IMMUNIZING COMPOSITION

Abstract

concerned with an antigenic composition comprising at least one antigen that comprises at least one antigenic epitope or antigenic determinant derived from a protein present in one or both of S. equi subsp. equi and subsp. zooepidemicus and use thereof for immunization of non-human mammals against S. equi subsp. equi and/or subsp. zooepidemicus. The present invention also discloses a vaccine composition comprising the aforesaid antigenic composition as immunizing component.

Description

BACKGROUND OF THE INVENTION

[0001]1. Field of the Invention

[0002]This invention is generally related to antigenic or immunogenic compositions and use thereof for immunization of non-human mammals, e.g. horses, against Streptococcus equi.

[0003]2. Background of the Invention

[0004]Streptococcal infections in horses are mainly caused by the species Streptococcus equi, which is classified as a Lancefield Group C Streptococcus and comprises two subspecies designated equi and zooepidemicus, respectively.

[0005]Streptococcus equi subsp. equi, which is virtually confined to horses, is the causative agent of strangles, a world-wide distributed and highly contagious serious disease of the upper respiratory tract of the Equidae. Strangles is one of the most frequently reported equine diseases world-wide and is characterized by fever, nasal discharge, and abscess formation in the retropharyngeal and mandibular lymph nodes. In some cases the disease shows a metastatic course in the body, so called "bastard strangles". The disease has a world-wide distribution and causes great economic losses. Moreover, since strangles is a highly contagious disease, not only infected animals but also all other members of e.g. an afflicted stud must be isolated for as long as up to three months.

[0006]S. equi subsp. zooepidemicus is considered as an opportunistic commensal often occurring in the upper respiratory tract of healthy horses. However, after stress or virus infection, it can cause a secondary infection, which results in strangles-like symptoms. Moreover, subsp. zooepidemicus infects not only horses but also a wide range of other animals, like pigs, dogs, cats, and cows. Even human cases of infection due to subsp. zooepidemicus have been reported. This subspecies has been implicated as the primary pathogen in conditions such as endometritis, cervicitis, abortion, mastitis, pneumonia, abscesses and joint infections.

[0007]Although it is possible to treat and cure these streptococcal infections with antibiotics, such as penicillin, tetracycline or gentamicin, an effective prophylactic agent that could prevent outbursts of such infections and obviate or reduce the risk for development of resistant strains associated with antibiotic treatment, would be appreciated.

[0008]3. Description of the Related Art

[0009]However, although many attempts have been made to develop prophylactic agents such as vaccines against S. equi, at the present time no efficient vaccines or immunizing preparations are available, neither for the subspecies equi nor for the subspecies zooepidemicus.

[0010]Existing vaccines against strangles are based on inactivated, e.g. heat-killed, or attenuated strains of S. equi subsp. equi or acid extracts/mutanolysin enriched in M-protein(s), i.e. immunogenic protein(s) produced by S. equi. A vaccine against S. equi subsp. zooepidemicus based on an M-like protein is disclosed in U.S. Pat. No. 5,583,014. In WO 87/00436, an avirulent strain of S. equi is disclosed for use as a vaccine against S. equi that stimulates an antibody response in the nasopharyngeal mucosa after administration thereof to a horse.

Recently, a commercial vaccine against strangles, Equilis StrepE from IntervetVET, UK, has been released in Great Britain (November 2004), which vaccine also has been used throughout Europe and in South Africa and South America. However, the safety and efficacy of this vaccine, which is based on an attenuated (living, deletion mutated) strain of S. equi subsp. equi, can be questioned.

[0011]Since the previously developed vaccines or immunizing preparations are hampered by side-effects and, moreover, provide insufficient protection, there is a need for efficient and safe prophylactic agents, such as vaccines, that protect against S. equi infections and/or prevent spread thereof without giving rise to undesirable side-effects.

[0012]It is well known that attachment to eukaryotic cell surfaces is an essential step in the establishment of infection and colonization by bacterial pathogens. Accordingly, streptococcal surface proteins, that interact with and/or bind to different components of the Extracellular Matrix (ECM) or plasma proteins of the host cell, are potential candidates for use as active component(s) for immunizing purposes.

[0013]This is illustrated by the vaccines based on M-like proteins mentioned above or disclosed in the literature, i.a. in WO 98/01561. The binding of fibrinogen and complement factor H to M-proteins is assumed to be important for the ability of streptococci to resist phagocytosis.

[0014]Another mechanism used by streptococci for attachment to host cells involves binding to the ECM component fibronectin (Fn) (Ref. 21, 22). Binding between Fn-binding bacterial cell-surface proteins and immobilized Fn promotes internalization of streptococci by epithelial cells (Ref. 2, 23, 24). Fibronectin is a dimeric glycoprotein found both in plasma and in a fibrillar form in the extracellular matrix. The main function of Fn is to mediate substrate adhesion of eukaryotic cells, which involves the binding of specific cell-surface receptors to certain domains of the Fn molecule. Furthermore, it also interacts with several other macromolecules, such as DNA, heparin, fibrin, and collagen.

[0015]Accordingly, Fn-binding proteins from different streptococcal species have been cloned and sequenced previously. For instance, from S. equi, one Fn-binding protein has been cloned and characterized, which is a Fn-binding cell-surface protein of subsp. zooepidemicus, that has been designated FNZ (Lindmark et al., 1996, Ref. 9). Another Fn-binding protein from S. equi subsp. equi, has been cloned and characterized by Lindmark and Guss (1999) (Ref. 12). This latter protein that is designated SFS and its potential use as an active component in a vaccine for protection of horses against strangles are disclosed in WO 00/37496.

[0016]In Jonsson et al. (1995) (Ref. 8), a protein designated ZAG has been cloned and characterized from S. equi subsp. zooepidemicus that mediates binding to the plasma proteinase inhibitor α₂M. It is speculated therein that this protein is similar in function to streptococcal M proteins. This protein, ZAG, is also disclosed in WO 95/07296, where its α₂M-binding properties are indicated. However, immunogenic properties or potential use thereof as an active component in a vaccine for protection of e.g. horses against strangles are not disclosed therein. The gene zag encoding ZAG is also disclosed in these references.

[0017]A gene that is similar to the aforesaid zag gene from S. equi subsp. zooepidemicus but is present in subsp. equi has been described by Lindmark et al. (1999) (Ref. 11) and Lindmark (1999) (Ref. 13). This gene is hereafter designated eag and encodes a protein designated EAG.

[0018]In WO 2004/032957 A1, antigenic compositions are disclosed which comprise at least one antigen derived from a protein designated EAG, which protein is present in S. equi, and which composition suitably comprises at least one further antigen selected from a group of proteins which are present in S. equi and are designated FNZ, SFS, SEC and ScIC, respectively.

[0019]In WO 2007/115059 A2, subunit immunogenic or vaccine compositions are disclosed which comprise at least one polypeptide of S. equi having a specific amino acid sequence as shown in the sequence listing attached to said publication or an analog thereof or a fragment thereof which is a part of said polypeptide and contains at least one epitope. However, no results as regards immunizing of horses against strangles are provided in this document.

[0020]In the study reported in Lannergard, J., Frykberg, L. and Guss, B. (2003) FEMS Microbiol Lett 222: 69-74, (Ref. 28), a new gene designated cne has been isolated and the corresponding protein CNE has been characterized.

[0021]In Flock, M., Jacobsson, K., Frykberg, L., Hirst, T., R., Franklin, A., Guss, B. and Flock, J.-I. (2004) Infect Immun 72:3228-3236 (Ref. 5), it is reported that in a mouse model of equine strangles, parts of the proteins designated FNZ, SFS and EAG, respectively, were used to immunize mice. FNZ and EAG were considered as promising candidates for development of a safe and efficacious vaccine against strangles.

[0022]In Lannergard, J. and Guss, B. (2006) FEMS Microbiol Lett 262: 230-235, (Ref. 26), two new proteins, IdeE and IdeZ, from S. equi subspecies equi and zooepidemicus, respectively, have been characterized as regards enzymatic activities.

[0023]In Vaccine (Timoney et al.; 2007) it is reported that a great number of recombinant extracellular proteins of S. equi, including CNE (also designated SEC) and Se 44.2 (also designated IdeE2) are useless as vaccine components. It is speculated therein that earlier results for SEC/CNE obtained for mice are not applicable to horses. Thus, it is not obvious that recombinant forms of surface localized proteins necessarily are likely candidates for vaccine components.

[0024]In Waller, A., Flock, M., Smith, K., Robinson, C., Mitchell, Z., Karlstrom, Å., Lannergard, J., Bergman, R., Guss, B. and Flock, J.-I. (2007) Vaccine 25: 3629-3635, (Ref. 27), vaccination of horses against strangles using the recombinant antigens EAG, CNE and ScIC from S. equi subspecies equi is reported. In this study, vaccinated horses showed, after challenge with S. equi subspecies equi, significantly reduced recovery of bacteria and significantly lower levels of nasal discharge.

[0025]Although many efforts have been made to develop efficient vaccines and some of the immunizing components of WO 2004/032957 A1 are promising candidates for use in a vaccine that protects against S. equi infection, development of safe vaccines having a high degree of immunogenicity and exhibiting limited or no side effects is still desirable.

BRIEF SUMMARY OF THE INVENTION

[0026]The present invention is based on an antigenic, suitably an immunogenic, composition comprising at least one antigen, suitably an immunogen, that comprises at least one antigenic epitope or antigenic determinant derived from a protein present in one or both of S. equi subsp. equi and subsp. zooepidemicus and use thereof for immunization of non-human mammals against S. equi subsp. equi and/or subsp. zooepidemicus.

[0027]The present invention is also directed to a vaccine composition comprising the afore-said antigenic composition as immunizing component; to methods to prepare said antigenic, suitably immunogenic, composition or vaccine composition; to methods to induce an immune response against S. equi in non-human mammals; and to methods for prophylactic or therapeutic treatment of S. equi infection in non-human mammals. When used generally, the expression "S. equi" refers to one or both of subsp. equi and subsp. zooepidemicus.

[0028]According to a suitable embodiment, the present invention is directed to a vaccine that protects equines, such as horses, against strangles.

[0029]In the context of infections caused by S. equi subsp. equi, the expression "non-human mammals" primarily refers to animals belonging to the family Equidae that consists of horses, donkeys and zebras and to hybrids thereof, such as mules and hinnies. Camels and dromedaries are also encompassed therein.

[0030]In connection with infections caused by S. equi subsp. zooepidemicus, the expression "non-human mammals" in addition refers also to other mammals such as cows, pigs, dogs and cats.

BRIEF DESCRIPTION OF THE DRAWINGS

[0031]In the following, the present invention is described in closer detail with reference to the drawings, where:

[0032]FIG. 1 shows weight loss of mice given experimental infection with S. equi subsp. equi strain 1866 after vaccination with the polypeptides Eq5 and Eq8 (open symbols) or non-vaccinated (filled symbols);

[0033]FIG. 2 shows nasal growth in mice given experimental infection with S. equi subsp. equi strain 1866 after vaccination with the polypeptides Eq5 and Eq8 (open symbols) or non-vaccinated (filled symbols);

[0034]FIG. 3 shows weight loss of mice given experimental infection with S. equi subsp. equi strain 1866 after vaccination with the polypeptide EAG (filled squares), the polypeptides EAG+IdeE+IdeE2 (open circles) or non-vaccinated controls (filled circles);

[0035]FIG. 4 shows nasal growth in mice given experimental infection with S. equi subsp. equi strain 1866 after vaccination with the polypeptide EAG (filled squares), the polypeptides EAG+IdeE+IdeE2 (open circles) or non-vaccinated controls (filled circles).

[0036]FIGS. 5a and 5b show weight loss and nasal growth in mice immunized with EAG+CNE+ScIC i.n. (filled squares), Eq5+Eq8 i.n. (filled circles) and the control (open circles).

[0037]In these FIGS. 1-5, mean values and standard errors are indicated.

[0038]FIG. 6 shows growth of challenge inoculum (S. equi subsp. equi strain 4047);

[0039]FIG. 7 shows mean pony temperatures during the vaccination phase;

[0040]FIG. 8 shows mean nasal score during the vaccination phase;

[0041]FIG. 9 shows mean lymph node score during the vaccination phase;

[0042]FIG. 10 shows mean counts of S. zooepidemicus in nasal washes during the vaccination phase;

[0043]FIG. 11 shows mean pony temperatures after challenge;

[0044]FIG. 12 shows mean fibrinogen levels during the challenge phase;

[0045]FIG. 13 shows mean neutrophil levels during the challenge phase;

[0046]FIG. 14 shows mean lymph node score during the challenge phase;

[0047]FIG. 15 shows mean nasal score during the challenge phase;

[0048]FIG. 16 shows mean S. zooepidemicus counts during challenge phase;

[0049]FIG. 17 shows mean pathology score on post mortem examination; and

[0050]FIG. 18 shows mean histopathology scores.

[0051]FIG. 19 shows ELISA measurements of IgG antibodies in nasal washings of seven immunized horses. The log dilution of sera required to give an absorbance value at a cut-off of 1.0 was calculated for each individual nasal wash sample. Mean values (n=7) with standard errors are shown. Samples taken before (pre imm. day 1) and twelve days after the third immunization are shown (day 86). The horses were immunized with EAG, CNE and ScIC.

[0052]FIG. 20 shows ELISA measurements of IgG antibodies in sera of seven immunized horses. The log dilution of sera required to give an absorbance value at a cut-off of 1.5 was calculated for each individual serum sample. Mean values (n=7) with standard errors are shown. Sample taken before (day 1), after V2 (day71), and after V3 (day 86) are shown.

[0053]FIG. 21 shows ELISA measurements of IgG antibodies in sera of immunized horses (Pentavac). The log dilution of sera required to give an absorbance value at a cut-off of 1.5 was calculated for each individual serum sample. Mean values (n=7) with standard errors are shown. Sample taken before (day 1), after V2 (day 71), and after V3 (day 86) and samples taken between V3 and V4 (day 270) are shown.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

[0054]SEQ ID NO 1 shows the amino acid sequence of the protein IdeE2.

[0055]SEQ ID NO 2 shows the amino acid sequence of the recombinant protein IdeE2.

[0056]SEQ ID NO 3 shows the amino acid sequence of the protein Eq5.

[0057]SEQ ID NO 4 shows the amino acid sequence of the recombinant protein Eq5.

[0058]SEQ ID NO 5 shows the amino acid sequence of the protein Eq8.

[0059]SEQ ID NO 6 shows the amino acid sequence of the recombinant protein Eq8.

[0060]SEQ ID NO 7 shows the amino acid sequence of the protein IdeZ2 from subsp. zooepidemicus.

[0061]SEQ ID NO 8 shows the amino acid sequence of the protein Eqz5 from subsp. zooepidemicus.

[0062]SEQ ID NO 9 shows the amino acid sequence of the protein Eqz8 from subsp. zooepidemicus.

[0063]SEQ ID NO 10 shows the amino acid sequence of the protein IdeE.

[0064]SEQ ID NO 11 shows the amino acid sequence of the protein IdeZ from subsp. zooepidemicus.

[0065]SEQ ID NOS 12 and 13 shows, respectively, the nucleotide sequence of the gene eag and the amino acid sequence of the protein EAG4B, which protein is usually designated EAG in connection with the present invention.

[0066]SEQ ID NO 14 shows the nucleotide sequence of the gene ideE2.

[0067]SEQ ID NO 15 shows the nucleotide sequence of the gene eq5.

[0068]SEQ ID NO 16 shows the nucleotide sequence of the gene eq8.

[0069]SEQ ID NO 17 shows the nucleotide sequence of the gene IdeZ2 from subsp. zooepidemicus.

[0070]SEQ ID NO 18 shows the nucleotide sequence of the gene eqz5 from subsp. zooepidemicus.

[0071]SEQ ID NO 19 shows the nucleotide sequence of the gene eqz8 from subsp. zooepidemicus.

[0072]SEQ ID NO 20 shows the nucleotide sequence of the gene ideE.

[0073]SEQ ID NO 21 shows the nucleotide sequence of the gene ideZ from subsp. zooepidemicus.

[0074]SEQ ID NOS 22-27 show nucleotide sequences of oligonucleotide primers.

[0075]SEQ ID NO 28 shows the amino acid sequence of the protein CNE (or SEC 2.16).

[0076]SEQ ID NO 29 shows the amino acid sequence of the protein ScIC.

[0077]SEQ ID NO 30 shows the amino acid sequence of the recombinant IdeE used for immunization.

[0078]SEQ ID NO 31-32 shows the nucleotide sequence of primers.

DETAILED DESCRIPTION OF THE INVENTION

[0079]The present invention is concerned with identification of polypeptides or proteins of S. equi that are able to elicit an antigenic, suitably an immunogenic, response, when administered to a non-human mammal; and to the identification of polynucleotides or genes encoding these polypeptides or proteins.

