcttca 180 accttaaaac aaaatcggaa acaagatatg ttaattccta ttaatttttt taattttgaa 240 tatatagtta aaaaacttaa caatcaaaac gcaataggtg tatatattct tccttctaat 300 cttactctta agcctgcatt atgtattcta gaatcacata aagaagactt tttaaataaa 360 tttcttctta ctatttcctc tgaaaattta aagcttcaat acaaatttaa tggacaaata 420 aaaaatccta agtccgtaaa tgaaatttgg acagatttat ttagcattgc tcatgttgac 480 atgaaactca gcacagatag aactttaagt tcatctatat ctcaattttg gttcagatta 540 gagttctgta aagaagataa ggattttatc ttatttccta cagctaacag atattctaga 600 aaactttgga agcactctat taaaaataat caattattta aagaaggcat acgaaactat 660 tcagaaatat cttcattacc ctatgaagaa gatcataatt ttgatattga tttagtattt 720 acttgggtca actcagaaga taagaattgg caagagttat ataaaaaata taagcccgac 780 tttaatagcg atgcaaccag tacatcaaga ttccttagta gagatgaatt aaaattcgca 840 ttacgctctt gggaaatgaa tggatccttc attcgaaaaa tttttattgt ctctaattgt 900 gctcccccag catggctaga tttaaataac cctaaaattc aatgggtata tcacgaagaa 960 attatgccac aaagtgccct tcctactttt agctcacatg ctattgaaac cagcttgcac 1020 catataccag gaattagtaa ctattttatt tacagcaatg acgacttcct attaactaaa 1080 ccattgaata aagacaattt cttctattcg aatggtattg caaagttaag attagaagca 1140 tggggaaatg ttaatggtga atgtactgaa ggagaacctg actacttaaa tggtgctcgc 1200 aatgcgaaca ctctcttaga aaaggaattt aaaaaattta ctactaaact acatactcac 1260 tcccctcaat ccatgagaac tgatatttta tttgagatgg aaaaaaaata tccagaagag 1320 tttaatagaa cactacataa taaattccga tctttagatg atattgcagt aacgggctat 1380 ctctatcatc attatgccct actctctgga cgagcactac aaagttctga caagacggaa 1440 cttgtacagc aaaatcatga tttcaaaaag aaactaaata atgtagtgac cttaactaaa 1500 gaaaggaatt ttgacaaact tcctttgagc gtatgtatca acgatggtgc tgatagtcac 1560 ttgaatgaag aatggaatgt tcaagttatt aagttcttag aaactctttt cccattacca 1620 tcatcatttg agaaa 1635 <210> SEQ ID NO 2 <211> LENGTH: 1635 <212> TYPE: DNA <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: DNA CP-A/CsaB codon optimized <400> SEQUENCE: 2 atgttcatct tgaacaaccg caaatggcgc aaattgaagc gtgacccaag cgcgtttttt 60 cgtgacagca aattcaactt tctgcgctat ttctccgcga aaaagtttgc gaagaatttc 120 aaaaacagct cgcatatcca taaaaccaac attagcaaag cgcagtccaa tatttccagc 180 accttgaagc agaaccgtaa gcaggatatg ctgatcccga tcaatttctt taattttgag 240 tacatcgtga agaaactgaa taaccaaaac gcaatcggcg tgtacattct gccgtctaat 300 ctgaccctga aaccagcatt gtgcatcttg gagtcgcaca aagaggactt cctgaacaaa 360 tttttgttga ccattagcag cgagaacctg aaactgcagt ataagttcaa tggtcagatc 420 aaaaatccga aaagcgtgaa cgaaatctgg accgacctgt ttagcattgc tcacgtcgac 480 atgaagctga gcaccgaccg tacgctgtcc tcgtccatca gccaattttg gtttcgcctg 540 gagttctgta aagaggacaa ggacttcatc ctgtttccga cggcaaatcg ttacagccgc 600 aagctgtgga agcacagcat caaaaataat cagctgttta aggaaggtat ccgtaactac 660 agcgagatta gctcgctgcc gtacgaggaa gaccataact tcgacatcga tctggtcttt 720 acctgggtca attcggaaga caaaaactgg caggaactgt acaagaaata taagccggat 780 tttaatagcg atgccacctc gacgagccgt tttctgagcc gtgacgagct gaagtttgcg 840 ctgcgctcgt gggaaatgaa cggtagcttc atccgtaaaa tctttatcgt cagcaactgc 900 gcgccgccgg cctggctgga tctgaacaat ccgaagatcc aatgggtgta tcacgaggag 960 atcatgccac agagcgccct gccaaccttc agcagccatg ctattgagac tagcttgcat 1020 cacattccgg gcatctccaa ctacttcatc tactctaatg acgattttct gttgaccaaa 1080 ccgctgaaca aagacaactt cttttactcc aacggtattg ctaaactgcg tctggaagcc 1140 tggggtaacg ttaacggtga atgtaccgaa ggcgagccgg attacctgaa cggcgcgcgt 1200 aacgcaaata cgctgctgga gaaagagttt aaaaagttta ccaccaagct gcacacccac 1260 agcccgcaga gcatgcgtac cgacatcctg ttcgagatgg agaaaaaata cccagaagag 1320 ttcaatcgca cgctgcacaa caagttccgc agcctggatg acatcgcggt taccggctac 1380 ctgtaccatc actacgcatt gctgtctggc cgcgctctgc aatccagcga taagaccgaa 1440 ctggtccagc agaatcacga ctttaaaaag aagctgaata atgttgtcac cctgaccaaa 1500 gagcgtaact ttgataagct gccgctgagc gtttgtatta atgacggtgc agacagccac 1560 ctgaatgagg agtggaatgt gcaagttatc aaattcttgg agaccttgtt cccgttgccg 1620 agctccttcg agaaa 1635 <210> SEQ ID NO 3 <211> LENGTH: 1425 <212> TYPE: DNA <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: DNA deltaN69-CP-A (codon optimized) (1425) <400> SEQUENCE: 3 ctgatcccga tcaatttctt taattttgag tacatcgtga agaaactgaa taaccaaaac 60 gcaatcggcg tgtacattct gccgtctaat ctgaccctga aaccagcatt gtgcatcttg 120 gagtcgcaca aagaggactt cctgaacaaa tttttgttga ccattagcag cgagaacctg 180 aaactgcagt ataagttcaa tggtcagatc aaaaatccga aaagcgtgaa cgaaatctgg 240 accgacctgt ttagcattgc tcacgtcgac atgaagctga gcaccgaccg tacgctgtcc 300 tcgtccatca gccaattttg gtttcgcctg gagttctgta aagaggacaa ggacttcatc 360 ctgtttccga cggcaaatcg ttacagccgc aagctgtgga agcacagcat caaaaataat 420 cagctgttta aggaaggtat ccgtaactac agcgagatta gctcgctgcc gtacgaggaa 480 gaccataact tcgacatcga tctggtcttt acctgggtca attcggaaga caaaaactgg 540 caggaactgt acaagaaata taagccggat tttaatagcg atgccacctc gacgagccgt 600 tttctgagcc gtgacgagct gaagtttgcg ctgcgctcgt gggaaatgaa cggtagcttc 660 atccgtaaaa tctttatcgt cagcaactgc gcgccgccgg cctggctgga tctgaacaat 720 ccgaagatcc aatgggtgta tcacgaggag atcatgccac agagcgccct gccaaccttc 780 agcagccatg ctattgagac tagcttgcat cacattccgg gcatctccaa ctacttcatc 840 tactctaatg acgattttct gttgaccaaa ccgctgaaca aagacaactt cttttactcc 900 aacggtattg ctaaactgcg tctggaagcc tggggtaacg ttaacggtga atgtaccgaa 960 ggcgagccgg attacctgaa cggcgcgcgt aacgcaaata cgctgctgga gaaagagttt 1020 aaaaagttta ccaccaagct gcacacccac agcccgcaga gcatgcgtac cgacatcctg 1080 ttcgagatgg agaaaaaata cccagaagag ttcaatcgca cgctgcacaa caagttccgc 1140 agcctggatg acatcgcggt taccggctac ctgtaccatc actacgcatt gctgtctggc 1200 cgcgctctgc aatccagcga taagaccgaa ctggtccagc agaatcacga ctttaaaaag 1260 aagctgaata atgttgtcac cctgaccaaa gagcgtaact ttgataagct gccgctgagc 1320 gtttgtatta atgacggtgc agacagccac ctgaatgagg agtggaatgt gcaagttatc 1380 aaattcttgg agaccttgtt cccgttgccg agctccttcg agaaa 1425 <210> SEQ ID NO 4 <211> LENGTH: 1344 <212> TYPE: DNA <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: DNA deltaN97-CP-A (1344) <400> SEQUENCE: 4 ccttctaatc ttactcttaa gcctgcatta tgtattctag aatcacataa agaagacttt 60 ttaaataaat ttcttcttac tatttcctct gaaaatttaa agcttcaata caaatttaat 120 ggacaaataa aaaatcctaa gtccgtaaat gaaatttgga cagatttatt tagcattgct 180 catgttgaca tgaaactcag cacagataga actttaagtt catctatatc tcaattttgg 240 ttcagattag agttctgtaa agaagataag gattttatct tatttcctac agctaacaga 300 tattctagaa aactttggaa gcactctatt aaaaataatc aattatttaa agaaggcata 360 cgaaactatt cagaaatatc ttcattaccc tatgaagaag atcataattt tgatattgat 420 ttagtattta cttgggtcaa ctcagaagat aagaattggc aagagttata taaaaaatat 480 aagcccgact ttaatagcga tgcaaccagt acatcaagat tccttagtag agatgaatta 540 aaattcgcat tacgctcttg ggaaatgaat ggatccttca ttcgaaaaat ttttattgtc 600 tctaattgtg ctcccccagc atggctagat ttaaataacc ctaaaattca atgggtatat 660 cacgaagaaa ttatgccaca aagtgccctt cctactttta gctcacatgc tattgaaacc 720 agcttgcacc atataccagg aattagtaac tattttattt acagcaatga cgacttccta 780 ttaactaaac cattgaataa agacaatttc ttctattcga atggtattgc aaagttaaga 840 ttagaagcat ggggaaatgt taatggtgaa tgtactgaag gagaacctga ctacttaaat 900 ggtgctcgca atgcgaacac tctcttagaa aaggaattta aaaaatttac tactaaacta 960 catactcact cccctcaatc catgagaact gatattttat ttgagatgga aaaaaaatat 1020 ccagaagagt ttaatagaac actacataat aaattccgat ctttagatga tattgcagta 1080 acgggctatc tctatcatca ttatgcccta ctctctggac gagcactaca aagttctgac 1140 aagacggaac ttgtacagca aaatcatgat ttcaaaaaga aactaaataa tgtagtgacc 1200 ttaactaaag aaaggaattt tgacaaactt cctttgagcg tatgtatcaa cgatggtgct 1260 gatagtcact tgaatgaaga atggaatgtt caagttatta agttcttaga aactcttttc 1320 ccattaccat catcatttga gaaa 1344

<210> SEQ ID NO 5 <211> LENGTH: 1134 <212> TYPE: DNA <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: DNA deltaN167-CP-A (1134) <400> SEQUENCE: 5 actttaagtt catctatatc tcaattttgg ttcagattag agttctgtaa agaagataag 60 gattttatct tatttcctac agctaacaga tattctagaa aactttggaa gcactctatt 120 aaaaataatc aattatttaa agaaggcata cgaaactatt cagaaatatc ttcattaccc 180 tatgaagaag atcataattt tgatattgat ttagtattta cttgggtcaa ctcagaagat 240 aagaattggc aagagttata taaaaaatat aagcccgact ttaatagcga tgcaaccagt 300 acatcaagat tccttagtag agatgaatta aaattcgcat tacgctcttg ggaaatgaat 360 ggatccttca ttcgaaaaat ttttattgtc tctaattgtg ctcccccagc atggctagat 420 ttaaataacc ctaaaattca atgggtatat cacgaagaaa ttatgccaca aagtgccctt 480 cctactttta gctcacatgc tattgaaacc agcttgcacc atataccagg aattagtaac 540 tattttattt acagcaatga cgacttccta ttaactaaac cattgaataa agacaatttc 600 ttctattcga atggtattgc aaagttaaga ttagaagcat ggggaaatgt taatggtgaa 660 tgtactgaag gagaacctga ctacttaaat ggtgctcgca atgcgaacac tctcttagaa 720 aaggaattta aaaaatttac tactaaacta catactcact cccctcaatc catgagaact 780 gatattttat ttgagatgga aaaaaaatat ccagaagagt ttaatagaac actacataat 840 aaattccgat ctttagatga tattgcagta acgggctatc tctatcatca ttatgcccta 900 ctctctggac gagcactaca aagttctgac aagacggaac ttgtacagca aaatcatgat 960 ttcaaaaaga aactaaataa tgtagtgacc ttaactaaag aaaggaattt tgacaaactt 1020 cctttgagcg tatgtatcaa cgatggtgct gatagtcact tgaatgaaga atggaatgtt 1080 caagttatta agttcttaga aactcttttc ccattaccat catcatttga gaaa 1134 <210> SEQ ID NO 6 <211> LENGTH: 933 <212> TYPE: DNA <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: DNA deltaN235-CP-A (933) <400> SEQUENCE: 6 gatattgatt tagtatttac ttgggtcaac tcagaagata agaattggca agagttatat 60 aaaaaatata agcccgactt taatagcgat gcaaccagta catcaagatt ccttagtaga 120 gatgaattaa aattcgcatt acgctcttgg gaaatgaatg gatccttcat tcgaaaaatt 180 tttattgtct ctaattgtgc tcccccagca tggctagatt taaataaccc taaaattcaa 240 tgggtatatc acgaagaaat tatgccacaa agtgcccttc ctacttttag ctcacatgct 300 attgaaacca gcttgcacca tataccagga attagtaact attttattta cagcaatgac 360 gacttcctat taactaaacc attgaataaa gacaatttct tctattcgaa tggtattgca 420 aagttaagat tagaagcatg gggaaatgtt aatggtgaat gtactgaagg agaacctgac 480 tacttaaatg gtgctcgcaa tgcgaacact ctcttagaaa aggaatttaa aaaatttact 540 actaaactac atactcactc ccctcaatcc atgagaactg atattttatt tgagatggaa 600 aaaaaatatc cagaagagtt taatagaaca ctacataata aattccgatc tttagatgat 660 attgcagtaa cgggctatct ctatcatcat tatgccctac tctctggacg agcactacaa 720 agttctgaca agacggaact tgtacagcaa aatcatgatt tcaaaaagaa actaaataat 780 gtagtgacct taactaaaga aaggaatttt gacaaacttc ctttgagcgt atgtatcaac 840 gatggtgctg atagtcactt gaatgaagaa tggaatgttc aagttattaa gttcttagaa 900 actcttttcc cattaccatc atcatttgag aaa 933 <210> SEQ ID NO 7 <211> LENGTH: 1497 <212> TYPE: DNA <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: DNA deltaC45-CP-A (1497) <400> SEQUENCE: 7 tttatactta ataacagaaa atggcgtaaa cttaaaagag accctagcgc tttctttcga 60 gatagtaaat ttaacttttt aagatatttt tctgctaaaa aatttgcaaa gaattttaaa 120 aattcatcac atatccataa aactaatata agtaaagctc aatcaaatat ttcttcaacc 180 ttaaaacaaa atcggaaaca agatatgtta attcctatta atttttttaa ttttgaatat 240 atagttaaaa aacttaacaa tcaaaacgca ataggtgtat atattcttcc ttctaatctt 300 actcttaagc ctgcattatg tattctagaa tcacataaag aagacttttt aaataaattt 360 cttcttacta tttcctctga aaatttaaag cttcaataca aatttaatgg acaaataaaa 420 aatcctaagt ccgtaaatga aatttggaca gatttattta gcattgctca tgttgacatg 480 aaactcagca cagatagaac tttaagttca tctatatctc aattttggtt cagattagag 540 ttctgtaaag aagataagga ttttatctta tttcctacag ctaacagata ttctagaaaa 600 ctttggaagc actctattaa aaataatcaa ttatttaaag aaggcatacg aaactattca 660 gaaatatctt cattacccta tgaagaagat cataattttg atattgattt agtatttact 720 tgggtcaact cagaagataa gaattggcaa gagttatata aaaaatataa gcccgacttt 780 aatagcgatg caaccagtac atcaagattc cttagtagag atgaattaaa attcgcatta 840 cgctcttggg aaatgaatgg atccttcatt cgaaaaattt ttattgtctc taattgtgct 900 cccccagcat ggctagattt aaataaccct aaaattcaat gggtatatca cgaagaaatt 960 atgccacaaa gtgcccttcc tacttttagc tcacatgcta ttgaaaccag cttgcaccat 1020 ataccaggaa ttagtaacta ttttatttac agcaatgacg acttcctatt aactaaacca 1080 ttgaataaag acaatttctt ctattcgaat ggtattgcaa agttaagatt agaagcatgg 1140 ggaaatgtta atggtgaatg tactgaagga gaacctgact acttaaatgg tgctcgcaat 1200 gcgaacactc tcttagaaaa ggaatttaaa aaatttacta ctaaactaca tactcactcc 1260 cctcaatcca tgagaactga tattttattt gagatggaaa aaaaatatcc agaagagttt 1320 aatagaacac tacataataa attccgatct ttagatgata ttgcagtaac gggctatctc 1380 tatcatcatt atgccctact ctctggacga gcactacaaa gttctgacaa gacggaactt 1440 gtacagcaaa atcatgattt caaaaagaaa ctaaataatg tagtgacctt aactaaa 1497 <210> SEQ ID NO 8 <211> LENGTH: 1557 <212> TYPE: DNA <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: DNA deltaC25-CP-A (1557) <400> SEQUENCE: 8 tttatactta ataacagaaa atggcgtaaa cttaaaagag accctagcgc tttctttcga 60 gatagtaaat ttaacttttt aagatatttt tctgctaaaa aatttgcaaa gaattttaaa 120 aattcatcac atatccataa aactaatata agtaaagctc aatcaaatat ttcttcaacc 180 ttaaaacaaa atcggaaaca agatatgtta attcctatta atttttttaa ttttgaatat 240 atagttaaaa aacttaacaa tcaaaacgca ataggtgtat atattcttcc ttctaatctt 300 actcttaagc ctgcattatg tattctagaa tcacataaag aagacttttt aaataaattt 360 cttcttacta tttcctctga aaatttaaag cttcaataca aatttaatgg acaaataaaa 420 aatcctaagt ccgtaaatga aatttggaca gatttattta gcattgctca tgttgacatg 480 aaactcagca cagatagaac tttaagttca tctatatctc aattttggtt cagattagag 540 ttctgtaaag aagataagga ttttatctta tttcctacag ctaacagata ttctagaaaa 600 ctttggaagc actctattaa aaataatcaa ttatttaaag aaggcatacg aaactattca 660 gaaatatctt cattacccta tgaagaagat cataattttg atattgattt agtatttact 720 tgggtcaact cagaagataa gaattggcaa gagttatata aaaaatataa gcccgacttt 780 aatagcgatg caaccagtac atcaagattc cttagtagag atgaattaaa attcgcatta 840 cgctcttggg aaatgaatgg atccttcatt cgaaaaattt ttattgtctc taattgtgct 900 cccccagcat ggctagattt aaataaccct aaaattcaat gggtatatca cgaagaaatt 960 atgccacaaa gtgcccttcc tacttttagc tcacatgcta ttgaaaccag cttgcaccat 1020 ataccaggaa ttagtaacta ttttatttac agcaatgacg acttcctatt aactaaacca 1080 ttgaataaag acaatttctt ctattcgaat ggtattgcaa agttaagatt agaagcatgg 1140 ggaaatgtta atggtgaatg tactgaagga gaacctgact acttaaatgg tgctcgcaat 1200 gcgaacactc tcttagaaaa ggaatttaaa aaatttacta ctaaactaca tactcactcc 1260 cctcaatcca tgagaactga tattttattt gagatggaaa aaaaatatcc agaagagttt 1320 aatagaacac tacataataa attccgatct ttagatgata ttgcagtaac gggctatctc 1380 tatcatcatt atgccctact ctctggacga gcactacaaa gttctgacaa gacggaactt 1440 gtacagcaaa atcatgattt caaaaagaaa ctaaataatg tagtgacctt aactaaagaa 1500 aggaattttg acaaacttcc tttgagcgta tgtatcaacg atggtgctga tagtcac 1557 <210> SEQ ID NO 9 <211> LENGTH: 545 <212> TYPE: PRT <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: AS CP-A/CsaB wildtyp/full-length (544 without M1) <400> SEQUENCE: 9 Met Phe Ile Leu Asn Asn Arg Lys Trp Arg Lys Leu Lys Arg Asp Pro 1 5 10 15 Ser Ala Phe Phe Arg Asp Ser Lys Phe Asn Phe Leu Arg Tyr Phe Ser 20 25 30 Ala Lys Lys Phe Ala Lys Asn Phe Lys Asn Ser Ser His Ile His Lys 35 40 45 Thr Asn Ile Ser Lys Ala Gln Ser Asn Ile Ser Ser Thr Leu Lys Gln 50 55 60 Asn Arg Lys Gln Asp Met Leu Ile Pro Ile Asn Phe Phe Asn Phe Glu 65 70 75 80 Tyr Ile Val Lys Lys Leu Asn Asn Gln Asn Ala Ile Gly Val Tyr Ile 85 90 95 Leu Pro Ser Asn Leu Thr Leu Lys Pro Ala Leu Cys Ile Leu Glu Ser 100 105 110 His Lys Glu Asp Phe Leu Asn Lys Phe Leu Leu Thr Ile Ser Ser Glu 115 120 125 Asn Leu Lys Leu Gln Tyr Lys Phe Asn Gly Gln Ile Lys Asn Pro Lys 130 135 140