[0080]The present invention is also concerned with fragments or analogs of said polypeptides or proteins or of said polynucleotides or genes.

[0081]More specifically, genes of S. equi encoding extracellular proteins were identified and, subsequently, the corresponding products were expressed and evaluated in vaccine studies. The present invention is at least partly based on such studies.

[0082]Accordingly, the present invention relates to an antigenic composition comprising at least one antigen, wherein said at least one antigen comprises at least part of a protein of Streptococcus equi subsp. equi or subsp. zooepidemicus, and said at least part of said protein comprises at least one antigenic epitope or antigenic determinant of Streptococcus equi.

[0083]According to one embodiment, the present invention is directed to an antigenic composition comprising at least one antigen, wherein said at least one antigen comprises at least part of a protein or polypeptide of Streptococcus equi subsp. equi or subsp. zooepidemicus and said at least part of said protein or polypeptide comprises at least one antigenic epitope or antigenic determinant of Streptococcus equi, and wherein said protein or polypeptide is selected from the group comprising:

[0084]a protein or polypeptide which is designated EAG and has an amino acid sequence as shown in SEQ ID NO: 13;

[0085]a protein or polypeptide which is designated IdeE and has an amino acid sequence as shown in SEQ ID NO: 10;

[0086]a protein or polypeptide which is designated IdeE2 and has an amino acid sequence as shown in SEQ ID NO: 1;

[0087]a protein or polypeptide which is designated Eq5 and has an amino acid sequence as shown in SEQ ID NO: 3;

[0088]a protein or polypeptide which is designated Eq8 and has an amino acid sequence as shown in SEQ ID NO: 5;

[0089]a protein or polypeptide which is designated IdeZ2 and has an amino acid sequence as shown in SEQ ID NO: 7;

[0090]a protein or polypeptide which is designated Eqz5 and has an amino acid sequence as shown in SEQ ID NO: 8; and

[0091]a protein or polypeptide which is designated Eqz8 and has an amino acid sequence as shown in SEQ ID NO: 9;

or an analog or a fragment thereof, and wherein a composition which comprises EAG, comprises at least one further antigen, which is a protein or polypeptide, which is selected from the group comprising IdeE, IdeE2, Eq5, Eq8, IdeZ2, Eqz5, and Eqz8.

[0092]For convenience, the polypeptides having amino acid sequences as shown in the sequence listing are frequently only designated EAG, IdeE, IdeE2, Eq5, Eq8, IdeZ2, Eqz5, and Eqz8, respectively. EAG, IdeE, IdeE2, Eq5, and Eq8 designate proteins that can be found in S. equi subsp. equi and IdeZ, IdeZ2, Eqz5, and Eqz8 designate proteins that can be found in S. equi subsp. zooepidemicus.

[0093]The antigens or immunogens of the present antigenic or immunogenic compositions may comprise the entire amino acid sequence of said protein or polypeptide or may comprise a fragment, e.g. a C-terminal or N-terminal fragment thereof, or an analog thereof. For instance, an N-terminal fragment of EAG is used according to various embodiments of the present invention.

[0094]According to one embodiment, the present invention is related to an antigenic or immunogenic composition which contains at least 2 or 3 antigens or immunogens selected from the group consisting of EAG, IdeE, IdeE2, Eq5, Eq8, IdeZ, IdeZ2, Eqz5, and Eqz8.

[0095]According to a specific embodiment, the present invention is related to an antigenic or immunogenic composition which contains at least 2 or 3 antigens or immunogens selected from the group consisting of EAG, IdeE, IdeE2, Eq5, and Eq8. Suitably this composition also comprises one or both of the previously described antigens ScIC (SEQ ID NO: 29) and CNE (SEQ ID NO: 28) (also designated SEC e.g. SEC 2.16). A further embodiment is related to an antigenic composition comprising EAG, ScIC, CNE, Eq5, and Eq8.

[0096]A suitable composition contains 2 antigens or immunogens which are comprised of Eq5 and Eq8, respectively. According to a further embodiment, the present invention is directed to a composition that contains 3 antigens or immunogens, which suitably are comprised of EAG, IdeE, and IdeE2. The present invention is also related to compositions that comprise one or both of IdeE and IdeE2.

[0097]The present invention is also related to an antigenic composition, wherein said at least one protein or polypeptide is selected from the group consisting of EAG, Eq5 and Eq8 and which composition further comprises at least one antigen, which is selected from the group comprising a protein or a polypeptide designated CNE (or SEC), which has an amino acid sequence as shown in SEQ ID NO: 28, and a protein or a polypeptide designated ScIC, which has an amino acid sequence as shown in SEQ ID NO: 29. Suitably, said at least one protein or polypeptide is selected from the group comprising IdeE and IdeE2.

[0098]Antigenic compositions of the present invention, which have been shown to be useful in vaccine compositions, comprise according to one embodiment, the antigens EAG, ScIC, CNE (or SEC), Eq5, Eq8, IdeE and IdeE2, and according to another embodiment, the antigens EAG, ScIC, CNE (or SEC), Eq5, and Eq8.

[0099]The present invention is also related to an antigenic composition, wherein said at least one protein or polypeptide is selected from the group consisting of EAG, Eq8, and IdeE2 and which composition comprises at least one further antigen which is selected from the group comprising IdeE, Eq5, IdeZ2, Eqz5 and Eqz8 and/or ScIC and CNE (or SEC).

[0100]According to the present invention, the antigenic composition suitably comprises at least one antigen which is recombinantly produced and/or at least one antigen which is an isolated or purified antigen.

[0101]From the above, it is evident that the present antigens or immunogens that are derived from proteins of Streptococcus equi may comprise the entire protein, a fragment of said protein or an analog of said protein which is antigenic or immunogenic. Thus, the present invention is not limited to the fragments of proteins that are specifically disclosed herein.

[0102]The antigenic composition of the present invention may comprise at least one recombinant vector and at least one polynucleotide inserted therein that encodes said at least one protein or polypeptide, and which vector is able to express said polypeptide in vivo in a non-human mammal susceptible to infection with S. equi.

[0103]According to one embodiment of the present invention, the vector is an expression vector which is a plasmid or a viral vector and wherein said polynucleotide has a nucleotide sequence that encodes an antigen of the present invention.

[0104]A further embodiment of the present invention is concerned with a vaccine composition for protecting non-human mammals against infection of Streptococcus equi, which comprises an antigenic composition as disclosed above as immunizing component, and a pharmaceutically acceptable carrier.

[0105]Suitably, the present vaccine composition comprises an antigenic or immunogenic composition that contains 2, 3 or more of the present antigens or immunogens as immunizing components. Optionally, one or more of these antigens or immunogens are comprised of analogs of said proteins or fragments thereof, e.g. N-terminal or C-terminal fragments.

[0106]The vaccine composition may comprise further components, such as an adjuvant. Suitably, said adjuvant stimulates systemic or mucosal immunity. Such adjuvants are well known in the art.

[0107]Suitable adjuvants for use according to the present invention comprise (1) polymers of acrylic or methacrylic acid, maleic anhydride and alkenyl derivative polymers, (2) immunostimulating sequences (ISS), (3) an oil in water emulsion, (4) cation lipids containing a quaternary ammonium salt, (5) cytokines, (6) aluminum hydroxide or aluminum phosphate, (7) saponin or (8) nanoparticles.

[0108]A suitable adjuvant for use according to the present invention is the adjuvant Abisco from Isconova AB, Sweden. The key components of ISCOMS are Quillaia saponins derived from the bark of the chilean soap bark tree Quillaia saporinaria molina. Quillaia saponins are well known for their ability to activate the immune system. Quillaia saponins mixed with cholesterol, and phospholipids under specific stoichiometry form spherical open cage like structures known as ISCOMS.

[0109]Another suitable adjuvant is Ginseng. Ginseng is a dry extract prepared from the root of the plant Panax ginseng, C. A. Meyer. Ginseng contains a number of active substances named ginsenosides that are a kind of saponins, chemically tri-terpenoid glycosides of the dammaran series. The ginsenosides have adjuvant properties and one of the most active adjuvant is the fraction named Rb1. It has been proved that the fraction Rb1 elicits a balanced Th1 and Th2 immune response as determined by measuring the levels of the cytokines IFN-γ, IL-2, IL-4, IL-10 secreted post vaccination with a Rb1 adjuvanted vaccine. In addition ginseng and the fraction Rb1 stimulates a strong antigen specific antibody response.

[0110]According to a suitable embodiment, the vaccine composition is a vaccine that protects susceptible mammals, suitably horses, against strangles caused by Streptococcus equi subsp. equi.

[0111]The vaccine composition of the present invention is provided in a physiologically administrable form. Suitably, it is administrable by subcutaneous, intramuscular or intranasal inoculation.

[0112]Suitably, the vaccine composition of the present invention stimulates serum, mucosal and/or bronchial lavage antibody responses directed to Streptococcus equi antigens in mammals susceptible to Streptococcus equi, suitably horses.

[0113]The present invention is also related to a method for producing an antigen or immunogen to be used in an antigenic or immunogenic composition of the present invention, which method comprises

[0114](a) providing a DNA fragment encoding said antigen and introducing said fragment into an expression vector;

[0115](b) introducing said vector, which contains said DNA fragment, into a compatible host cell;

[0116](c) culturing said host cell provided in step (b) under conditions required for expression of the product encoded by said DNA fragment; and

[0117](d) isolating the expressed product from the cultured host cell.

[0118]Preferably, said method further comprises a step (e) wherein the isolated product from step (d) is purified, e.g. by affinity chromatography or other chromatographic methods known in the art.

[0119]Accordingly, the antigens of the present invention are usually produced according to recombinant technique.

[0120]A further embodiment of the present invention is concerned with a method for preparation of a vaccine of the present invention, which vaccine contains as immunizing component an antigenic or immunogenic composition as disclosed above, said method comprising mixing said antigenic composition and a pharmaceutically acceptable carrier.

[0121]The present invention is also related to a method for the production of an antiserum, said method comprising administering an antigenic preparation of the present invention to an animal host to produce antibodies in said animal host and recovering antiserum containing said antibodies produced in said animal host.

[0122]Moreover, the present invention is concerned with a method of prophylactic or therapeutic treatment of S. equi infection in non-human mammals, suitably horses, comprising administering to said mammal an immunologically effective amount of a vaccine or an antiserum of the present invention.

[0123]Accordingly, the present invention is related to a method for protecting horses against Streptococcus equi infection, which method comprises inoculating a horse intramuscular, subcutaneously or intranasally, or a combination of e.g. both subcutaneously and intranasally, with a vaccine composition of the present invention to induce an immune response against Streptococcus equi in said horse. Suitably, an immune response, in the form of IgG and/or IgA and/or IgM antibodies in the nasopharyngeal mucus, is induced in said horse.

[0124]The present invention also relates to an antibody preparation comprising at least one, and suitably at least two, antibodies specific for a protein or a polypeptide of the present antigenic composition, which antibody/antibodies is/are polyclonal or monoclonal; or which preparation comprises a fragment of said antibodies.

[0125]The antibody preparation of the present invention could be used prophylactically or therapeutically against strangles and provides passive immunization when administered to a non-human mammal susceptible to infection by Streptococcus equi or infected by Streptococcus equi.

[0126]The present invention describes a vaccine composition comprising one or several antigen components which have been prepared according to the present method using E. coli as host cells. The source of these antigens might also be the native bacteria, if methods are developed for expression and purification thereof. Alternatively, the antigens of the present invention can also be produced according to methods that are based on fusion strategies where various parts of the respective antigen are recombined resulting in a fusion can in protein consisting of parts from different antigens. This fusion strategy could also be suitable for introducing immune reactive part(s), e.g. T-cell epitopes or attenuated toxins (or parts thereof), thereby introducing other features suitable for optimizing the antigen presentation or localization. Furthermore, other hosts for expressing the recombinant antigens addition to E. coli also be other suitable species of bacteria and viruses. Today many different systems for expression of heterologus expression are well known in the field of molecular biology.

[0127]Yet another implication of this invention is that it can be used to design specific attenuated mutants of S. equi that lack or have inactivated genes important for survival (i.e. mutations causing deficiency in metabolic pathways) in the host but retain or overproduce the antigens of the present invention.

EXPERIMENTAL PART

[0128]The DNA sequence of the genome of S. equi subsp. equi and subsp. zooepidemicus have been determined (www.sanger.ac.uk/) but not yet annotated. By screening open reading frames a great number of genes encoding extracellular proteins were identified. Among these genes a selected number were chosen and recombinant proteins were produced and evaluated in vaccine studies. The cloning and expression of these genes is described below. Furthermore, the use of these proteins as antigens will also be described.

Example 1

Constructions of Clones Harboring the Genes ideE, ideE2, eq5 and eq8 from Subsp. equi.

[0129]Chromosomal DNA from S. equi subspecies equi strain 1866 (PCT/SE03/01587, Lannergard and Guss 2007) was used as a template to amplify potential genes encoding IdeE2, Eq5 and Eq8 (the nucleotide- and protein-sequences are presented in the sequence listing further below). To identify the predicted signal sequences, the computer program SignalP (http://www.cbs.dtu.dk/services/SignalP/) was used. The sequences of primers used to amplify the genes or part of the genes ideE, ideE2, eq5 and eq8 are listed in the Primer Table. Cleavage sites for the restriction enzymes NcoI and XhoI were included in the primer sequences to match the cloning sites in the plasmid vector pTYB4 (New England Biolabs). The PCR amplifications were performed using the primers (20 pmol/μl) and the ReadyToGo® PCR beads (GE Healthcare) using the following programme: Step 1, pre-heat 1 minute at 95° C., DNA strand separation; Step 2, 30 seconds at 95° C.; Step 3, annealing 15 seconds at 46° C.; and Step 4, elongation for 2 minutes at 72° C., Steps 2-4 were run for 26 cycles. The PCR products were analysed on a 1 agarose gel, and thereafter purified using the QIAquick PCR Purification Kit® (Qiagen). Cleavage with the restriction enzymes was performed over night whereupon the fragments were purified one additional time using the same kit.

Primer Table: The primer sequences used to PCR amplify the genes ideE, ideE2, eq5 and eq8. The nucleotides underlined correspond to the introduced restriction cleavage sites NcoI and XhoI.

TABLE-US-00001 Gene Primer Primer sequence ideE2 Forward primer 5'-CATGCCATGGAGGTAGTTGAAGTTTGGCCTAAT-3' (SEQ ID NO: 22) ideE2 Reverse primer 5'-CCGCTCGAGTTTTTCTGTCTTGTTGAAGTAATCTGC-3' (SEQ ID NO: 23) eq5 Forward primer Eqp51: 5'-GTAGCCATGGAAACGACTACTGCTAGTGCA-3' (SEQ ID NO: 24) eq5 Reverse primer Eqp52: 5'CTGGCTCGAGCGGTTTAGCAACCAAGGCT-3' (SEQ ID NO: 25) eq8 Forward primer Eqp81: 5' CATGCCATGGCGACTACCCTAGCAGGACAAA-3' (SEQ ID NO: 26) eq8 Reverse primer Eqp82: 5'CTAGCTCGAGGTGCTTAAGCTTTTCAATCTG-3' (SEQ ID NO: 27) ideE Forward primer IdEG1: 5'-TACTGGATCCGACGATTACCAAAGGAATGCTAC-3' (SeQ ID NO: 31) ideE Reverse primer IdEG2: TGATCTCGAGTTAGCTCAGTTTCTGCCATATG (SEQ ID NO: 32)

To clone and produce recombinant proteins in E. coli the IMPACT® Protein Purification System (New England Biolabs) was used. E. coli strain ER2566 containing the pTYB4 vector (New England Biolabs) was grown according to the manufacturer's instructions, and the vector was purified using the QIAprep Spin Miniprep (Qiagen). Purified vector was digested using restriction endonucleases NcoI and XhoI. After digestion, the vector was treated with the enzyme alkaline phosphatase to reduce the background of re-ligated vector in the later ligation step. For the ligation of the vector and the respective PCR product, the ReadyToGo T4DNA Ligase (GE Healthcare) was used. After ligation, the respective sample were transformed into competent cells of E. coli strain ER2566 using electroporation, and spread on LA-Amp plates (Luria-Bertani broth agar plates supplemented with ampicillin, final conc. 50 μg/ml) and incubated over night at 37° C. Next day colonies were counted and four colonies per construct were cultivated and used for further experiments. To verify the presence of an insert in the respective constructs, plasmids were purified and additional PCR analyses were performed using the respective primer combination. The sequence of the respective insert was also determined by DNA sequencing using primers that hybridise in the vector (T7 universal forward primer and a reverse primer located in the intein coding region).

[0130]Cloning of the ideE gene of S. equi subsp. equi strain 1866 has been reported previously by Lannergard and Guss (2006). The GenBank accession number of ideE is DQ508733. The part of the gene used to obtain the recombinant IdeE protein used for immunization was cloned using the primers IdEG1 and IdEG2 listed in the Primer Table. After PCR amplification the DNA fragment was digested with restriction enzymes BamHI and XhoI and ligated into the vector pGEX6-P-1 (GE Healthcare), previously digested with the same enzymes.

Example 2

Preparation of Antigens CNE, ScIC, EAG4B, IdeE, IdeE2, Eq5 and Eq8

[0131]The vector used is a part of an E. coli expression and purification system called IMPACT® T7 (NEB Inc.) Briefly, following the manufacturer's instructions the clones expressing recombinant IdeE2, Eq5 and Eq8, respectively were grown at 37° C. in Luria Bertani growth medium supplemented with ampicillin (final conc. 50 μg/ml). At an optical density (OD₆₀₀)˜0.6, the growth medium was supplemented with IPTG (final conc. 0.4 mM) and the growth temperature shifted to 20° C. After incubation over night the cells were harvested and resuspended in a buffer [20 mM Tris-HCl (pH 8.0), 500 mM NaCl, 0.1 mM EDTA, and 0.1% Triton X100] and lysed by freezing and thawing. After centrifugation, the supernatant was sterile filtrated and applied onto a chitin column. The columns were extensively washed using the same buffer and subsequently treated with cleavage buffer [20 mM Tris-HCl (pH 8.0), 50 mM NaCl, 0.1 mM EDTA, and 30 mM dithiothreitol (DTT)]. In the cleavage buffer, the reducing conditions induce an intein-mediated self-cleavage that releases the antigen part from the column while the intein-chitin-binding part is still bound. The eluted samples containing the antigens were dialysed against phosphate-buffered saline [PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.4 mM KH₂PO₄ (pH 7.4)] and concentrated. The amounts of antigens obtained were determined and the quality was checked using SDS-PAGE. The recombinant IdeE protein was produced and purified using the GST-affinity chromatography system according to the procedure recommended by the manufacturer (GE Healthcare). The description of and production of the recombinant proteins CNE(SEC), ScIC, and EAG4B antigens have been described previously (WO 2004/032957 (PCT/SE03/01587), Waller et al 2007). In the following examples, the EAG4B protein is simply called EAG.

Example 3

Recombinant IdE2 Cleaves IgG

[0132]IdE has previously been shown to be a protease that specifically cleaves IgG from various species (Lannegard and Guss 2006). To test if recombinant IdeE2 also cleaves antibodies, IgG from human, horse and mouse were incubated in PBS at 37° C. for one hour. Purified recombinant IdeE was used as a positive control and the negative control was pure IgG. After cleavage, the samples were analysed using 8-25% gradient SDS-PAGE. The result showed that recombinant IdeE2 cleaves cleaves horse IgG much more efficiently than IdeE does.

Example 4

Presence of the Genes ideE, ideE2, eq5, and eq8 in S. equi subsp. zooepidemicus

[0133]Previously the presence of a homologous subsp. equi ideE gene in subsp. zooepidemicus has been reported (Lannegard and Guss 2006). Using the S. zooepidemicus genome database (www.sanger.ac.uk/), the presence of similar genes to ideE2, eq5 and eq8 in subspecies zooepidemicus was analysed using BLAST search. The results showed that genes encoding similar proteins were detected. The sequence of these genes called ideZ2, eqz5 and eqz8 along with amino acid sequences IdeZ2, Eqz5 and Eqz8 are shown in the list of sequences in the experimental part of this specification.

Example 5

Immunisation of Mice with Eq5 and Eq8

[0134]Mice (NMRI) weighting approximately 23-25 g were kept in cages of five animals in each. The mice were immunised intranasally with 12 micrograms of each antigen and 10 microgram of Abisco 300 (Isconova AB, Sweden). Fifteen animals were immunised with antigen (Eq5 and Eq8) and 15 were only given Abisco 300 adjuvant to serve as a negative control. The total volume was kept to less than 27 μl and applied into the nostrils twice with 30 minutes interval of mice anaesthetized with Isoflovet (Abbot Laboratories, England). Immunisations were given on days 0, 13 and 32.

Example 6

Immunisation of Mice with EAG, IdeE and IdeE2

[0135]Immunisation with EAG, IdeE and IdeE2 was performed essentially as for Eq5 and Eq8. However, animals were divided into three groups, with ten mice in each group. These were given EAG+IdeE+IdeE2 or EAG only and one group with only adjuvans, Abisco 300, as negative control. Immunisations were given on days 0, 21 and 53. Experimental infection was given on day 60.

Example 7

Experimental Infection with Streptococcus equi subsp. equi

[0136]Experimental infection was given on day 43 (10 days after last time of immunisation) for Eq5+Eq8 and on day 60 (10 days after last immunisation) for EAG+/-IdeE+IdeE2. S. equi subsp. equi strain 1866 from a clinical case of strangles was used. The strain was first passed through an animal by inoculating ca 10⁶ CFU into the nostrils of an anaesthetized mouse. Bacteria were recovered after 7 days from the nose of the mouse and grown on BG plates at 37° C. in 5% CO₂. A single colony was grown on BG plates overnight at 37° C. and resuspended in Todd Hewitt Broth (THB) with 1% yeast extract (THY). The culture was kept at -80° C. in vials and a new vial was used for each experiment. To infect mice, bacteria were grown on BG plates at 37° C. in 5% CO₂ overnight, followed by inoculation into THY and grown without shaking over night. The cultures was then diluted 10 times into THY and 10% horse serum (Sigma) and grown for 4 hours at 37° C. in 5% CO₂. The culture was centrifuged and resuspended in THB. A dose containing 1×10⁶ CFU in 10 μl was used for all S. equi infections of mice. The animals were followed daily. Bacterial nasal growth was scored on a four-graded scale from 0 to +++ by gently pressing the nose of the animal onto a blood agar plate in a reproducible manner. The nasal sample was then spread out onto the entire surface of the plate. One + means 5-100 colonies; two + means more than 100 and three + means confluent growth. The weight was determined every day and the percentage of weight-loss was calculated.

Example 8

Experimental Results of Vaccination

[0137]Mice were immunised with both Eq5 and Eq8 and the percentage weight loss over time was determined. FIG. 1 shows that vaccinated animals (n=15) lost less weight that control animals (n=15). P-values=0.0001 for all days (Student's t-test). Nasal growth of S. equi was also determined daily on a four graded scale. FIG. 2 shows that the vaccinated animals had much less nasal growth than the control group. The frequency of animals grossly colonised nasally with bacteria (scoring 2-3) on day 5 was significantly different between the two groups; p=0.002 (Fisher's exact test).

[0138]In the next experiment, mice were vaccinated with EAG (n=10), with EAG+IdeE+IdeE2 (n=10) or non-vaccinated (n=10). The percentage weight loss over time was determined. FIG. 3 shows that animals vaccinated with EAG+IdeE+IdeE2 lost less weight that control animals. P values were 0.0013, 0.0008 and 0.0009 for days 3, 5 and 6 respectively (Student's t-test). Animals vaccinated with EAG alone also lost weight to a similar magnitude as control animals. Nasal growth of S. equi was also determined daily on a four graded scale. FIG. 4 shows that the animals vaccinated with EAG+IdeE+IdeE2 had much less nasal growth than the control group. Again, vaccination with only EAG showed no protection.

Example 9

Immunisation of Mice with Eq5, Eq8, and EAG, CNE, ScIC

[0139]Immunisation i.n. with Eq5+Eq8 and EAG+CNE+ScIC was performed as above with three groups with ten mice in each group. One group with Eq5+Eq8 and one with EAG+CNE+ScIC. The third group was the control with Abisco-300. Immunisations were given on days 0, 14 and 22. Challenge was given on day 29. The experimental results are shown in FIG. 5a and FIG. 5b. FIG. 5a and b show significant protection for EAG+CNE+ScIC (n=10). P-values were 0.04 and 0.09 for day 2 and 5. The protection with Eq5+Eq8 was even more pronounced where p-values were 0.005 and 0.009 for these days.

List of Sequences

[0140](1) SEQ ID NO: 1 and SEQ ID NO: 14 are combined to show the amino acid sequence of the IdeE2 protein (SEQ ID NO: 1) under the nucleotide sequence of ideE2 (SEQ ID NO: 14)

TABLE-US-00002 atgatgaaaaaacaa M M K K Q tcattcacacactcacgtaaacctaaattcggtatgagaaaattatctattggccttgcc S F T H S R K P K F G M R K L S I G L A tcatgtatgctaggaatgatgttcctaacaacaggacatgtttctggtgaggtagttgaa S C M L G M M F L T T G H V S G E V V E gtttggcctaatgggcaaaatcctaatggtaaaatagaaattctaagtcaaactgagcac V W P N G Q N P N G K I E I L S Q T E H tctgagcatttacagaaattacgcgatattgaagatttccaagctcaaaagcaagctgat S E H L Q K L R D I E D F Q A Q K Q A D catgttcgttacactaaatggttagatggggtaactgttgatgagcatgaattcagaaaa H V R Y T K W L D G V T V D E H E F R K atcaaggaatatgacacagaatattatgtaacacctcttttaagtggtaaaggttactat I K E Y D T E Y Y V T P L L S G K G Y Y gatatcaataaagatttcaatcaagatagtgataaatgtgctgccgctgtagcggctaat D I N K D F N Q D S D K C A A A V A A N atgttccattattggtttgatagaaatagagacagtattaatcgtttcttaagtcaaagt M F H Y W F D R N R D S I N R F L S Q S ccaggtgaaaatggtgttattaaacttgaaaatgaaaaaacaatagaagtatcaaaattt P G E N G V I K L E N E K T I E V S K F ttagaaacttaccgtagtgatggtgattatcttgataaaagtccgttttttgaccttatc L E T Y R S D G D Y L D K S P F F D L I agtaacagctttaaaggtcctgtttgggcaaataagctattggatgcttacattaacggc S N S F K G P V W A N K L L D A Y I N G tatggttatatccataaatttgctaaaaatactccacattctaaaaataataatagtaaa Y G Y I H K F A K N T P H S K N N N S K tttaatttctttaaaaaagtatttgatggtaatctcttgacagatattcaccaaattttt F N F F K K V F D G N L L T D I H Q I F gattataacactttttcagataaattaagtgaggctctctatactggtaaagccattgga D Y N T F S D K L S E A L Y T G K A I G ttggcctacggacctggagacttgcgtcgttcactaggtcatattatttctgtctgggga L A Y G P G D L R R S L G H I I S V W G gctgatcttgacgatcagaatcgcgtggtagctatttatgtaactgattctgatgataaa A D L D D Q N R V V A I Y V T D S D D K aagttaactataggaaatgagagagttggtttgaagcgatataaagtatctagcgatgat K L T I G N E R V G L K R Y K V S S D D caaggtcgtgctcgtctgacgactcgtgataaagataacacaggtggtgaaattcgatct Q G R A R L T T R D K D N T G G E I R S attgaaacattagatatgggtacacaagagtgggcagattacttcaacaagacagaaaaa I E T L D M G T Q E W A D Y F N K T E K taa -

(2) SEQ ID NO: 2 shows the recombinant IdeE2 protein sequence. The amino acids in bold are those that corresponds to the amino acids encoded by the pTYB4 vector while the rest originates from the IdeE2 protein.

TABLE-US-00003 MEVVEVWPNGQNPNGKIEILSQTEHSEHLQKLRDIEDFQAQKQADHVRYT KWLDGVTVDEHEFRKIKEYDTEYYVTPLLSGKGYYDINKDFNQDSDKCAA AVAANMFHYWFDRNRDSINRFLSQSPGENGVIKLENEKTIEVSKFLETYR SDGDYLDKSPFFDLISNSFKGPVWANKLLDAYINGYGYIHKFAKNTPHSK NNNSKFNFFKKVFDGNLLTDIHQIFDYNTFSDKLSEALYTGKAIGLAYGP GDLRRSLGHIISVWGADLDDQNRVVAIYVTDSDDKKLTIGNERVGLKRYK VSSDDQGRARLTTRDKDNTGGEIRSIETLDMGTQEWADYFNKTEKLEPG

(3) SEQ ID NO: 3 and SEQ ID NO: 15 are combined to show the amino acid sequence of the Eq5 protein (SEQ ID NO: 3) under the nucleotide sequence of eq5 gene (SEQ ID NO: 15)

TABLE-US-00004 atgaagaaattcacgaaacggtgtcttaagggctgtggtcttgttggattagttttcagc M K K F T K R C L K G C G L V G L V F S acaggattggttgccttgtcggataatattgatagcgctttaacagtaggggcggaaacg T G L V A L S D N I D S A L T V G A E T actactgctagtgcatttgaaaataatgggacaggtcaacatctgaactggcacatagat T T A S A F E N N G T G Q H L N W H I D attccacaagaatatacagttgaattaggagaaccaattactatctcagatcttatgagt I P Q E Y T V E L G E P I T I S D L M S caaattacggttactcgtaaaggtagtaatgggactgttaatgatggagatacttttgac Q I T V T R K G S N G T V N D G D T F D tttatttcgaatggagatggttcaagaggaattgatacccctggagtaaaaatatggttt F I S N G D G S R G I D T P G V K I W F gacttttacaatgctgcgggtacttcctttttaactgatgaaatgttagcttcgcctaca D F Y N A A G T S F L T D E M L A S P T tatgctgtaccggggggatcttatactattaaagcttgggtattctatgggaaaaatgat Y A V P G G S Y T I K A W V F Y G K N D accaaaaagctcttcacatttaaactaaaaaattccaacagcaataaaactgagttaagg T K K L F T F K L K N S N S N K T E L R aagtcgttagaggaggctaagctaaaactcagccagcctgaaggaacgtattctgatgaa K S L E E A K L K L S Q P E G T Y S D E tcactgcaagccttgcaatcagcggttactcttggtaagacctatttaaacagtgaccct S L Q A L Q S A V T L G K T Y L N S D P gatcaaaatacagtagatcaatctgttactactattgattccgctattactagtcttgtt D Q N T V D Q S V T T I D S A I T S L V aatcttaatgctttaaatgaagctattaatcaagctacaccttttataacagatggcaaa N L N A L N E A I N Q A T P F I T D G K gagtatcctaaagaagcgtatgacggtcttgtgcaaaagcttgcagcggcagctaagctt E Y P K E A Y D G L V Q K L A A A A K L caaaattcatttggtccttcacaaggagatgttgataaggctgcgactgatttaacgcaa Q N S F G P S Q G D V D K A A T D L T Q gctcttacgacgcttaagactgctgtagcgcatgaagccttagatcaagccttggctaag A L T T L K T A V A H E A L D Q A L A K ctgttagagctttaccgagaaaatccaaatcttgctttgacatcagagtctttgaaggaa L L E L Y R E N P N L A L T S E S L K E ttgtacaataaggccattgaagcagcaggtaccttctatagaactgttaacaaggataaa L Y N K A I E A A G T F Y R T V N K D K gagagaaaagacatttccctttatgagctagagcgctacactacagaaacaaattcagtt E R K D I S L Y E L E R Y T T E T N S V gttgatactattttaaaggtaaaggctgcgattgccgaagaaggaaaggcaaaattgcgt V D T I L K V K A A I A E E G K A K L R tctgctttagaccaattaaatgctcttatcggagaaaatctagacctatctccatataca S A L D Q L N A L I G E N L D L S P Y T gcagcttctgctcaagcctatacagaccagctagctaaggctaaggaggtcgcagcagcg A A S A Q A Y T D Q L A K A K E V A A A ggtgagacagcttatgctcaggagacagaaccgacagctattactaacagcttggttaag G E T A Y A Q E T E P T A I T N S L V K gtgttaaatgctaagaaatccctctcagatgccaaggcagccttggttgctaaaccggtc V L N A K K S L S D A K A A L V A K P V gatccagtagatccagtagacccagtggatccggtagacccagtagatccggtagaccca D P V D P V D P V D P V D P V D P V D P gtggatccggtagacccagtggatccagtagacccagtagacccagtagacccagtggat V D P V D P V D P V D P V D P V D P V D ccggtagacccagtggatccggtagaccaggtcgatccaatcgacccagoggatccagta P V D P V D P V D P V D P I D P A D P V aaaccatcagatcctgaggttaagccagagcctaaaccagaatctaagcctgaagctaag K P S D P E V K P E P K P E S K P E A K aaggaggacaagaaagcagctgataagcagcaagtgcttccggcaactgctgatacagct K E D K K A A D K Q Q V L P A T A D T A aatccattctttacagcagcagctcttgcagttattgcttgtgcaggccagcttgctatt N P F F T A A A L A V I A C A G Q L A I gtgtcaagacgcaaagaatcaaattaactgtaggcgatgattttccccctttaattaatt V S R R K E S N - L - A M I F P L - L I

(4) SEQ ID NO: 4 shows the recombinant Eq5 protein sequence: The amino acids in bold are those that corresponds to the amino acids encoded by the pTYB4 vector while the rest originates from the Eq5 protein.