Ser Val Asn Glu Ile Trp Thr Asp Leu Phe Ser Ile Ala His Val Asp 145 150 155 160 Met Lys Leu Ser Thr Asp Arg Thr Leu Ser Ser Ser Ile Ser Gln Phe 165 170 175 Trp Phe Arg Leu Glu Phe Cys Lys Glu Asp Lys Asp Phe Ile Leu Phe 180 185 190 Pro Thr Ala Asn Arg Tyr Ser Arg Lys Leu Trp Lys His Ser Ile Lys 195 200 205 Asn Asn Gln Leu Phe Lys Glu Gly Ile Arg Asn Tyr Ser Glu Ile Ser 210 215 220 Ser Leu Pro Tyr Glu Glu Asp His Asn Phe Asp Ile Asp Leu Val Phe 225 230 235 240 Thr Trp Val Asn Ser Glu Asp Lys Asn Trp Gln Glu Leu Tyr Lys Lys 245 250 255 Tyr Lys Pro Asp Phe Asn Ser Asp Ala Thr Ser Thr Ser Arg Phe Leu 260 265 270 Ser Arg Asp Glu Leu Lys Phe Ala Leu Arg Ser Trp Glu Met Asn Gly 275 280 285 Ser Phe Ile Arg Lys Ile Phe Ile Val Ser Asn Cys Ala Pro Pro Ala 290 295 300 Trp Leu Asp Leu Asn Asn Pro Lys Ile Gln Trp Val Tyr His Glu Glu 305 310 315 320 Ile Met Pro Gln Ser Ala Leu Pro Thr Phe Ser Ser His Ala Ile Glu 325 330 335 Thr Ser Leu His His Ile Pro Gly Ile Ser Asn Tyr Phe Ile Tyr Ser 340 345 350 Asn Asp Asp Phe Leu Leu Thr Lys Pro Leu Asn Lys Asp Asn Phe Phe 355 360 365 Tyr Ser Asn Gly Ile Ala Lys Leu Arg Leu Glu Ala Trp Gly Asn Val 370 375 380 Asn Gly Glu Cys Thr Glu Gly Glu Pro Asp Tyr Leu Asn Gly Ala Arg 385 390 395 400 Asn Ala Asn Thr Leu Leu Glu Lys Glu Phe Lys Lys Phe Thr Thr Lys 405 410 415 Leu His Thr His Ser Pro Gln Ser Met Arg Thr Asp Ile Leu Phe Glu 420 425 430 Met Glu Lys Lys Tyr Pro Glu Glu Phe Asn Arg Thr Leu His Asn Lys 435 440 445 Phe Arg Ser Leu Asp Asp Ile Ala Val Thr Gly Tyr Leu Tyr His His 450 455 460 Tyr Ala Leu Leu Ser Gly Arg Ala Leu Gln Ser Ser Asp Lys Thr Glu 465 470 475 480 Leu Val Gln Gln Asn His Asp Phe Lys Lys Lys Leu Asn Asn Val Val 485 490 495 Thr Leu Thr Lys Glu Arg Asn Phe Asp Lys Leu Pro Leu Ser Val Cys 500 505 510 Ile Asn Asp Gly Ala Asp Ser His Leu Asn Glu Glu Trp Asn Val Gln 515 520 525 Val Ile Lys Phe Leu Glu Thr Leu Phe Pro Leu Pro Ser Ser Phe Glu 530 535 540 Lys 545 <210> SEQ ID NO 10 <211> LENGTH: 475 <212> TYPE: PRT <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: AS deltaN69-CP-A (codon optimized) (475) <400> SEQUENCE: 10 Leu Ile Pro Ile Asn Phe Phe Asn Phe Glu Tyr Ile Val Lys Lys Leu 1 5 10 15 Asn Asn Gln Asn Ala Ile Gly Val Tyr Ile Leu Pro Ser Asn Leu Thr 20 25 30 Leu Lys Pro Ala Leu Cys Ile Leu Glu Ser His Lys Glu Asp Phe Leu 35 40 45 Asn Lys Phe Leu Leu Thr Ile Ser Ser Glu Asn Leu Lys Leu Gln Tyr 50 55 60 Lys Phe Asn Gly Gln Ile Lys Asn Pro Lys Ser Val Asn Glu Ile Trp 65 70 75 80 Thr Asp Leu Phe Ser Ile Ala His Val Asp Met Lys Leu Ser Thr Asp 85 90 95 Arg Thr Leu Ser Ser Ser Ile Ser Gln Phe Trp Phe Arg Leu Glu Phe 100 105 110 Cys Lys Glu Asp Lys Asp Phe Ile Leu Phe Pro Thr Ala Asn Arg Tyr 115 120 125 Ser Arg Lys Leu Trp Lys His Ser Ile Lys Asn Asn Gln Leu Phe Lys 130 135 140 Glu Gly Ile Arg Asn Tyr Ser Glu Ile Ser Ser Leu Pro Tyr Glu Glu 145 150 155 160 Asp His Asn Phe Asp Ile Asp Leu Val Phe Thr Trp Val Asn Ser Glu 165 170 175 Asp Lys Asn Trp Gln Glu Leu Tyr Lys Lys Tyr Lys Pro Asp Phe Asn 180 185 190 Ser Asp Ala Thr Ser Thr Ser Arg Phe Leu Ser Arg Asp Glu Leu Lys 195 200 205 Phe Ala Leu Arg Ser Trp Glu Met Asn Gly Ser Phe Ile Arg Lys Ile 210 215 220 Phe Ile Val Ser Asn Cys Ala Pro Pro Ala Trp Leu Asp Leu Asn Asn 225 230 235 240 Pro Lys Ile Gln Trp Val Tyr His Glu Glu Ile Met Pro Gln Ser Ala 245 250 255 Leu Pro Thr Phe Ser Ser His Ala Ile Glu Thr Ser Leu His His Ile 260 265 270 Pro Gly Ile Ser Asn Tyr Phe Ile Tyr Ser Asn Asp Asp Phe Leu Leu 275 280 285 Thr Lys Pro Leu Asn Lys Asp Asn Phe Phe Tyr Ser Asn Gly Ile Ala 290 295 300 Lys Leu Arg Leu Glu Ala Trp Gly Asn Val Asn Gly Glu Cys Thr Glu 305 310 315 320 Gly Glu Pro Asp Tyr Leu Asn Gly Ala Arg Asn Ala Asn Thr Leu Leu 325 330 335 Glu Lys Glu Phe Lys Lys Phe Thr Thr Lys Leu His Thr His Ser Pro 340 345 350 Gln Ser Met Arg Thr Asp Ile Leu Phe Glu Met Glu Lys Lys Tyr Pro 355 360 365 Glu Glu Phe Asn Arg Thr Leu His Asn Lys Phe Arg Ser Leu Asp Asp 370 375 380 Ile Ala Val Thr Gly Tyr Leu Tyr His His Tyr Ala Leu Leu Ser Gly 385 390 395 400 Arg Ala Leu Gln Ser Ser Asp Lys Thr Glu Leu Val Gln Gln Asn His 405 410 415 Asp Phe Lys Lys Lys Leu Asn Asn Val Val Thr Leu Thr Lys Glu Arg 420 425 430 Asn Phe Asp Lys Leu Pro Leu Ser Val Cys Ile Asn Asp Gly Ala Asp 435 440 445 Ser His Leu Asn Glu Glu Trp Asn Val Gln Val Ile Lys Phe Leu Glu 450 455 460 Thr Leu Phe Pro Leu Pro Ser Ser Phe Glu Lys 465 470 475 <210> SEQ ID NO 11 <211> LENGTH: 448 <212> TYPE: PRT <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: AS deltaN97-CP-A (448) <400> SEQUENCE: 11 Pro Ser Asn Leu Thr Leu Lys Pro Ala Leu Cys Ile Leu Glu Ser His 1 5 10 15 Lys Glu Asp Phe Leu Asn Lys Phe Leu Leu Thr Ile Ser Ser Glu Asn 20 25 30 Leu Lys Leu Gln Tyr Lys Phe Asn Gly Gln Ile Lys Asn Pro Lys Ser 35 40 45 Val Asn Glu Ile Trp Thr Asp Leu Phe Ser Ile Ala His Val Asp Met 50 55 60 Lys Leu Ser Thr Asp Arg Thr Leu Ser Ser Ser Ile Ser Gln Phe Trp 65 70 75 80 Phe Arg Leu Glu Phe Cys Lys Glu Asp Lys Asp Phe Ile Leu Phe Pro 85 90 95 Thr Ala Asn Arg Tyr Ser Arg Lys Leu Trp Lys His Ser Ile Lys Asn 100 105 110 Asn Gln Leu Phe Lys Glu Gly Ile Arg Asn Tyr Ser Glu Ile Ser Ser 115 120 125 Leu Pro Tyr Glu Glu Asp His Asn Phe Asp Ile Asp Leu Val Phe Thr 130 135 140 Trp Val Asn Ser Glu Asp Lys Asn Trp Gln Glu Leu Tyr Lys Lys Tyr 145 150 155 160 Lys Pro Asp Phe Asn Ser Asp Ala Thr Ser Thr Ser Arg Phe Leu Ser 165 170 175 Arg Asp Glu Leu Lys Phe Ala Leu Arg Ser Trp Glu Met Asn Gly Ser 180 185 190 Phe Ile Arg Lys Ile Phe Ile Val Ser Asn Cys Ala Pro Pro Ala Trp 195 200 205 Leu Asp Leu Asn Asn Pro Lys Ile Gln Trp Val Tyr His Glu Glu Ile 210 215 220 Met Pro Gln Ser Ala Leu Pro Thr Phe Ser Ser His Ala Ile Glu Thr 225 230 235 240 Ser Leu His His Ile Pro Gly Ile Ser Asn Tyr Phe Ile Tyr Ser Asn 245 250 255 Asp Asp Phe Leu Leu Thr Lys Pro Leu Asn Lys Asp Asn Phe Phe Tyr 260 265 270 Ser Asn Gly Ile Ala Lys Leu Arg Leu Glu Ala Trp Gly Asn Val Asn 275 280 285 Gly Glu Cys Thr Glu Gly Glu Pro Asp Tyr Leu Asn Gly Ala Arg Asn 290 295 300 Ala Asn Thr Leu Leu Glu Lys Glu Phe Lys Lys Phe Thr Thr Lys Leu 305 310 315 320 His Thr His Ser Pro Gln Ser Met Arg Thr Asp Ile Leu Phe Glu Met 325 330 335

Glu Lys Lys Tyr Pro Glu Glu Phe Asn Arg Thr Leu His Asn Lys Phe 340 345 350 Arg Ser Leu Asp Asp Ile Ala Val Thr Gly Tyr Leu Tyr His His Tyr 355 360 365 Ala Leu Leu Ser Gly Arg Ala Leu Gln Ser Ser Asp Lys Thr Glu Leu 370 375 380 Val Gln Gln Asn His Asp Phe Lys Lys Lys Leu Asn Asn Val Val Thr 385 390 395 400 Leu Thr Lys Glu Arg Asn Phe Asp Lys Leu Pro Leu Ser Val Cys Ile 405 410 415 Asn Asp Gly Ala Asp Ser His Leu Asn Glu Glu Trp Asn Val Gln Val 420 425 430 Ile Lys Phe Leu Glu Thr Leu Phe Pro Leu Pro Ser Ser Phe Glu Lys 435 440 445 <210> SEQ ID NO 12 <211> LENGTH: 378 <212> TYPE: PRT <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: AS deltaN167-CP-A (378) <400> SEQUENCE: 12 Thr Leu Ser Ser Ser Ile Ser Gln Phe Trp Phe Arg Leu Glu Phe Cys 1 5 10 15 Lys Glu Asp Lys Asp Phe Ile Leu Phe Pro Thr Ala Asn Arg Tyr Ser 20 25 30 Arg Lys Leu Trp Lys His Ser Ile Lys Asn Asn Gln Leu Phe Lys Glu 35 40 45 Gly Ile Arg Asn Tyr Ser Glu Ile Ser Ser Leu Pro Tyr Glu Glu Asp 50 55 60 His Asn Phe Asp Ile Asp Leu Val Phe Thr Trp Val Asn Ser Glu Asp 65 70 75 80 Lys Asn Trp Gln Glu Leu Tyr Lys Lys Tyr Lys Pro Asp Phe Asn Ser 85 90 95 Asp Ala Thr Ser Thr Ser Arg Phe Leu Ser Arg Asp Glu Leu Lys Phe 100 105 110 Ala Leu Arg Ser Trp Glu Met Asn Gly Ser Phe Ile Arg Lys Ile Phe 115 120 125 Ile Val Ser Asn Cys Ala Pro Pro Ala Trp Leu Asp Leu Asn Asn Pro 130 135 140 Lys Ile Gln Trp Val Tyr His Glu Glu Ile Met Pro Gln Ser Ala Leu 145 150 155 160 Pro Thr Phe Ser Ser His Ala Ile Glu Thr Ser Leu His His Ile Pro 165 170 175 Gly Ile Ser Asn Tyr Phe Ile Tyr Ser Asn Asp Asp Phe Leu Leu Thr 180 185 190 Lys Pro Leu Asn Lys Asp Asn Phe Phe Tyr Ser Asn Gly Ile Ala Lys 195 200 205 Leu Arg Leu Glu Ala Trp Gly Asn Val Asn Gly Glu Cys Thr Glu Gly 210 215 220 Glu Pro Asp Tyr Leu Asn Gly Ala Arg Asn Ala Asn Thr Leu Leu Glu 225 230 235 240 Lys Glu Phe Lys Lys Phe Thr Thr Lys Leu His Thr His Ser Pro Gln 245 250 255 Ser Met Arg Thr Asp Ile Leu Phe Glu Met Glu Lys Lys Tyr Pro Glu 260 265 270 Glu Phe Asn Arg Thr Leu His Asn Lys Phe Arg Ser Leu Asp Asp Ile 275 280 285 Ala Val Thr Gly Tyr Leu Tyr His His Tyr Ala Leu Leu Ser Gly Arg 290 295 300 Ala Leu Gln Ser Ser Asp Lys Thr Glu Leu Val Gln Gln Asn His Asp 305 310 315 320 Phe Lys Lys Lys Leu Asn Asn Val Val Thr Leu Thr Lys Glu Arg Asn 325 330 335 Phe Asp Lys Leu Pro Leu Ser Val Cys Ile Asn Asp Gly Ala Asp Ser 340 345 350 His Leu Asn Glu Glu Trp Asn Val Gln Val Ile Lys Phe Leu Glu Thr 355 360 365 Leu Phe Pro Leu Pro Ser Ser Phe Glu Lys 370 375 <210> SEQ ID NO 13 <211> LENGTH: 311 <212> TYPE: PRT <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: AS deltaN235-CP-A (311) <400> SEQUENCE: 13 Asp Ile Asp Leu Val Phe Thr Trp Val Asn Ser Glu Asp Lys Asn Trp 1 5 10 15 Gln Glu Leu Tyr Lys Lys Tyr Lys Pro Asp Phe Asn Ser Asp Ala Thr 20 25 30 Ser Thr Ser Arg Phe Leu Ser Arg Asp Glu Leu Lys Phe Ala Leu Arg 35 40 45 Ser Trp Glu Met Asn Gly Ser Phe Ile Arg Lys Ile Phe Ile Val Ser 50 55 60 Asn Cys Ala Pro Pro Ala Trp Leu Asp Leu Asn Asn Pro Lys Ile Gln 65 70 75 80 Trp Val Tyr His Glu Glu Ile Met Pro Gln Ser Ala Leu Pro Thr Phe 85 90 95 Ser Ser His Ala Ile Glu Thr Ser Leu His His Ile Pro Gly Ile Ser 100 105 110 Asn Tyr Phe Ile Tyr Ser Asn Asp Asp Phe Leu Leu Thr Lys Pro Leu 115 120 125 Asn Lys Asp Asn Phe Phe Tyr Ser Asn Gly Ile Ala Lys Leu Arg Leu 130 135 140 Glu Ala Trp Gly Asn Val Asn Gly Glu Cys Thr Glu Gly Glu Pro Asp 145 150 155 160 Tyr Leu Asn Gly Ala Arg Asn Ala Asn Thr Leu Leu Glu Lys Glu Phe 165 170 175 Lys Lys Phe Thr Thr Lys Leu His Thr His Ser Pro Gln Ser Met Arg 180 185 190 Thr Asp Ile Leu Phe Glu Met Glu Lys Lys Tyr Pro Glu Glu Phe Asn 195 200 205 Arg Thr Leu His Asn Lys Phe Arg Ser Leu Asp Asp Ile Ala Val Thr 210 215 220 Gly Tyr Leu Tyr His His Tyr Ala Leu Leu Ser Gly Arg Ala Leu Gln 225 230 235 240 Ser Ser Asp Lys Thr Glu Leu Val Gln Gln Asn His Asp Phe Lys Lys 245 250 255 Lys Leu Asn Asn Val Val Thr Leu Thr Lys Glu Arg Asn Phe Asp Lys 260 265 270 Leu Pro Leu Ser Val Cys Ile Asn Asp Gly Ala Asp Ser His Leu Asn 275 280 285 Glu Glu Trp Asn Val Gln Val Ile Lys Phe Leu Glu Thr Leu Phe Pro 290 295 300 Leu Pro Ser Ser Phe Glu Lys 305 310 <210> SEQ ID NO 14 <211> LENGTH: 499 <212> TYPE: PRT <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: AS deltaC45-CP-A (499) <400> SEQUENCE: 14 Phe Ile Leu Asn Asn Arg Lys Trp Arg Lys Leu Lys Arg Asp Pro Ser 1 5 10 15 Ala Phe Phe Arg Asp Ser Lys Phe Asn Phe Leu Arg Tyr Phe Ser Ala 20 25 30 Lys Lys Phe Ala Lys Asn Phe Lys Asn Ser Ser His Ile His Lys Thr 35 40 45 Asn Ile Ser Lys Ala Gln Ser Asn Ile Ser Ser Thr Leu Lys Gln Asn 50 55 60 Arg Lys Gln Asp Met Leu Ile Pro Ile Asn Phe Phe Asn Phe Glu Tyr 65 70 75 80 Ile Val Lys Lys Leu Asn Asn Gln Asn Ala Ile Gly Val Tyr Ile Leu 85 90 95 Pro Ser Asn Leu Thr Leu Lys Pro Ala Leu Cys Ile Leu Glu Ser His 100 105 110 Lys Glu Asp Phe Leu Asn Lys Phe Leu Leu Thr Ile Ser Ser Glu Asn 115 120 125 Leu Lys Leu Gln Tyr Lys Phe Asn Gly Gln Ile Lys Asn Pro Lys Ser 130 135 140 Val Asn Glu Ile Trp Thr Asp Leu Phe Ser Ile Ala His Val Asp Met 145 150 155 160 Lys Leu Ser Thr Asp Arg Thr Leu Ser Ser Ser Ile Ser Gln Phe Trp 165 170 175 Phe Arg Leu Glu Phe Cys Lys Glu Asp Lys Asp Phe Ile Leu Phe Pro 180 185 190 Thr Ala Asn Arg Tyr Ser Arg Lys Leu Trp Lys His Ser Ile Lys Asn 195 200 205 Asn Gln Leu Phe Lys Glu Gly Ile Arg Asn Tyr Ser Glu Ile Ser Ser 210 215 220 Leu Pro Tyr Glu Glu Asp His Asn Phe Asp Ile Asp Leu Val Phe Thr 225 230 235 240 Trp Val Asn Ser Glu Asp Lys Asn Trp Gln Glu Leu Tyr Lys Lys Tyr 245 250 255 Lys Pro Asp Phe Asn Ser Asp Ala Thr Ser Thr Ser Arg Phe Leu Ser 260 265 270 Arg Asp Glu Leu Lys Phe Ala Leu Arg Ser Trp Glu Met Asn Gly Ser 275 280 285 Phe Ile Arg Lys Ile Phe Ile Val Ser Asn Cys Ala Pro Pro Ala Trp 290 295 300 Leu Asp Leu Asn Asn Pro Lys Ile Gln Trp Val Tyr His Glu Glu Ile 305 310 315 320 Met Pro Gln Ser Ala Leu Pro Thr Phe Ser Ser His Ala Ile Glu Thr 325 330 335 Ser Leu His His Ile Pro Gly Ile Ser Asn Tyr Phe Ile Tyr Ser Asn 340 345 350 Asp Asp Phe Leu Leu Thr Lys Pro Leu Asn Lys Asp Asn Phe Phe Tyr

355 360 365 Ser Asn Gly Ile Ala Lys Leu Arg Leu Glu Ala Trp Gly Asn Val Asn 370 375 380 Gly Glu Cys Thr Glu Gly Glu Pro Asp Tyr Leu Asn Gly Ala Arg Asn 385 390 395 400 Ala Asn Thr Leu Leu Glu Lys Glu Phe Lys Lys Phe Thr Thr Lys Leu 405 410 415 His Thr His Ser Pro Gln Ser Met Arg Thr Asp Ile Leu Phe Glu Met 420 425 430 Glu Lys Lys Tyr Pro Glu Glu Phe Asn Arg Thr Leu His Asn Lys Phe 435 440 445 Arg Ser Leu Asp Asp Ile Ala Val Thr Gly Tyr Leu Tyr His His Tyr 450 455 460 Ala Leu Leu Ser Gly Arg Ala Leu Gln Ser Ser Asp Lys Thr Glu Leu 465 470 475 480 Val Gln Gln Asn His Asp Phe Lys Lys Lys Leu Asn Asn Val Val Thr 485 490 495 Leu Thr Lys <210> SEQ ID NO 15 <211> LENGTH: 519 <212> TYPE: PRT <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: AS deltaC25-CP-A (519) <400> SEQUENCE: 15 Phe Ile Leu Asn Asn Arg Lys Trp Arg Lys Leu Lys Arg Asp Pro Ser 1 5 10 15 Ala Phe Phe Arg Asp Ser Lys Phe Asn Phe Leu Arg Tyr Phe Ser Ala 20 25 30 Lys Lys Phe Ala Lys Asn Phe Lys Asn Ser Ser His Ile His Lys Thr 35 40 45 Asn Ile Ser Lys Ala Gln Ser Asn Ile Ser Ser Thr Leu Lys Gln Asn 50 55 60 Arg Lys Gln Asp Met Leu Ile Pro Ile Asn Phe Phe Asn Phe Glu Tyr 65 70 75 80 Ile Val Lys Lys Leu Asn Asn Gln Asn Ala Ile Gly Val Tyr Ile Leu 85 90 95 Pro Ser Asn Leu Thr Leu Lys Pro Ala Leu Cys Ile Leu Glu Ser His 100 105 110 Lys Glu Asp Phe Leu Asn Lys Phe Leu Leu Thr Ile Ser Ser Glu Asn 115 120 125 Leu Lys Leu Gln Tyr Lys Phe Asn Gly Gln Ile Lys Asn Pro Lys Ser 130 135 140 Val Asn Glu Ile Trp Thr Asp Leu Phe Ser Ile Ala His Val Asp Met 145 150 155 160 Lys Leu Ser Thr Asp Arg Thr Leu Ser Ser Ser Ile Ser Gln Phe Trp 165 170 175 Phe Arg Leu Glu Phe Cys Lys Glu Asp Lys Asp Phe Ile Leu Phe Pro 180 185 190 Thr Ala Asn Arg Tyr Ser Arg Lys Leu Trp Lys His Ser Ile Lys Asn 195 200 205 Asn Gln Leu Phe Lys Glu Gly Ile Arg Asn Tyr Ser Glu Ile Ser Ser 210 215 220 Leu Pro Tyr Glu Glu Asp His Asn Phe Asp Ile Asp Leu Val Phe Thr 225 230 235 240 Trp Val Asn Ser Glu Asp Lys Asn Trp Gln Glu Leu Tyr Lys Lys Tyr 245 250 255 Lys Pro Asp Phe Asn Ser Asp Ala Thr Ser Thr Ser Arg Phe Leu Ser 260 265 270 Arg Asp Glu Leu Lys Phe Ala Leu Arg Ser Trp Glu Met Asn Gly Ser 275 280 285 Phe Ile Arg Lys Ile Phe Ile Val Ser Asn Cys Ala Pro Pro Ala Trp 290 295 300 Leu Asp Leu Asn Asn Pro Lys Ile Gln Trp Val Tyr His Glu Glu Ile 305 310 315 320 Met Pro Gln Ser Ala Leu Pro Thr Phe Ser Ser His Ala Ile Glu Thr 325 330 335 Ser Leu His His Ile Pro Gly Ile Ser Asn Tyr Phe Ile Tyr Ser Asn 340 345 350 Asp Asp Phe Leu Leu Thr Lys Pro Leu Asn Lys Asp Asn Phe Phe Tyr 355 360 365 Ser Asn Gly Ile Ala Lys Leu Arg Leu Glu Ala Trp Gly Asn Val Asn 370 375 380 Gly Glu Cys Thr Glu Gly Glu Pro Asp Tyr Leu Asn Gly Ala Arg Asn 385 390 395 400 Ala Asn Thr Leu Leu Glu Lys Glu Phe Lys Lys Phe Thr Thr Lys Leu 405 410 415 His Thr His Ser Pro Gln Ser Met Arg Thr Asp Ile Leu Phe Glu Met 420 425 430 Glu Lys Lys Tyr Pro Glu Glu Phe Asn Arg Thr Leu His Asn Lys Phe 435 440 445 Arg Ser Leu Asp Asp Ile Ala Val Thr Gly Tyr Leu Tyr His His Tyr 450 455 460 Ala Leu Leu Ser Gly Arg Ala Leu Gln Ser Ser Asp Lys Thr Glu Leu 465 470 475 480 Val Gln Gln Asn His Asp Phe Lys Lys Lys Leu Asn Asn Val Val Thr 485 490 495 Leu Thr Lys Glu Arg Asn Phe Asp Lys Leu Pro Leu Ser Val Cys Ile 500 505 510 Asn Asp Gly Ala Asp Ser His 515 <210> SEQ ID NO 16 <211> LENGTH: 1455 <212> TYPE: DNA <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: DNA CP-X/CsxA full-length (1455) <400> SEQUENCE: 16 attatgagca aaattagcaa attggtaacc cacccaaacc ttttctttcg agattatttc 60 ttaaaaaaag caccgttaaa ttatggcgaa aatattaaac ctttaccagt cgaaacctct 120 tctcatagca aaaaaaatac agcccataaa acacccgtat catccgacca accaattgaa 180 gatccatacc cagtaacatt tccaattgat gtagtttata cttgggtaga ttcagatgat 240 gaaaaattca atgaagaacg cctaaagttt caaaattcaa gcacatctga gactctacaa 300 ggcaaagcag aaagcaccga tattgcaaga ttccaatcac gcgacgaatt aaaatattcg 360 attcgaagcc tgatgaagta tgccccatgg gtaaatcata tttacattgt aacaaatggt 420 caaataccaa aatggttaga taccaacaat acaaaggtaa cgattatccc tcactcaact 480 attatcgaca gtcaatttct ccctactttt aattctcacg tcattgaatc ctctctatat 540 aaaatcccag gattatcaga gcattacatt tatttcaatg atgatgtcat gctagctaga 600 gatttaagcc catcttattt ctttacaagc agcggattag caaaactgtt tattaccaac 660 tctcgtctac caaatggcta taagaatgtg aaagacacac caacccaatg ggcctcaaaa 720 aattcccgtg agcttttaca tgcagaaaca ggattttggg ctgaagccat gtttgcacat 780 acttttcatc cacaacgtaa aagtgtacat gaatctattg aacacctatg gcatgaacaa 840 ttaaatgttt gtcgtcaaaa ccgtttccgt gatatttcag atattaacat ggcgacattc 900 ctgcaccacc attttgccat tttgacaggc caagctcttg ctacacgcac taaatgtatt 960 tactttaaca ttcgctctcc tcaagcagct cagcattaca aaacattatt agctcgaaaa 1020 ggaagcgaat acagcccaca ttctatctgc ttaaatgatc atacatcgag caataaaaat 1080 attttatcta attacgaagc caaattacaa agctttttag aaacatacta tccagatgta 1140 tcagaagcag aaattctcct tcctactaaa tctgaagtag ctgaattagt taaacataaa 1200 gattatttaa ctgtatatac taaattatta cctattatca ataagcagct ggtcaataaa 1260 tataataaac cttattcata tcttttctat tatttaggtt tatctgcccg gtttttattt 1320 gaagaaacgc aacaagaaca ctaccgggaa actgctgaag aaaatttaca aatcttttgt 1380 ggcctaaacc caaaacatac actagccctc aaatacttag cggatgtcac cctcacatca 1440 cagcctagtg gacaa 1455 <210> SEQ ID NO 17 <211> LENGTH: 1284 <212> TYPE: DNA <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: DNA deltaN58-CP-X (1284) <400> SEQUENCE: 17 ccaattgaag atccataccc agtaacattt ccaattgatg tagtttatac ttgggtagat 60 tcagatgatg aaaaattcaa tgaagaacgc ctaaagtttc aaaattcaag cacatctgag 120 actctacaag gcaaagcaga aagcaccgat attgcaagat tccaatcacg cgacgaatta 180 aaatattcga ttcgaagcct gatgaagtat gccccatggg taaatcatat ttacattgta 240 acaaatggtc aaataccaaa atggttagat accaacaata caaaggtaac gattatccct 300 cactcaacta ttatcgacag tcaatttctc cctactttta attctcacgt cattgaatcc 360 tctctatata aaatcccagg attatcagag cattacattt atttcaatga tgatgtcatg 420 ctagctagag atttaagccc atcttatttc tttacaagca gcggattagc aaaactgttt 480 attaccaact ctcgtctacc aaatggctat aagaatgtga aagacacacc aacccaatgg 540 gcctcaaaaa attcccgtga gcttttacat gcagaaacag gattttgggc tgaagccatg 600 tttgcacata cttttcatcc acaacgtaaa agtgtacatg aatctattga acacctatgg 660 catgaacaat taaatgtttg tcgtcaaaac cgtttccgtg atatttcaga tattaacatg 720 gcgacattcc tgcaccacca ttttgccatt ttgacaggcc aagctcttgc tacacgcact 780 aaatgtattt actttaacat tcgctctcct caagcagctc agcattacaa aacattatta 840 gctcgaaaag gaagcgaata cagcccacat tctatctgct taaatgatca tacatcgagc 900 aataaaaata ttttatctaa ttacgaagcc aaattacaaa gctttttaga aacatactat 960 ccagatgtat cagaagcaga aattctcctt cctactaaat ctgaagtagc tgaattagtt 1020 aaacataaag attatttaac tgtatatact aaattattac ctattatcaa taagcagctg 1080 gtcaataaat ataataaacc ttattcatat cttttctatt atttaggttt atctgcccgg 1140 tttttatttg aagaaacgca acaagaacac taccgggaaa ctgctgaaga aaatttacaa 1200 atcttttgtg gcctaaaccc aaaacataca ctagccctca aatacttagc ggatgtcacc 1260 ctcacatcac agcctagtgg acaa 1284