TABLE-US-00005 METTTASAFENNGTGQHLNWHIDIPQEYTVELGEPITISDLMSQITVTRK GSNGTVNDGDTFDFISNGDGSRGIDTPGVKIWFDFYNAAGTSFLTDEMLA SPTYAVPGGSYTIKAWVFYGKNDTKKLFTFKLKNSNSNKTELRKSLEEAK LKLSQPEGTYSDESLQALQSAVTLGKTYLNSDPDQNTVDQSVTTIDSAIT SLVNLNALNEAINQATPFITDGKEYPKEAYDGLVQKLAAAAKLQNSFGPS QGDVDKAATDLTQALTTLKTAVAHEALDQALAKLLELYRENPNLALTSES LKELYNKAIEAAGTFYRTVNKDKERKDISLYELERYTTETNSVVDTILKV KAAIAEEGKAKLRSALDQLNALIGENLDLSPYTAASAQAYTDQLAKAKEV AAAGETAYAQETEPTAITNSLVKVLNAKKSLSDAKAALVAKPLEPG

(5) SEQ ID NO: 5 and SEQ ID NO: 16 are combined to show the amino acid sequence of the Eq8 protein (SEQ ID NO: 5) under the nucleotide sequence of eq8 gene (SEQ ID NO: 16)

TABLE-US-00006 atgaacaaaaaatcagcaagacgcaggcgtaagaatcttattacgaagcttgcgatgaca M N K K S A R R R R K N L I T K L A M T agtgccttaaccctgggtgtaggcgcagcgactaccctagcaggacaaacagaagtacgg S A L T L G V G A A T T L A G Q T E V R gctgataatatcttacgcttagatatgacagataaagaagcagttgaaaaattcgctaac A D N I L R L D M T D K E A V E K F A N gagcttaaaaatgaagtccataaaaactatcgtggtagtaatacttggcaaaagcttacc E L K N E V H K N Y R G S N T W Q K L T cttatacttaatggttatcaaaaccttagagaacaaatagagaccgagctaaaaaatagt L I L N G Y Q N L R E Q I E T E L K N S gaacaaaaagtaaaagagcttaatgataaggttaatagtgaaactcaaggaaaacaagag E Q K V K E L N D K V N S E T Q G K Q E ttacagaatcagcttgagaaagaaaaagaagagttagaaacactaaaaaaagagcttgaa L Q N Q L E K E K E E L E T L K K E L E gctgagaaggctaaaggaactggagaaacagagaagcttcaaaaggaaattgaagcaaaa A E K A K G T G E T E K L Q K E I E A K aatgcaatgatttctgacctacaaaaacagcttgaggaaactaagcaaagggttcaagag N A M I S D L Q K Q L E E T K Q R V Q E tttgaagctgaagtaggtaaattaatggccgaaaaggcagacctacaaacaaaattaaat F E A E V G K L M A E K A D L Q T K L N gaacaagagcagcttaacgctaagcttcaaaaagaaattgaagacttaaaggctcagatt E Q E Q L N A K L Q K E I E D L K A Q I gaaaagcttaagcactgtcaagatacacctaagccagagcctaagccagagcctaagcca E K L K H C Q D T P K P E P K P E P K P gagcctaagccagagcctaagccagagcctaagccagagcctaagccagagcctaagcca E P K P E P K P E P K P E P K P E P K P gagcctaagccagggcctaagccagagcctaagccagagcctaagccagggcctaagcca E P K P G P K P E P K P E P K P G P K P gagcctaagccagagcctaagccagggcctaagccagggcctaagccagagcctaagcca E P K P E P K P G P K P G P K P E P K P gggcctaagccagagcctaagccagagcctaagccagagcctaagcctgaagctaagaag G P K P E P K P E P K P E P K P E A K K cctgaacaacctaaaccaatgactaaaccaggagctaagaagcctgagcaatcacttcca P E Q P K P M T K P G A K K P E Q S L P tcaactggtgacatcagaaatccattcttcacgcctgcagctattgctattatgatcgca S T G D I R N P F F T P A A I A I M I A gcaggtaccattgccattccaaaacgcaaggaagaagattaaacaaattaacaatcccca A G T I A I P K R K E E D - T N - Q S P

(6) SEQ ID NO: 6 shows the recombinant Eq8 protein sequence: The amino acids in bold are those that corresponds to the amino acids encoded by the pTYB4 vector while the rest originates from the Eq8 protein.

TABLE-US-00007 MATTLAGQTEVRADNILRLDMTDKEAVEKFANELKNEVHKNYRGSNTWQK LTLILNGYQNLREQIETELKNSEQKVKELNDKVNSETQGKQELQNQLEKE KEELETLKKELEAEKAKGTGETEKLQKEIEAKNAMISDLQKQLEETKQRV QEFEAEVGKLMAEKADLQTKLNEQEQLNAKLQKEIEDLKAQIEKLKHLEP G

(7) SEQ ID NO: 7 and SEQ ID NO: 17 are combined to show the amino acid sequence of the IdeZ2 protein (SEQ ID NO: 7) under the nucleotide sequence of the ideZ2 gene (SEQ ID NO: 17) from S. equi subsp. zooepidemicus

TABLE-US-00008 atgatgaaaaaacaatcattcacacactcacgtaaacctaaattcggtatgagaaaatta M M K K Q S F T H S R K P K F G M R K L tctattggccttgcctcatgtatgctaggaatgatgttcctaacaacaagccatgtttct S I G L A S C M L G M M F L T T S H V S ggtgaggtagttgaagtttggccttatgggcaagatcctaatgataaaatagaagtttta G E V V E V W P Y G Q D P N D K I E V L agtcaatctgagtattccgaatatttacagagattacacgatgttgaagatttccaagct S Q S E Y S E Y L Q R L H D V E D F Q A gaaaagaaaaaagaaggagttgtccgtacacaatggttagagggtgtgaacgttactgac E K K K E G V V R T Q W L E G V N V T D catgacttccggaaaatcactgatggtggtagtgtttattatgccacacctcttttaaat H D F R K I T D G G S V Y Y A T P L L N gatagaggctattatgatatcaacaagaatttcaatcaagacagtgataaatgtgctgct D R G Y Y D I N K N F N Q D S D K C A A gctgtggcagttaatatgttccattattggcttgataggaataaagataatgtagctaag A V A V N M F H Y W L D R N K D N V A K tttcttagtcaaagtccagaccatggttttgttgaaggtgaacctacttttaacttagta F L S Q S P D H G F V E G E P T F N L V gattttcaatatacatatgcatctccatatgaagaaggaggatatagggacaatagtaaa D F Q Y T Y A S P Y E E G G Y R D N S K ctcttcgactttattagcaaggcttttaataagcctctttgggcaaataaattgttagat L F D F I S K A F N K P L W A N K L L D gcttacattaatggctatggctatatcgacagatacgttaaaaataccccgcattctgga A Y I N G Y G Y I D R Y V K N T P H S G caaaataatagtaaatttaatttctttaaaaaagtatttgatggcaagctcttgacagat Q N N S K F N F F K K V F D G K L L T D attcaacaaatttttgattattatactttatcgtctgagctacgtgaagctcttgatact I Q Q I F D Y Y T L S S E L R E A L D T ggcaaagctattggtttagcctatggacctggagatttacgccgttctctgggacatatt G K A I G L A Y G P G D L R R S L G H I atctccgtctggggagctgacattaatgaagatggaaatgtcgtggctatttatgtgact I S V W G A D I N E D G N V V A I Y V T gattccgatgataaaaaattaactatagggaataaaaaagaccgaattggtttgaagcga D S D D K K L T I G N K K D R I G L K R tacaaactgtattctgataacgtgggacgagctcgcctaacagcctatgctacagaaaac Y K L Y S D N V G R A R L T A Y A T E N caacaaactggtggtgaagttcgagggattgaaactttagatatggctacacaagattgg Q Q T G G E V R G I E T L D M A T Q D W gcagattattttagcaggacagacgaagcagaacaataa A D Y F S R T D E A E Q -

(8) SEQ ID NO: 8 and SEQ ID NO: 18 are combined to show the amino acid sequence of the Eqz5 protein (SEQ ID NO: 8) under the nucleotide sequence of the eqz5 gene (SEQ ID NO: 18) from S. equi subsp. zooepidemicus

TABLE-US-00009 atgaagaaattcacgaaacggtgtctt M K K F T K R C L aagggctgcggtcttgttggattagttttcagcacaggattggttgccttgtcggataat K G C G L V G L V F S T G L V A L S D N attgatagcgctttaacagtaggggcggaaacggctactactgctaatgcatttgaagaa I D S A L T V G A E T A T T A N A F E E agtggtgaccaacaacataaaaattggcatatttatattccagaggtttatactgttaaa S G D Q Q H K N W H I Y I P E V Y T V K gtcggtcagccaatcaccattgaggatatcttaagtcagattacgattactcgtaaggga V G Q P I T I E D I L S Q I T I T R K G gaagattcgcaaggtaaaacatctcccggaatgatctatacttatgaagaataccctaaa E D S Q G K T S P G M I Y T Y E E Y P K gtacgaggaattgaagtttcagcaggaactatttggtttgatttttataattctggaaac V R G I E V S A G T I W F D F Y N S G N tgggtaaataatgatgttttagctaccttcaacgaacctggaggaacttataccttatct W V N N D V L A T F N E P G G T Y T L S gcttgggcatactatgctaacgaaaatgtaaaaaaacaatttgttttcaaacttcaagtt A W A Y Y A N E N V K K Q F V F K L Q V gaaaatagtgataagcgtgcattagaacaatctottgctactgctaacgaaaagttacag E N S D K R A L E Q S L A T A N E K L Q gctcctgaaggaacgtattctgatgaatcactgcaacgtttacaagaatcagttttcctt A P E G T Y S D E S L Q R L Q E S V F L ggtcaaacttatttgaacagggatcctgagcaacaagaagtggacgatatgaaggcaact G Q T Y L N R D P E Q Q E V D D M K A T attgattctgctgtttctggtcttgttgatcttactgtcttaaataccgcagttgaaaca I D S A V S G L V D L T V L N T A V E T gcaacaccattgttaacagatggtaaggagtatcctaaagaagcgtatgatagccttgtt A T P L L T D G K E Y P K E A Y D S L V caaaagcttgcagcagcagctaagcttcaaaattcctttaacccatcacaagaagaagtt Q K L A A A A K L Q N S F N P S Q E E V aacgaggctgcgactgatttaacgcaagctcttacgacgcttaagactgctgtagcgcat N E A A T D L T Q A L T T L K T A V A H gaagccttagatcaagccttggctaagctgttagagctttaccgagaaaatccaaacctt E A L D Q A L A K L L E L Y R E N P N L gctttgacatcagagcctttgaaggaattgtacaataaggccattgaagcagcaggcacc A L T S E P L K E L Y N K A I E A A G T ttctatagaactgttagcaaggataaagagagaaaaggcatttccctttatgagctagag F Y R T V S K D K E R K G I S L Y E L E cgttacactacagaaacaaactcagttgttgatactattttaaaggtaaaggctgcaatt R Y T T E T N S V V D T I L K V K A A I gccgaagaaggaaaggcaaaattgcgttctgctttagaccaattaaatgctcttatcgga A E E G K A K L R S A L D Q L N A L I G gaaaatctagacctatctccatatacagcagcttctgctcaagcctatacagaccagcta E N L D L S P Y T A A S A Q A Y T D Q L gctaaggctaaggaggttgcagcagcgggtgagacagcttatgctcaggagacagaaccg A K A K E V A A A G E T A Y A Q E T E P acagctattactaacagcttgattaaggtgctaaatgctaagaaatccctctcagatgcc T A I T N S L I K V L N A K K S L S D A aaggcagcattggttgctaaaccggtagatccggtagacccagtagatccggtagaccca K A A L V A K P V D P V D P V D P V D P gtggatccggtagacccaattgatccagtagatccagtaaaaccagtcgatcctgaggtt V D P V D P I D P V D P V K P V D P E V aagccagagcctaaaccagaatctaagcctgaagctaagaaggaggacaagaaagcagct K P E P K P E S K P E A K K E D K K A A gataagcagcaagtgcttccggcaactgctgatacagctaacccattctttacagcagca D K Q Q V L P A T A D T A N P F F T A A gctcttgcagttattgcttgtgcaggccagcttgctattgtgtcaagacgcaaagaatca A L A V A I C A G Q L A I V S R R K E S aattaa N -

(9) SEQ ID NO: 9 and SEQ ID NO: 19 are combined to show the amino acid sequence of the Eqz8 protein (SEQ ID NO: 9) under the nucleotide sequence of the eqz8 gene (SEQ ID NO: 19) from S. equi subsp. zooepidemicus

TABLE-US-00010 atgaacaaaaaatcagca M N K K S A agacgcaagcgtaaggatcttatcacgaagcttgcgatgacaagtgccttaaccctgggt R R K R K D L I T K L A M T S A L T L G gtaggcgcagcagctaccatagcaggacaaacagaagtacgggctgaggttctaacctta V G A A A T I A G Q T E V R A E V L T L aatatgaaagataaagctaaagttgaagaattcgctaataagcttaaagattacgcaaag N M K D K A K V E E F A N K L K D Y A K caaaagaaatctggccaaattactttgcaagaactttcccttatacttgatgggtacaga Q K K S G Q I T L Q E L S L I L D G Y R aatattagggagcagatagaacaagacttagctactacagaaaaaactaaaaatttctat N I R E Q I E Q D L A T T E K T K N F Y ggagaacagttaattcttactgataaactttatcagtctgaaaaagaaaagaaagaaaag G E Q L I L T D K L Y Q S E K E K K E K ctagaagctgagctacaactaagccaacaaaaaattcatgaccttgatgaaaaacatcaa L E A E L Q L S Q Q K I H D L D E K H Q aaagagaaattagagctacaagaacaacttgaggcttcaaatcaaaagattaaagagctt K E K L E L Q E Q L E A S N Q K I K E L gaaatggcaaagagcacagctgaagctgaaataaatagactaacagatgaaaaaaatgga E M A K S T A E A E I N R L T A E K N G ttacaagaaaaattaaataatcaagaaaagcttaatgctgagttacaagcaaaattagct L Q E K L N N Q E K L N A E L Q A K L A aagcaagaagagcttaacgctaagcttcaaaaggaaattgacgaattaaatgctcagctt K Q E E L N A K L Q K E I D E L N A Q L gaaaagcttaagcattgtcaagatacacctaagccagagcctaagccagagcctaagcca E K L K H C Q D T P K P E P K P E P K P gagcctaagccagagcctaagccagagcctaagccagagcctaagccagagcctaagcca E P K P E P K P E P K P E P K P E P K P gagcctaagccagagcctaagccagagcctaagccagagcctaagccagagcctaagcca E P K P E P K P E P K P E P K P E P K P gagcctaagccagagcctaagccagagcctaagccagagcctaagccagagcctaagcca E P K P E P K P E P K P E P K P E P K P gagcctaagccagagcctaagccagagcctaagccagagcctaagccagagcctaagcca E P K P E P K P E P K P E P K P E P K P gagcctaagccagagcctaagccagagcctaagccagagcctaagccagagcctaagcca E P K P E P K P E P K P E P K P E P K P gagcctaagccagagcctaagcctgaagctaaaaagcctgaacaacctaaaccaatgact E P K P E P K P E A K K P E Q P K P M T aaaccaggggctaagaagcctgagcaatcacttccatcaactggtgacatcagaaatcca K P G A K K P E Q S L P S T G D I R N P ttcttcacacctgcagctattgctattatgatcgcagcaggtaccattgcaattccaaaa F F T P A A I A I M I A A G T I A I P K cgcaaggaagaagactaa R K E E D -

(10) SEQ ID NO: 10 and SEQ ID NO: 20 are combined to show the amino acid sequence of the IdeE protein (SEQ ID NO: 10) under the nucleotide sequence of the ideE gene (SEQ ID NO: 20).

[0141]The nucleotide sequence of the ideE gene (GenBank DQ508733) and the amino acid sequence of the IdeE protein from S. equi subsp. equi are shown.

TABLE-US-00011 atgaaaacaatagcttatccaaataaacctcactccttatcagctggtctcttaactgct M K T I A Y P N K P H S L S A G L L T A atagctatttttagcctggcgagttcaaacattacttatgctgacgattaccaaaggaat I A I F S L A S S N I T Y A D D Y Q R N gctacggaagottatgccaaagaagtaccacatcagatcacttctgtatggaccaaaggt A T E A Y A K E V P H Q I T S V W T K G gttacaccactaacacccgagcagtttcgatataataacgaagatgtgatccatgcgcca V T P L T P E Q F R Y N N E D V I H A P tatcttgctcatcaaggctggtacgatatcaccaaggccttcgatgggaaggataatctc Y L A H Q G W Y D I T K A F D G K D N L ttgtgtggcgcagcaacggcaggtaatatgctgcattggtggtttgatcaaaataaaaca L C G A A T A G N M L H W W F D Q N K T gagattgaagcctatttaagtaaacaccctgaaaagcaaaaaatcatttttaacaaccaa E I E A Y L S K H P E K Q K I I F N N Q gagctatttgatttgaaagctgctatcgataccaaggacagtcaaaccaatagtcagctt E L F D L K A A I D T K D S Q T N S Q L tttaattattttagagataaagcctttccaaatctatcagcacgtcaactcggggttatg F N Y F R D K A F P N L S A R Q L G V M cctgatcttgttctagacatgtttatcaatggttactacttaaatgtgtttaaaacacag P D L V L D M F I N G Y Y L N V F K T Q tctactgatgtcaatcgaccttatcaggacaaggacaaacgaggtggtattttcgatgct S T D V N R P Y Q D K D K R G G I F D A gttttcaccagaggagatcagacaacgctcttgacagctcgtcatgatttaaaaaataaa V F T R G D Q T T L L T A R H D L K N K ggactaaatgacatcagcaccattatcaagcaagaactgactgaaggaagagcccttgct G L N D I S T I I K Q E L T E G R A L A ttatcacatacctacgccaatgttagcattagccatgtgattaacttgtggggagctgat L S H T Y A N V S I S H V I N L W G A D tttaatgctgaaggaaaccttgaggccatctatgtcacagactcagatgctaatgcgtct F N A E G N L E A I Y V T D S D A N A S attggtatgaaaaaatattttgtcggcattaatgctcatagacatgtcgccatttctgcc I G M K K Y F V G I N A H R H V A I S A aagaaaatagaaggagaaaacattggcgctcaagtattaggcttatttacgctttccagt K K I E G E N I G A Q V L G L F T L S S ggcaaggacatatggcagaaactgagctaa G K D I W Q K L S -

(11) SEQ ID NO: 11 and SEQ ID NO: 21 are combined to show the amino acid sequence of the IdeZ protein (SEQ ID NO: 11) under the nucleotide sequence of the ideZ gene (SEQ ID NO: 21).

[0142]The nucleotide sequence of the ideZ gene (Genbank DQ826037) and the amino acid sequence of the IdeZ protein from S. equi subsp. zooepidemicus are shown.

TABLE-US-00012 atgaaaacaatagcttatccaaataaacctcactccttatcagctggtctcttaactgct M K T I A Y P N K P H S L S A G L L T A atagctatttttagcctggcgagttcaaacattacttatgctgacgattaccaaaggaat I A I F S L A S S N I T Y A D D Y Q R N gctgcggaagtttatgccaaagaagtaccacatcagatcacttctgtatggaccaaaggt A A E V Y A K E V P H Q I T S V W T K G gttacaccactaacacccgagcagtttcgatataataacgaagatgtgatccatgcgcca V T P L T P E Q F R Y N N E D V I H A P tatcttgctcatcaaggctggtacgatatcaccaaggtcttcgatgggaaggataatctc Y L A H Q G W Y D I T K V F D G K D N L ttgtgtggcgcagcaacggcaggtaatatgctgcattggtggtttgatcaaaataaaaca L C G A A T A G N M L H W W F D Q N K T gagattgaagcctatttaagtaaacaccctgaaaagcaaaaaatcatttttaacaaccaa E I E A Y L S K H P E K Q K I I F N N Q gagctatttgatttgaaagctgctatcgataccaaggacagtcaaaccaatagtcagctt E L F D L K A A I D T K D S Q T N S Q L tttaattattttagagataaagcctttccaaatctatcagcacgtcaactcggggttatg F N Y F R D K A F P N L S A R Q L G V M cctgatcttgttctagacatgtttatcaatggttactacttaaatgtgtttaaaacacag P D L V L D M F I N G Y Y L N V F K T Q tctactgatgtcaatcgaccttatcaggacaaggacaaacgaggtggtattttcgatgct S T D V N R P Y Q D K D K R G G I F D A gttttcaccagaggagatcagacaacgctcttgacagctcgtcatgatttaaaaaataaa V F T R G D Q T T L L T A R H D L K N K ggactaaatgacatcagcaccattatcaagcaggaactgactgaaggaagagcccttgct G L N D I S T I I K Q E L T E G R A L A ttatcacatacctacgccaatgttagcattagccatgtgattaacttgtggggagctgat L S H T Y A N V S I S H V I N L W G A D tttaatgctgaaggaaaccttgaggccatctatgtcacagactcagatgctaatgcgtct F N A E G N L E A I Y V T D S D A N A S attggtatgaaaaaatattttgtcggcattaatgctcatggacatgtcgccatttctgcc I G M K K Y F V G I N A H G H V A I S A aagaaaatagaaggagaaaacattggcgctcaagtattaggcttatttacgctttccagt K K I E G E N I G A Q V L G L F T L S S ggcaaggacatatggcagaaactgagctaa G K D I W Q K L S -

(12) SEQ ID NO: 12

[0143]Nucleotide sequence of the eag gene

TABLE-US-00013 1 aaataattttgtttaactttaagaaggagatataaccatggctctag atg 51 ctacaacggtgttagagcctacaacagccttcattagagaagctgtt agg 101 gaaatcaatcagctgagtgatgactacgctgacaatcaagagcttca ggc 151 tgttcttgctaatgctggagttgaggcacttgctgcagatactgttg atc 201 aggctaaagcagctcttgacaaagcaaaggcagctgttgctggtgtt cag 251 cttgatgaagcaagacgtgaggcttacagaacaatcaatgccttaag tga 301 tcagcacaaaagcgatcaaaaggttcagctagctctagttgctgcag cag 351 ctaaggtggcagatgctgcttcagttgatcaagtgaatgcagccatt aat 401 gatgctcatacagctattgcggacattacaggagcagccttgttgga ggc 451 taaagaagctgctatcaatgaactaaagcagtatggcattagtgatt act 501 atgtgaccttaatcaacaaagccaaaactgttgaaggtgtcaatgcg ctt 551 aaggcaaagattttatcagctctaccgtagctcgagcccgggtgctt tgc

(13) SEQ ID NO: 13

[0144]Amino acid sequence of the EAG4B protein

TABLE-US-00014 1 MALDATTVLE PTTAFIREAV REINQLSDDY ADNQELQAVL ANAGVEALAA DTVDQAKAAL 61 DKAKAAVAGV QLDEARREAY RTINALSDQH KSDQKVQLAL VAAAAKVADA ASVDQVNAAI 121 NDAHTAIADI TGAALLEAKE AAINELKQYG ISDYYVTLIN KAKTVEGVNA LKAKILSALP

(14) SEQ ID NO: 28

[0145]Protein sequence of SEC2.16 (CNE)

TABLE-US-00015 Met Ala Thr Asn Leu Ser Asp Asn Ile Thr Ser Leu Thr Val Ala Ser 1 5 10 15 Ser Ser Leu Arg Asp Gly Glu Arg Thr Thr Val Lys Val Ala Phe Asp 20 25 30 Asp Lys Lys Gln Lys Ile Lys Ala Gly Asp Thr Ile Glu Val Thr Trp 35 40 45 Pro Thr Ser Gly Asn Val Tyr Ile Gln Gly Phe Asn Lys Thr Ile Pro 50 55 60 Leu Asn Ile Arg Gly Val Asp Val Gly Thr Leu Glu Val Thr Leu Asp 65 70 75 80 Lys Ala Val Phe Thr Phe Asn Gln Asn Ile Glu Thr Met His Asp Val 85 90 95 Ser Gly Trp Gly Glu Phe Asp Ile Thr Val Arg Asn Val Thr Gln Thr 100 105 110 Thr Ala Glu Thr Ser Gly Thr Thr Thr Val Lys Val Gly Asn Arg Thr 115 120 125 Ala Thr Ile Thr Val Thr Lys Pro Glu Ala Gly Thr Gly Thr Ser Ser 130 135 140 Phe Tyr Tyr Lys Thr Gly Asp Ile Gln Pro Asn Asp Thr Glu Arg Val 145 150 155 160 Arg Trp Phe Leu Leu Ile Asn Asn Asn Lys Glu Trp Val Ala Asn Thr 165 170 175 Val Thr Val Glu Asp Asp Ile Gln Gly Gly Gln Thr Leu Asp Met Ser 180 185 190 Ser Phe Asp Ile Thr Val Ser Gly Tyr Arg Asn Glu Arg Phe Val Gly 195 200 205 Glu Asn Ala Leu Thr Glu Phe His Thr Thr Phe Pro Asn Ser Val Ile 210 215 220 Thr Ala Thr Asp Asn His Ile Ser Val Arg Leu Asp Gln Tyr Asp Ala 225 230 235 240 Ser Gln Asn Thr Val Asn Ile Ala Tyr Lys Thr Lys Ile Thr Asp Phe 245 250 255 Asp Gln Lys Glu Phe Ala Asn Asn Ser Lys Ile Trp Tyr Gln Ile Leu 260 265 270 Tyr Lys Asp Gln Val Ser Gly Gln Glu Ser Asn His Gln Val Ala Asn 275 280 285 Ile Asn Ala Asn Gly Gly Val Asp Gly Ser Arg Tyr Thr Ser Phe Thr 290 295 300 Val Lys Lys Ile Trp Asn Asp Lys Glu Asn Gln Asp Gly Lys Arg Pro 305 310 315 320 Lys Thr Ile Thr Val Gln Leu Tyr Ala Asn Asp Gln Lys Val Asn Asp 325 330 335 Lys Thr Ile Glu Leu Ser Asp Thr Asn Ser Trp Gln Ala Ser Phe Gly 340 345 350 Lys Leu Asp Lys Tyr Asp Ser Gln Asn Gln Lys Ile Thr Tyr Ser Val 355 360 365 Lys Glu Val Met Val Pro Val Gly Tyr Gln Ser Gln Val Glu Gly Asp 370 375 380 Ser Gly Val Gly Phe Thr Ile Thr Asn Thr Tyr Thr Pro Glu Val Ile 385 390 395 400 Ser Ile Thr Gly Gln Lys Thr Trp Asp Asp Arg Glu Asn Gln Asp Gly 405 410 415 Lys Arg Pro Lys Glu Ile Thr Val Arg Leu Leu Ala Asn Asp Ala Ala 420 425 430 Thr Asp Lys Val Ala Thr Ala Ser Glu Gln Thr Gly Trp Lys Tyr Thr 435 440 445 Phe Thr Asn Leu Pro Lys Tyr Lys Asp Gly Lys Gln Ile Thr Tyr Thr 450 455 460 Ile Gln Glu Asp Pro Val Ala Asp Tyr Thr Thr Thr Ile Gln Gly Phe 465 470 475 480 Asp Ile Thr Asn His His Glu Val Ala Leu Thr Ser Leu Lys Val Ile 485 490 495 Lys Val Trp Asn Asp Lys Asp Asp Tyr Tyr His Lys Arg Pro Lys Glu 500 505 510 Ile Thr Ile Leu Leu Lys Ala Asp Gly Lys Val Ile Arg Glu His Gln 515 520 525 Met Thr Pro Asp Gln Gln Gly Lys Trp Glu Tyr Thr Phe Asp Gln Leu 530 535 540 Pro Val Tyr Gln Ala Gly Lys Lys Ile Ser Tyr Ser Ile Glu Glu Lys 545 550 555 560 Gln Val Ala Gly Tyr Gln Ala Pro Val Tyr Glu Val Asp Glu Gly Leu 565 570 575 Lys Gln Val Thr Val Thr Asn Thr Leu Asn Pro Ser Tyr Lys Leu Glu 580 585 590 Pro Gly

(15) SEQ ID NO 29

[0146]Protein sequence of ScIC

TABLE-US-00016 Met Thr Asn Lys Thr Lys Arg Thr Gly Leu Val Arg Lys Tyr Gly Ala 1 5 10 15 Cys Ser Ala Ala Ile Ala Leu Ala Ala Leu Ala Ser Leu Gly Ala Gly 20 25 30 Lys Ala Val Lys Ala Asp Gln Pro Ala Ala Leu Lys Tyr Pro Glu Pro 35 40 45 Arg Asp Tyr Phe Leu His Thr Arg Glu Gly Asp Val Ile Tyr Asp Glu 50 55 60 Asp Ile Lys Arg Tyr Phe Glu Asp Leu Glu Ala Tyr Leu Thr Ala Arg 65 70 75 80 Leu Gly Gly Ile Asp Lys Lys Val Glu Glu Ala Ala Gln Lys Pro Gly 85 90 95 Ile Pro Gly Pro Thr Gly Pro Gln Gly Pro Lys Gly Asp Lys Gly Asp 100 105 110 Pro Gly Ala Pro Gly Glu Arg Gly Pro Ala Gly Pro Lys Gly Asp Thr 115 120 125 Gly Glu Ala Gly Pro Arg Gly Glu Gln Gly Pro Ala Gly Gln Ala Gly 130 135 140 Glu Arg Gly Pro Lys Gly Asp Pro Gly Ala Pro Gly Pro Lys Gly Glu 145 150 155 160 Lys Gly Asp Thr Gly Ala Val Gly Pro Lys Gly Glu Lys Gly Asp Thr 165 170 175 Gly Ala Thr Gly Pro Lys Gly Asp Lys Gly Glu Arg Gly Glu Lys Gly 180 185 190 Glu Gln Gly Gln Arg Gly Glu Lys Gly Glu Gln Gly Gln Arg Gly Glu 195 200 205 Lys Gly Glu Gln Lys Pro Lys Gly Asp Gln Gly Lys Asp Thr Lys Pro 210 215 220 Ser Ala Pro Lys Ala Pro Glu Lys Ala Pro Ala Pro Lys Ala Pro Lys 225 230 235 240 Ala Ser Glu Gln Ser Ser Asn Pro Lys Ala Pro Ala Pro Lys Ser Ala 245 250 255 Pro Ser Lys Ser Ala Ala Pro Thr Gly Gln Lys Ala Ala Leu Pro Ala 260 265 270 Thr Gly Glu Ile Asn His Pro Phe Phe Thr Leu Ala Ala Leu Ser Val 275 280 285 Ile Ala Ser Val Gly Val Leu Thr Leu Lys Gly Lys Lys Asp 290 295 300

(16) SEQ ID NO 30. Recombinant protein IdeE

TABLE-US-00017 GPLGSDDYQRNATEAYAKEVPHQITSVWTKGVTPLTPEQFRYNNEDVIHA PYLAHQGWYDITKAFDGKDNLLCGAATAGNMLHWWFDQNKTEIEAYLSKH PEKQKIIFNNQELFDLKAAIDTKDSQTNSQLFNYFRDKAFPNLSARQLGV MPDLVLDMFINGYYLNVFKTQSTDVNRPYQDKDKRGGIFDAVFTRGDQTT LLTARHDLKNKGLNDISTIIKQELTEGRALALSHTYANVSISHVINLWGA DFNAEGNLEAIYVTDSDANASIGMKKYFVGINAHRHVAISAKKIEGENIG AQVLGLFTLSSGKDIWQKLS

Amino acids in bold originates from the vector.

Example 10

Vaccination Study

[0147]The objective of this study was to determine the level of protection conferred on vaccination with Intervacc's new multi-component subunit vaccine following intranasal challenge with wild type S. equi strain 4047 in Welsh Mountain ponies. The study has been performed by Animal Health Trust, UK. The vaccines used therein, which are designated Nordostrep Septavac or Nordostrep Pentavac A (or only Septavac or Pentavac) are disclosed below.

Methods

[0148]The ponies were initially randomised into 3 groups for the vaccination period.

TABLE-US-00018 TABLE 1 Vaccination groups. Group Vaccine No per group Route 1 Nordostrep Septavac 7 IN + SC 2 Nordostrep Pentavac A 7 IN + SC 3 Placebo 7 IN + SC

In the first trial groups 1 and 3 were taken through to challenge. (The challenge of second trial group 2 (Pentavac A) is described in section 9). The decision as to which vaccine group to challenge was taken by Intervacc one week prior to challenge.

The Nordostrep Pentavac A Formulation

[0149]The Pentavac vaccine consisted of the following five S. equi recombinant proteins: EAG, ScIC, CNE, Eq5 and Eq8. For subcutaneous vaccination, the five proteins were mixed in PBS (50 μg/ml of the respective protein), divided in aliquots of 1 ml in vials and stored at -20° C. Immediately before vaccination, the vial was thawed and mixed with 1 ml adjuvant (Abisco 200, 375 μg/dose, Isconova AB, Sweden). For intranasal vaccination the five proteins were mixed in PBS (150 μg/ml of respective protein) and divided in aliquots of 2 ml in vials and stored at -20° C. Immediately before vaccination the vial was thawed and mixed with 2 ml adjuvant (Abisco 300, 500 μg/dose, Isconova AB, Sweden). In the placebo formulations the S. equi proteins were omitted. Thus, the placebo for subcutaneous vaccination only contained PBS and Abisco 200, 375 μg/dose and for intranasal vaccination, the placebo contained only PBS and Abisco 300, 500 μg/dose.