<210> SEQ ID NO 18 <211> LENGTH: 1146 <212> TYPE: DNA <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: DNA deltaN104-CP-X (1146) <400> SEQUENCE: 18 gaaagcaccg atattgcaag attccaatca cgcgacgaat taaaatattc gattcgaagc 60 ctgatgaagt atgccccatg ggtaaatcat atttacattg taacaaatgg tcaaatacca 120 aaatggttag ataccaacaa tacaaaggta acgattatcc ctcactcaac tattatcgac 180 agtcaatttc tccctacttt taattctcac gtcattgaat cctctctata taaaatccca 240 ggattatcag agcattacat ttatttcaat gatgatgtca tgctagctag agatttaagc 300 ccatcttatt tctttacaag cagcggatta gcaaaactgt ttattaccaa ctctcgtcta 360 ccaaatggct ataagaatgt gaaagacaca ccaacccaat gggcctcaaa aaattcccgt 420 gagcttttac atgcagaaac aggattttgg gctgaagcca tgtttgcaca tacttttcat 480 ccacaacgta aaagtgtaca tgaatctatt gaacacctat ggcatgaaca attaaatgtt 540 tgtcgtcaaa accgtttccg tgatatttca gatattaaca tggcgacatt cctgcaccac 600 cattttgcca ttttgacagg ccaagctctt gctacacgca ctaaatgtat ttactttaac 660 attcgctctc ctcaagcagc tcagcattac aaaacattat tagctcgaaa aggaagcgaa 720 tacagcccac attctatctg cttaaatgat catacatcga gcaataaaaa tattttatct 780 aattacgaag ccaaattaca aagcttttta gaaacatact atccagatgt atcagaagca 840 gaaattctcc ttcctactaa atctgaagta gctgaattag ttaaacataa agattattta 900 actgtatata ctaaattatt acctattatc aataagcagc tggtcaataa atataataaa 960 ccttattcat atcttttcta ttatttaggt ttatctgccc ggtttttatt tgaagaaacg 1020 caacaagaac actaccggga aactgctgaa gaaaatttac aaatcttttg tggcctaaac 1080 ccaaaacata cactagccct caaatactta gcggatgtca ccctcacatc acagcctagt 1140 ggacaa 1146 <210> SEQ ID NO 19 <211> LENGTH: 933 <212> TYPE: DNA <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: DNA deltaC174-CP-X (933) <400> SEQUENCE: 19 attatgagca aaattagcaa attggtaacc cacccaaacc ttttctttcg agattatttc 60 ttaaaaaaag caccgttaaa ttatggcgaa aatattaaac ctttaccagt cgaaacctct 120 tctcatagca aaaaaaatac agcccataaa acacccgtat catccgacca accaattgaa 180 gatccatacc cagtaacatt tccaattgat gtagtttata cttgggtaga ttcagatgat 240 gaaaaattca atgaagaacg cctaaagttt caaaattcaa gcacatctga gactctacaa 300 ggcaaagcag aaagcaccga tattgcaaga ttccaatcac gcgacgaatt aaaatattcg 360 attcgaagcc tgatgaagta tgccccatgg gtaaatcata tttacattgt aacaaatggt 420 caaataccaa aatggttaga taccaacaat acaaaggtaa cgattatccc tcactcaact 480 attatcgaca gtcaatttct ccctactttt aattctcacg tcattgaatc ctctctatat 540 aaaatcccag gattatcaga gcattacatt tatttcaatg atgatgtcat gctagctaga 600 gatttaagcc catcttattt ctttacaagc agcggattag caaaactgtt tattaccaac 660 tctcgtctac caaatggcta taagaatgtg aaagacacac caacccaatg ggcctcaaaa 720 aattcccgtg agcttttaca tgcagaaaca ggattttggg ctgaagccat gtttgcacat 780 acttttcatc cacaacgtaa aagtgtacat gaatctattg aacacctatg gcatgaacaa 840 ttaaatgttt gtcgtcaaaa ccgtttccgt gatatttcag atattaacat ggcgacattc 900 ctgcaccacc attttgccat tttgacaggc caa 933 <210> SEQ ID NO 20 <211> LENGTH: 1158 <212> TYPE: DNA <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: DNA deltaC99-CP-X (1158) <400> SEQUENCE: 20 attatgagca aaattagcaa attggtaacc cacccaaacc ttttctttcg agattatttc 60 ttaaaaaaag caccgttaaa ttatggcgaa aatattaaac ctttaccagt cgaaacctct 120 tctcatagca aaaaaaatac agcccataaa acacccgtat catccgacca accaattgaa 180 gatccatacc cagtaacatt tccaattgat gtagtttata cttgggtaga ttcagatgat 240 gaaaaattca atgaagaacg cctaaagttt caaaattcaa gcacatctga gactctacaa 300 ggcaaagcag aaagcaccga tattgcaaga ttccaatcac gcgacgaatt aaaatattcg 360 attcgaagcc tgatgaagta tgccccatgg gtaaatcata tttacattgt aacaaatggt 420 caaataccaa aatggttaga taccaacaat acaaaggtaa cgattatccc tcactcaact 480 attatcgaca gtcaatttct ccctactttt aattctcacg tcattgaatc ctctctatat 540 aaaatcccag gattatcaga gcattacatt tatttcaatg atgatgtcat gctagctaga 600 gatttaagcc catcttattt ctttacaagc agcggattag caaaactgtt tattaccaac 660 tctcgtctac caaatggcta taagaatgtg aaagacacac caacccaatg ggcctcaaaa 720 aattcccgtg agcttttaca tgcagaaaca ggattttggg ctgaagccat gtttgcacat 780 acttttcatc cacaacgtaa aagtgtacat gaatctattg aacacctatg gcatgaacaa 840 ttaaatgttt gtcgtcaaaa ccgtttccgt gatatttcag atattaacat ggcgacattc 900 ctgcaccacc attttgccat tttgacaggc caagctcttg ctacacgcac taaatgtatt 960 tactttaaca ttcgctctcc tcaagcagct cagcattaca aaacattatt agctcgaaaa 1020 ggaagcgaat acagcccaca ttctatctgc ttaaatgatc atacatcgag caataaaaat 1080 attttatcta attacgaagc caaattacaa agctttttag aaacatacta tccagatgta 1140 tcagaagcag aaattctc 1158 <210> SEQ ID NO 21 <211> LENGTH: 987 <212> TYPE: DNA <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: DNA deltaN58 deltaC99-CP-X (987) <400> SEQUENCE: 21 ccaattgaag atccataccc agtaacattt ccaattgatg tagtttatac ttgggtagat 60 tcagatgatg aaaaattcaa tgaagaacgc ctaaagtttc aaaattcaag cacatctgag 120 actctacaag gcaaagcaga aagcaccgat attgcaagat tccaatcacg cgacgaatta 180 aaatattcga ttcgaagcct gatgaagtat gccccatggg taaatcatat ttacattgta 240 acaaatggtc aaataccaaa atggttagat accaacaata caaaggtaac gattatccct 300 cactcaacta ttatcgacag tcaatttctc cctactttta attctcacgt cattgaatcc 360 tctctatata aaatcccagg attatcagag cattacattt atttcaatga tgatgtcatg 420 ctagctagag atttaagccc atcttatttc tttacaagca gcggattagc aaaactgttt 480 attaccaact ctcgtctacc aaatggctat aagaatgtga aagacacacc aacccaatgg 540 gcctcaaaaa attcccgtga gcttttacat gcagaaacag gattttgggc tgaagccatg 600 tttgcacata cttttcatcc acaacgtaaa agtgtacatg aatctattga acacctatgg 660 catgaacaat taaatgtttg tcgtcaaaac cgtttccgtg atatttcaga tattaacatg 720 gcgacattcc tgcaccacca ttttgccatt ttgacaggcc aagctcttgc tacacgcact 780 aaatgtattt actttaacat tcgctctcct caagcagctc agcattacaa aacattatta 840 gctcgaaaag gaagcgaata cagcccacat tctatctgct taaatgatca tacatcgagc 900 aataaaaata ttttatctaa ttacgaagcc aaattacaaa gctttttaga aacatactat 960 ccagatgtat cagaagcaga aattctc 987 <210> SEQ ID NO 22 <211> LENGTH: 1233 <212> TYPE: DNA <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: DNA deltaN65deltaC10-CP-X (1233) <400> SEQUENCE: 22 gtaacatttc caattgatgt agtttatact tgggtagatt cagatgatga aaaattcaat 60 gaagaacgcc taaagtttca aaattcaagc acatctgaga ctctacaagg caaagcagaa 120 agcaccgata ttgcaagatt ccaatcacgc gacgaattaa aatattcgat tcgaagcctg 180 atgaagtatg ccccatgggt aaatcatatt tacattgtaa caaatggtca aataccaaaa 240 tggttagata ccaacaatac aaaggtaacg attatccctc actcaactat tatcgacagt 300 caatttctcc ctacttttaa ttctcacgtc attgaatcct ctctatataa aatcccagga 360 ttatcagagc attacattta tttcaatgat gatgtcatgc tagctagaga tttaagccca 420 tcttatttct ttacaagcag cggattagca aaactgttta ttaccaactc tcgtctacca 480 aatggctata agaatgtgaa agacacacca acccaatggg cctcaaaaaa ttcccgtgag 540 cttttacatg cagaaacagg attttgggct gaagccatgt ttgcacatac ttttcatcca 600 caacgtaaaa gtgtacatga atctattgaa cacctatggc atgaacaatt aaatgtttgt 660 cgtcaaaacc gtttccgtga tatttcagat attaacatgg cgacattcct gcaccaccat 720 tttgccattt tgacaggcca agctcttgct acacgcacta aatgtattta ctttaacatt 780 cgctctcctc aagcagctca gcattacaaa acattattag ctcgaaaagg aagcgaatac 840 agcccacatt ctatctgctt aaatgatcat acatcgagca ataaaaatat tttatctaat 900 tacgaagcca aattacaaag ctttttagaa acatactatc cagatgtatc agaagcagaa 960 attctccttc ctactaaatc tgaagtagct gaattagtta aacataaaga ttatttaact 1020 gtatatacta aattattacc tattatcaat aagcagctgg tcaataaata taataaacct 1080 tattcatatc ttttctatta tttaggttta tctgcccggt ttttatttga agaaacgcaa 1140 caagaacact accgggaaac tgctgaagaa aatttacaaa tcttttgtgg cctaaaccca 1200 aaacatacac tagccctcaa atacttagcg gat 1233 <210> SEQ ID NO 23 <211> LENGTH: 960 <212> TYPE: DNA <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: DNA deltaN67deltaC99-CP-X (960) <400> SEQUENCE: 23 tttccaattg atgtagttta tacttgggta gattcagatg atgaaaaatt caatgaagaa 60 cgcctaaagt ttcaaaattc aagcacatct gagactctac aaggcaaagc agaaagcacc 120

gatattgcaa gattccaatc acgcgacgaa ttaaaatatt cgattcgaag cctgatgaag 180 tatgccccat gggtaaatca tatttacatt gtaacaaatg gtcaaatacc aaaatggtta 240 gataccaaca atacaaaggt aacgattatc cctcactcaa ctattatcga cagtcaattt 300 ctccctactt ttaattctca cgtcattgaa tcctctctat ataaaatccc aggattatca 360 gagcattaca tttatttcaa tgatgatgtc atgctagcta gagatttaag cccatcttat 420 ttctttacaa gcagcggatt agcaaaactg tttattacca actctcgtct accaaatggc 480 tataagaatg tgaaagacac accaacccaa tgggcctcaa aaaattcccg tgagctttta 540 catgcagaaa caggattttg ggctgaagcc atgtttgcac atacttttca tccacaacgt 600 aaaagtgtac atgaatctat tgaacaccta tggcatgaac aattaaatgt ttgtcgtcaa 660 aaccgtttcc gtgatatttc agatattaac atggcgacat tcctgcacca ccattttgcc 720 attttgacag gccaagctct tgctacacgc actaaatgta tttactttaa cattcgctct 780 cctcaagcag ctcagcatta caaaacatta ttagctcgaa aaggaagcga atacagccca 840 cattctatct gcttaaatga tcatacatcg agcaataaaa atattttatc taattacgaa 900 gccaaattac aaagcttttt agaaacatac tatccagatg tatcagaagc agaaattctc 960 <210> SEQ ID NO 24 <211> LENGTH: 485 <212> TYPE: PRT <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: AS CP-X/CsxA full-length (485) <400> SEQUENCE: 24 Ile Met Ser Lys Ile Ser Lys Leu Val Thr His Pro Asn Leu Phe Phe 1 5 10 15 Arg Asp Tyr Phe Leu Lys Lys Ala Pro Leu Asn Tyr Gly Glu Asn Ile 20 25 30 Lys Pro Leu Pro Val Glu Thr Ser Ser His Ser Lys Lys Asn Thr Ala 35 40 45 His Lys Thr Pro Val Ser Ser Asp Gln Pro Ile Glu Asp Pro Tyr Pro 50 55 60 Val Thr Phe Pro Ile Asp Val Val Tyr Thr Trp Val Asp Ser Asp Asp 65 70 75 80 Glu Lys Phe Asn Glu Glu Arg Leu Lys Phe Gln Asn Ser Ser Thr Ser 85 90 95 Glu Thr Leu Gln Gly Lys Ala Glu Ser Thr Asp Ile Ala Arg Phe Gln 100 105 110 Ser Arg Asp Glu Leu Lys Tyr Ser Ile Arg Ser Leu Met Lys Tyr Ala 115 120 125 Pro Trp Val Asn His Ile Tyr Ile Val Thr Asn Gly Gln Ile Pro Lys 130 135 140 Trp Leu Asp Thr Asn Asn Thr Lys Val Thr Ile Ile Pro His Ser Thr 145 150 155 160 Ile Ile Asp Ser Gln Phe Leu Pro Thr Phe Asn Ser His Val Ile Glu 165 170 175 Ser Ser Leu Tyr Lys Ile Pro Gly Leu Ser Glu His Tyr Ile Tyr Phe 180 185 190 Asn Asp Asp Val Met Leu Ala Arg Asp Leu Ser Pro Ser Tyr Phe Phe 195 200 205 Thr Ser Ser Gly Leu Ala Lys Leu Phe Ile Thr Asn Ser Arg Leu Pro 210 215 220 Asn Gly Tyr Lys Asn Val Lys Asp Thr Pro Thr Gln Trp Ala Ser Lys 225 230 235 240 Asn Ser Arg Glu Leu Leu His Ala Glu Thr Gly Phe Trp Ala Glu Ala 245 250 255 Met Phe Ala His Thr Phe His Pro Gln Arg Lys Ser Val His Glu Ser 260 265 270 Ile Glu His Leu Trp His Glu Gln Leu Asn Val Cys Arg Gln Asn Arg 275 280 285 Phe Arg Asp Ile Ser Asp Ile Asn Met Ala Thr Phe Leu His His His 290 295 300 Phe Ala Ile Leu Thr Gly Gln Ala Leu Ala Thr Arg Thr Lys Cys Ile 305 310 315 320 Tyr Phe Asn Ile Arg Ser Pro Gln Ala Ala Gln His Tyr Lys Thr Leu 325 330 335 Leu Ala Arg Lys Gly Ser Glu Tyr Ser Pro His Ser Ile Cys Leu Asn 340 345 350 Asp His Thr Ser Ser Asn Lys Asn Ile Leu Ser Asn Tyr Glu Ala Lys 355 360 365 Leu Gln Ser Phe Leu Glu Thr Tyr Tyr Pro Asp Val Ser Glu Ala Glu 370 375 380 Ile Leu Leu Pro Thr Lys Ser Glu Val Ala Glu Leu Val Lys His Lys 385 390 395 400 Asp Tyr Leu Thr Val Tyr Thr Lys Leu Leu Pro Ile Ile Asn Lys Gln 405 410 415 Leu Val Asn Lys Tyr Asn Lys Pro Tyr Ser Tyr Leu Phe Tyr Tyr Leu 420 425 430 Gly Leu Ser Ala Arg Phe Leu Phe Glu Glu Thr Gln Gln Glu His Tyr 435 440 445 Arg Glu Thr Ala Glu Glu Asn Leu Gln Ile Phe Cys Gly Leu Asn Pro 450 455 460 Lys His Thr Leu Ala Leu Lys Tyr Leu Ala Asp Val Thr Leu Thr Ser 465 470 475 480 Gln Pro Ser Gly Gln 485 <210> SEQ ID NO 25 <211> LENGTH: 428 <212> TYPE: PRT <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: AS deltaN58-CP-X (428) <400> SEQUENCE: 25 Pro Ile Glu Asp Pro Tyr Pro Val Thr Phe Pro Ile Asp Val Val Tyr 1 5 10 15 Thr Trp Val Asp Ser Asp Asp Glu Lys Phe Asn Glu Glu Arg Leu Lys 20 25 30 Phe Gln Asn Ser Ser Thr Ser Glu Thr Leu Gln Gly Lys Ala Glu Ser 35 40 45 Thr Asp Ile Ala Arg Phe Gln Ser Arg Asp Glu Leu Lys Tyr Ser Ile 50 55 60 Arg Ser Leu Met Lys Tyr Ala Pro Trp Val Asn His Ile Tyr Ile Val 65 70 75 80 Thr Asn Gly Gln Ile Pro Lys Trp Leu Asp Thr Asn Asn Thr Lys Val 85 90 95 Thr Ile Ile Pro His Ser Thr Ile Ile Asp Ser Gln Phe Leu Pro Thr 100 105 110 Phe Asn Ser His Val Ile Glu Ser Ser Leu Tyr Lys Ile Pro Gly Leu 115 120 125 Ser Glu His Tyr Ile Tyr Phe Asn Asp Asp Val Met Leu Ala Arg Asp 130 135 140 Leu Ser Pro Ser Tyr Phe Phe Thr Ser Ser Gly Leu Ala Lys Leu Phe 145 150 155 160 Ile Thr Asn Ser Arg Leu Pro Asn Gly Tyr Lys Asn Val Lys Asp Thr 165 170 175 Pro Thr Gln Trp Ala Ser Lys Asn Ser Arg Glu Leu Leu His Ala Glu 180 185 190 Thr Gly Phe Trp Ala Glu Ala Met Phe Ala His Thr Phe His Pro Gln 195 200 205 Arg Lys Ser Val His Glu Ser Ile Glu His Leu Trp His Glu Gln Leu 210 215 220 Asn Val Cys Arg Gln Asn Arg Phe Arg Asp Ile Ser Asp Ile Asn Met 225 230 235 240 Ala Thr Phe Leu His His His Phe Ala Ile Leu Thr Gly Gln Ala Leu 245 250 255 Ala Thr Arg Thr Lys Cys Ile Tyr Phe Asn Ile Arg Ser Pro Gln Ala 260 265 270 Ala Gln His Tyr Lys Thr Leu Leu Ala Arg Lys Gly Ser Glu Tyr Ser 275 280 285 Pro His Ser Ile Cys Leu Asn Asp His Thr Ser Ser Asn Lys Asn Ile 290 295 300 Leu Ser Asn Tyr Glu Ala Lys Leu Gln Ser Phe Leu Glu Thr Tyr Tyr 305 310 315 320 Pro Asp Val Ser Glu Ala Glu Ile Leu Leu Pro Thr Lys Ser Glu Val 325 330 335 Ala Glu Leu Val Lys His Lys Asp Tyr Leu Thr Val Tyr Thr Lys Leu 340 345 350 Leu Pro Ile Ile Asn Lys Gln Leu Val Asn Lys Tyr Asn Lys Pro Tyr 355 360 365 Ser Tyr Leu Phe Tyr Tyr Leu Gly Leu Ser Ala Arg Phe Leu Phe Glu 370 375 380 Glu Thr Gln Gln Glu His Tyr Arg Glu Thr Ala Glu Glu Asn Leu Gln 385 390 395 400 Ile Phe Cys Gly Leu Asn Pro Lys His Thr Leu Ala Leu Lys Tyr Leu 405 410 415 Ala Asp Val Thr Leu Thr Ser Gln Pro Ser Gly Gln 420 425 <210> SEQ ID NO 26 <211> LENGTH: 382 <212> TYPE: PRT <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: AS deltaN104-CP-X (382) <400> SEQUENCE: 26 Glu Ser Thr Asp Ile Ala Arg Phe Gln Ser Arg Asp Glu Leu Lys Tyr 1 5 10 15 Ser Ile Arg Ser Leu Met Lys Tyr Ala Pro Trp Val Asn His Ile Tyr 20 25 30 Ile Val Thr Asn Gly Gln Ile Pro Lys Trp Leu Asp Thr Asn Asn Thr 35 40 45 Lys Val Thr Ile Ile Pro His Ser Thr Ile Ile Asp Ser Gln Phe Leu 50 55 60 Pro Thr Phe Asn Ser His Val Ile Glu Ser Ser Leu Tyr Lys Ile Pro 65 70 75 80 Gly Leu Ser Glu His Tyr Ile Tyr Phe Asn Asp Asp Val Met Leu Ala 85 90 95