The Nordostrep Septavac Formulation

[0150]The Septavac vaccine consisted of the following seven S. equi recombinant proteins: EAG, ScIC, CNE, Eq5, Eq8, IdeE and IdeE2. For subcutaneous vaccination, the seven proteins were mixed in PBS (50 μg/ml of respective protein) and divided in aliquots of 1 ml in vials and stored at -20° C. Immediately before vaccination the vial was thawed and mixed with 1 ml adjuvant (Abisco 200, 375 μg/dose, Isconova AB, Sweden). For intranasal vaccination, the seven proteins were mixed in PBS (150 μg/ml of the respective protein) and divided in aliquots of 2 ml in vials and stored at -20° C. Immediately before vaccination, the vial was thawed and mixed with 2 ml adjuvant (Abisco 300, 500 μg/dose, Isconova AB, Sweden). In the placebo formulations, the S. equi proteins were omitted. Thus, the placebo for subcutaneous vaccination only contained PBS and Abisco 200, 375 μg/dose, and for intranasal vaccination, it only contained PBS and Abisco 300, 500 μg/dose.

[0151]In these formulations, EAG is comprised of the fragment EAG4B and CNE is the fragment designated 2.16.

Short Summary of Results

[0152]This study evaluated the efficacy of a new multi-component subunit vaccine for the prevention of strangles. The Septavac vaccine induced pyrexia in ponies for one day after first and second vaccinations. However, there were no other adverse reactions and this vaccine appears to be very well tolerated.

[0153]All ponies were challenged with an identical dose of 1×10⁸ cfu of S. equi strain 4047, which was split and administered via both nostrils. All seven control ponies developed pyrexia and multiple lymph node abscesses (100%). Only one vaccinated pony developed pyrexia (which could have been due to an ongoing S. zooepidemicus infection) and only one developed lymph node abscesses (14%). Statistically, vaccinated ponies were significantly protected from S. equi as measured by temperature, post mortem score, and fibrinogen and neutrophil levels.

[0154]Overall, the Septavac vaccine was a safe and effective vaccine for the prevention of strangles. However, the invention is not restricted to the Septavac and Pentavac vaccines which have been studied in this Example but many combinations of the present antigens/immunogens are possible candidates for use in vaccine compositions for prevention of strangles.

1 Procedure

[0155]Two earlier studies (WO 2004/032957 A1 and ref. 27) demonstrated that Intervacc vaccines conferred some protection against S. equi challenge. All four vaccinated groups across the two studies showed reduced guttural pouch empyema. The present study was designed to compare the immunogenicity of two Nordvacc vaccines: one containing five (Pentavac) and one containing seven (Septavac) S. equi proteins.

[0156]Blood and nasal wash samples were taken according to the protocol to determine the equine immune responses to the vaccine subunits. Based on immunogenicity data, one vaccinated group was challenged to quantify the level of protection conferred.

[0157]Each pony was challenged with a total challenge dose of 1×10⁸ cfu of S. equi strain 4047 administered via the spraying of a 2 ml culture containing 5×10⁷ cfu into both nostrils. This dose regime is believed to optimise the infection rate whilst avoiding overwhelming the host immune response.

[0158]Ponies were carefully monitored for the onset of clinical signs of disease over a period of three weeks post challenge by regular checks, daily physical examination, monitoring of body temperature, the taking of sera to determine seroconversion and the taking of nasal washes for bacteriological analysis. All ponies were subjected to post mortem examination following abscessation or reaching the study endpoint at 3 weeks post challenge to determine the severity of disease pathology according to a scoring system developed at the AHT. Histopathological examination of tissues recovered from the study ponies was used to identify early signs of S. equi infection that were not obvious on post mortem (PM) examination.

TABLE-US-00019 TABLE 2 Sampling Schedule Day Volume of Day of of sera to be study week date procedure taken Sample/Analysis/comment day -10 Thurs 31/01/2008 veterinary examination day 1 Mon 11/02/2008 Obs/temps, 40 ml normal, IgG and CFU from NW; ELISA for IgG from sera NW, BL 20 ml EDTA day 2 Tues 12/02/2008 Obs/temps day 3 Wed 13/02/2008 Obs/temps day 4 Thurs 14/02/2008 Obs/temps, V1 7 contr and 7 vaccinated day 5 to 18 Fri 15/02/2008 to Obs/temps 28/02/2008 day 50 Mon 31/03/2008 Obs/temps, 20 ml normal IgG and CFU from NW; ELISA for IgG from sera NW, BL day 51 Tues 01/04/2008 Obs/temps day 52 to 59 Wed 02/04/2008 to Obs/temps 09/04/2008 day 60 Thurs 10/04/2008 Obs/temps, V2 7 contr and 7 vaccinated day 61 to 68 Fri 11/04/2008 to Obs/temps 18/04/2008 day 71 Mon 21/04/2008 Obs/temps, 20 ml normal IgG and CFU from NW; ELISA for IgG from sera NW, BL day 72 Tues 22/04/2008 Obs/temps day 73 Wed 23/04/2008 Obs/temps day 74 Thurs 24/04/2008 Obs/temps, V3 7 contr and 7 Vaccinated day 75 to 81 Fri 25/04/2008 to Obs/temps 2/05/2008 day 86 Tues 06/05/2008 Obs/temps, 40 ml normal, IgG and CFU from NW; ELISA for IgG from sera; fibrinogen and NW, BL 20 ml EDTA neutrophil levels to be quantified Move to Allen Centre day 87 Wed 07/05/2008 Obs/temps day 88 Thurs 08/05/2008 Obs/temps, Challenge day 89 Fri 09/05/2008 Obs/temps, 20 ml normal, IgG and CFU from NW; ELISA for IgG from sera; fibrinogen and NW, BL 10 ml EDTA neutrophil levels to be quantified day 90 Sat 10/05/2008 Obs/temps day 91 Sun 11/05/2008 Obs/temps day 92 Mon 12/05/2008 Obs/temps, 20 ml normal, IgG and CFU from NW; ELISA for IgG from sera; fibrinogen and NW, BL 10 ml EDTA neutrophil levels to be quantified day 93 Tues 13/05/2008 Obs/temps day 94 Wed 14/05/2008 Obs/temps, 20 ml normal, IgG and CFU from NW; ELISA for IgG from sera; fibrinogen and NW, BL 10 ml EDTA neutrophil levels to be quantified day 95 Thurs 15/05/2008 Obs/temps day 96 Fri 16/05/2008 Obs/temps, 20 ml normal, IgG and CFU from NW; ELISA for IgG from sera; fibrinogen and NW, BL 10 ml EDTA neutrophil levels to be quantified day 97 Sat 17/05/2008 Obs/temps day 98 Sun 18/05/2008 Obs/temps day 99 Mon 19/05/2008 Obs/temps, 20 ml normal, IgG and CFU from NW; ELISA for IgG from sera; fibrinogen and NW, BL 10 ml EDTA neutrophil levels to be quantified day 100 Tues 20/05/2008 Obs/temps day 101 Wed 21/05/2008 Obs/temps, 20 ml normal, IgG and CFU from NW; ELISA for IgG from sera; fibrinogen and NW, BL 10 ml EDTA neutrophil levels to be quantified day 102 Thurs 22/05/2008 Obs/temps day 103 Fri 23/05/2008 Obs/temps, 20 ml normal, IgG and CFU from NW; ELISA for IgG from sera; fibrinogen and NW, BL 10 ml EDTA neutrophil levels to be quantified day 104 Sat 24/05/2008 Obs/temps day 105 Sun 25/05/2008 Obs/temps day 106 Mon 26/05/2008 Obs/temps, 30 ml IgG and CFU from NW; ELISA for IgG from sera; fibrinogen and NW, BL neutrophil levels to be quantified day 107 Tues 27/05/2008 Obs/temps day 108 Wed 28/05/2008 Obs/temps, 30 ml IgG and CFU from NW; ELISA for IgG from sera; fibrinogen and NW, BL neutrophil levels to be quantified day 109 Thurs 29/05/2008 Obs/temps Comments: NW = Nasal washings BL = Blood sample 1. Nasal immunisation in both nostrils at all three occasions (2 × 2 ml in each nostril = 4 ml/vaccination) 2. Subcutaneous immunisation near submandibular lymph nodes (1 ml) 3. Two groups of seven (7) horses will be vaccinated and seven (7) unvaccinated/controls 4. IgG in sera analysed by Intervacc AS; IgG in NW by AHT (7 antigens)

2.1 Vaccine

[0159]Nordostrep Vaccines for Horses [0160]Group 1: 7 ponies vaccinated with Nordostrep Pentavac A [0161]2 ml subcutaneous injection (1 ml on each side of the head) [0162]4 ml intranasal injection (2 ml in each nostril) [0163]Day 4; 60; 74 [0164]Group 2: 7 ponies vaccinated with Nordostrep Septavac [0165]2 ml subcutaneous injection (1 ml on each side of the head) [0166]4 ml intranasal injection (2 ml in each nostril) [0167]Day 4; 60; 74 [0168]Group 3: 7 ponies vaccinated with Placebo [0169]2 ml subcutaneous injection (1 ml on each side of the head) [0170]4 ml intranasal injection (2 ml in each nostril) [0171]Day 4; 60; 74

[0172]The vaccine vials were received by the AHT prior to the first vaccination and stored at -20° C. until use in freezer number EQ No. 2305. Placebo (containing no antigens) and adjuvant vials were stored at 4° C. until use in fridge number EQ No. 44.

[0173]At the time of vaccination, vaccines and adjuvants were mixed as stated in the protocol in situ by A Waller, L Prowse or C Robinson at AHT.

2.2 Challenge Bacterium

[0174]S. equi 4047 was prepared from fresh plates as described in SOP/BACT/25.

[0175]The bacteria grew as expected and the 1:40 diluted culture was harvested when the OD₆₀₀nm reached 0.3. The growth of the challenge inoculum is shown in FIG. 6. The following results were obtained. [0176]Plating results: 1/10⁵ dilution 37 colonies [0177]35 colonies [0178]33 colonies [0179]32 colonies [0180]Mean=34.25 in 100 μl [0181]Therefore actual dose per pony=4×34.25×10⁵×10 [0182]=1.37×10⁸ cfu/dose

3 Animal Management

3.1 Supply

[0183]Twenty one Welsh Mountain ponies originally supplied by Mr Beedles, Shropshire, UK, were used. Ponies were approximately 8 months of age at the time of the first vaccination.

3.2 Identification/allocation

[0184]Ponies were identified by a microchip in the neck. The 21 ponies were randomly assigned to vaccination groups (Table 3).

TABLE-US-00020 TABLE 3 Vaccination groups and pony chip IDs Group Vaccine Pony Chip ID's 1 Septavac 00012, 00159, 00833, 00976, 99123, 99668, 99794 2 Pentavac 01298, 01605, 01724, 99223, 99229, 99773, 99919 3 Placebo 00173, 00427, 01635, 02078, 99549, 99776, 99886

3.3 Husbandry

[0185]Prior to challenge, ponies were kept at pasture on grass at Lanwades Park, Kentford, UK and Kirtling, Newmarket, UK. These sites have been approved by the Home Office for this type of work. Drinking water was available ad libitum.

[0186]Ponies in groups 1 and 3 were transferred to the ACVS (Allen Centre), three days prior to challenge to allow acclimatisation. Ponies were separated into two animal rooms according to their vaccination groups, so that ponies from each vaccination group were kept together.

Methods

4.1 Vaccination

[0187]Vaccinations were given by subcutaneous injection near the retropharyngeal lymph nodes according to AHT SOP/EQU/03 or via intranasal spray according to AHT SOP/EQU/07.

4.1.2 Preliminary Clinical Examination

[0188]A veterinarian clinically examined all ponies before the first vaccination, before V2 (due to S. zoo infection) and before V3. Only healthy ponies in good clinical condition were included in the study (SOP/EQU/08).

4.1.3 Vaccination

[0189]Ponies received vaccinations according to Table 4. With the exception that pony 9229 was pyrexic on Feb. 14, 2008 due to an ongoing S. zooepidemicus infection. This pony recovered over the weekend and was vaccinated on Apr. 18, 2008.

TABLE-US-00021 TABLE 4 Vaccination routes and dates Group Vaccine V1 V2* V3* A Septavac 14/2/08 10/4/08 24/4/08 B Pentavac 14/2/08 10/4/08 24/4/08 C Placebo 14/2/08 10/4/08 24/4/08 *Delayed by 7 days due to S. zooepidemicus infection.

4.1.4 Clinical Observations Around Vaccinations

[0190]Clinical observations were performed daily after vaccination. If adverse reactions occurred, then additional checks were made as required.

4.2 Experimental Challenge with S. equi 4047

4.2.1 Preliminary Clinical Examination

[0191]Prior to transfer to the ACVS, a veterinarian clinically examined the challenge ponies. Only healthy ponies in good clinical condition were subjected to the challenge.

4.2.2 Challenge

[0192]Two weeks after the third vaccination (Aug. 5, 2008), each pony was challenged by intranasal administration of 2 ml of a fresh S. equi 4047 culture into both nostrils using a flexible tube and spray nozzle according to AHT SOP/BACT32. Such a challenge dose was predicted to contain a total of 1×10⁸ cfu of S. equi 4047.

[0193]No problems were encountered during the administration of the challenge dose. Spare inocula were used to quantify the actual challenge dose administered, which was found to be 1.37×10⁸ cfu/dose.

4.3 Post Challenge Monitoring

4.3.1 Clinical Examination

[0194]Ponies were examined according to AHT SOP/EQU/02. Each pony was examined clinically on the day of challenge, and on each of the following 21 days for the occurrence of symptoms associated with S. equi infection (demeanor, nasal discharge, lymph node swelling and abscessation, signs of coughing, difficulty swallowing and feeding, and ocular signs).

4.3.2 Rectal Temperatures

[0195]Individual rectal temperatures were taken at around 9.00 am from the day of challenge through to day 21 after challenge.

4.4 Blood Sampling

[0196]Blood samples were taken from the jugular vein according to AHT SOP/EQU/01 and according to the study protocol schedule. Serum was prepared according to AHT SOP/EQU/01 and stored frozen at -20° C. or below until use.

4.5 Processing of Blood Samples

[0197]Processing of blood samples was carried out by Leah Prowse under the responsibility of Andrew Waller at the Animal Health Trust.

4.6 Processing of Nasal Wash Samples

[0198]Individual nasal washes were taken according to AHT SOP/EQU/02 as stated in the study protocol schedule.

[0199]A 500 μl sample of the nasal wash was added to 500 μl of Todd-Hewitt Broth in situ at the time of sampling for transportation to the lab to allow quantification of the number of β-haemolytic streptococci per ml according to AHT SOP/BACT/02. The remaining nasal wash sample was centrifuged and the supernatant decanted into a clean 5 ml polypropylene tube and stored at

-70° C. until use for quantification of mucosal antibodies.

4.7 Post Mortem Examination

[0200]Provision was made for a complete post mortem examination to be carried out by the Animal Health Trust on all ponies following euthanasia as a result of abscessation or on reaching the study end point 21 days post challenge.

[0201]Tissue samples were preserved in phosphate buffered formalin and subjected to microscopic examination according to standard techniques and provision of a full and formal report. Tissue swabs were taken and the results recorded and used to evaluate the level of S. equi infection. Charcoal swabs were taken from each of the areas as stated in the protocol and processed on COBA Streptococcal selective plates to determine the presence of S. equi.

[0202]Strangles pathology was scored using the system in Table 5.

TABLE-US-00022 TABLE 5 Pathology scoring system Pathology Score Retropharyngeal or submandibular lymph node abscess: 15 Retropharyngeal or submandibular lymph node 10 microabscess: Empyaema of guttural pouch: 5 Scarring of guttural pouch: 5 Enlarged lymph node: 1 Follicular hyperplasia of guttural pouch: 1

4.8 Histopathological Examination

[0203]Tissue samples taken from ponies at post mortem examination were fixed in formalin, cut into sections and sent to Professor Ken Smith at the Royal Veterinary College for analysis. Professor Smith prepared a report for the samples from each pony and his observations were scored according to Table 6.