Arg Asp Leu Ser Pro Ser Tyr Phe Phe Thr Ser Ser Gly Leu Ala Lys 100 105 110 Leu Phe Ile Thr Asn Ser Arg Leu Pro Asn Gly Tyr Lys Asn Val Lys 115 120 125 Asp Thr Pro Thr Gln Trp Ala Ser Lys Asn Ser Arg Glu Leu Leu His 130 135 140 Ala Glu Thr Gly Phe Trp Ala Glu Ala Met Phe Ala His Thr Phe His 145 150 155 160 Pro Gln Arg Lys Ser Val His Glu Ser Ile Glu His Leu Trp His Glu 165 170 175 Gln Leu Asn Val Cys Arg Gln Asn Arg Phe Arg Asp Ile Ser Asp Ile 180 185 190 Asn Met Ala Thr Phe Leu His His His Phe Ala Ile Leu Thr Gly Gln 195 200 205 Ala Leu Ala Thr Arg Thr Lys Cys Ile Tyr Phe Asn Ile Arg Ser Pro 210 215 220 Gln Ala Ala Gln His Tyr Lys Thr Leu Leu Ala Arg Lys Gly Ser Glu 225 230 235 240 Tyr Ser Pro His Ser Ile Cys Leu Asn Asp His Thr Ser Ser Asn Lys 245 250 255 Asn Ile Leu Ser Asn Tyr Glu Ala Lys Leu Gln Ser Phe Leu Glu Thr 260 265 270 Tyr Tyr Pro Asp Val Ser Glu Ala Glu Ile Leu Leu Pro Thr Lys Ser 275 280 285 Glu Val Ala Glu Leu Val Lys His Lys Asp Tyr Leu Thr Val Tyr Thr 290 295 300 Lys Leu Leu Pro Ile Ile Asn Lys Gln Leu Val Asn Lys Tyr Asn Lys 305 310 315 320 Pro Tyr Ser Tyr Leu Phe Tyr Tyr Leu Gly Leu Ser Ala Arg Phe Leu 325 330 335 Phe Glu Glu Thr Gln Gln Glu His Tyr Arg Glu Thr Ala Glu Glu Asn 340 345 350 Leu Gln Ile Phe Cys Gly Leu Asn Pro Lys His Thr Leu Ala Leu Lys 355 360 365 Tyr Leu Ala Asp Val Thr Leu Thr Ser Gln Pro Ser Gly Gln 370 375 380 <210> SEQ ID NO 27 <211> LENGTH: 311 <212> TYPE: PRT <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: AS deltaC174-CP-X (311) <400> SEQUENCE: 27 Ile Met Ser Lys Ile Ser Lys Leu Val Thr His Pro Asn Leu Phe Phe 1 5 10 15 Arg Asp Tyr Phe Leu Lys Lys Ala Pro Leu Asn Tyr Gly Glu Asn Ile 20 25 30 Lys Pro Leu Pro Val Glu Thr Ser Ser His Ser Lys Lys Asn Thr Ala 35 40 45 His Lys Thr Pro Val Ser Ser Asp Gln Pro Ile Glu Asp Pro Tyr Pro 50 55 60 Val Thr Phe Pro Ile Asp Val Val Tyr Thr Trp Val Asp Ser Asp Asp 65 70 75 80 Glu Lys Phe Asn Glu Glu Arg Leu Lys Phe Gln Asn Ser Ser Thr Ser 85 90 95 Glu Thr Leu Gln Gly Lys Ala Glu Ser Thr Asp Ile Ala Arg Phe Gln 100 105 110 Ser Arg Asp Glu Leu Lys Tyr Ser Ile Arg Ser Leu Met Lys Tyr Ala 115 120 125 Pro Trp Val Asn His Ile Tyr Ile Val Thr Asn Gly Gln Ile Pro Lys 130 135 140 Trp Leu Asp Thr Asn Asn Thr Lys Val Thr Ile Ile Pro His Ser Thr 145 150 155 160 Ile Ile Asp Ser Gln Phe Leu Pro Thr Phe Asn Ser His Val Ile Glu 165 170 175 Ser Ser Leu Tyr Lys Ile Pro Gly Leu Ser Glu His Tyr Ile Tyr Phe 180 185 190 Asn Asp Asp Val Met Leu Ala Arg Asp Leu Ser Pro Ser Tyr Phe Phe 195 200 205 Thr Ser Ser Gly Leu Ala Lys Leu Phe Ile Thr Asn Ser Arg Leu Pro 210 215 220 Asn Gly Tyr Lys Asn Val Lys Asp Thr Pro Thr Gln Trp Ala Ser Lys 225 230 235 240 Asn Ser Arg Glu Leu Leu His Ala Glu Thr Gly Phe Trp Ala Glu Ala 245 250 255 Met Phe Ala His Thr Phe His Pro Gln Arg Lys Ser Val His Glu Ser 260 265 270 Ile Glu His Leu Trp His Glu Gln Leu Asn Val Cys Arg Gln Asn Arg 275 280 285 Phe Arg Asp Ile Ser Asp Ile Asn Met Ala Thr Phe Leu His His His 290 295 300 Phe Ala Ile Leu Thr Gly Gln 305 310 <210> SEQ ID NO 28 <211> LENGTH: 386 <212> TYPE: PRT <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: AS deltaC99-CP-X (386) <400> SEQUENCE: 28 Ile Met Ser Lys Ile Ser Lys Leu Val Thr His Pro Asn Leu Phe Phe 1 5 10 15 Arg Asp Tyr Phe Leu Lys Lys Ala Pro Leu Asn Tyr Gly Glu Asn Ile 20 25 30 Lys Pro Leu Pro Val Glu Thr Ser Ser His Ser Lys Lys Asn Thr Ala 35 40 45 His Lys Thr Pro Val Ser Ser Asp Gln Pro Ile Glu Asp Pro Tyr Pro 50 55 60 Val Thr Phe Pro Ile Asp Val Val Tyr Thr Trp Val Asp Ser Asp Asp 65 70 75 80 Glu Lys Phe Asn Glu Glu Arg Leu Lys Phe Gln Asn Ser Ser Thr Ser 85 90 95 Glu Thr Leu Gln Gly Lys Ala Glu Ser Thr Asp Ile Ala Arg Phe Gln 100 105 110 Ser Arg Asp Glu Leu Lys Tyr Ser Ile Arg Ser Leu Met Lys Tyr Ala 115 120 125 Pro Trp Val Asn His Ile Tyr Ile Val Thr Asn Gly Gln Ile Pro Lys 130 135 140 Trp Leu Asp Thr Asn Asn Thr Lys Val Thr Ile Ile Pro His Ser Thr 145 150 155 160 Ile Ile Asp Ser Gln Phe Leu Pro Thr Phe Asn Ser His Val Ile Glu 165 170 175 Ser Ser Leu Tyr Lys Ile Pro Gly Leu Ser Glu His Tyr Ile Tyr Phe 180 185 190 Asn Asp Asp Val Met Leu Ala Arg Asp Leu Ser Pro Ser Tyr Phe Phe 195 200 205 Thr Ser Ser Gly Leu Ala Lys Leu Phe Ile Thr Asn Ser Arg Leu Pro 210 215 220 Asn Gly Tyr Lys Asn Val Lys Asp Thr Pro Thr Gln Trp Ala Ser Lys 225 230 235 240 Asn Ser Arg Glu Leu Leu His Ala Glu Thr Gly Phe Trp Ala Glu Ala 245 250 255 Met Phe Ala His Thr Phe His Pro Gln Arg Lys Ser Val His Glu Ser 260 265 270 Ile Glu His Leu Trp His Glu Gln Leu Asn Val Cys Arg Gln Asn Arg 275 280 285 Phe Arg Asp Ile Ser Asp Ile Asn Met Ala Thr Phe Leu His His His 290 295 300 Phe Ala Ile Leu Thr Gly Gln Ala Leu Ala Thr Arg Thr Lys Cys Ile 305 310 315 320 Tyr Phe Asn Ile Arg Ser Pro Gln Ala Ala Gln His Tyr Lys Thr Leu 325 330 335 Leu Ala Arg Lys Gly Ser Glu Tyr Ser Pro His Ser Ile Cys Leu Asn 340 345 350 Asp His Thr Ser Ser Asn Lys Asn Ile Leu Ser Asn Tyr Glu Ala Lys 355 360 365 Leu Gln Ser Phe Leu Glu Thr Tyr Tyr Pro Asp Val Ser Glu Ala Glu 370 375 380 Ile Leu 385 <210> SEQ ID NO 29 <211> LENGTH: 329 <212> TYPE: PRT <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: AS deltaN58deltaC99-CP-X (329) <400> SEQUENCE: 29 Pro Ile Glu Asp Pro Tyr Pro Val Thr Phe Pro Ile Asp Val Val Tyr 1 5 10 15 Thr Trp Val Asp Ser Asp Asp Glu Lys Phe Asn Glu Glu Arg Leu Lys 20 25 30 Phe Gln Asn Ser Ser Thr Ser Glu Thr Leu Gln Gly Lys Ala Glu Ser 35 40 45 Thr Asp Ile Ala Arg Phe Gln Ser Arg Asp Glu Leu Lys Tyr Ser Ile 50 55 60 Arg Ser Leu Met Lys Tyr Ala Pro Trp Val Asn His Ile Tyr Ile Val 65 70 75 80 Thr Asn Gly Gln Ile Pro Lys Trp Leu Asp Thr Asn Asn Thr Lys Val 85 90 95 Thr Ile Ile Pro His Ser Thr Ile Ile Asp Ser Gln Phe Leu Pro Thr 100 105 110 Phe Asn Ser His Val Ile Glu Ser Ser Leu Tyr Lys Ile Pro Gly Leu 115 120 125 Ser Glu His Tyr Ile Tyr Phe Asn Asp Asp Val Met Leu Ala Arg Asp 130 135 140 Leu Ser Pro Ser Tyr Phe Phe Thr Ser Ser Gly Leu Ala Lys Leu Phe 145 150 155 160 Ile Thr Asn Ser Arg Leu Pro Asn Gly Tyr Lys Asn Val Lys Asp Thr 165 170 175

Pro Thr Gln Trp Ala Ser Lys Asn Ser Arg Glu Leu Leu His Ala Glu 180 185 190 Thr Gly Phe Trp Ala Glu Ala Met Phe Ala His Thr Phe His Pro Gln 195 200 205 Arg Lys Ser Val His Glu Ser Ile Glu His Leu Trp His Glu Gln Leu 210 215 220 Asn Val Cys Arg Gln Asn Arg Phe Arg Asp Ile Ser Asp Ile Asn Met 225 230 235 240 Ala Thr Phe Leu His His His Phe Ala Ile Leu Thr Gly Gln Ala Leu 245 250 255 Ala Thr Arg Thr Lys Cys Ile Tyr Phe Asn Ile Arg Ser Pro Gln Ala 260 265 270 Ala Gln His Tyr Lys Thr Leu Leu Ala Arg Lys Gly Ser Glu Tyr Ser 275 280 285 Pro His Ser Ile Cys Leu Asn Asp His Thr Ser Ser Asn Lys Asn Ile 290 295 300 Leu Ser Asn Tyr Glu Ala Lys Leu Gln Ser Phe Leu Glu Thr Tyr Tyr 305 310 315 320 Pro Asp Val Ser Glu Ala Glu Ile Leu 325 <210> SEQ ID NO 30 <211> LENGTH: 411 <212> TYPE: PRT <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: AS deltaN65deltaC10-CP-X (411) <400> SEQUENCE: 30 Val Thr Phe Pro Ile Asp Val Val Tyr Thr Trp Val Asp Ser Asp Asp 1 5 10 15 Glu Lys Phe Asn Glu Glu Arg Leu Lys Phe Gln Asn Ser Ser Thr Ser 20 25 30 Glu Thr Leu Gln Gly Lys Ala Glu Ser Thr Asp Ile Ala Arg Phe Gln 35 40 45 Ser Arg Asp Glu Leu Lys Tyr Ser Ile Arg Ser Leu Met Lys Tyr Ala 50 55 60 Pro Trp Val Asn His Ile Tyr Ile Val Thr Asn Gly Gln Ile Pro Lys 65 70 75 80 Trp Leu Asp Thr Asn Asn Thr Lys Val Thr Ile Ile Pro His Ser Thr 85 90 95 Ile Ile Asp Ser Gln Phe Leu Pro Thr Phe Asn Ser His Val Ile Glu 100 105 110 Ser Ser Leu Tyr Lys Ile Pro Gly Leu Ser Glu His Tyr Ile Tyr Phe 115 120 125 Asn Asp Asp Val Met Leu Ala Arg Asp Leu Ser Pro Ser Tyr Phe Phe 130 135 140 Thr Ser Ser Gly Leu Ala Lys Leu Phe Ile Thr Asn Ser Arg Leu Pro 145 150 155 160 Asn Gly Tyr Lys Asn Val Lys Asp Thr Pro Thr Gln Trp Ala Ser Lys 165 170 175 Asn Ser Arg Glu Leu Leu His Ala Glu Thr Gly Phe Trp Ala Glu Ala 180 185 190 Met Phe Ala His Thr Phe His Pro Gln Arg Lys Ser Val His Glu Ser 195 200 205 Ile Glu His Leu Trp His Glu Gln Leu Asn Val Cys Arg Gln Asn Arg 210 215 220 Phe Arg Asp Ile Ser Asp Ile Asn Met Ala Thr Phe Leu His His His 225 230 235 240 Phe Ala Ile Leu Thr Gly Gln Ala Leu Ala Thr Arg Thr Lys Cys Ile 245 250 255 Tyr Phe Asn Ile Arg Ser Pro Gln Ala Ala Gln His Tyr Lys Thr Leu 260 265 270 Leu Ala Arg Lys Gly Ser Glu Tyr Ser Pro His Ser Ile Cys Leu Asn 275 280 285 Asp His Thr Ser Ser Asn Lys Asn Ile Leu Ser Asn Tyr Glu Ala Lys 290 295 300 Leu Gln Ser Phe Leu Glu Thr Tyr Tyr Pro Asp Val Ser Glu Ala Glu 305 310 315 320 Ile Leu Leu Pro Thr Lys Ser Glu Val Ala Glu Leu Val Lys His Lys 325 330 335 Asp Tyr Leu Thr Val Tyr Thr Lys Leu Leu Pro Ile Ile Asn Lys Gln 340 345 350 Leu Val Asn Lys Tyr Asn Lys Pro Tyr Ser Tyr Leu Phe Tyr Tyr Leu 355 360 365 Gly Leu Ser Ala Arg Phe Leu Phe Glu Glu Thr Gln Gln Glu His Tyr 370 375 380 Arg Glu Thr Ala Glu Glu Asn Leu Gln Ile Phe Cys Gly Leu Asn Pro 385 390 395 400 Lys His Thr Leu Ala Leu Lys Tyr Leu Ala Asp 405 410 <210> SEQ ID NO 31 <211> LENGTH: 320 <212> TYPE: PRT <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: AS deltaN67deltaC99-CP-X (320) <400> SEQUENCE: 31 Phe Pro Ile Asp Val Val Tyr Thr Trp Val Asp Ser Asp Asp Glu Lys 1 5 10 15 Phe Asn Glu Glu Arg Leu Lys Phe Gln Asn Ser Ser Thr Ser Glu Thr 20 25 30 Leu Gln Gly Lys Ala Glu Ser Thr Asp Ile Ala Arg Phe Gln Ser Arg 35 40 45 Asp Glu Leu Lys Tyr Ser Ile Arg Ser Leu Met Lys Tyr Ala Pro Trp 50 55 60 Val Asn His Ile Tyr Ile Val Thr Asn Gly Gln Ile Pro Lys Trp Leu 65 70 75 80 Asp Thr Asn Asn Thr Lys Val Thr Ile Ile Pro His Ser Thr Ile Ile 85 90 95 Asp Ser Gln Phe Leu Pro Thr Phe Asn Ser His Val Ile Glu Ser Ser 100 105 110 Leu Tyr Lys Ile Pro Gly Leu Ser Glu His Tyr Ile Tyr Phe Asn Asp 115 120 125 Asp Val Met Leu Ala Arg Asp Leu Ser Pro Ser Tyr Phe Phe Thr Ser 130 135 140 Ser Gly Leu Ala Lys Leu Phe Ile Thr Asn Ser Arg Leu Pro Asn Gly 145 150 155 160 Tyr Lys Asn Val Lys Asp Thr Pro Thr Gln Trp Ala Ser Lys Asn Ser 165 170 175 Arg Glu Leu Leu His Ala Glu Thr Gly Phe Trp Ala Glu Ala Met Phe 180 185 190 Ala His Thr Phe His Pro Gln Arg Lys Ser Val His Glu Ser Ile Glu 195 200 205 His Leu Trp His Glu Gln Leu Asn Val Cys Arg Gln Asn Arg Phe Arg 210 215 220 Asp Ile Ser Asp Ile Asn Met Ala Thr Phe Leu His His His Phe Ala 225 230 235 240 Ile Leu Thr Gly Gln Ala Leu Ala Thr Arg Thr Lys Cys Ile Tyr Phe 245 250 255 Asn Ile Arg Ser Pro Gln Ala Ala Gln His Tyr Lys Thr Leu Leu Ala 260 265 270 Arg Lys Gly Ser Glu Tyr Ser Pro His Ser Ile Cys Leu Asn Asp His 275 280 285 Thr Ser Ser Asn Lys Asn Ile Leu Ser Asn Tyr Glu Ala Lys Leu Gln 290 295 300 Ser Phe Leu Glu Thr Tyr Tyr Pro Asp Val Ser Glu Ala Glu Ile Leu 305 310 315 320 <210> SEQ ID NO 32 <211> LENGTH: 1119 <212> TYPE: DNA <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: UDP-GlcNAc-Epimerase (NmA) cloned from Neisseria meningitidis serogroup A, coding sequence >UDP-GlcNAc-Epimerase-NmA (AF019760 REGION: 479..1597) <400> SEQUENCE: 32 atgaaagtct taaccgtctt tggcactcgc cctgaagcta ttaaaatggc gcctgtaatt 60 ctagagttac aaaaacataa cacaattact tcaaaagttt gcattactgc acagcatcgt 120 gaaatgctag atcaggtttt gagcctattc gaaatcaaag ctgattatga tttaaatatc 180 atgaaaccca accagagcct acaagaaatc acaacaaata tcatctcaag ccttaccgat 240 gttcttgaag atttcaaacc tgactgcgtc cttgctcacg gagacaccac aacaactttt 300 gcagctagcc ttgctgcatt ctatcaaaaa atacctgttg gccacattga agcaggcctg 360 agaacttata atttatactc tccttggcca gaggaagcaa ataggcgttt aacaagcgtt 420 ctaagccagt ggcattttgc acctactgaa gattctaaaa ataacttact atctgaatca 480 ataccttctg acaaagttat tgttactgga aatactgtca tagatgcact aatggtatct 540 ctagaaaaac taaaaataac tacaattaaa aaacaaatgg aacaagcttt tccatttatt 600 caggacaact ctaaagtaat tttaattacc gctcatagaa gagaaaatca tggggaaggt 660 attaaaaata ttggactttc tatcttagaa ttagctaaaa aatacccaac attctctttt 720 gtgattccgc tccatttaaa tcctaacgtt agaaaaccaa ttcaagattt attatcctct 780 gtgcacaatg ttcatcttat tgagccacaa gaatacttac cattcgtata tttaatgtct 840 aaaagccata taatattaag tgattcaggc ggcatacaag aagaagctcc atccctagga 900 aaaccagttc ttgtattaag agatactaca gaacgtcctg aagctgtagc tgcaggaact 960 gtaaaattag taggttctga aactcaaaat attattgaga gctttacaca actaattgaa 1020 taccctgaat attatgaaaa aatggctaat attgaaaacc cttacgggat aggtaatgcc 1080 tcaaaaatca ttgtagaaac tttattaaag aatagataa 1119 <210> SEQ ID NO 33 <211> LENGTH: 372 <212> TYPE: PRT <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: UDP-GlcNAc-Epimerase (NmA) cloned from Neisseria meningitidis serogroup A amino acid sequence >UDP-GlcNAc-Epimerase-NmA (AAC38285).pro <400> SEQUENCE: 33

Met Lys Val Leu Thr Val Phe Gly Thr Arg Pro Glu Ala Ile Lys Met 1 5 10 15 Ala Pro Val Ile Leu Glu Leu Gln Lys His Asn Thr Ile Thr Ser Lys 20 25 30 Val Cys Ile Thr Ala Gln His Arg Glu Met Leu Asp Gln Val Leu Ser 35 40 45 Leu Phe Glu Ile Lys Ala Asp Tyr Asp Leu Asn Ile Met Lys Pro Asn 50 55 60 Gln Ser Leu Gln Glu Ile Thr Thr Asn Ile Ile Ser Ser Leu Thr Asp 65 70 75 80 Val Leu Glu Asp Phe Lys Pro Asp Cys Val Leu Ala His Gly Asp Thr 85 90 95 Thr Thr Thr Phe Ala Ala Ser Leu Ala Ala Phe Tyr Gln Lys Ile Pro 100 105 110 Val Gly His Ile Glu Ala Gly Leu Arg Thr Tyr Asn Leu Tyr Ser Pro 115 120 125 Trp Pro Glu Glu Ala Asn Arg Arg Leu Thr Ser Val Leu Ser Gln Trp 130 135 140 His Phe Ala Pro Thr Glu Asp Ser Lys Asn Asn Leu Leu Ser Glu Ser 145 150 155 160 Ile Pro Ser Asp Lys Val Ile Val Thr Gly Asn Thr Val Ile Asp Ala 165 170 175 Leu Met Val Ser Leu Glu Lys Leu Lys Ile Thr Thr Ile Lys Lys Gln 180 185 190 Met Glu Gln Ala Phe Pro Phe Ile Gln Asp Asn Ser Lys Val Ile Leu 195 200 205 Ile Thr Ala His Arg Arg Glu Asn His Gly Glu Gly Ile Lys Asn Ile 210 215 220 Gly Leu Ser Ile Leu Glu Leu Ala Lys Lys Tyr Pro Thr Phe Ser Phe 225 230 235 240 Val Ile Pro Leu His Leu Asn Pro Asn Val Arg Lys Pro Ile Gln Asp 245 250 255 Leu Leu Ser Ser Val His Asn Val His Leu Ile Glu Pro Gln Glu Tyr 260 265 270 Leu Pro Phe Val Tyr Leu Met Ser Lys Ser His Ile Ile Leu Ser Asp 275 280 285 Ser Gly Gly Ile Gln Glu Glu Ala Pro Ser Leu Gly Lys Pro Val Leu 290 295 300 Val Leu Arg Asp Thr Thr Glu Arg Pro Glu Ala Val Ala Ala Gly Thr 305 310 315 320 Val Lys Leu Val Gly Ser Glu Thr Gln Asn Ile Ile Glu Ser Phe Thr 325 330 335 Gln Leu Ile Glu Tyr Pro Glu Tyr Tyr Glu Lys Met Ala Asn Ile Glu 340 345 350 Asn Pro Tyr Gly Ile Gly Asn Ala Ser Lys Ile Ile Val Glu Thr Leu 355 360 365 Leu Lys Asn Arg 370 <210> SEQ ID NO 34 <211> LENGTH: 1713 <212> TYPE: DNA <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: DNA StrepII-Thrombin-BamHI/BglII-CsaB-XhoI-His6 <400> SEQUENCE: 34 atggctagct ggagccaccc gcagttcgaa aaaggcgccc tggttccgcg tggatctttt 60 atacttaata acagaaaatg gcgtaaactt aaaagagacc ctagcgcttt ctttcgagat 120 agtaaattta actttttaag atatttttct gctaaaaaat ttgcaaagaa ttttaaaaat 180 tcatcacata tccataaaac taatataagt aaagctcaat caaatatttc ttcaacctta 240 aaacaaaatc ggaaacaaga tatgttaatt cctattaatt tttttaattt tgaatatata 300 gttaaaaaac ttaacaatca aaacgcaata ggtgtatata ttcttccttc taatcttact 360 cttaagcctg cattatgtat tctagaatca cataaagaag actttttaaa taaatttctt 420 cttactattt cctctgaaaa tttaaagctt caatacaaat ttaatggaca aataaaaaat 480 cctaagtccg taaatgaaat ttggacagat ttatttagca ttgctcatgt tgacatgaaa 540 ctcagcacag atagaacttt aagttcatct atatctcaat tttggttcag attagagttc 600 tgtaaagaag ataaggattt tatcttattt cctacagcta acagatattc tagaaaactt 660 tggaagcact ctattaaaaa taatcaatta tttaaagaag gcatacgaaa ctattcagaa 720 atatcttcat taccctatga agaagatcat aattttgata ttgatttagt atttacttgg 780 gtcaactcag aagataagaa ttggcaagag ttatataaaa aatataagcc cgactttaat 840 agcgatgcaa ccagtacatc aagattcctt agtagagatg aattaaaatt cgcattacgc 900 tcttgggaaa tgaatggatc cttcattcga aaaattttta ttgtctctaa ttgtgctccc 960 ccagcatggc tagatttaaa taaccctaaa attcaatggg tatatcacga agaaattatg 1020 ccacaaagtg cccttcctac ttttagctca catgctattg aaaccagctt gcaccatata 1080 ccaggaatta gtaactattt tatttacagc aatgacgact tcctattaac taaaccattg 1140 aataaagaca atttcttcta ttcgaatggt attgcaaagt taagattaga agcatgggga 1200 aatgttaatg gtgaatgtac tgaaggagaa cctgactact taaatggtgc tcgcaatgcg 1260 aacactctct tagaaaagga atttaaaaaa tttactacta aactacatac tcactcccct 1320 caatccatga gaactgatat tttatttgag atggaaaaaa aatatccaga agagtttaat 1380 agaacactac ataataaatt ccgatcttta gatgatattg cagtaacggg ctatctctat 1440 catcattatg ccctactctc tggacgagca ctacaaagtt ctgacaagac ggaacttgta 1500 cagcaaaatc atgatttcaa aaagaaacta aataatgtag tgaccttaac taaagaaagg 1560 aattttgaca aacttccttt gagcgtatgt atcaacgatg gtgctgatag tcacttgaat 1620 gaagaatgga atgttcaagt tattaagttc ttagaaactc ttttcccatt accatcatca 1680 tttgagaaac tcgagcacca ccaccaccac cac 1713 <210> SEQ ID NO 35 <211> LENGTH: 57 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: DNA StrepII-Thrombin-BamHI/BglII <400> SEQUENCE: 35 atggctagct ggagccaccc gcagttcgaa aaaggcgccc tggttccgcg tggatct 57 <210> SEQ ID NO 36 <211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: DNA XhoI-His6 <400> SEQUENCE: 36 ctcgagcacc accaccacca ccac 24 <210> SEQ ID NO 37 <211> LENGTH: 571 <212> TYPE: PRT <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: AS StrepII-Thrombin-BamHI/BglII-CsaB-XhoI-His6 <400> SEQUENCE: 37 Met Ala Ser Trp Ser His Pro Gln Phe Glu Lys Gly Ala Leu Val Pro 1 5 10 15 Arg Gly Ser Phe Ile Leu Asn Asn Arg Lys Trp Arg Lys Leu Lys Arg 20 25 30 Asp Pro Ser Ala Phe Phe Arg Asp Ser Lys Phe Asn Phe Leu Arg Tyr 35 40 45 Phe Ser Ala Lys Lys Phe Ala Lys Asn Phe Lys Asn Ser Ser His Ile 50 55 60 His Lys Thr Asn Ile Ser Lys Ala Gln Ser Asn Ile Ser Ser Thr Leu 65 70 75 80 Lys Gln Asn Arg Lys Gln Asp Met Leu Ile Pro Ile Asn Phe Phe Asn 85 90 95 Phe Glu Tyr Ile Val Lys Lys Leu Asn Asn Gln Asn Ala Ile Gly Val 100 105 110 Tyr Ile Leu Pro Ser Asn Leu Thr Leu Lys Pro Ala Leu Cys Ile Leu 115 120 125 Glu Ser His Lys Glu Asp Phe Leu Asn Lys Phe Leu Leu Thr Ile Ser 130 135 140 Ser Glu Asn Leu Lys Leu Gln Tyr Lys Phe Asn Gly Gln Ile Lys Asn 145 150 155 160 Pro Lys Ser Val Asn Glu Ile Trp Thr Asp Leu Phe Ser Ile Ala His 165 170 175 Val Asp Met Lys Leu Ser Thr Asp Arg Thr Leu Ser Ser Ser Ile Ser 180 185 190 Gln Phe Trp Phe Arg Leu Glu Phe Cys Lys Glu Asp Lys Asp Phe Ile 195 200 205 Leu Phe Pro Thr Ala Asn Arg Tyr Ser Arg Lys Leu Trp Lys His Ser 210 215 220 Ile Lys Asn Asn Gln Leu Phe Lys Glu Gly Ile Arg Asn Tyr Ser Glu 225 230 235 240 Ile Ser Ser Leu Pro Tyr Glu Glu Asp His Asn Phe Asp Ile Asp Leu 245 250 255 Val Phe Thr Trp Val Asn Ser Glu Asp Lys Asn Trp Gln Glu Leu Tyr 260 265 270 Lys Lys Tyr Lys Pro Asp Phe Asn Ser Asp Ala Thr Ser Thr Ser Arg 275 280 285 Phe Leu Ser Arg Asp Glu Leu Lys Phe Ala Leu Arg Ser Trp Glu Met 290 295 300 Asn Gly Ser Phe Ile Arg Lys Ile Phe Ile Val Ser Asn Cys Ala Pro 305 310 315 320 Pro Ala Trp Leu Asp Leu Asn Asn Pro Lys Ile Gln Trp Val Tyr His 325 330 335 Glu Glu Ile Met Pro Gln Ser Ala Leu Pro Thr Phe Ser Ser His Ala 340 345 350 Ile Glu Thr Ser Leu His His Ile Pro Gly Ile Ser Asn Tyr Phe Ile 355 360 365 Tyr Ser Asn Asp Asp Phe Leu Leu Thr Lys Pro Leu Asn Lys Asp Asn 370 375 380 Phe Phe Tyr Ser Asn Gly Ile Ala Lys Leu Arg Leu Glu Ala Trp Gly 385 390 395 400