TABLE-US-00023 TABLE 6 Histopathology scoring system Histopathology Score Rhinitis 1 Lymphadenitis 1 Pharyngitis 1 Lymph node abscessation 5 Guttural pouch empyema 5

Deviations

[0204]The study was performed in accordance with the study protocol no. 08.C001.P and subsequent amendments, with the following deviations from the agreed study protocol: [0205]Pony 9229 was pyrexic on Feb. 14, 2008 due to an ongoing S. zooepidemicus infection. This pony recovered over the weekend and was vaccinated on Apr. 18, 2008. [0206]Date of V2 delayed 7 days due to S. zoo infection in 45% of ponies. This had a knock on effect on V3 and challenge which were also delayed 7 days. [0207]A delay of one day occurred on sampling ponies due to staff shortages. Ponies due to be sampled on day 85 were actually sampled on day 86. [0208]20 ml of EDTA blood was taken on day 86 instead of 10 ml to enable purification of the ponies' DNA for archiving. [0209]Nordvacc decided to retain the unchallenged Pentavac group (2) for a 6-month period to monitor the duration of antibody response.

6 Fate of Ponies at the End of the Study

[0210]All ponies in groups 1 and 3 were euthanased and subjected to post mortem examination. Ponies in group 2 were retained for 6 months to monitor the duration of antibody responses.

7 Archiving

[0211]The raw data have been archived by Animal Health Trust, Lanwades Park, Kentford, Newmarket, Suffolk, CB8 7UU.

8 Summary of Results

8.1 Responses Following the First and Second Vaccinations

8.1.1 Clinical Responses

[0212]All ponies responded well to first vaccination. No injection site reactions were observed in any of the groups. However, a rise in rectal temperature was observed in the vaccinated groups (FIG. 7). This was most pronounced in the Septavac group with 4 of 7 ponies developing pyrexia (temperature>38.9° C.) one day post V1 and 5 of 7 ponies developing pyrexia one day post V2. In comparison, 2 of 7 ponies of the Pentavac group and none of the controls were pyrexic post V1, and 3 ponies of the Pentavac group and no controls were pyrexic post V2. Interestingly, only 1 Septavac, 2 Pentavac and 1 control pony developed pyrexia post V3. This could be due to the high level of antibodies induced post V2, which may have neutralized the antigens in the vaccine more effectively.

[0213]There were no obvious differences in nasal score (FIG. 8), lymph node score (FIG. 9) or S. zooepidemicus counts (FIG. 10) between the study groups during the vaccination phase, with the exception of some ponies that had ongoing S. zooepidemicus infections typical of ponies of this age. This resulted in a rise in mean rectal temperature around the original date for V2 (Apr. 3, 2008) as demonstrated in FIG. 7. Ponies were allowed to recover from this S. zooepidemicus infection and all ponies were vaccinated on Apr. 10, 2008.

8.2 Responses Following Challenge

[0214]The preparation and conduct of both challenges went extremely well and all ponies received the required dose of S. equi without incident on the May 8, 2008.

[0215]Earliest onset of pyrexia was at day 4 post challenge in control pony 2078. Two more ponies developed pyrexia on day 5, another on day 6 and 7 and the final control pony developed pyrexia on day 10 (FIG. 11). The mean number of days that control ponies were pyrexic was 4.2 days compared with 0.7 days for vaccinated ponies (Table 7). However, it should be noted that control ponies were euthanased on welfare grounds from day 8 post challenge and all control ponies had been euthanased by day 13 post challenge. This has had a knock on effect on the mean temperatures, observation scores, fibrinogen and neutrophil levels and observation scores for control ponies, which decline as ponies succumbing to S. equi infection were euthanased.

[0216]Overall, there was a significant difference in the mean temperatures of the two groups from day 5 to day 11 post challenge (FIG. 11). Of the Septavac ponies only pony 0976 developed pyrexia on day 8 (Table 7). However, this may have been due to the ongoing S. zooepidemicus infection that was evident in this pony.

[0217]Fibrinogen levels were significantly different between the two study groups on days 6, 8 and 11 post challenge (FIG. 12). All controls developed elevated fibrinogen levels, but only 2 vaccinates (ponies 0976 and 9794) had higher levels.

[0218]Neutrophil levels were also significantly different between the two study groups on days 6, 8 and 11 post challenge (FIG. 13). All controls developed elevated neutrophil levels, but only 1 vaccinate (pony 9794) had higher levels.

[0219]There was an increased level of submandibular lymph node swelling in control ponies, although this did not appear to be statistically significant (FIG. 14). There were no differences in nasal discharge (FIG. 15) or S. zooepidemicus counts (FIG. 16) between the study groups.

[0220]On post mortem examination, all controls were found to have multiple lymph node abscesses, while only one vaccinated pony, 9794, was found to have lymph node abscesses (Tables 8 and 9). Overall the mean pathology score for controls and 11.7, respectively indicating that a significant level of protection had been induced by the Septavac vaccine (FIG. 17). S. equi was isolated from the lymph nodes of all control ponies, but only 2 vaccinates (0976 and 9794) (Table 10). These findings were strengthened by histopathological examination, which confirmed that only one Septavac pony had developed abscesses in at least two of their lymph nodes (Table 11 and FIG. 18).

[0221]Furthermore, the IgG levels in nasal washings and serum samples of the septavac group were measured using ELISA (FIGS. 19 and 20) showing that the antigens generate mucosal and serum antibodies.

TABLE-US-00024 TABLE 7 Number of days pyrexic after challenge Pony Number of Group ID days Septavac 0012 0 Septavac 0159 0 Septavac 0833 0 Septavac 0976 5 Septavac 9123 0 Septavac 9668 0 Septavac 9794 0 Control 0173 2 Control 0427 4 Control 1635 5 Control 2078 4 Control 9549 4 Control 9776 5 Control 9886 6 Mean Septavac = 0.7 days Mean control = 4.2 days* *All control ponies were euthanased by day 13 post-challenge, but most would have continued to have elevated temperatures had they not been euthanased on welfare grounds.

TABLE-US-00025 TABLE 8 Post Mortem Analysis after Challenge Total PM Group Pony ID score Septavac 0012 6 Septavac 0159 3 Septavac 0833 5 Septavac 0976 6 Septavac 9123 4 Septavac 9668 1 Septavac 9794 57 Control 0173 42 Control 0427 53 Control 1635 66 Control 2078 49 Control 9549 57 Control 9776 43 Control 9886 42

TABLE-US-00026 TABLE 9 Number and Location of Abscesses on Post Morte SMLN RPLN Group Pony ID L R L R Septavac 0012 -- -- -- -- Septavac 0159 -- -- -- -- Septavac 0833 -- -- -- -- Septavac 0976 -- -- -- -- Septavac 9123 -- -- -- -- Septavac 9668 -- -- -- -- Septavac 9794 -- Control 0173 -- -- Control 0427 -- Control 1635 Control 2078 -- Control 9549 -- Control 9776 -- -- Control 9886 -- -- SMLN--Submandibular Lymph Node RPLN--Retropharyngeal Lymph Node = abscess

TABLE-US-00027 TABLE 10 S. equi Counts Found in the Lymph Nodes on Post Mortem S. equi Tracheal/ Confirmed Pony SMLN RPLN Cervical Broncheal by sugar ID L R L R LN LN test 0012 -- -- -- -- -- -- -- 0159 -- -- -- -- -- -- -- 0833 -- -- -- -- -- -- -- 0976 -- Sparse -- -- -- -- Yes 9123 -- -- -- -- -- -- -- 9668 -- -- -- -- -- -- -- 9794 -- Con Con Con -- -- Yes 0173 Sparse Sparse Con Con -- -- Yes 0427 Con Con Con Con -- -- Yes 1635 Con Con Con Con -- -- Yes 2078 Con Con Con Con Con -- Yes 9549 Con Con Con Con -- -- Yes 9776 Sparse Sparse Con Con -- -- Yes 9886 -- Sparse Con Con -- -- Yes

[0222]Ponies 0833 and 0159 showed sparse S. equi in areas other than the lymph node. Ponies 0012, 9123 and 9668 showed no S. equi.

Con--confluent

SMLN--Submandibular Lymph Node

RPLN--Retropharyngeal Lymph Node

TABLE-US-00028 [0223]TABLE 11 Histopathology Scores Pony Chip ID 0012 0159 0833 0976 9123 9668 9794 0173 0427 1635 2078 9549 9776 9886 Identity Sep Sep Sep Sep Sep Sep Sep Con Con Con Con Con Con Con Nasal 0 1 1 0 0 0 0 0 0 0 0 0 1 0 turbinate Nasopharynx 0 0 0 1 0 0 0 0 0 0 0 0 0 0 SMLN-L 0 0 0 0 0 0 0 0 5 5 0 5 0 0 SMLN-R 0 0 0 0 0 0 5 0 5 5 5 0 0 0 RPLN-L 0 0 0 1 0 0 5 5 5 5 5 5 5 5 RPLN-R 0 0 0 1 0 0 5 5 5 5 5 5 5 5 Gut pouch-L 5 5 0 0 0 0 5 5 5 5 5 5 5 5 Gut pouch-R 5 0 0 0 0 0 5 5 5 5 5 5 5 5 Lung 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Brain 0 0 0 0 0 0 0 0 0 0 0 0 0 0 TOTAL 10 6 1 3 0 0 25 20 30 30 25 25 21 20 Rhinitis; 1 Guttural pouch empyaema: 5 Pharyngitis: 1 Lymphadenitis: 1 Sep = Septavac Lymph node abscessation: 5 Con = Control

9. Pentavac A Vaccination Study

[0224]In the second trial the seven horses of group 2 (section 3.2, table 3) where after vaccination V3 (Table 4) kept at pasture on grass and blood samples where taken regularly to measure IgG antibody titers in ELISA against the five antigens present in the Pentavac A formulation (FIG. 21). In Day 270 (Nov. 6, 2008) a booster dose of Pentavac A was given according to the procedure described in section 4.1. Before challenge the group was transferred to ACVS and fourteen days post booster the group was experimentally challenged with S. equi 4047 as described in section 4.2 and monitored essentially as described in section 4.3.

9.1 Brief Summary of the Pentavac A Vaccination Study

[0225]The Pentavac A study revealed that after vaccination a significant antibody response against the individual antigens remains for at least six months (FIG. 21).

The Pentavac A vaccine delayed the onset of infection upon challenge with S. equi and that one of the ponies in the group did not developed strangles.

Further Applications

[0226]One implication of the present invention is that enzymes degrading immunoglobulins can be used as antigens in a vaccine to protect the target animal from infection. Therefore one embodiment of the present invention is that concerning the human pathogenic group A streptococci (GAS) it is possible to construct a vaccine composition which protects humans from infections caused by this bacterium. In strains of GAS there are several reported extracellular immunoglobulin degrading proteins (called Sib35, IdeS or Mac-proteins) which share amino acid sequence homologies to IdeE and IdeE2 and therefore in light of the present invention can be purified and used as antigens in a vaccine separately or in combination with other purified extracellular proteins (like M-proteins or M-like proteins or fragments thereof) from group A strains. As in the present invention another implication is that the invention can be used to develop specific antisera, polyclonal or monoclonal antibodies to be used for diagnostic purposes or to be used in passive immunisations of the target animal including humans.

REFERENCES

[0227]1. Courtney, H. S., Y. Li, J. B. Dale, and D. L. Hasty. 1994. Cloning, sequencing and expression of a fibronectin/fibrinogen-binding protein from group A streptococci. Infect. Immun. 62:3937-3946. [0228]2. Cue, D., P. E. Dombek, H. Lam, and P. P. Cleary. 1998. Streptococcus pyogenes serotype M1 encodes multiple pathways for entry into human epithelial cells. Infect. Immun. 66:4593-4601. [0229]3. Barnham, M., A. Ljunggren, and M. McIntyre. 1987. Human infection with Streptococcus zooepidemicus (Lancefield group C): three case reports. Epidem. Inf. 98: 183-190. [0230]4. Galan, J. E., and J. F. Timoney. 1988. Immunologic and geneticcomparison of Streptococcus equi isolates from the United States and Europe. J. Clin. Microbiol. 26:1142-1146. [0231]5. Flock, M., Jacobsson, K., Frykberg, L., Hirst, T., R., Franklin, A., Guss, B. and Flock, J.-I. (2004) Infect Immun 72:3228-3236. [0232]6. Engvall, E., E. Ruoslahti, and J. M. Miller. 1978. Affinity of fibronectin to collagen of different genetic types and to fibronogen. J. Exp. Med. 147:1584-1595. [0233]7. Patti, J. M., Jonsson, H., Guss, B., Switalski, L. M., Wiberg, K., Lindberg, M., and Hoook, M. (1992) Molecular characterization and expression of a gene encoding a Staphylococcus aureus collagen adhesin. J. Biol. Chem. 267:4766-4772. [0234]8. Jonsson, H., Lindmark, H., and Guss. B. (1995) A protein G related cell surface protein in Streptococcus zooepidemicus. Infect Immun 63:2968-2975. [0235]9. Lindmark, H., Jacobsson, K., Frykberg, L., and Guss, B. (1996) Fibronectin-binding protein of Streptococcus equi subspecies zooepidemicus. Infect Immun 64:3993-3999. [0236]10. Jacobsson, K., Jonsson, H., Lindmark, H., Guss, B., Lindberg, M., and Frykberg. L. (1997) Shot-gun phage display mapping of two streptococcal cell-surface proteins. Microbiol. Res. 152:1-8. [0237]11. Lindmark, H., Jonsson, P., Olsson-Engvall, E., and Guss, B. (1999) Pulsed-field gel electrophoresis and distribution of the genes zag and fnz in isolates of Streptococcus equi. Res Vet Sci. 66:93-99. [0238]12. Lindmark, H., and Guss, B. (1999) SFS, a novel fibronectin-binding protein from Streptococcus equi, inhibits the binding between fibronectin and collagen. Infect. Immun. 67: 2383-2388. [0239]13. Lindmark, H. (1999) Characterization of adhesive extracellular proteins from Streptococcus equi. (Doctoral thesis) Acta Universitatis Agriculturae Sueciae, Agraria 139. ISBN 91-576-5488-3. [0240]14. Lindmark, H., Nilsson, M., and Guss, B. (2001) Comparison of the fibronectin-binding protein FNE from Streptococcus equi subspecies equi with FNZ from S. equi subspecies zooepidemicus reveals a major and conserved difference. 69: 3159-3163. [0241]15. Schneewind, O., Fowler, A. and Faull, K. F. (1995) Structure of the cell wall anchor of surface proteins in Staphylococcus aureus. Science 268:103-106. [0242]16. Anton Mayr et al. Handbuch der Shutzimpfungen in der Tiermedizin.3.3.4. p. 196-200. Verlag Paul Parey. Berlin and Hamburg. 11984 [0243]17. Elson, C. O., and M. T. Dertzbaugh. 1999. p. 817-838, Nucosal immunology, 2^nd ed. Of Academic Press, New York, N.Y. [0244]18. Winner et al. 1991. Infect. Immun. 59:997-982 [0245]19. Ogra et al. Clinical Microbiology Reviews, April 2002, p. 430-445. [0246]20. B. Morein and Karin Lovgren Bengtsson 1998, 76:295-299. Immunology and Cellbiology [0247]21. Hanski, E., and M. G. Caparon. 1992. Protein F, a fibonectin-binding protein, is an adhesin of the group A streptococcus Streptococcus pyogenes. Proc. Natl. Acad. Sci. USA 89:6172-6176. [0248]22. Hanski, E., P. A. Horwitz, and M. G. Caparon. 1992. Expression of protein F, the Fibronectin-binding protein of Streptococcus pyogenes JRS4, in heterologous streptococcal and enterococcal strains promotes their adherence to respiratory epithelial cells. Infect. Immun. 60:5119-5125. [0249]23. Jadoun, J., V. Ozeri, E. Burstein, E. Skuteisky, E. Hanski, and S. Sela. 1998. Protein F1 required for efficient entry of Streptococcus pyogenes into epithelial cells. J. Infect. Dis. 178:147-158. [0250]24. Molinri, G., S. R. Talay, P. Valentin-Weigand, M. Rohde, and G. S. Chhatwal. 1997. The fibronectin-binding protein of Streptococcus pyogenes Sfbl, is involved in the internalization of group A streptococci by epithelial cells. Infect. Immun. 65:1357-1363.