Asn Val Asn Gly Glu Cys Thr Glu Gly Glu Pro Asp Tyr Leu Asn Gly 405 410 415 Ala Arg Asn Ala Asn Thr Leu Leu Glu Lys Glu Phe Lys Lys Phe Thr 420 425 430 Thr Lys Leu His Thr His Ser Pro Gln Ser Met Arg Thr Asp Ile Leu 435 440 445 Phe Glu Met Glu Lys Lys Tyr Pro Glu Glu Phe Asn Arg Thr Leu His 450 455 460 Asn Lys Phe Arg Ser Leu Asp Asp Ile Ala Val Thr Gly Tyr Leu Tyr 465 470 475 480 His His Tyr Ala Leu Leu Ser Gly Arg Ala Leu Gln Ser Ser Asp Lys 485 490 495 Thr Glu Leu Val Gln Gln Asn His Asp Phe Lys Lys Lys Leu Asn Asn 500 505 510 Val Val Thr Leu Thr Lys Glu Arg Asn Phe Asp Lys Leu Pro Leu Ser 515 520 525 Val Cys Ile Asn Asp Gly Ala Asp Ser His Leu Asn Glu Glu Trp Asn 530 535 540 Val Gln Val Ile Lys Phe Leu Glu Thr Leu Phe Pro Leu Pro Ser Ser 545 550 555 560 Phe Glu Lys Leu Glu His His His His His His 565 570 <210> SEQ ID NO 38 <211> LENGTH: 19 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: AS StrepII-Thrombin-BamHI <400> SEQUENCE: 38 Met Ala Ser Trp Ser His Pro Gln Phe Glu Lys Gly Ala Leu Val Pro 1 5 10 15 Arg Gly Ser <210> SEQ ID NO 39 <211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: AS XhoI-His6 <400> SEQUENCE: 39 Leu Glu His His His His His His 1 5 <210> SEQ ID NO 40 <211> LENGTH: 1455 <212> TYPE: DNA <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: DNA NdeI-dN69CsaB(co)-XhoI-His6 <400> SEQUENCE: 40 catatgctga tcccgatcaa tttctttaat tttgagtaca tcgtgaagaa actgaataac 60 caaaacgcaa tcggcgtgta cattctgccg tctaatctga ccctgaaacc agcattgtgc 120 atcttggagt cgcacaaaga ggacttcctg aacaaatttt tgttgaccat tagcagcgag 180 aacctgaaac tgcagtataa gttcaatggt cagatcaaaa atccgaaaag cgtgaacgaa 240 atctggaccg acctgtttag cattgctcac gtcgacatga agctgagcac cgaccgtacg 300 ctgtcctcgt ccatcagcca attttggttt cgcctggagt tctgtaaaga ggacaaggac 360 ttcatcctgt ttccgacggc aaatcgttac agccgcaagc tgtggaagca cagcatcaaa 420 aataatcagc tgtttaagga aggtatccgt aactacagcg agattagctc gctgccgtac 480 gaggaagacc ataacttcga catcgatctg gtctttacct gggtcaattc ggaagacaaa 540 aactggcagg aactgtacaa gaaatataag ccggatttta atagcgatgc cacctcgacg 600 agccgttttc tgagccgtga cgagctgaag tttgcgctgc gctcgtggga aatgaacggt 660 agcttcatcc gtaaaatctt tatcgtcagc aactgcgcgc cgccggcctg gctggatctg 720 aacaatccga agatccaatg ggtgtatcac gaggagatca tgccacagag cgccctgcca 780 accttcagca gccatgctat tgagactagc ttgcatcaca ttccgggcat ctccaactac 840 ttcatctact ctaatgacga ttttctgttg accaaaccgc tgaacaaaga caacttcttt 900 tactccaacg gtattgctaa actgcgtctg gaagcctggg gtaacgttaa cggtgaatgt 960 accgaaggcg agccggatta cctgaacggc gcgcgtaacg caaatacgct gctggagaaa 1020 gagtttaaaa agtttaccac caagctgcac acccacagcc cgcagagcat gcgtaccgac 1080 atcctgttcg agatggagaa aaaataccca gaagagttca atcgcacgct gcacaacaag 1140 ttccgcagcc tggatgacat cgcggttacc ggctacctgt accatcacta cgcattgctg 1200 tctggccgcg ctctgcaatc cagcgataag accgaactgg tccagcagaa tcacgacttt 1260 aaaaagaagc tgaataatgt tgtcaccctg accaaagagc gtaactttga taagctgccg 1320 ctgagcgttt gtattaatga cggtgcagac agccacctga atgaggagtg gaatgtgcaa 1380 gttatcaaat tcttggagac cttgttcccg ttgccgagct ccttcgagaa actcgagcac 1440 caccaccacc accac 1455 <210> SEQ ID NO 41 <400> SEQUENCE: 41 000 <210> SEQ ID NO 42 <211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: DNA XhoI-His6 <400> SEQUENCE: 42 ctcgagcacc accaccacca ccac 24 <210> SEQ ID NO 43 <211> LENGTH: 484 <212> TYPE: PRT <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: AS NdeI-dN69CsaB(co)-XhoI-His6 <400> SEQUENCE: 43 Met Leu Ile Pro Ile Asn Phe Phe Asn Phe Glu Tyr Ile Val Lys Lys 1 5 10 15 Leu Asn Asn Gln Asn Ala Ile Gly Val Tyr Ile Leu Pro Ser Asn Leu 20 25 30 Thr Leu Lys Pro Ala Leu Cys Ile Leu Glu Ser His Lys Glu Asp Phe 35 40 45 Leu Asn Lys Phe Leu Leu Thr Ile Ser Ser Glu Asn Leu Lys Leu Gln 50 55 60 Tyr Lys Phe Asn Gly Gln Ile Lys Asn Pro Lys Ser Val Asn Glu Ile 65 70 75 80 Trp Thr Asp Leu Phe Ser Ile Ala His Val Asp Met Lys Leu Ser Thr 85 90 95 Asp Arg Thr Leu Ser Ser Ser Ile Ser Gln Phe Trp Phe Arg Leu Glu 100 105 110 Phe Cys Lys Glu Asp Lys Asp Phe Ile Leu Phe Pro Thr Ala Asn Arg 115 120 125 Tyr Ser Arg Lys Leu Trp Lys His Ser Ile Lys Asn Asn Gln Leu Phe 130 135 140 Lys Glu Gly Ile Arg Asn Tyr Ser Glu Ile Ser Ser Leu Pro Tyr Glu 145 150 155 160 Glu Asp His Asn Phe Asp Ile Asp Leu Val Phe Thr Trp Val Asn Ser 165 170 175 Glu Asp Lys Asn Trp Gln Glu Leu Tyr Lys Lys Tyr Lys Pro Asp Phe 180 185 190 Asn Ser Asp Ala Thr Ser Thr Ser Arg Phe Leu Ser Arg Asp Glu Leu 195 200 205 Lys Phe Ala Leu Arg Ser Trp Glu Met Asn Gly Ser Phe Ile Arg Lys 210 215 220 Ile Phe Ile Val Ser Asn Cys Ala Pro Pro Ala Trp Leu Asp Leu Asn 225 230 235 240 Asn Pro Lys Ile Gln Trp Val Tyr His Glu Glu Ile Met Pro Gln Ser 245 250 255 Ala Leu Pro Thr Phe Ser Ser His Ala Ile Glu Thr Ser Leu His His 260 265 270 Ile Pro Gly Ile Ser Asn Tyr Phe Ile Tyr Ser Asn Asp Asp Phe Leu 275 280 285 Leu Thr Lys Pro Leu Asn Lys Asp Asn Phe Phe Tyr Ser Asn Gly Ile 290 295 300 Ala Lys Leu Arg Leu Glu Ala Trp Gly Asn Val Asn Gly Glu Cys Thr 305 310 315 320 Glu Gly Glu Pro Asp Tyr Leu Asn Gly Ala Arg Asn Ala Asn Thr Leu 325 330 335 Leu Glu Lys Glu Phe Lys Lys Phe Thr Thr Lys Leu His Thr His Ser 340 345 350 Pro Gln Ser Met Arg Thr Asp Ile Leu Phe Glu Met Glu Lys Lys Tyr 355 360 365 Pro Glu Glu Phe Asn Arg Thr Leu His Asn Lys Phe Arg Ser Leu Asp 370 375 380 Asp Ile Ala Val Thr Gly Tyr Leu Tyr His His Tyr Ala Leu Leu Ser 385 390 395 400 Gly Arg Ala Leu Gln Ser Ser Asp Lys Thr Glu Leu Val Gln Gln Asn 405 410 415 His Asp Phe Lys Lys Lys Leu Asn Asn Val Val Thr Leu Thr Lys Glu 420 425 430 Arg Asn Phe Asp Lys Leu Pro Leu Ser Val Cys Ile Asn Asp Gly Ala 435 440 445 Asp Ser His Leu Asn Glu Glu Trp Asn Val Gln Val Ile Lys Phe Leu 450 455 460 Glu Thr Leu Phe Pro Leu Pro Ser Ser Phe Glu Lys Leu Glu His His 465 470 475 480 His His His His <210> SEQ ID NO 44 <400> SEQUENCE: 44 000 <210> SEQ ID NO 45

<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: AS XhoI-His6 <400> SEQUENCE: 45 Leu Glu His His His His His His 1 5 <210> SEQ ID NO 46 <211> LENGTH: 1425 <212> TYPE: DNA <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: DNA deltaN69-CP-A (wt) (1425) <400> SEQUENCE: 46 ttaattccta ttaatttttt taattttgaa tatatagtta aaaaacttaa caatcaaaac 60 gcaataggtg tatatattct tccttctaat cttactctta agcctgcatt atgtattcta 120 gaatcacata aagaagactt tttaaataaa tttcttctta ctatttcctc tgaaaattta 180 aagcttcaat acaaatttaa tggacaaata aaaaatccta agtccgtaaa tgaaatttgg 240 acagatttat ttagcattgc tcatgttgac atgaaactca gcacagatag aactttaagt 300 tcatctatat ctcaattttg gttcagatta gagttctgta aagaagataa ggattttatc 360 ttatttccta cagctaacag atattctaga aaactttgga agcactctat taaaaataat 420 caattattta aagaaggcat acgaaactat tcagaaatat cttcattacc ctatgaagaa 480 gatcataatt ttgatattga tttagtattt acttgggtca actcagaaga taagaattgg 540 caagagttat ataaaaaata taagcccgac tttaatagcg atgcaaccag tacatcaaga 600 ttccttagta gagatgaatt aaaattcgca ttacgctctt gggaaatgaa tggatccttc 660 attcgaaaaa tttttattgt ctctaattgt gctcccccag catggctaga tttaaataac 720 cctaaaattc aatgggtata tcacgaagaa attatgccac aaagtgccct tcctactttt 780 agctcacatg ctattgaaac cagcttgcac catataccag gaattagtaa ctattttatt 840 tacagcaatg acgacttcct attaactaaa ccattgaata aagacaattt cttctattcg 900 aatggtattg caaagttaag attagaagca tggggaaatg ttaatggtga atgtactgaa 960 ggagaacctg actacttaaa tggtgctcgc aatgcgaaca ctctcttaga aaaggaattt 1020 aaaaaattta ctactaaact acatactcac tcccctcaat ccatgagaac tgatatttta 1080 tttgagatgg aaaaaaaata tccagaagag tttaatagaa cactacataa taaattccga 1140 tctttagatg atattgcagt aacgggctat ctctatcatc attatgccct actctctgga 1200 cgagcactac aaagttctga caagacggaa cttgtacagc aaaatcatga tttcaaaaag 1260 aaactaaata atgtagtgac cttaactaaa gaaaggaatt ttgacaaact tcctttgagc 1320 gtatgtatca acgatggtgc tgatagtcac ttgaatgaag aatggaatgt tcaagttatt 1380 aagttcttag aaactctttt cccattacca tcatcatttg agaaa 1425 <210> SEQ ID NO 47 <211> LENGTH: 2628 <212> TYPE: DNA <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: DNA MBP-S3N10-BamHI-CsxA-Xho1-His6 <400> SEQUENCE: 47 atgaaaactg aagaaggtaa actggtaatc tggattaacg gcgataaagg ctataacggt 60 ctcgctgaag tcggtaagaa attcgagaaa gataccggaa ttaaagtcac cgttgagcat 120 ccggataaac tggaagagaa attcccacag gttgcggcaa ctggcgatgg ccctgacatt 180 atcttctggg cacacgaccg ctttggtggc tacgctcaat ctggcctgtt ggctgaaatc 240 accccggaca aagcgttcca ggacaagctg tatccgttta cctgggatgc cgtacgttac 300 aacggcaagc tgattgctta cccgatcgct gttgaagcgt tatcgctgat ttataacaaa 360 gatctgctgc cgaacccgcc aaaaacctgg gaagagatcc cggcgctgga taaagaactg 420 aaagcgaaag gtaagagcgc gctgatgttc aacctgcaag aaccgtactt cacctggccg 480 ctgattgctg ctgacggggg ttatgcgttc aagtatgaaa acggcaagta cgacattaaa 540 gacgtgggcg tggataacgc tggcgcgaaa gcgggtctga ccttcctggt tgacctgatt 600 aaaaacaaac acatgaatgc agacaccgat tactccatcg cagaagctgc ctttaataaa 660 ggcgaaacag cgatgaccat caacggcccg tgggcatggt ccaacatcga caccagcaaa 720 gtgaattatg gtgtaacggt actgccgacc ttcaagggtc aaccatccaa accgttcgtt 780 ggcgtgctga gcgcaggtat taacgccgcc agtccgaaca aagagctggc gaaagagttc 840 ctcgaaaact atctgctgac tgatgaaggt ctggaagcgg ttaataaaga caaaccgctg 900 ggtgccgtag cgctgaagtc ttacgaggaa gagttggcga aagatccacg tattgccgcc 960 accatggaaa acgcccagaa aggtgaaatc atgccgaaca tcccgcagat gtccgctttc 1020 tggtatgccg tgcgtactgc ggtgatcaac gccgccagcg gtcgtcagac tgtcgatgaa 1080 gccctgaaag acgcgcagac taattcgagc tccaataaca ataacaacaa caataacaat 1140 aacggatcca ttatgagcaa aattagcaaa ttggtaaccc acccaaacct tttctttcga 1200 gattatttct taaaaaaagc accgttaaat tatggcgaaa atattaaacc tttaccagtc 1260 gaaacctctt ctcatagcaa aaaaaataca gcccataaaa cacccgtatc atccgaccaa 1320 ccaattgaag atccataccc agtaacattt ccaattgatg tagtttatac ttgggtagat 1380 tcagatgatg aaaaattcaa tgaagaacgc ctaaagtttc aaaattcaag cacatctgag 1440 actctacaag gcaaagcaga aagcaccgat attgcaagat tccaatcacg cgacgaatta 1500 aaatattcga ttcgaagcct gatgaagtat gccccatggg taaatcatat ttacattgta 1560 acaaatggtc aaataccaaa atggttagat accaacaata caaaggtaac gattatccct 1620 cactcaacta ttatcgacag tcaatttctc cctactttta attctcacgt cattgaatcc 1680 tctctatata aaatcccagg attatcagag cattacattt atttcaatga tgatgtcatg 1740 ctagctagag atttaagccc atcttatttc tttacaagca gcggattagc aaaactgttt 1800 attaccaact ctcgtctacc aaatggctat aagaatgtga aagacacacc aacccaatgg 1860 gcctcaaaaa attcccgtga gcttttacat gcagaaacag gattttgggc tgaagccatg 1920 tttgcacata cttttcatcc acaacgtaaa agtgtacatg aatctattga acacctatgg 1980 catgaacaat taaatgtttg tcgtcaaaac cgtttccgtg atatttcaga tattaacatg 2040 gcgacattcc tgcaccacca ttttgccatt ttgacaggcc aagctcttgc tacacgcact 2100 aaatgtattt actttaacat tcgctctcct caagcagctc agcattacaa aacattatta 2160 gctcgaaaag gaagcgaata cagcccacat tctatctgct taaatgatca tacatcgagc 2220 aataaaaata ttttatctaa ttacgaagcc aaattacaaa gctttttaga aacatactat 2280 ccagatgtat cagaagcaga aattctcctt cctactaaat ctgaagtagc tgaattagtt 2340 aaacataaag attatttaac tgtatatact aaattattac ctattatcaa taagcagctg 2400 gtcaataaat ataataaacc ttattcatat cttttctatt atttaggttt atctgcccgg 2460 tttttatttg aagaaacgca acaagaacac taccgggaaa ctgctgaaga aaatttacaa 2520 atcttttgtg gcctaaaccc aaaacataca ctagccctca aatacttagc ggatgtcacc 2580 ctcacatcac agcctagtgg acaactcgag caccaccacc accaccac 2628 <210> SEQ ID NO 48 <211> LENGTH: 1149 <212> TYPE: DNA <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: DNA MBP-S3N10-BamHI <400> SEQUENCE: 48 atgaaaactg aagaaggtaa actggtaatc tggattaacg gcgataaagg ctataacggt 60 ctcgctgaag tcggtaagaa attcgagaaa gataccggaa ttaaagtcac cgttgagcat 120 ccggataaac tggaagagaa attcccacag gttgcggcaa ctggcgatgg ccctgacatt 180 atcttctggg cacacgaccg ctttggtggc tacgctcaat ctggcctgtt ggctgaaatc 240 accccggaca aagcgttcca ggacaagctg tatccgttta cctgggatgc cgtacgttac 300 aacggcaagc tgattgctta cccgatcgct gttgaagcgt tatcgctgat ttataacaaa 360 gatctgctgc cgaacccgcc aaaaacctgg gaagagatcc cggcgctgga taaagaactg 420 aaagcgaaag gtaagagcgc gctgatgttc aacctgcaag aaccgtactt cacctggccg 480 ctgattgctg ctgacggggg ttatgcgttc aagtatgaaa acggcaagta cgacattaaa 540 gacgtgggcg tggataacgc tggcgcgaaa gcgggtctga ccttcctggt tgacctgatt 600 aaaaacaaac acatgaatgc agacaccgat tactccatcg cagaagctgc ctttaataaa 660 ggcgaaacag cgatgaccat caacggcccg tgggcatggt ccaacatcga caccagcaaa 720 gtgaattatg gtgtaacggt actgccgacc ttcaagggtc aaccatccaa accgttcgtt 780 ggcgtgctga gcgcaggtat taacgccgcc agtccgaaca aagagctggc gaaagagttc 840 ctcgaaaact atctgctgac tgatgaaggt ctggaagcgg ttaataaaga caaaccgctg 900 ggtgccgtag cgctgaagtc ttacgaggaa gagttggcga aagatccacg tattgccgcc 960 accatggaaa acgcccagaa aggtgaaatc atgccgaaca tcccgcagat gtccgctttc 1020 tggtatgccg tgcgtactgc ggtgatcaac gccgccagcg gtcgtcagac tgtcgatgaa 1080 gccctgaaag acgcgcagac taattcgagc tccaataaca ataacaacaa caataacaat 1140 aacggatcc 1149 <210> SEQ ID NO 49 <211> LENGTH: 876 <212> TYPE: PRT <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: AS MBP-S3N10-BamHI-CsxA-Xho1-His6 <400> SEQUENCE: 49 Met Lys Thr Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp Lys 1 5 10 15 Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp Thr 20 25 30 Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys Phe 35 40 45 Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp Ala 50 55 60 His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu Ile 65 70 75 80 Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp Asp 85 90 95 Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val Glu 100 105 110 Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro Lys 115 120 125

Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys Gly 130 135 140 Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp Pro 145 150 155 160 Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly Lys 165 170 175 Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala Gly 180 185 190 Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala Asp 195 200 205 Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala 210 215 220 Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser Lys 225 230 235 240 Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro Ser 245 250 255 Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser Pro 260 265 270 Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr Asp 275 280 285 Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val Ala 290 295 300 Leu Lys Ser Tyr Glu Glu Glu Leu Ala Lys Asp Pro Arg Ile Ala Ala 305 310 315 320 Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro Gln 325 330 335 Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala Ala 340 345 350 Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr Asn 355 360 365 Ser Ser Ser Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Gly Ser Ile 370 375 380 Met Ser Lys Ile Ser Lys Leu Val Thr His Pro Asn Leu Phe Phe Arg 385 390 395 400 Asp Tyr Phe Leu Lys Lys Ala Pro Leu Asn Tyr Gly Glu Asn Ile Lys 405 410 415 Pro Leu Pro Val Glu Thr Ser Ser His Ser Lys Lys Asn Thr Ala His 420 425 430 Lys Thr Pro Val Ser Ser Asp Gln Pro Ile Glu Asp Pro Tyr Pro Val 435 440 445 Thr Phe Pro Ile Asp Val Val Tyr Thr Trp Val Asp Ser Asp Asp Glu 450 455 460 Lys Phe Asn Glu Glu Arg Leu Lys Phe Gln Asn Ser Ser Thr Ser Glu 465 470 475 480 Thr Leu Gln Gly Lys Ala Glu Ser Thr Asp Ile Ala Arg Phe Gln Ser 485 490 495 Arg Asp Glu Leu Lys Tyr Ser Ile Arg Ser Leu Met Lys Tyr Ala Pro 500 505 510 Trp Val Asn His Ile Tyr Ile Val Thr Asn Gly Gln Ile Pro Lys Trp 515 520 525 Leu Asp Thr Asn Asn Thr Lys Val Thr Ile Ile Pro His Ser Thr Ile 530 535 540 Ile Asp Ser Gln Phe Leu Pro Thr Phe Asn Ser His Val Ile Glu Ser 545 550 555 560 Ser Leu Tyr Lys Ile Pro Gly Leu Ser Glu His Tyr Ile Tyr Phe Asn 565 570 575 Asp Asp Val Met Leu Ala Arg Asp Leu Ser Pro Ser Tyr Phe Phe Thr 580 585 590 Ser Ser Gly Leu Ala Lys Leu Phe Ile Thr Asn Ser Arg Leu Pro Asn 595 600 605 Gly Tyr Lys Asn Val Lys Asp Thr Pro Thr Gln Trp Ala Ser Lys Asn 610 615 620 Ser Arg Glu Leu Leu His Ala Glu Thr Gly Phe Trp Ala Glu Ala Met 625 630 635 640 Phe Ala His Thr Phe His Pro Gln Arg Lys Ser Val His Glu Ser Ile 645 650 655 Glu His Leu Trp His Glu Gln Leu Asn Val Cys Arg Gln Asn Arg Phe 660 665 670 Arg Asp Ile Ser Asp Ile Asn Met Ala Thr Phe Leu His His His Phe 675 680 685 Ala Ile Leu Thr Gly Gln Ala Leu Ala Thr Arg Thr Lys Cys Ile Tyr 690 695 700 Phe Asn Ile Arg Ser Pro Gln Ala Ala Gln His Tyr Lys Thr Leu Leu 705 710 715 720 Ala Arg Lys Gly Ser Glu Tyr Ser Pro His Ser Ile Cys Leu Asn Asp 725 730 735 His Thr Ser Ser Asn Lys Asn Ile Leu Ser Asn Tyr Glu Ala Lys Leu 740 745 750 Gln Ser Phe Leu Glu Thr Tyr Tyr Pro Asp Val Ser Glu Ala Glu Ile 755 760 765 Leu Leu Pro Thr Lys Ser Glu Val Ala Glu Leu Val Lys His Lys Asp 770 775 780 Tyr Leu Thr Val Tyr Thr Lys Leu Leu Pro Ile Ile Asn Lys Gln Leu 785 790 795 800 Val Asn Lys Tyr Asn Lys Pro Tyr Ser Tyr Leu Phe Tyr Tyr Leu Gly 805 810 815 Leu Ser Ala Arg Phe Leu Phe Glu Glu Thr Gln Gln Glu His Tyr Arg 820 825 830 Glu Thr Ala Glu Glu Asn Leu Gln Ile Phe Cys Gly Leu Asn Pro Lys 835 840 845 His Thr Leu Ala Leu Lys Tyr Leu Ala Asp Val Thr Leu Thr Ser Gln 850 855 860 Pro Ser Gly Gln Leu Glu His His His His His His 865 870 875 <210> SEQ ID NO 50 <211> LENGTH: 383 <212> TYPE: PRT <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: AS MBP-S3N10-BamHI <400> SEQUENCE: 50 Met Lys Thr Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp Lys 1 5 10 15 Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp Thr 20 25 30 Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys Phe 35 40 45 Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp Ala 50 55 60 His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu Ile 65 70 75 80 Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp Asp 85 90 95 Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val Glu 100 105 110 Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro Lys 115 120 125 Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys Gly 130 135 140 Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp Pro 145 150 155 160 Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly Lys 165 170 175 Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala Gly 180 185 190 Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala Asp 195 200 205 Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala 210 215 220 Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser Lys 225 230 235 240 Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro Ser 245 250 255 Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser Pro 260 265 270 Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr Asp 275 280 285 Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val Ala 290 295 300 Leu Lys Ser Tyr Glu Glu Glu Leu Ala Lys Asp Pro Arg Ile Ala Ala 305 310 315 320 Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro Gln 325 330 335 Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala Ala 340 345 350 Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr Asn 355 360 365 Ser Ser Ser Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Gly Ser 370 375 380 <210> SEQ ID NO 51 <211> LENGTH: 1263 <212> TYPE: DNA <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: DNA NdeI-dN65dC10-CsxA-XhoI-His6 <400> SEQUENCE: 51 catatggtaa catttccaat tgatgtagtt tatacttggg tagattcaga tgatgaaaaa 60 ttcaatgaag aacgcctaaa gtttcaaaat tcaagcacat ctgagactct acaaggcaaa 120 gcagaaagca ccgatattgc aagattccaa tcacgcgacg aattaaaata ttcgattcga 180 agcctgatga agtatgcccc atgggtaaat catatttaca ttgtaacaaa tggtcaaata 240 ccaaaatggt tagataccaa caatacaaag gtaacgatta tccctcactc aactattatc 300 gacagtcaat ttctccctac ttttaattct cacgtcattg aatcctctct atataaaatc 360 ccaggattat cagagcatta catttatttc aatgatgatg tcatgctagc tagagattta 420 agcccatctt atttctttac aagcagcgga ttagcaaaac tgtttattac caactctcgt 480 ctaccaaatg gctataagaa tgtgaaagac acaccaaccc aatgggcctc aaaaaattcc 540

cgtgagcttt tacatgcaga aacaggattt tgggctgaag ccatgtttgc acatactttt 600 catccacaac gtaaaagtgt acatgaatct attgaacacc tatggcatga acaattaaat 660 gtttgtcgtc aaaaccgttt ccgtgatatt tcagatatta acatggcgac attcctgcac 720 caccattttg ccattttgac aggccaagct cttgctacac gcactaaatg tatttacttt 780 aacattcgct ctcctcaagc agctcagcat tacaaaacat tattagctcg aaaaggaagc 840 gaatacagcc cacattctat ctgcttaaat gatcatacat cgagcaataa aaatatttta 900 tctaattacg aagccaaatt acaaagcttt ttagaaacat actatccaga tgtatcagaa 960 gcagaaattc tccttcctac taaatctgaa gtagctgaat tagttaaaca taaagattat 1020 ttaactgtat atactaaatt attacctatt atcaataagc agctggtcaa taaatataat 1080 aaaccttatt catatctttt ctattattta ggtttatctg cccggttttt atttgaagaa 1140 acgcaacaag aacactaccg ggaaactgct gaagaaaatt tacaaatctt ttgtggccta 1200 aacccaaaac atacactagc cctcaaatac ttagcggatc tcgagcacca ccaccaccac 1260 cac 1263 <210> SEQ ID NO 52 <211> LENGTH: 420 <212> TYPE: PRT <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: AS NdeI-dN65dC10-CsxA-XhoI-His6 <400> SEQUENCE: 52 Met Val Thr Phe Pro Ile Asp Val Val Tyr Thr Trp Val Asp Ser Asp 1 5 10 15 Asp Glu Lys Phe Asn Glu Glu Arg Leu Lys Phe Gln Asn Ser Ser Thr 20 25 30 Ser Glu Thr Leu Gln Gly Lys Ala Glu Ser Thr Asp Ile Ala Arg Phe 35 40 45 Gln Ser Arg Asp Glu Leu Lys Tyr Ser Ile Arg Ser Leu Met Lys Tyr 50 55 60 Ala Pro Trp Val Asn His Ile Tyr Ile Val Thr Asn Gly Gln Ile Pro 65 70 75 80 Lys Trp Leu Asp Thr Asn Asn Thr Lys Val Thr Ile Ile Pro His Ser 85 90 95 Thr Ile Ile Asp Ser Gln Phe Leu Pro Thr Phe Asn Ser His Val Ile 100 105 110 Glu Ser Ser Leu Tyr Lys Ile Pro Gly Leu Ser Glu His Tyr Ile Tyr 115 120 125 Phe Asn Asp Asp Val Met Leu Ala Arg Asp Leu Ser Pro Ser Tyr Phe 130 135 140 Phe Thr Ser Ser Gly Leu Ala Lys Leu Phe Ile Thr Asn Ser Arg Leu 145 150 155 160 Pro Asn Gly Tyr Lys Asn Val Lys Asp Thr Pro Thr Gln Trp Ala Ser 165 170 175 Lys Asn Ser Arg Glu Leu Leu His Ala Glu Thr Gly Phe Trp Ala Glu 180 185 190 Ala Met Phe Ala His Thr Phe His Pro Gln Arg Lys Ser Val His Glu 195 200 205 Ser Ile Glu His Leu Trp His Glu Gln Leu Asn Val Cys Arg Gln Asn 210 215 220 Arg Phe Arg Asp Ile Ser Asp Ile Asn Met Ala Thr Phe Leu His His 225 230 235 240 His Phe Ala Ile Leu Thr Gly Gln Ala Leu Ala Thr Arg Thr Lys Cys 245 250 255 Ile Tyr Phe Asn Ile Arg Ser Pro Gln Ala Ala Gln His Tyr Lys Thr 260 265 270 Leu Leu Ala Arg Lys Gly Ser Glu Tyr Ser Pro His Ser Ile Cys Leu 275 280 285 Asn Asp His Thr Ser Ser Asn Lys Asn Ile Leu Ser Asn Tyr Glu Ala 290 295 300 Lys Leu Gln Ser Phe Leu Glu Thr Tyr Tyr Pro Asp Val Ser Glu Ala 305 310 315 320 Glu Ile Leu Leu Pro Thr Lys Ser Glu Val Ala Glu Leu Val Lys His 325 330 335 Lys Asp Tyr Leu Thr Val Tyr Thr Lys Leu Leu Pro Ile Ile Asn Lys 340 345 350 Gln Leu Val Asn Lys Tyr Asn Lys Pro Tyr Ser Tyr Leu Phe Tyr Tyr 355 360 365 Leu Gly Leu Ser Ala Arg Phe Leu Phe Glu Glu Thr Gln Gln Glu His 370 375 380 Tyr Arg Glu Thr Ala Glu Glu Asn Leu Gln Ile Phe Cys Gly Leu Asn 385 390 395 400 Pro Lys His Thr Leu Ala Leu Lys Tyr Leu Ala Asp Leu Glu His His 405 410 415 His His His His 420 <210> SEQ ID NO 53 <211> LENGTH: 741 <212> TYPE: DNA <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: DNA CsaC <400> SEQUENCE: 53 atgttatcta atttaaaaac aggaaataat atcttaggat tacctgaatt tgagttgaat 60 ggctgccgat tcttatataa aaaaggtata gaaaaaacaa ttattacttt ttcagcattt 120 cctcctaaag atattgctca aaaatataat tatataaaag attttttaag ttctaattat 180 acttttttag cattcttaga taccaaatat ccagaagatg atgctagagg cacttattac 240 attactaatg agttagataa tggatattta caaaccatac attgtattat tcaattatta 300 tcgaatacaa atcaagaaga tacctacctt ttgggttcaa gtaaaggtgg cgttggcgca 360 cttctactcg gtcttacata taattatcct aatataatta ttaatgctcc tcaagccaaa 420 ttagcagatt atatcaaaac acgctcgaaa accattcttt catatatgct tggaacctct 480 aaaagatttc aagatattaa ttacgattat atcaatgact tcttactatc taaaattaag 540 acttgcgact cctcacttaa atggaatatt catataactt gcggaaaaga tgattcatat 600 catttaaatg aattagaaat tctaaaaaat gaatttaata taaaagctat tacgattaaa 660 accaaactaa tttctggcgg gcatgataat gaagcaattg cccactatag agaatacttt 720 aaaaccataa tccaaaatat a 741 <210> SEQ ID NO 54 <211> LENGTH: 247 <212> TYPE: PRT <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: AS CsaC <400> SEQUENCE: 54 Met Leu Ser Asn Leu Lys Thr Gly Asn Asn Ile Leu Gly Leu Pro Glu 1 5 10 15 Phe Glu Leu Asn Gly Cys Arg Phe Leu Tyr Lys Lys Gly Ile Glu Lys 20 25 30 Thr Ile Ile Thr Phe Ser Ala Phe Pro Pro Lys Asp Ile Ala Gln Lys 35 40 45 Tyr Asn Tyr Ile Lys Asp Phe Leu Ser Ser Asn Tyr Thr Phe Leu Ala 50 55 60 Phe Leu Asp Thr Lys Tyr Pro Glu Asp Asp Ala Arg Gly Thr Tyr Tyr 65 70 75 80 Ile Thr Asn Glu Leu Asp Asn Gly Tyr Leu Gln Thr Ile His Cys Ile 85 90 95 Ile Gln Leu Leu Ser Asn Thr Asn Gln Glu Asp Thr Tyr Leu Leu Gly 100 105 110 Ser Ser Lys Gly Gly Val Gly Ala Leu Leu Leu Gly Leu Thr Tyr Asn 115 120 125 Tyr Pro Asn Ile Ile Ile Asn Ala Pro Gln Ala Lys Leu Ala Asp Tyr 130 135 140 Ile Lys Thr Arg Ser Lys Thr Ile Leu Ser Tyr Met Leu Gly Thr Ser 145 150 155 160 Lys Arg Phe Gln Asp Ile Asn Tyr Asp Tyr Ile Asn Asp Phe Leu Leu 165 170 175 Ser Lys Ile Lys Thr Cys Asp Ser Ser Leu Lys Trp Asn Ile His Ile 180 185 190 Thr Cys Gly Lys Asp Asp Ser Tyr His Leu Asn Glu Leu Glu Ile Leu 195 200 205 Lys Asn Glu Phe Asn Ile Lys Ala Ile Thr Ile Lys Thr Lys Leu Ile 210 215 220 Ser Gly Gly His Asp Asn Glu Ala Ile Ala His Tyr Arg Glu Tyr Phe 225 230 235 240 Lys Thr Ile Ile Gln Asn Ile 245 <210> SEQ ID NO 55 <211> LENGTH: 34 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer KS422/KS273 <400> SEQUENCE: 55 gcatctcata tggctgttat tatatttgtt aacg 34 <210> SEQ ID NO 56 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer wt NmW-135 <400> SEQUENCE: 56 ccgctcgagt ttttcttggc caaaaaactg 30 <210> SEQ ID NO 57 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer wt NmY <400> SEQUENCE: 57 ccgctcgagt ttttcttggc caaaaaactg 30 <210> SEQ ID NO 58 <211> LENGTH: 45 <212> TYPE: DNA

<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer KS350/KS351 <400> SEQUENCE: 58 ctgatcatga catcagaaag tgcgggattt ccatatatat ttatg 45 <210> SEQ ID NO 59 <211> LENGTH: 45 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer E307A <400> SEQUENCE: 59 cataaatata tatggaaatc ccgcactttc tgatgtcatg atcag 45 <210> SEQ ID NO 60 <211> LENGTH: 41 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer KS370/KS371 <400> SEQUENCE: 60 atctcgcgtt gctgtaggtg tttatgcaac tagcttattt g 41 <210> SEQ ID NO 61 <211> LENGTH: 41 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer S972A <400> SEQUENCE: 61 caaataagct agttgcataa acacctacag caacgcgaga t 41 <210> SEQ ID NO 62 <211> LENGTH: 51 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer AR11/AR12 <400> SEQUENCE: 62 atacagatat cctaatcatg acatctcaaa gcgcaggctt tggttatata t 51 <210> SEQ ID NO 63 <211> LENGTH: 51 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer E307A <400> SEQUENCE: 63 atatataacc aaagcctgcg ctttgagatg tcatgattag gatatctgta t 51 <210> SEQ ID NO 64 <211> LENGTH: 34 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer KS422/KS421 <400> SEQUENCE: 64 gcatctcata tggctgttat tatatttgtt aacg 34 <210> SEQ ID NO 65 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer C-delta639 <400> SEQUENCE: 65 ccgctcgagg ctgcgcggaa gaatagtg 28 <210> SEQ ID NO 66 <211> LENGTH: 35 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer AR2/KS273 <400> SEQUENCE: 66 gcatctcata tgtttaataa cgtatcatta tcgtc 35 <210> SEQ ID NO 67 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer N-delta398 <400> SEQUENCE: 67 ccgctcgagt ttttcttggc caaaaaactg 30 <210> SEQ ID NO 68 <211> LENGTH: 35 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer AR1/KS273 <400> SEQUENCE: 68 gcatctcata tgactgatga taatttaata cctat 35 <210> SEQ ID NO 69 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer N-delta562 <400> SEQUENCE: 69 ccgctcgagt ttttcttggc caaaaaactg 30 <210> SEQ ID NO 70 <211> LENGTH: 35 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer AR9/AR273 <400> SEQUENCE: 70 gcatctcata tgaaatattc ttataaatat atcta 35 <210> SEQ ID NO 71 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer N-delta609 <400> SEQUENCE: 71 ccgctcgagt ttttcttggc caaaaaactg 30 <210> SEQ ID NO 72 <211> LENGTH: 35 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer AR10/KS273 <400> SEQUENCE: 72 gcatctcata tgtcttggga acttattcgt gcctc 35 <210> SEQ ID NO 73 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer N-delta639 <400> SEQUENCE: 73 ccgctcgagt ttttcttggc caaaaaactg 30 <210> SEQ ID NO 74 <211> LENGTH: 31 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer KS433/KS273 <400> SEQUENCE: 74 gcatctcata tgggtaagcg ttcgatggat g 31 <210> SEQ ID NO 75 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer N-delta676 <400> SEQUENCE: 75 ccgctcgagt ttttcttggc caaaaaactg 30 <210> SEQ ID NO 76 <211> LENGTH: 34 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer KS434/KS273 <400> SEQUENCE: 76 gcatctcata tgtcactgaa aagtaatgta gttg 34 <210> SEQ ID NO 77 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer N-delta729 <400> SEQUENCE: 77 ccgctcgagt ttttcttggc caaaaaactg 30 <210> SEQ ID NO 78 <211> LENGTH: 35 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer KS435/KS273 <400> SEQUENCE: 78 gcatctcata tgaatatcga agcatttcta aaacc 35 <210> SEQ ID NO 79 <211> LENGTH: 30

<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer N-delta777 <400> SEQUENCE: 79 ccgctcgagt ttttcttggc caaaaaactg 30 <210> SEQ ID NO 80 <211> LENGTH: 34 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer KS422/KS421 <400> SEQUENCE: 80 gcatctcata tggctgttat tatatttgtt aacg 34 <210> SEQ ID NO 81 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer C-delta639 <400> SEQUENCE: 81 ccgctcgagg ctgcgcggaa gaatagtg 28 <210> SEQ ID NO 82 <211> LENGTH: 34 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer KS422/KS374 <400> SEQUENCE: 82 gcatctcata tggctgttat tatatttgtt aacg 34 <210> SEQ ID NO 83 <211> LENGTH: 29 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer C-delta479 <400> SEQUENCE: 83 ccgctcgagc gtttgcatgt tgggtaaag 29 <210> SEQ ID NO 84 <211> LENGTH: 35 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer AR2/KS273 <400> SEQUENCE: 84 gcatctcata tgtttaataa cgtatcatta tcgtc 35 <210> SEQ ID NO 85 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer N-delta398 <400> SEQUENCE: 85 ccgctcgagt ttttcttggc caaaaaactg 30 <210> SEQ ID NO 86 <211> LENGTH: 35 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer AR1/KS273 <400> SEQUENCE: 86 gcatctcata tgactgatga taatttaata cctat 35 <210> SEQ ID NO 87 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer N-delta562 <400> SEQUENCE: 87 ccgctcgagt ttttcttggc caaaaaactg 30 <210> SEQ ID NO 88 <211> LENGTH: 31 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer KS433/KS273 <400> SEQUENCE: 88 gcatctcata tgggtaagcg ttcgatggat g 31 <210> SEQ ID NO 89 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer N-delta 676 <400> SEQUENCE: 89 ccgctcgagt ttttcttggc caaaaaactg 30 <210> SEQ ID NO 90 <211> LENGTH: 34 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer KS434/KS273 <400> SEQUENCE: 90 gcatctcata tgtcactgaa aagtaatgta gttg 34 <210> SEQ ID NO 91 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: N-delta 729 <400> SEQUENCE: 91 ccgctcgagt ttttcttggc caaaaaactg 30 <210> SEQ ID NO 92 <211> LENGTH: 31 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Recloning primer <400> SEQUENCE: 92 gcatctcata tgggtaagcg ttcgatggat g 31 <210> SEQ ID NO 93 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Recloning Primer <400> SEQUENCE: 93 ccgctcgagt ttttcttggc caaaaaactg 30 <210> SEQ ID NO 94 <211> LENGTH: 29 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer StrepII-CsaA-His6 <400> SEQUENCE: 94 gcggatccaa agtcttaacc gtctttggc 29 <210> SEQ ID NO 95 <211> LENGTH: 34 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer StrepII-CsaA-His6 <400> SEQUENCE: 95 ccgctcgagt ctattcttta ataaagtttc taca 34 <210> SEQ ID NO 96 <211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer StrepII-CsaB-His6 <400> SEQUENCE: 96 gcagatcttt tatacttaat aacagaaaat ggc 33 <210> SEQ ID NO 97 <211> LENGTH: 32 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer StrepII-CsaB-His6 <400> SEQUENCE: 97 ccgctcgagt ttctcaaatg atgatggtaa tg 32 <210> SEQ ID NO 98 <211> LENGTH: 32 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer StrepII-delta69-CsaB-His6 <400> SEQUENCE: 98 ccgctcgagt ttctcaaatg atgatggtaa tg 32 <210> SEQ ID NO 99 <211> LENGTH: 34 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer StrepII-delta69-CsaB-His6 <400> SEQUENCE: 99 gcagatctat gttaattcct attaattttt ttaa 34 <210> SEQ ID NO 100

<211> LENGTH: 32 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer delta69-CsaB-His6 <400> SEQUENCE: 100 ccgctcgagt ttctcaaatg atgatggtaa tg 32 <210> SEQ ID NO 101 <211> LENGTH: 39 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer delta69-CsaB-His6 <400> SEQUENCE: 101 gcatctcata tgttaattcc tattaatttt ttttaattt 39 <210> SEQ ID NO 102 <211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer delta69-CsaBCo-His6 <400> SEQUENCE: 102 gcatctcata tgctgatccc gatcaatttc ttt 33 <210> SEQ ID NO 103 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer delta69-CsaBCo-His6 <400> SEQUENCE: 103 ccgctcgagt ttctcgaagg agctcggc 28 <210> SEQ ID NO 104 <211> LENGTH: 27 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer StrepII-CsaC-His6 <400> SEQUENCE: 104 ccgctcgagt atattttgga ttatggt 27 <210> SEQ ID NO 105 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer StrepII-CsaC-His6 <400> SEQUENCE: 105 gcggatcctt atctaattta aaaacagg 28 <210> SEQ ID NO 106 <211> LENGTH: 366 <212> TYPE: PRT <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: NmL (CsIA); Homologue of CsxA/CsaB <400> SEQUENCE: 106 Met Lys Arg Leu Lys Lys Leu Thr Arg Glu Pro Gly Val Phe Phe Arg 1 5 10 15 Asp Tyr Phe Asn Lys Lys Tyr Pro Val Arg Asn Ile Glu Gln Arg Ile 20 25 30 Asn Glu Ile Glu Glu Pro Ala Ile Ile Ala Asn Ser Leu His Leu Ala 35 40 45 Ala Val Glu Ser Ala Ile His Leu Ser Pro Phe Lys Ile Asp Val Val 50 55 60 Phe Thr Trp Val Asp Asn Ser Asp Thr Gln Trp Gln Gln Arg His Gln 65 70 75 80 Gln Tyr Cys His Ala Ala Ser Pro Asn Asn Leu Tyr Ser Asn Asp Glu 85 90 95 Thr Arg Phe Ala Asn His Asn Glu Leu Tyr Tyr Ser Leu His Ser Val 100 105 110 Arg Ser Phe Leu Pro Trp Val Asn His Ile Tyr Ile Ile Thr Asp Ser 115 120 125 Gln Thr Pro Lys Trp Phe Lys Ser Ala Glu Tyr Pro Asn Val Ser Ile 130 135 140 Ile Asp His Ser Glu Ile Ile Asp Lys Gln Tyr Leu Pro Thr Phe Asn 145 150 155 160 Ser His Val Ile Glu Ala His Leu His Asn Ile Pro Asn Leu Ser Glu 165 170 175 His Phe Ile Tyr Phe Asn Asp Asp Val Phe Val Ala Arg Pro Leu His 180 185 190 Arg Glu His Phe Phe His Ala Asn Gly Ile Ala Ser Leu Phe Ile Ala 195 200 205 Asp Lys Ser Leu Gln Lys Met Ala Thr Lys Gly Thr Ile Thr Pro Thr 210 215 220 Leu Ser Ala Ser Gln Asn Cys Ile Arg Leu Leu Asn Gln Arg Tyr Asp 225 230 235 240 Cys Asn Leu Asp His Pro Leu Val His Thr Tyr Val Pro Leu Arg Lys 245 250 255 Ser Gly Phe Gln Thr Ala Trp Gln Tyr Tyr Arg Glu Glu Ile Lys Ala 260 265 270 Phe Leu Pro Asn Lys Phe Arg Thr Asn Gln Asp Leu Asn Leu Ala Thr 275 280 285 Phe Leu Val Pro Trp Leu Met Tyr Leu Asp Gly Lys Ser Ile Pro Asn 290 295 300 Asn Asp Ile Cys Tyr Tyr Phe Asn Ile Arg Ser Asn Lys Ala Pro Thr 305 310 315 320 Gln Tyr Leu Lys Leu Leu Gln Lys Asn Glu Asp Asn Gln Gln Pro His 325 330 335 Ser Phe Cys Ala Asn Asp Phe His Ser Glu Gln Gln Leu Tyr Asp Tyr 340 345 350 His Ala Lys Leu Ile Ala Met Leu Lys Asp Tyr Phe Lys Ile 355 360 365 <210> SEQ ID NO 107 <211> LENGTH: 1037 <212> TYPE: PRT <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: polymerase of serogroup Y <400> SEQUENCE: 107 Met Ala Val Ile Ile Phe Val Asn Gly Ile Arg Ala Val Asn Gly Leu 1 5 10 15 Val Lys Ser Ser Ile Asn Thr Ala Asn Ala Phe Ala Glu Glu Gly Leu 20 25 30 Asp Val His Leu Ile Asn Phe Val Gly Asn Ile Thr Gly Ala Glu His 35 40 45 Leu Ser Pro Pro Phe His Leu His Pro Asn Val Lys Thr Ser Ser Ile 50 55 60 Ile Asp Leu Phe Asn Asp Ile Pro Glu Asn Val Ser Cys Arg Asn Ile 65 70 75 80 Pro Phe Tyr Ser Ile His Gln Gln Phe Phe Lys Ala Glu Tyr Ser Ala 85 90 95 His Tyr Lys His Val Leu Met Lys Ile Glu Ser Leu Leu Ser Glu Glu 100 105 110 Asp Ser Ile Ile Phe Thr His Pro Leu Gln Leu Glu Met Tyr Arg Leu 115 120 125 Ala Asn Asn Asn Ile Lys Ser Lys Ala Lys Leu Ile Val Gln Ile His 130 135 140 Gly Asn Tyr Met Glu Glu Ile His Asn Tyr Glu Ile Trp Ala Arg Asn 145 150 155 160 Ile Asp Tyr Val Asp Tyr Leu Gln Thr Val Ser Asp Glu Met Leu Glu 165 170 175 Glu Met His Ser His Phe Lys Ile Lys Lys Asp Lys Leu Val Phe Ile 180 185 190 Pro Asn Ile Thr Tyr Pro Ile Ser Leu Glu Lys Lys Glu Ala Asp Phe 195 200 205 Phe Ile Lys Asp Asn Glu Asp Ile Asp Asn Ala Gln Lys Phe Lys Arg 210 215 220 Ile Ser Ile Val Gly Ser Ile Gln Pro Arg Lys Asn Gln Leu Asp Ala 225 230 235 240 Ile Lys Ile Ile Asn Lys Ile Lys Asn Glu Asn Tyr Ile Leu Gln Ile 245 250 255 Tyr Gly Lys Ser Ile Asn Lys Asp Tyr Phe Glu Leu Ile Lys Lys Tyr 260 265 270 Ile Lys Asp Asn Lys Leu Gln Asn Arg Ile Leu Phe Lys Gly Glu Ser 275 280 285 Ser Glu Gln Glu Ile Tyr Glu Asn Thr Asp Ile Leu Ile Met Thr Ser 290 295 300 Gln Ser Glu Gly Phe Gly Tyr Ile Phe Leu Glu Gly Met Val Tyr Asp 305 310 315 320 Ile Pro Ile Leu Ala Tyr Asn Phe Lys Tyr Gly Ala Asn Asp Phe Ser 325 330 335 Asn Tyr Asn Glu Asn Ala Ser Val Phe Lys Thr Gly Asp Ile Ser Gly 340 345 350 Met Ala Lys Lys Ile Ile Glu Leu Leu Asn Asn Pro Glu Lys Tyr Lys 355 360 365 Glu Leu Val Gln Tyr Asn His Asn Arg Phe Leu Lys Glu Tyr Ala Lys 370 375 380 Asp Val Val Met Ala Lys Tyr Phe Thr Ile Leu Pro Arg Ser Phe Asn 385 390 395 400 Asn Val Ser Leu Ser Ser Ala Phe Ser Arg Lys Glu Leu Asp Glu Phe 405 410 415 Gln Asn Ile Thr Phe Ser Ile Glu Asp Ser Asn Asp Leu Ala His Ile 420 425 430 Trp Asn Phe Glu Leu Thr Asn Pro Ala Gln Asn Met Asn Phe Phe Ala 435 440 445 Leu Val Gly Lys Arg Lys Phe Pro Met Asp Ala His Ile Gln Gly Thr 450 455 460 Gln Cys Thr Ile Lys Ile Ala His Lys Lys Thr Gly Asn Leu Leu Ser 465 470 475 480 Leu Leu Leu Lys Lys Arg Asn Gln Leu Asn Leu Ser Arg Gly Tyr Thr 485 490 495

Leu Ile Ala Glu Asp Asn Ser Tyr Glu Lys Tyr Ile Gly Ala Ile Ser 500 505 510 Asn Lys Gly Asn Phe Glu Ile Ile Ala Asn Lys Lys Asn Ser Leu Val 515 520 525 Thr Ile Asn Lys Ser Thr Leu Glu Leu His Glu Ile Pro His Glu Leu 530 535 540 His Gln Asn Lys Leu Leu Ile Ala Leu Pro Asn Met Gln Thr Pro Leu 545 550 555 560 Lys Ile Thr Asp Asp Asn Leu Ile Pro Ile Gln Ala Ser Ile Lys Leu 565 570 575 Glu Lys Ile Gly Asn Thr Tyr Tyr Pro Cys Phe Leu Pro Ser Gly Ile 580 585 590 Phe Asn Asn Ile Cys Leu Asp Tyr Gly Glu Glu Ser Lys Ile Ile Asn 595 600 605 Phe Ser Lys Tyr Ser Tyr Lys Tyr Ile Tyr Asp Ser Ile Arg His Ile 610 615 620 Glu Gln His Thr Asp Ile Ser Asp Ile Ile Val Cys Asn Val Tyr Ser 625 630 635 640 Trp Glu Leu Ile Arg Ala Ser Val Ile Glu Ser Leu Met Glu Phe Thr 645 650 655 Gly Lys Trp Glu Lys His Phe Gln Thr Ser Pro Lys Ile Asp Tyr Arg 660 665 670 Phe Asp His Glu Gly Lys Arg Ser Met Asp Asp Val Phe Ser Glu Glu 675 680 685 Thr Phe Ile Met Glu Phe Pro Arg Lys Asn Gly Ile Asp Lys Lys Thr 690 695 700 Ala Ala Phe Gln Asn Ile Pro Asn Ser Ile Val Met Glu Tyr Pro Gln 705 710 715 720 Thr Asn Gly Tyr Ser Met Arg Ser His Ser Leu Lys Ser Asn Val Val 725 730 735 Ala Ala Lys His Phe Leu Glu Lys Leu Asn Lys Ile Lys Val Asp Ile 740 745 750 Lys Phe Lys Lys His Asp Leu Ala Asn Ile Lys Lys Met Asn Arg Ile 755 760 765 Ile Tyr Glu His Leu Gly Ile Asn Ile Asn Ile Glu Ala Phe Leu Lys 770 775 780 Pro Arg Leu Glu Lys Phe Lys Arg Glu Glu Lys Tyr Phe His Asp Phe 785 790 795 800 Phe Lys Arg Asn Asn Phe Lys Glu Val Ile Phe Pro Ser Thr Tyr Trp 805 810 815 Asn Pro Gly Ile Ile Cys Ala Ala His Lys Gln Gly Ile Lys Val Ser 820 825 830 Asp Ile Gln Tyr Ala Ala Ile Thr Pro Tyr His Pro Ala Tyr Phe Lys 835 840 845 Ser Pro Lys Ser His Tyr Val Ala Asp Lys Leu Phe Leu Trp Ser Glu 850 855 860 Tyr Trp Asn His Glu Leu Leu Pro Asn Pro Thr Arg Glu Ile Gly Ser 865 870 875 880 Gly Ala Ala Tyr Trp Tyr Ala Leu Asp Asp Val Arg Phe Ser Glu Lys 885 890 895 Leu Asn Tyr Asp Tyr Ile Phe Leu Ser Gln Ser Arg Ile Ser Ser Arg 900 905 910 Leu Leu Ser Phe Ala Ile Glu Phe Ala Leu Lys Asn Pro Gln Leu Gln 915 920 925 Leu Leu Phe Ser Lys His Leu Asp Glu Asn Ile Asp Leu Lys Asn Arg 930 935 940 Ile Ile Pro Asp Asn Leu Ile Ile Ser Thr Glu Ser Ser Ile Gln Gly 945 950 955 960 Ile Asn Glu Ser Arg Val Ala Val Gly Val Tyr Ser Thr Ser Leu Phe 965 970 975 Glu Ala Leu Ala Cys Gly Lys Gln Thr Phe Val Val Lys Tyr Pro Gly 980 985 990 Tyr Glu Ile Met Ser Asn Glu Ile Asp Ser Gly Leu Phe Phe Ala Val 995 1000 1005 Glu Thr Pro Glu Glu Met Leu Glu Lys Thr Ser Pro Asn Trp Val Ala 1010 1015 1020 Val Ala Asp Ile Glu Asn Gln Phe Phe Gly Gln Glu Lys 1025 1030 1035 <210> SEQ ID NO 108 <211> LENGTH: 1037 <212> TYPE: PRT <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: polymerase of serogroup W-135 <400> SEQUENCE: 108 Met Ala Val Ile Ile Phe Val Asn Gly Ile Arg Ala Val Asn Gly Leu 1 5 10 15 Val Lys Ser Ser Ile Asn Thr Ala Asn Ala Phe Ala Glu Glu Gly Leu 20 25 30 Asp Val His Leu Ile Asn Phe Val Gly Asn Ile Thr Gly Ala Glu His 35 40 45 Leu Tyr Pro Pro Phe His Leu His Pro Asn Val Lys Thr Ser Ser Ile 50 55 60 Ile Asp Leu Phe Asn Asp Ile Pro Glu Asn Val Ser Cys Arg Asn Thr 65 70 75 80 Pro Phe Tyr Ser Ile His Gln Gln Phe Phe Lys Ala Glu Tyr Ser Ala 85 90 95 His Tyr Lys His Val Leu Met Lys Ile Glu Ser Leu Leu Ser Ala Glu 100 105 110 Asp Ser Ile Ile Phe Thr His Pro Leu Gln Leu Glu Met Tyr Arg Leu 115 120 125 Ala Asn Asn Asp Ile Lys Ser Lys Ala Lys Leu Ile Val Gln Ile His 130 135 140 Gly Asn Tyr Met Glu Glu Ile His Asn Tyr Glu Ile Leu Ala Arg Asn 145 150 155 160 Ile Asp Tyr Val Asp Tyr Leu Gln Thr Val Ser Asp Glu Met Leu Glu 165 170 175 Glu Met His Ser His Phe Lys Ile Lys Lys Asp Lys Leu Val Phe Ile 180 185 190 Pro Asn Ile Thr Tyr Pro Ile Ser Leu Glu Lys Lys Glu Ala Asp Phe 195 200 205 Phe Ile Lys Asp Asn Glu Asp Ile Asp Asn Ala Gln Lys Phe Lys Arg 210 215 220 Ile Ser Ile Val Gly Ser Ile Gln Pro Arg Lys Asn Gln Leu Asp Ala 225 230 235 240 Ile Lys Ile Ile Asn Lys Ile Lys Asn Glu Asn Tyr Ile Leu Gln Ile 245 250 255 Tyr Gly Lys Ser Ile Asn Lys Asp Tyr Phe Glu Leu Ile Lys Lys Tyr 260 265 270 Ile Lys Asp Asn Lys Leu Gln Asn Arg Ile Leu Phe Lys Gly Glu Ser 275 280 285 Ser Glu Gln Glu Ile Tyr Glu Asn Thr Asp Ile Leu Ile Met Thr Ser 290 295 300 Glu Ser Glu Gly Phe Pro Tyr Ile Phe Met Glu Gly Met Val Tyr Asp 305 310 315 320 Ile Pro Ile Val Val Tyr Asp Phe Lys Tyr Gly Ala Asn Asp Tyr Ser 325 330 335 Asn Tyr Asn Glu Asn Gly Cys Val Phe Lys Thr Gly Asp Ile Ser Gly 340 345 350 Met Ala Lys Lys Ile Ile Glu Leu Leu Asn Asn Pro Glu Lys Tyr Lys 355 360 365 Glu Leu Val Gln Tyr Asn His Asn Arg Phe Leu Lys Glu Tyr Ala Lys 370 375 380 Asp Val Val Met Ala Lys Tyr Phe Thr Ile Leu Pro Arg Ser Phe Asn 385 390 395 400 Asn Val Ser Leu Ser Ser Ala Phe Ser Arg Lys Glu Leu Asp Glu Phe 405 410 415 Gln Asn Ile Thr Phe Ser Ile Glu Asp Ser Asn Asp Leu Ala His Ile 420 425 430 Trp Asn Phe Glu Leu Thr Asn Pro Ala Gln Asn Met Asn Phe Phe Ala 435 440 445 Leu Val Gly Lys Arg Lys Phe Pro Met Asp Ala His Ile Gln Gly Thr 450 455 460 Gln Cys Thr Ile Lys Ile Ala His Lys Lys Thr Gly Asn Leu Leu Ser 465 470 475 480 Leu Leu Leu Lys Lys Arg Asn Gln Leu Asn Leu Ser Arg Gly Tyr Thr 485 490 495 Leu Ile Ala Glu Asp Asn Ser Tyr Glu Lys Tyr Ile Gly Ala Ile Ser 500 505 510 Asn Lys Gly Asn Phe Glu Ile Ile Ala Asn Lys Lys Ser Ser Leu Val 515 520 525 Thr Ile Asn Lys Ser Thr Leu Glu Leu His Glu Ile Pro His Glu Leu 530 535 540 His Gln Asn Lys Leu Leu Ile Ala Leu Pro Asn Met Gln Thr Pro Leu 545 550 555 560 Lys Ile Thr Asp Asp Asn Leu Ile Pro Ile Gln Ala Ser Ile Lys Leu 565 570 575 Glu Lys Ile Gly Asn Thr Tyr Tyr Pro Cys Phe Leu Pro Ser Gly Ile 580 585 590 Phe Asn Asn Ile Cys Leu Asp Tyr Gly Glu Glu Ser Lys Ile Ile Asn 595 600 605 Phe Ser Lys Tyr Ser Tyr Lys Tyr Ile Tyr Asp Ser Ile Arg His Ile 610 615 620 Glu Gln His Thr Asp Ile Ser Asp Ile Ile Val Cys Asn Val Tyr Ser 625 630 635 640 Trp Glu Leu Ile Arg Ala Ser Val Ile Glu Ser Leu Met Glu Phe Thr 645 650 655 Gly Lys Trp Glu Lys His Phe Gln Thr Ser Pro Lys Ile Asp Tyr Arg 660 665 670 Phe Asp His Glu Gly Lys Arg Ser Met Asp Asp Val Phe Ser Glu Glu 675 680 685 Thr Phe Ile Met Glu Phe Pro Arg Lys Asn Gly Ile Asp Lys Lys Thr 690 695 700 Ala Ala Phe Gln Asn Ile Pro Asn Ser Ile Val Met Glu Tyr Pro Gln 705 710 715 720 Thr Asn Gly Tyr Ser Met Arg Ser His Ser Leu Lys Ser Asn Val Val 725 730 735

Ala Ala Lys His Phe Leu Glu Lys Leu Asn Lys Ile Lys Val Asp Ile 740 745 750 Lys Phe Lys Lys His Asp Leu Ala Asn Ile Lys Lys Met Asn Arg Ile 755 760 765 Ile Tyr Glu His Leu Gly Ile Asn Ile Asn Ile Glu Ala Phe Leu Lys 770 775 780 Pro Arg Leu Glu Lys Phe Lys Arg Glu Glu Lys Tyr Phe His Asp Phe 785 790 795 800 Phe Lys Arg Asn Asn Phe Lys Glu Val Ile Phe Pro Ser Thr Tyr Trp 805 810 815 Asn Pro Gly Ile Ile Cys Ala Ala His Lys Gln Gly Ile Lys Val Ser 820 825 830 Asp Ile Gln Tyr Ala Ala Ile Thr Pro Tyr His Pro Ala Tyr Phe Lys 835 840 845 Ser Pro Lys Ser His Tyr Val Ala Asp Lys Leu Phe Leu Trp Ser Glu 850 855 860 Tyr Trp Asn His Glu Leu Leu Pro Asn Pro Thr Arg Glu Ile Gly Ser 865 870 875 880 Gly Ala Ala Tyr Trp Tyr Ala Leu Asp Asp Val Arg Phe Ser Glu Lys 885 890 895 Leu Asn Tyr Asp Tyr Ile Phe Leu Ser Gln Ser Arg Ile Ser Ser Arg 900 905 910 Leu Leu Ser Phe Ala Ile Glu Phe Ala Leu Lys Asn Pro Gln Leu Gln 915 920 925 Leu Leu Phe Ser Lys His Pro Asp Glu Asn Ile Asp Leu Lys Asn Arg 930 935 940 Ile Ile Pro Asp Asn Leu Ile Ile Ser Thr Glu Ser Ser Ile Gln Gly 945 950 955 960 Ile Asn Glu Ser Arg Val Ala Val Gly Val Tyr Ser Thr Ser Leu Phe 965 970 975 Glu Ala Leu Ala Cys Gly Lys Gln Thr Phe Val Val Lys Tyr Pro Gly 980 985 990 Tyr Glu Ile Met Ser Asn Glu Ile Asp Ser Gly Leu Phe Phe Ala Val 995 1000 1005 Glu Thr Pro Glu Glu Met Leu Glu Lys Thr Ser Pro Asn Trp Val Ala 1010 1015 1020 Val Ala Asp Ile Glu Asn Gln Phe Phe Gly Gln Glu Lys 1025 1030 1035 <210> SEQ ID NO 109 <211> LENGTH: 495 <212> TYPE: PRT <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: Polysialyltransferase of NmB <400> SEQUENCE: 109 Met Leu Lys Lys Ile Lys Lys Ala Leu Phe Gln Pro Lys Lys Phe Phe 1 5 10 15 Gln Asp Ser Met Trp Leu Thr Thr Ser Pro Phe Tyr Leu Thr Pro Pro 20 25 30 Arg Asn Asn Leu Phe Val Ile Ser Asn Leu Gly Gln Leu Asn Gln Val 35 40 45 Gln Ser Leu Ile Lys Ile Gln Lys Leu Thr Asn Asn Leu Leu Val Ile 50 55 60 Leu Tyr Thr Ser Lys Asn Leu Lys Met Pro Lys Leu Val His Gln Ser 65 70 75 80 Ala Asn Lys Asn Leu Phe Glu Ser Ile Tyr Leu Phe Glu Leu Pro Arg 85 90 95 Ser Pro Asn Asn Ile Thr Pro Lys Lys Leu Leu Tyr Ile Tyr Arg Ser 100 105 110 Tyr Lys Lys Ile Leu Asn Ile Ile Gln Pro Ala His Leu Tyr Met Leu 115 120 125 Ser Phe Thr Gly His Tyr Ser Tyr Leu Ile Ser Ile Ala Lys Lys Lys 130 135 140 Asn Ile Thr Thr His Leu Ile Asp Glu Gly Thr Gly Thr Tyr Ala Pro 145 150 155 160 Leu Leu Glu Ser Phe Ser Tyr His Pro Thr Lys Leu Glu Arg Tyr Leu 165 170 175 Ile Gly Asn Asn Leu Asn Ile Lys Gly Tyr Ile Asp His Phe Asp Ile 180 185 190 Leu His Val Pro Phe Pro Glu Tyr Ala Lys Lys Ile Phe Asn Ala Lys 195 200 205 Lys Tyr Asn Arg Phe Phe Ala His Ala Gly Gly Ile Ser Ile Asn Asn 210 215 220 Asn Ile Ala Asn Leu Gln Lys Lys Tyr Gln Ile Ser Lys Asn Asp Tyr 225 230 235 240 Ile Phe Val Ser Gln Arg Tyr Pro Ile Ser Asp Asp Leu Tyr Tyr Lys 245 250 255 Ser Ile Val Glu Ile Leu Asn Ser Ile Ser Leu Gln Ile Lys Gly Lys 260 265 270 Ile Phe Ile Lys Leu His Pro Lys Glu Met Gly Asn Asn Tyr Val Met 275 280 285 Ser Leu Phe Leu Asn Met Val Glu Ile Asn Pro Arg Leu Val Val Ile 290 295 300 Asn Glu Pro Pro Phe Leu Ile Glu Pro Leu Ile Tyr Leu Thr Asn Pro 305 310 315 320 Lys Gly Ile Ile Gly Leu Ala Ser Ser Ser Leu Ile Tyr Thr Pro Leu 325 330 335 Leu Ser Pro Ser Thr Gln Cys Leu Ser Ile Gly Glu Leu Ile Ile Asn 340 345 350 Leu Ile Gln Lys Tyr Ser Met Val Glu Asn Thr Glu Met Ile Gln Glu 355 360 365 His Leu Glu Ile Ile Lys Lys Phe Asn Phe Ile Asn Ile Leu Asn Asp 370 375 380 Leu Asn Gly Val Ile Ser Asn Pro Leu Phe Lys Thr Glu Glu Thr Phe 385 390 395 400 Glu Thr Leu Leu Lys Ser Ala Glu Phe Ala Tyr Lys Ser Lys Asn Tyr 405 410 415 Phe Gln Ala Ile Phe Tyr Trp Gln Leu Ala Ser Lys Asn Asn Ile Thr 420 425 430 Leu Leu Gly His Lys Ala Leu Trp Tyr Tyr Asn Ala Leu Tyr Asn Val 435 440 445 Lys Gln Ile Tyr Lys Met Glu Tyr Ser Asp Ile Phe Tyr Ile Asp Asn 450 455 460 Ile Ser Val Asp Phe His Ser Lys Asp Lys Leu Thr Trp Glu Lys Ile 465 470 475 480 Lys His Tyr Tyr Tyr Ser Ala Asp Asn Arg Ile Gly Arg Asp Arg 485 490 495 <210> SEQ ID NO 110 <211> LENGTH: 492 <212> TYPE: PRT <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: Polysialyltransferase of NmC <400> SEQUENCE: 110 Met Leu Gln Lys Ile Arg Lys Ala Leu Phe His Pro Lys Lys Phe Phe 1 5 10 15 Gln Asp Ser Gln Trp Phe Ala Thr Pro Leu Phe Ser Ser Phe Ala Pro 20 25 30 Lys Ser Asn Leu Phe Ile Ile Ser Thr Phe Ala Gln Leu Asn Gln Ala 35 40 45 His Ser Leu Thr Lys Met Gln Lys Leu Lys Asn Asn Leu Leu Val Ile 50 55 60 Leu Tyr Thr Thr Gln Asn Met Lys Met Pro Lys Leu Ile Gln Lys Ser 65 70 75 80 Val Asp Lys Glu Leu Phe Ser Val Thr Tyr Met Phe Glu Leu Pro Arg 85 90 95 Lys Pro Gly Ile Val Ser Pro Lys Lys Phe Leu Tyr Ile Gln Arg Gly 100 105 110 Tyr Lys Lys Leu Leu Lys Thr Ile Gln Pro Ala His Leu Tyr Val Met 115 120 125 Ser Phe Ala Gly His Tyr Ser Ser Leu Leu Ser Leu Ala Lys Lys Met 130 135 140 Asn Ile Thr Thr His Leu Val Glu Glu Gly Thr Ala Thr Tyr Ala Pro 145 150 155 160 Leu Leu Glu Ser Phe Thr Tyr Lys Pro Thr Lys Phe Glu Gln Arg Phe 165 170 175 Val Gly Asn Asn Leu His Gln Lys Gly Tyr Phe Asp Lys Phe Asp Ile 180 185 190 Leu His Val Ala Phe Pro Glu Tyr Ala Lys Lys Ile Phe Asn Ala Asn 195 200 205 Glu Tyr His Arg Phe Phe Ala His Ser Gly Gly Ile Ser Thr Ser Gln 210 215 220 Ser Ile Ala Lys Ile Gln Asp Lys Tyr Arg Ile Ser Gln Asn Asp Tyr 225 230 235 240 Ile Phe Val Ser Gln Arg Tyr Pro Val Ser Asp Glu Val Tyr Tyr Lys 245 250 255 Thr Ile Val Glu Thr Leu Asn Gln Met Ser Leu Arg Ile Glu Gly Lys 260 265 270 Ile Phe Ile Lys Leu His Pro Lys Glu Met Glu Asn Lys Asn Ile Met 275 280 285 Ser Leu Phe Leu Asn Met Val Thr Ile Asn Pro Arg Leu Val Val Ile 290 295 300 Asn Glu Pro Pro Phe Leu Ile Asp Pro Leu Ile Tyr Leu Thr Thr Pro 305 310 315 320 Lys Gly Ile Ile Gly Leu Thr Ser Thr Ser Ile Val Tyr Thr Pro Leu 325 330 335 Leu Ser Pro Thr Thr Gln Cys Leu Ser Ile Gly Gln Ile Val Ile Asp 340 345 350 Ser Ile His His Thr Ala Gln Gln Glu Asn Thr Ala Leu Ile Glu Glu 355 360 365 His Leu Glu Ile Val Lys Gln Phe Asp Phe Ile Lys Ile Leu Ser Ser 370 375 380 Ile Glu Asp Gly Ile Asp Thr Asn Ser Phe Lys Thr Glu Glu Thr Leu 385 390 395 400 Glu Met Leu Leu Lys Ser Ala Glu Tyr Ala Tyr Lys Asn Lys Asn Phe 405 410 415 Tyr Gln Ala Ile Phe Tyr Trp Gln Leu Ala Ser Asn Asn Asp Leu Ser 420 425 430

Val Leu Gly Tyr Lys Ser Leu Trp Tyr Tyr Asn Ala Leu Asn Lys Val 435 440 445 Lys Gln Asn Tyr Lys Met Lys Tyr Leu Glu Ile Asn Tyr Ile Glu Arg 450 455 460 Ile Ser Leu Tyr Phe Asn Asp Lys Asp Lys Met Ile Trp Gln Asn Ile 465 470 475 480 Lys Asn Asp Phe Phe Lys Tyr Ser Leu Cys Asn Gln 485 490 <210> SEQ ID NO 111 <211> LENGTH: 409 <212> TYPE: PRT <213> ORGANISM: Escherichia coli <220> FEATURE: <223> OTHER INFORMATION: Escherichia coli K1 <400> SEQUENCE: 111 Met Ile Phe Asp Ala Ser Leu Lys Lys Leu Arg Lys Leu Phe Val Asn 1 5 10 15 Pro Ile Gly Phe Phe Arg Asp Ser Trp Phe Phe Asn Ser Lys Asn Lys 20 25 30 Ala Glu Glu Leu Leu Ser Pro Leu Lys Ile Lys Ser Lys Asn Ile Phe 35 40 45 Ile Ile Ser Asn Leu Gly Gln Leu Lys Lys Ala Glu Ser Phe Val Gln 50 55 60 Lys Phe Ser Lys Arg Ser Asn Tyr Leu Ile Val Leu Ala Thr Glu Lys 65 70 75 80 Asn Thr Glu Met Pro Lys Ile Ile Val Glu Gln Ile Asn Asn Lys Leu 85 90 95 Phe Ser Ser Tyr Lys Val Leu Phe Ile Pro Thr Phe Pro Asn Val Phe 100 105 110 Ser Leu Lys Lys Val Ile Trp Phe Tyr Asn Val Tyr Asn Tyr Leu Val 115 120 125 Leu Asn Ser Lys Ala Lys Asp Ala Tyr Phe Met Ser Tyr Ala Gln His 130 135 140 Tyr Ala Ile Phe Val Tyr Leu Phe Lys Lys Asn Asn Ile Arg Cys Ser 145 150 155 160 Leu Ile Glu Glu Gly Thr Gly Thr Tyr Lys Thr Glu Lys Glu Asn Pro 165 170 175 Val Val Asn Ile Asn Phe Tyr Ser Glu Ile Ile Asn Ser Ile Ile Leu 180 185 190 Phe His Tyr Pro Asp Leu Lys Phe Glu Asn Val Tyr Gly Thr Tyr Pro 195 200 205 Ile Leu Leu Lys Lys Lys Phe Asn Ala Gln Lys Phe Val Glu Phe Lys 210 215 220 Gly Ala Pro Ser Val Lys Ser Ser Thr Arg Ile Asp Asn Val Ile His 225 230 235 240 Lys Tyr Ser Ile Thr Arg Asp Asp Ile Ile Tyr Ala Asn Gln Lys Tyr 245 250 255 Leu Ile Glu His Thr Leu Phe Ala Asp Ser Leu Ile Ser Ile Leu Leu 260 265 270 Arg Ile Asp Lys Pro Asp Asn Ala Arg Ile Phe Ile Lys Pro His Pro 275 280 285 Lys Glu Pro Lys Lys Asn Ile Asn Ala Ile Gln Lys Ala Ile Lys Lys 290 295 300 Ala Lys Cys Arg Asp Ile Ile Leu Ile Thr Glu Pro Asp Phe Leu Ile 305 310 315 320 Glu Pro Val Ile Lys Lys Ala Lys Ile Lys His Leu Ile Gly Leu Thr 325 330 335 Ser Ser Ser Leu Val Tyr Ala Pro Leu Val Ser Lys Arg Cys Gln Ser 340 345 350 Tyr Ser Ile Ala Pro Leu Met Ile Lys Leu Cys Asp Asn Asp Lys Ser 355 360 365 Gln Lys Gly Ile Asn Thr Leu Arg Leu His Phe Asp Ile Leu Lys Asn 370 375 380 Phe Asp Asn Val Lys Ile Leu Ser Asp Asp Ile Thr Ser Pro Ser Leu 385 390 395 400 His Asp Lys Arg Ile Phe Leu Gly Glu 405 <210> SEQ ID NO 112 <211> LENGTH: 409 <212> TYPE: PRT <213> ORGANISM: Escherichia coli <220> FEATURE: <223> OTHER INFORMATION: Escherichia coli K92 <400> SEQUENCE: 112 Met Ile Phe Asp Ala Ser Leu Lys Lys Leu Arg Lys Leu Phe Val Asn 1 5 10 15 Pro Ile Gly Phe Phe Arg Asp Ser Trp Phe Phe Asn Ser Lys Asn Lys 20 25 30 Ala Glu Glu Leu Leu Ser Pro Leu Lys Ile Lys Ser Lys Asn Ile Phe 35 40 45 Ile Val Ala His Leu Gly Gln Leu Lys Lys Ala Glu Leu Phe Ile Gln 50 55 60 Lys Phe Ser Arg Arg Ser Asn Phe Leu Ile Val Leu Ala Thr Lys Lys 65 70 75 80 Asn Thr Glu Met Pro Arg Leu Ile Leu Glu Gln Met Asn Lys Lys Leu 85 90 95 Phe Ser Ser Tyr Lys Leu Leu Phe Ile Pro Thr Glu Pro Asn Thr Phe 100 105 110 Ser Leu Lys Lys Val Ile Trp Phe Tyr Asn Val Tyr Lys Tyr Ile Val 115 120 125 Leu Asn Ser Lys Ala Lys Asp Ala Tyr Phe Met Ser Tyr Ala Gln His 130 135 140 Tyr Ala Ile Phe Ile Trp Leu Phe Lys Lys Asn Asn Ile Arg Cys Ser 145 150 155 160 Leu Ile Glu Glu Gly Thr Gly Thr Tyr Lys Thr Glu Lys Lys Lys Pro 165 170 175 Leu Val Asn Ile Asn Phe Tyr Ser Trp Ile Ile Asn Ser Ile Ile Leu 180 185 190 Phe His Tyr Pro Asp Leu Lys Phe Glu Asn Val Tyr Gly Thr Phe Pro 195 200 205 Asn Leu Leu Lys Glu Lys Phe Asp Ala Lys Lys Ile Phe Glu Phe Lys 210 215 220 Thr Ile Pro Leu Val Lys Ser Ser Thr Arg Met Asp Asn Leu Ile His 225 230 235 240 Lys Tyr Arg Ile Thr Arg Asp Asp Ile Ile Tyr Val Ser Gln Arg Tyr 245 250 255 Trp Ile Asp Asn Glu Leu Tyr Ala His Leu Leu Ile Ser Thr Leu Met 260 265 270 Arg Ile Asp Lys Ser Asp Asn Ala Arg Val Phe Ile Lys Pro His Pro 275 280 285 Lys Glu Thr Lys Lys Tyr Ile Asn Ala Ile Gln Gly Ala Ile Asn Lys 290 295 300 Ala Lys Arg Arg Asp Ile Ile Ile Ile Val Glu Lys Asp Phe Leu Ile 305 310 315 320 Glu Ser Ile Ile Lys Lys Cys Lys Ile Lys His Leu Ile Gly Leu Ala 325 330 335 Ser Ser Ser Leu Val Tyr Ala Pro Leu Val Tyr Lys Glu Cys Lys Thr 340 345 350 Tyr Ser Ile Ala Pro Ile Ile Ile Lys Leu Cys Asn Asn Glu Lys Ser 355 360 365 Gln Lys Gly Ile Asn Thr Leu Arg Leu His Phe Asp Ile Leu Lys Asn 370 375 380 Phe Asp Asn Val Lys Ile Leu Ser Asp Asp Ile Thr Ser Pro Ser Leu 385 390 395 400 His Asp Lys Arg Ile Phe Leu Gly Glu 405 <210> SEQ ID NO 113 <211> LENGTH: 29 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer StrepII-deltaN235-CsaB-His6 <400> SEQUENCE: 113 gcagatctat tgatttagta tttacttgg 29 <210> SEQ ID NO 114 <211> LENGTH: 32 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer StrepII-deltaN235-CsaB-His6 <400> SEQUENCE: 114 ccgctcgagt ttctcaaatg atgatggtaa tg 32 <210> SEQ ID NO 115 <211> LENGTH: 29 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer StrepII-deltaN167-CsaB-His6 <400> SEQUENCE: 115 gcagatctac tttaagttca tctatatct 29 <210> SEQ ID NO 116 <211> LENGTH: 32 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer StrepII-deltaN167-CsaB-His6 <400> SEQUENCE: 116 ccgctcgagt ttctcaaatg atgatggtaa tg 32 <210> SEQ ID NO 117 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer StrepII-deltaN97-CsaB-His6 <400> SEQUENCE: 117 gcagatctcc ttctaatctt actcttaagc 30 <210> SEQ ID NO 118 <211> LENGTH: 32

<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer StrepII-deltaN97-CsaB-His6 <400> SEQUENCE: 118 ccgctcgagt ttctcaaatg atgatggtaa tg 32 <210> SEQ ID NO 119 <211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer StrepII-deltaC41-CsaB-His6 <400> SEQUENCE: 119 gcagatcttt tatacttaat aacagaaaat ggc 33 <210> SEQ ID NO 120 <211> LENGTH: 32 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer StrepII-deltaC41-CsaB-His6 <400> SEQUENCE: 120 cgcctcgaga aaattccttt ctttagttaa gg 32 <210> SEQ ID NO 121 <211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer StrepII-deltaC25-CsaB-His6 <400> SEQUENCE: 121 gcagatcttt tatacttaat aacagaaaat ggc 33 <210> SEQ ID NO 122 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer StrepII-deltaC25-CsaB-His6 <400> SEQUENCE: 122 cgcctcgagg tgactatcag caccatcg 28 <210> SEQ ID NO 123 <211> LENGTH: 32 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer MBP-deltaN58-CsxA-His6 <400> SEQUENCE: 123 cgggatcccc aattgaagat ccatacccag ta 32 <210> SEQ ID NO 124 <211> LENGTH: 29 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer MBP-deltaN58-CsxA-His6 <400> SEQUENCE: 124 ccgctcgagt tgtccactag gctgtgatg 29 <210> SEQ ID NO 125 <211> LENGTH: 32 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer MBP-deltaC99-CsxA-His6 <400> SEQUENCE: 125 gcggatccat tatgagcaaa attagcaaat tg 32 <210> SEQ ID NO 126 <211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer MBP-deltaC99-CsxA-His6 <400> SEQUENCE: 126 ccgctcgagg agaatttctg cttctgatac atc 33 <210> SEQ ID NO 127 <211> LENGTH: 32 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer MBP-deltaN58deltaC99-CsxA-His6 <400> SEQUENCE: 127 cgggatcccc aattgaagat ccatacccag ta 32 <210> SEQ ID NO 128 <211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer MBP-deltaN58deltaC99-CsxA-His6 <400> SEQUENCE: 128 ccgctcgagg agaatttctg cttctgatac atc 33 <210> SEQ ID NO 129 <211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer MBP-deltaN104-CsxA-His6 <400> SEQUENCE: 129 cgggatccga aagcaccgat attgcaagat tcc 33 <210> SEQ ID NO 130 <211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer MBP-deltaN104-CsxA-His6 <400> SEQUENCE: 130 ccgctcgagg agaatttctg cttctgatac atc 33 <210> SEQ ID NO 131 <211> LENGTH: 32 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer MBP-deltaC174-CsxA-His6 <400> SEQUENCE: 131 gcggatccat tatgagcaaa attagcaaat tg 32 <210> SEQ ID NO 132 <211> LENGTH: 37 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer MBP-deltaC174-CsxA-His6 <400> SEQUENCE: 132 ccgctcgagt tggcctgtca aaatggcaaa atggtgg 37 <210> SEQ ID NO 133 <211> LENGTH: 58 <212> TYPE: PRT <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: Fragment Figure 7 CslA <400> SEQUENCE: 133 Thr Phe Leu Val Pro Trp Leu Met Tyr Leu Asp Gly Lys Ser Ile Pro 1 5 10 15 Asn Asn Asp Ile Cys Tyr Tyr Phe Asn Ile Arg Ser Asn Lys Ala Pro 20 25 30 Thr Gln Tyr Leu Lys Leu Leu Gln Lys Asn Glu Asp Asn Gln Gln Pro 35 40 45 His Ser Phe Cys Ala Asn Asp Phe His Ser 50 55 <210> SEQ ID NO 134 <211> LENGTH: 60 <212> TYPE: PRT <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: Fragment Figure 7 CsaB <400> SEQUENCE: 134 Gly Tyr Leu Tyr His His Tyr Ala Leu Leu Ser Gly Arg Ala Leu Gln 1 5 10 15 Ser Ser Asp Lys Thr Glu Leu Val Gln Gln Asn His Asp Phe Lys Lys 20 25 30 Lys Leu Asn Asn Val Val Thr Leu Thr Lys Glu Arg Asn Phe Asp Lys 35 40 45 Leu Pro Leu Ser Val Cys Ile Asn Asp Gly Ala Asp 50 55 60 <210> SEQ ID NO 135 <211> LENGTH: 58 <212> TYPE: PRT <213> ORGANISM: Neisseria meningitidis <220> FEATURE: <223> OTHER INFORMATION: Fragment Figure 7 CsxA <400> SEQUENCE: 135 Thr Phe Leu His His His Phe Ala Ile Leu Thr Gly Gln Ala Leu Ala 1 5 10 15 Thr Arg Thr Lys Cys Ile Tyr Phe Asn Ile Arg Ser Pro Gln Ala Ala 20 25 30 Gln His Tyr Lys Thr Leu Leu Ala Arg Lys Gly Ser Glu Tyr Ser Pro 35 40 45 His Ser Ile Cys Leu Asn Asp His Thr Ser 50 55

Claims

1. In vitro method for producing Neisseria meningitidis capsular polysaccharides which have a defined length, said method comprising the steps: (a) incubating at least one capsule polymerase with at least one donor carbohydrate and at least one acceptor carbohydrate; wherein the ratio of donor carbohydrate to acceptor carbohydrate is a ratio from 10:1 to 400:1; and (b) isolating the resulting capsular polysaccharides, wherein the capsule polymerase is the capsule polymerase of Neisseria meningitidis serogroup A or a truncated version of the capsule polymerase of Neisseria meningitidis serogroup X.

2. In vitro method for producing Neisseria meningitidis capsular polysaccharides which have a defined length, said method comprising the steps: (a) incubating at least one capsule polymerase with at least one donor carbohydrate and at least one acceptor carbohydrate; wherein the incubation time ranges from 3 to 45 minutes; and (b) isolating the resulting capsular polysaccharides, wherein the capsule polymerase is a truncated version of the capsule polymerase of Neisseria meningitidis serogroup X.

3. In vitro method for producing Neisseria meningitidis capsular polysaccharides which have a defined length, said method comprising the steps: (a) incubating at least one capsule polymerase with at least one donor carbohydrate and at least one acceptor carbohydrate; wherein (i) the ratio of donor carbohydrate to acceptor carbohydrate is a ratio from 10:1 to 10000:1, and (ii) the incubation time ranges from 3 to 45 minutes; and (b) isolating the resulting capsular polysaccharides, wherein the capsule polymerase is a truncated version of the capsule polymerase of Neisseria meningitidis serogroup X.

4. A composition comprising: (i) at least one capsule polymerase; (ii) at least one donor carbohydrate; and (iii) at least one acceptor carbohydrate, wherein the ratio of donor carbohydrate to acceptor carbohydrate is a ratio from 10:1 to 10000:1, and wherein the capsule polymerase is the capsule polymerase of Neisseria meningitidis serogroup A or a truncated version of the capsule polymerase of Neisseria meningitidis serogroup X.

5. The composition of claim 4, wherein the ratio of donor carbohydrate to acceptor carbohydrate is a ratio from 20:1 to 240:1.

6. The method of claim 1 or the composition of claim 4 or 5, wherein the capsule polymerase of Neisseria meningitidis serogroup A is the polypeptide of any one of (a) to (f): (a) a polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule having the nucleic acid sequence of any one of SEQ ID NO: 1 to 3; (b) a polypeptide comprising the amino acid sequence of SEQ ID NO: 9 or 10; (c) a polypeptide encoded by a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 9 or 10 or of a functional fragment thereof, wherein the function comprises the ability to transfer ManNAc-1P or a derivative thereof from UDP-ManNAc or a derivative thereof onto an acceptor carbohydrate; (d) a polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule hybridizing under stringent conditions to the complementary strand of a nucleic acid molecule as defined in (a) or (c) and encoding a functional polypeptide; or a functional fragment thereof, wherein the function comprises the ability to transfer ManNAc-1P or a derivative thereof from UDP-ManNAc or a derivative thereof onto an acceptor carbohydrate; (e) a polypeptide having at least 80% identity to the polypeptide of any one of (a) to (d), whereby said polypeptide is functional; or a functional fragment thereof, wherein the function comprises the ability to transfer ManNAc-1P or a derivative thereof from UDP-ManNAc or a derivative thereof onto an acceptor carbohydrate; and (f) a polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule being degenerate as a result of the genetic code to the nucleotide sequence of a nucleic acid molecule as defined in (a), (c), and (d).

7. A polypeptide comprising: (a) an amino acid sequence encoded by a nucleic acid molecule which is a terminally truncated version of the nucleic acid sequence of SEQ ID NO: 16 and comprises maximal 50-95% of the nucleic acid sequence of SEQ ID NO: 16; (b) an amino acid sequence which is a terminally truncated version of the amino acid sequence of SEQ ID NO: 24 and comprises maximal 50-95% of the amino acid sequence of SEQ ID NO: 24; (c) a polypeptide encoded by a nucleic acid molecule encoding a polypeptide having an amino acid sequence which is a terminally truncated version of the amino acid sequence of SEQ ID NO: 24 and comprises maximal 50-95% of the amino acid sequence of SEQ ID NO: 24; (d) a polypeptide encoded by a nucleic acid molecule hybridizing under stringent conditions to the complementary strand of a nucleic acid molecule as defined in (a) or (c) and encoding a functional polypeptide; wherein the function comprises the ability to transfer GlcNAc-1P or a derivative thereof from UDP-GlcNAc or a derivative thereof onto an acceptor carbohydrate; (e) a polypeptide having at least 80% identity to the polypeptide of any one of (a) to (d), whereby said polypeptide is functional; wherein the function comprises the ability to transfer GlcNAc-1P or a derivative thereof from UDP-GlcNAc or a derivative thereof onto an acceptor carbohydrate; or (f) a polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule being degenerate as a result of the genetic code to the nucleotide sequence of a nucleic acid molecule as defined in (a), (c) or (d).

8. The polypeptide of claim 7, comprising: (a) an amino acid sequence which is a C-terminally or C- and N-terminally truncated version of the amino acid sequence of SEQ ID NO: 24 and comprises maximal 50-95% of the amino acid sequence of SEQ ID NO: 24; (b) a polypeptide encoded by a nucleic acid molecule encoding a polypeptide having an amino acid sequence which is a C-terminally or C- and N-terminally truncated version of the amino acid sequence of SEQ ID NO: 24 and comprises maximal 50-95% of the amino acid sequence of SEQ ID NO: 24; or (c) a polypeptide having at least 80% identity to the polypeptide of (a) or (b), whereby said polypeptide is functional; wherein the function comprises the ability to transfer GlcNAc-1P or a derivative thereof from UDP-GlcNAc or a derivative thereof onto an acceptor carbohydrate.

9. The polypeptide of claim 7 or 8, wherein the polypeptide comprises: (i) an amino acid sequence encoded by a nucleic acid molecule which comprises the nucleic acid sequence of SEQ ID NO: 17, 20 or 21; or a nucleic acid sequence having at least 80% identity to the nucleic acid sequence of any one of SEQ ID NO: 17, 20 or 21 and encoding a functional polypeptide; wherein the function comprises the ability to transfer GlcNAc-1P or a derivative thereof from UDP-GlcNAc or a derivative thereof onto an acceptor carbohydrate; or (ii) the amino acid sequence of any one of SEQ ID NO: 25, 28 or 29; or an amino acid sequence having at least 80% identity to SEQ ID NO: 25, 28 or 29 and being functional, wherein the function comprises the ability to transfer GlcNAc-1P or a derivative thereof from UDP-GlcNAc or a derivative thereof onto an acceptor carbohydrate.

10. The polypeptide of any one of claims 7 to 9, wherein the function comprises the ability to transfer GlcNAc-1P or a derivative thereof from UDP-GlcNAc or a derivative thereof onto an acceptor carbohydrate; wherein at least 60% of the the produced polymers have a DP between 10 and 60 or an avDP between 15 and 20, when the ratio of donor carbohydrate to acceptor carbohydrate is in the range of 10:1 to 1000:1.

11. Nucleic acid molecule encoding the polypeptide of any one of claims 7 to 10.

12. Vector comprising the nucleic acid molecule of claim 11.

13. Host cell comprising the vector of claim 12.

14. The method of any one of claims 1 to 3 and 6, or the composition of any one of claims 4 to 6, wherein the truncated version of the capsule polymerase of Neisseria meningitidis serogroup X is the polypeptide as defined in any one of claims 7 to 10.

15. The method of any one of claims 1 to 3, 6 and 14, wherein at least 60% of the produced capsular polysaccharides have a DP of 10 to 60 or an avDP of 15 to 20.

16. The method of any one of claims 1, 3, 6, 14 and 15, or the composition of any one of claims 4 to 6, 14 and 15, wherein (i) the ratio of donor carbohydrate to acceptor carbohydrate is a ratio from 20:1 to 80:1; and the capsule polymerase is either a polypeptide comprising maximal 80% of the amino acid sequence of SEQ ID NO: 24 and comprising the amino acid sequence of SEQ ID NO: 28, or a polypeptide comprising the amino acid sequence of SEQ ID NO: 9; or (ii) the ratio of donor carbohydrate to acceptor carbohydrate is a ratio from 100:1 to 400:1; and the capsule polymerase is a polypeptide comprising maximal 68% of the amino acid sequence of SEQ ID NO: 24 and comprising the amino acid sequence of SEQ ID NO: 29.

17. The method of any one of claims 1 to 3, 6 and 14 to 16, wherein said donor carbohydrate is activated or wherein said donor carbohydrate is activated during step (a).

18. The method of claim 17, wherein said donor carbohydrate is activated during step (a) by contacting said donor carbohydrate with an activating enzyme.

19. The method of claim 18, wherein in step (a) said donor carbohydrate is further contacted with PEP and/or at least one activating nucleotide.

20. The composition of any one of claims 4 to 6, 14 and 16, wherein said donor carbohydrate is activated.

21. The method of any one of claims 17 to 19 or the composition of claim 20, wherein said donor carbohydrate is activated by linkage to an activating nucleotide.

22. The method of claim 19 or 21 or the composition of claim 21, wherein said activating nucleotide is CMP, CDP, CTP, UMP, UDP, TDP, AMP or UTP.

23. The method of any one of claims 1 to 3, 6, 14 to 19, 21 and 22 or the composition of any one of claims 4 to 6, 14, 16 and 20 to 22, wherein the capsule polymerase is a truncated version of the capsule polymerase of Neisseria meningitidis serogroup X and wherein at least one donor carbohydrate is UDP-GlcNAc.

24. The method of any one of claims 1 to 3, 6, 14 to 19 and 21 to 23 or the composition of any one of claims 4 to 6, 14, 16 and 20 to 23, wherein the capsule polymerase is a truncated version of the capsule polymerase of Neisseria meningitidis serogroup X and wherein at least one donor carbohydrate is GlcNAc-1-P.

25. The method of any one of claims 1, 6, 14 to 19, 21 and 22 or the composition of any one of claims 4 to 6, 14, 16 and 20 to 22, wherein the capsule polymerase is the capsule polymerase of Neisseria meningitidis serogroup A and wherein at least one donor carbohydrate is UDP-ManNAc.

26. The method of any one of claims 1, 6, 14 to 19, 21, 22 and 25 or the composition of any one of claims 4 to 6, 14, 16, 20 to 22 and 25, wherein the capsule polymerase is the capsule polymerase of Neisseria meningitidis serogroup A and wherein at least one donor carbohydrate is UDP-GlcNAc.

27. The method of claim 26, wherein in step (a) the capsule polymerase is further incubated with the UDP-GlcNAc-epimerase.

28. The composition of claim 26, further comprising the UDP-GlcNAc-epimerase.

29. The method of any one of claims 1, 6, 14 to 19, 21, 22 and 25 to 27 or the composition of any one of claims 4 to 6, 14, 16, 29 to 22, 25, 26 and 28, wherein the capsule polymerase is the capsule polymerase of Neisseria meningitidis serogroup A and wherein at least one donor carbohydrate is ManNAc-1-P.

30. The method of any one of claims 1 to 3, 6, 14 to 19, 21 to 27 and 29 or the composition of any one of claims 4 to 6, 14, 16, 20 to 26, 28 and 29, wherein said acceptor carbohydrate is capsule polysaccharide of Neisseria meningitidis serogroup A or X or a carbohydrate structure containing terminal GlcNAc residues.

31. The method of claim 30 or the composition of claim 30, wherein the carbohydrate structure containing terminal GlcNAc-residues is hyaluronic acid, heparin sulphate, heparan sulphate or protein-linked oligosaccharides.

32. The method of any one of claims 1 to 3, 6, 14 to 19, 21 to 27 and 29 or the composition of any one of claims 4 to 6, 14, 16, 20 to 26, and 28-30, wherein at least 80% of the acceptor carbohydrates are polysaccharides with a DP of .gtoreq.4.

33. The method of any one of claims 1 to 3, 6, 14 to 19, 21 to 27 and 29 to 32 or composition of any one of claims 4 to 6, 14, 16, 20 to 26 and 28 to 32, wherein the acceptor carbohydrate is non-acetylated and/or dephosphorylated, preferably non-acetylated and dephosphorylated.

34. The method of any one of claims 1 to 3, 6, 14 to 19, 21 to 27 and 29 to 33 or the composition of any one of claims 4 to 6, 14, 16, 20 to 26 and 28 to 33, wherein the acceptor carbohydrate carries one or more additional functional groups at its reducing end.

35. The method of any one of claims 1 to 3, 6, 14 to 19, 21 to 27 and 29 to 31, and 33 or the composition of any one of claims 4 to 6, 14, 16, 20 to 26 and 28 to 31, and 33, wherein said acceptor carbohydrate is a dimer of ManNAc carrying a phosphodiester at the reducing end.

36. The method of claim 35 or the composition of claim 35, wherein the phosphate group at the reducing end is extended with alkyl-azides, alkyl-amindes or sulfhydryl-groups.

37. The method of claim 35 or 36 or the composition of claim 35 or 36, wherein the acceptor carbohydrate is a disaccharide carrying a decyl-phosphate-ester at the reducing end.

38. The method of any one of claims 1 to 3, 6, 14 to 19, 21 to 27 and 29 to 37 or the composition of any one of claims 4 to 6, 14, 16, 20 to 26 and 28 to 37, wherein said acceptor carbohydrate is purified.

39. The method of any one of claims 1 to 3, 6, 14 to 19, 21 to 27 and 29 to 38, further comprising O-acetylation of the produced capsule polysaccharides.

40. The method of claim 39, wherein the O-acetylation is performed by contacting the produced capsule polysaccharides with an O-acetyltransferase.

41. The composition of any one of claims 4 to 6, 14, 16, 20 to 26 and 28 to 38, further comprising an O-acetyltransferase.

42. The method of claim 40 or composition of claim 41, wherein the O-acetyltransferase is the polypeptide of any one of (a) to (f): (a) a polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 53; (b) a polypeptide comprising the amino acid sequence of SEQ ID NO: 54; (c) a polypeptide encoded by a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 54 or of a functional fragment thereof, wherein the function comprises the ability to transfer Acetyl-groups from the donor Acetyl-Coenzyme A onto hydroxyl-groups of UDP-ManNAc or oligo- and polymeric structures consisting of ManNAc-1-phosphate units linked together by phosphodiester linkages; (d) a polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule hybridizing under stringent conditions to the complementary strand of a nucleic acid molecule as defined in (a) or (c) and encoding a functional polypeptide; or a functional fragment thereof, wherein the function comprises the ability to transfer Acetyl-groups from the donor Acetyl-Coenzyme A onto hydroxyl-groups of UDP-ManNAc or oligo- and polymeric structures consisting of ManNAc-1-phosphate units linked together by phosphodiester linkages; (e) a polypeptide having at least 80% identity to the polypeptide of any one of (a) to (d), whereby said polypeptide is functional; or a functional fragment thereof, wherein the function comprises the ability to transfer Acetyl-groups from the donor Acetyl-Coenzyme A onto hydroxyl-groups of UDP-ManNAc or oligo- and polymeric structures consisting of ManNAc-1-phosphate units linked together by phosphodiester linkages; and (f) a polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule being degenerate as a result of the genetic code to the nucleotide sequence of a nucleic acid molecule as defined in (a), (c), and (d).

43. The method of any one of claims 35 to 40 and 42 or the composition of any one of claims 35 to 38 and 41, wherein the capsule polymerase is the capsule polymerase of Neisseria meningitidis serogroup A.

44. The method of claim 43, wherein the produced capsule polysaccharides are O-acetylated in positions 3 and 4 of ManNAc.

45. The method of any one of claims 1 to 3, 6, 14 to 19, 21 to 27, 29 to 39 and 41 to 44, further comprising covalently attaching the produced capsular polysaccharides to a carrier molecule.

46. The method of claim 45, wherein the carrier molecule is tetanus toxoid tetanus toxoid and CRM.sub.197 an inactive form of diphteria-toxin.

47. The method of any one of claims 1, 6, 14, 15, 17 to 19, 21 to 27, 29 to 39, and 41 to 46, wherein the ratio of donor carbohydrate to acceptor carbohydrate is a ratio from 20:1 to 400:1.

48. The method of any one of claims 3, 6, 14, 15, 17 to 19, 21 to 27, 29 to 39, and 41 to 46, wherein the ratio of donor carbohydrate to acceptor carbohydrate is a ratio from 200:1 to 1000:1.

49. The composition of any one of claims 4 to 6, 14, 16, 20 to 26, 28 to 38, and 41 to 43, wherein the ratio of donor carbohydrate to acceptor carbohydrate is a ratio from 20:1 to 1000:1.

50. The method of any one of claims 1 to 3, 6, 14 to 19, 21 to 27, 29 to 39 and 41 to 48, wherein the capsule polymerase is immobilized on a solid phase.

51. A capsular polysaccharide which has been produced by the method of any one of claims 1 to 3, 6, 14 to 19, 21 to 27, 29 to 39, 41 to 48 and 50.

52. The capsular polysaccharide of claim 51 for use as a medicament.

53. A vaccine comprising the capsular polysaccharide of claim 51, optionally further comprising a pharmaceutically acceptable carrier.

54. A method for producing a vaccine comprising the method of any one of claims 1 to 3, 6, 14 to 19, 21 to 27, 29 to 39 and 41 to 48 and 50.

55. The capsular polysaccharide of claim 51 or 52 or the vaccine of claim 53 for use in vaccination of a subject.

56. The capsular polysaccharide of claim 55 or the vaccine of claim 55, wherein the subject is a human being.

57. The capsular polysaccharide of claim 555 or 56 or the vaccine of claim 55 or 56, wherein the vaccination is against meningococcal meningitidis caused by Neisseria meningitidis serogroup A or X.