[0251]25. Lannergard, J., Frykberg, L. and Guss B. (2003) CNE, a collagen-binding protein of Streptrococcus equi. FEMS Microbiol. Lett. 222:69-74. [0252]26. Lannergard, J. and Guss, B. (2006) FEMS Microbiol Lett 262: 230-235. [0253]27. Waller, A., Flock, M., Smith, K., Robinson, C., Mitchell, Z., Karlstrom, Å., Lannergard, J., Bergman, R., Guss, B. and Flock, J.-I. (2007) Vaccine 25: 3629-3635. [0254]28. WO 92/07002 [0255]29. WO 00/37496 [0256]30. WO 2007/115059

Sequence CWU 1

321385PRTStreptococcus equi 1Met Met Lys Lys Gln Ser Phe Thr His Ser Arg Lys Pro Lys Phe Gly1 5 10 15Met Arg Lys Leu Ser Ile Gly Leu Ala Ser Cys Met Leu Gly Met Met 20 25 30Phe Leu Thr Thr Gly His Val Ser Gly Glu Val Val Glu Val Trp Pro 35 40 45Asn Gly Gln Asn Pro Asn Gly Lys Ile Glu Ile Leu Ser Gln Thr Glu 50 55 60His Ser Glu His Leu Gln Lys Leu Arg Asp Ile Glu Asp Phe Gln Ala65 70 75 80Gln Lys Gln Ala Asp His Val Arg Tyr Thr Lys Trp Leu Asp Gly Val 85 90 95Thr Val Asp Glu His Glu Phe Arg Lys Ile Lys Glu Tyr Asp Thr Glu 100 105 110Tyr Tyr Val Thr Pro Leu Leu Ser Gly Lys Gly Tyr Tyr Asp Ile Asn 115 120 125Lys Asp Phe Asn Gln Asp Ser Asp Lys Cys Ala Ala Ala Val Ala Ala 130 135 140Asn Met Phe His Tyr Trp Phe Asp Arg Asn Arg Asp Ser Ile Asn Arg145 150 155 160Phe Leu Ser Gln Ser Pro Gly Glu Asn Gly Val Ile Lys Leu Glu Asn 165 170 175Glu Lys Thr Ile Glu Val Ser Lys Phe Leu Glu Thr Tyr Arg Ser Asp 180 185 190Gly Asp Tyr Leu Asp Lys Ser Pro Phe Phe Asp Leu Ile Ser Asn Ser 195 200 205Phe Lys Gly Pro Val Trp Ala Asn Lys Leu Leu Asp Ala Tyr Ile Asn 210 215 220Gly Tyr Gly Tyr Ile His Lys Phe Ala Lys Asn Thr Pro His Ser Lys225 230 235 240Asn Asn Asn Ser Lys Phe Asn Phe Phe Lys Lys Val Phe Asp Gly Asn 245 250 255Leu Leu Thr Asp Ile His Gln Ile Phe Asp Tyr Asn Thr Phe Ser Asp 260 265 270Lys Leu Ser Glu Ala Leu Tyr Thr Gly Lys Ala Ile Gly Leu Ala Tyr 275 280 285Gly Pro Gly Asp Leu Arg Arg Ser Leu Gly His Ile Ile Ser Val Trp 290 295 300Gly Ala Asp Leu Asp Asp Gln Asn Arg Val Val Ala Ile Tyr Val Thr305 310 315 320Asp Ser Asp Asp Lys Lys Leu Thr Ile Gly Asn Glu Arg Val Gly Leu 325 330 335Lys Arg Tyr Lys Val Ser Ser Asp Asp Gln Gly Arg Ala Arg Leu Thr 340 345 350Thr Arg Asp Lys Asp Asn Thr Gly Gly Glu Ile Arg Ser Ile Glu Thr 355 360 365Leu Asp Met Gly Thr Gln Glu Trp Ala Asp Tyr Phe Asn Lys Thr Glu 370 375 380Lys3852349PRTArtificial SequenceChemically synthesized Ide E2 protein having one N-terminal and four C-terminal amino acids originating from the pTYB4 vector 2Met Glu Val Val Glu Val Trp Pro Asn Gly Gln Asn Pro Asn Gly Lys1 5 10 15Ile Glu Ile Leu Ser Gln Thr Glu His Ser Glu His Leu Gln Lys Leu 20 25 30Arg Asp Ile Glu Asp Phe Gln Ala Gln Lys Gln Ala Asp His Val Arg 35 40 45Tyr Thr Lys Trp Leu Asp Gly Val Thr Val Asp Glu His Glu Phe Arg 50 55 60Lys Ile Lys Glu Tyr Asp Thr Glu Tyr Tyr Val Thr Pro Leu Leu Ser65 70 75 80Gly Lys Gly Tyr Tyr Asp Ile Asn Lys Asp Phe Asn Gln Asp Ser Asp 85 90 95Lys Cys Ala Ala Ala Val Ala Ala Asn Met Phe His Tyr Trp Phe Asp 100 105 110Arg Asn Arg Asp Ser Ile Asn Arg Phe Leu Ser Gln Ser Pro Gly Glu 115 120 125Asn Gly Val Ile Lys Leu Glu Asn Glu Lys Thr Ile Glu Val Ser Lys 130 135 140Phe Leu Glu Thr Tyr Arg Ser Asp Gly Asp Tyr Leu Asp Lys Ser Pro145 150 155 160Phe Phe Asp Leu Ile Ser Asn Ser Phe Lys Gly Pro Val Trp Ala Asn 165 170 175Lys Leu Leu Asp Ala Tyr Ile Asn Gly Tyr Gly Tyr Ile His Lys Phe 180 185 190Ala Lys Asn Thr Pro His Ser Lys Asn Asn Asn Ser Lys Phe Asn Phe 195 200 205Phe Lys Lys Val Phe Asp Gly Asn Leu Leu Thr Asp Ile His Gln Ile 210 215 220Phe Asp Tyr Asn Thr Phe Ser Asp Lys Leu Ser Glu Ala Leu Tyr Thr225 230 235 240Gly Lys Ala Ile Gly Leu Ala Tyr Gly Pro Gly Asp Leu Arg Arg Ser 245 250 255Leu Gly His Ile Ile Ser Val Trp Gly Ala Asp Leu Asp Asp Gln Asn 260 265 270Arg Val Val Ala Ile Tyr Val Thr Asp Ser Asp Asp Lys Lys Leu Thr 275 280 285Ile Gly Asn Glu Arg Val Gly Leu Lys Arg Tyr Lys Val Ser Ser Asp 290 295 300Asp Gln Gly Arg Ala Arg Leu Thr Thr Arg Asp Lys Asp Asn Thr Gly305 310 315 320Gly Glu Ile Arg Ser Ile Glu Thr Leu Asp Met Gly Thr Gln Glu Trp 325 330 335Ala Asp Tyr Phe Asn Lys Thr Glu Lys Leu Glu Pro Gly 340 3453608PRTStreptococcus equi 3Met Lys Lys Phe Thr Lys Arg Cys Leu Lys Gly Cys Gly Leu Val Gly1 5 10 15Leu Val Phe Ser Thr Gly Leu Val Ala Leu Ser Asp Asn Ile Asp Ser 20 25 30Ala Leu Thr Val Gly Ala Glu Thr Thr Thr Ala Ser Ala Phe Glu Asn 35 40 45Asn Gly Thr Gly Gln His Leu Asn Trp His Ile Asp Ile Pro Gln Glu 50 55 60Tyr Thr Val Glu Leu Gly Glu Pro Ile Thr Ile Ser Asp Leu Met Ser65 70 75 80Gln Ile Thr Val Thr Arg Lys Gly Ser Asn Gly Thr Val Asn Asp Gly 85 90 95Asp Thr Phe Asp Phe Ile Ser Asn Gly Asp Gly Ser Arg Gly Ile Asp 100 105 110Thr Pro Gly Val Lys Ile Trp Phe Asp Phe Tyr Asn Ala Ala Gly Thr 115 120 125Ser Phe Leu Thr Asp Glu Met Leu Ala Ser Pro Thr Tyr Ala Val Pro 130 135 140Gly Gly Ser Tyr Thr Ile Lys Ala Trp Val Phe Tyr Gly Lys Asn Asp145 150 155 160Thr Lys Lys Leu Phe Thr Phe Lys Leu Lys Asn Ser Asn Ser Asn Lys 165 170 175Thr Glu Leu Arg Lys Ser Leu Glu Glu Ala Lys Leu Lys Leu Ser Gln 180 185 190Pro Glu Gly Thr Tyr Ser Asp Glu Ser Leu Gln Ala Leu Gln Ser Ala 195 200 205Val Thr Leu Gly Lys Thr Tyr Leu Asn Ser Asp Pro Asp Gln Asn Thr 210 215 220Val Asp Gln Ser Val Thr Thr Ile Asp Ser Ala Ile Thr Ser Leu Val225 230 235 240Asn Leu Asn Ala Leu Asn Glu Ala Ile Asn Gln Ala Thr Pro Phe Ile 245 250 255Thr Asp Gly Lys Glu Tyr Pro Lys Glu Ala Tyr Asp Gly Leu Val Gln 260 265 270Lys Leu Ala Ala Ala Ala Lys Leu Gln Asn Ser Phe Gly Pro Ser Gln 275 280 285Gly Asp Val Asp Lys Ala Ala Thr Asp Leu Thr Gln Ala Leu Thr Thr 290 295 300Leu Lys Thr Ala Val Ala His Glu Ala Leu Asp Gln Ala Leu Ala Lys305 310 315 320Leu Leu Glu Leu Tyr Arg Glu Asn Pro Asn Leu Ala Leu Thr Ser Glu 325 330 335Ser Leu Lys Glu Leu Tyr Asn Lys Ala Ile Glu Ala Ala Gly Thr Phe 340 345 350Tyr Arg Thr Val Asn Lys Asp Lys Glu Arg Lys Asp Ile Ser Leu Tyr 355 360 365Glu Leu Glu Arg Tyr Thr Thr Glu Thr Asn Ser Val Val Asp Thr Ile 370 375 380Leu Lys Val Lys Ala Ala Ile Ala Glu Glu Gly Lys Ala Lys Leu Arg385 390 395 400Ser Ala Leu Asp Gln Leu Asn Ala Leu Ile Gly Glu Asn Leu Asp Leu 405 410 415Ser Pro Tyr Thr Ala Ala Ser Ala Gln Ala Tyr Thr Asp Gln Leu Ala 420 425 430Lys Ala Lys Glu Val Ala Ala Ala Gly Glu Thr Ala Tyr Ala Gln Glu 435 440 445Thr Glu Pro Thr Ala Ile Thr Asn Ser Leu Val Lys Val Leu Asn Ala 450 455 460Lys Lys Ser Leu Ser Asp Ala Lys Ala Ala Leu Val Ala Lys Pro Val465 470 475 480Asp Pro Val Asp Pro Val Asp Pro Val Asp Pro Val Asp Pro Val Asp 485 490 495Pro Val Asp Pro Val Asp Pro Val Asp Pro Val Asp Pro Val Asp Pro 500 505 510Val Asp Pro Val Asp Pro Val Asp Pro Val Asp Pro Val Asp Pro Val 515 520 525Asp Pro Val Asp Pro Ile Asp Pro Ala Asp Pro Val Lys Pro Ser Asp 530 535 540Pro Glu Val Lys Pro Glu Pro Lys Pro Glu Ser Lys Pro Glu Ala Lys545 550 555 560Lys Glu Asp Lys Lys Ala Ala Asp Lys Gln Gln Val Leu Pro Ala Thr 565 570 575Ala Asp Thr Ala Asn Pro Phe Phe Thr Ala Ala Ala Leu Ala Val Ile 580 585 590Ala Cys Ala Gly Gln Leu Ala Ile Val Ser Arg Arg Lys Glu Ser Asn 595 600 6054446PRTArtificial SequenceChemically synthesized Eq5 protein having one N-terminal and four C-terminal amino acids originating from the pTYB4 vector 4Met Glu Thr Thr Thr Ala Ser Ala Phe Glu Asn Asn Gly Thr Gly Gln1 5 10 15His Leu Asn Trp His Ile Asp Ile Pro Gln Glu Tyr Thr Val Glu Leu 20 25 30Gly Glu Pro Ile Thr Ile Ser Asp Leu Met Ser Gln Ile Thr Val Thr 35 40 45Arg Lys Gly Ser Asn Gly Thr Val Asn Asp Gly Asp Thr Phe Asp Phe 50 55 60Ile Ser Asn Gly Asp Gly Ser Arg Gly Ile Asp Thr Pro Gly Val Lys65 70 75 80Ile Trp Phe Asp Phe Tyr Asn Ala Ala Gly Thr Ser Phe Leu Thr Asp 85 90 95Glu Met Leu Ala Ser Pro Thr Tyr Ala Val Pro Gly Gly Ser Tyr Thr 100 105 110Ile Lys Ala Trp Val Phe Tyr Gly Lys Asn Asp Thr Lys Lys Leu Phe 115 120 125Thr Phe Lys Leu Lys Asn Ser Asn Ser Asn Lys Thr Glu Leu Arg Lys 130 135 140Ser Leu Glu Glu Ala Lys Leu Lys Leu Ser Gln Pro Glu Gly Thr Tyr145 150 155 160Ser Asp Glu Ser Leu Gln Ala Leu Gln Ser Ala Val Thr Leu Gly Lys 165 170 175Thr Tyr Leu Asn Ser Asp Pro Asp Gln Asn Thr Val Asp Gln Ser Val 180 185 190Thr Thr Ile Asp Ser Ala Ile Thr Ser Leu Val Asn Leu Asn Ala Leu 195 200 205Asn Glu Ala Ile Asn Gln Ala Thr Pro Phe Ile Thr Asp Gly Lys Glu 210 215 220Tyr Pro Lys Glu Ala Tyr Asp Gly Leu Val Gln Lys Leu Ala Ala Ala225 230 235 240Ala Lys Leu Gln Asn Ser Phe Gly Pro Ser Gln Gly Asp Val Asp Lys 245 250 255Ala Ala Thr Asp Leu Thr Gln Ala Leu Thr Thr Leu Lys Thr Ala Val 260 265 270Ala His Glu Ala Leu Asp Gln Ala Leu Ala Lys Leu Leu Glu Leu Tyr 275 280 285Arg Glu Asn Pro Asn Leu Ala Leu Thr Ser Glu Ser Leu Lys Glu Leu 290 295 300Tyr Asn Lys Ala Ile Glu Ala Ala Gly Thr Phe Tyr Arg Thr Val Asn305 310 315 320Lys Asp Lys Glu Arg Lys Asp Ile Ser Leu Tyr Glu Leu Glu Arg Tyr 325 330 335Thr Thr Glu Thr Asn Ser Val Val Asp Thr Ile Leu Lys Val Lys Ala 340 345 350Ala Ile Ala Glu Glu Gly Lys Ala Lys Leu Arg Ser Ala Leu Asp Gln 355 360 365Leu Asn Ala Leu Ile Gly Glu Asn Leu Asp Leu Ser Pro Tyr Thr Ala 370 375 380Ala Ser Ala Gln Ala Tyr Thr Asp Gln Leu Ala Lys Ala Lys Glu Val385 390 395 400Ala Ala Ala Gly Glu Thr Ala Tyr Ala Gln Glu Thr Glu Pro Thr Ala 405 410 415Ile Thr Asn Ser Leu Val Lys Val Leu Asn Ala Lys Lys Ser Leu Ser 420 425 430Asp Ala Lys Ala Ala Leu Val Ala Lys Pro Leu Glu Pro Gly 435 440 4455373PRTStreptococcus equi 5Met Asn Lys Lys Ser Ala Arg Arg Arg Arg Lys Asn Leu Ile Thr Lys1 5 10 15Leu Ala Met Thr Ser Ala Leu Thr Leu Gly Val Gly Ala Ala Thr Thr 20 25 30Leu Ala Gly Gln Thr Glu Val Arg Ala Asp Asn Ile Leu Arg Leu Asp 35 40 45Met Thr Asp Lys Glu Ala Val Glu Lys Phe Ala Asn Glu Leu Lys Asn 50 55 60Glu Val His Lys Asn Tyr Arg Gly Ser Asn Thr Trp Gln Lys Leu Thr65 70 75 80Leu Ile Leu Asn Gly Tyr Gln Asn Leu Arg Glu Gln Ile Glu Thr Glu 85 90 95Leu Lys Asn Ser Glu Gln Lys Val Lys Glu Leu Asn Asp Lys Val Asn 100 105 110Ser Glu Thr Gln Gly Lys Gln Glu Leu Gln Asn Gln Leu Glu Lys Glu 115 120 125Lys Glu Glu Leu Glu Thr Leu Lys Lys Glu Leu Glu Ala Glu Lys Ala 130 135 140Lys Gly Thr Gly Glu Thr Glu Lys Leu Gln Lys Glu Ile Glu Ala Lys145 150 155 160Asn Ala Met Ile Ser Asp Leu Gln Lys Gln Leu Glu Glu Thr Lys Gln 165 170 175Arg Val Gln Glu Phe Glu Ala Glu Val Gly Lys Leu Met Ala Glu Lys 180 185 190Ala Asp Leu Gln Thr Lys Leu Asn Glu Gln Glu Gln Leu Asn Ala Lys 195 200 205Leu Gln Lys Glu Ile Glu Asp Leu Lys Ala Gln Ile Glu Lys Leu Lys 210 215 220His Cys Gln Asp Thr Pro Lys Pro Glu Pro Lys Pro Glu Pro Lys Pro225 230 235 240Glu Pro Lys Pro Glu Pro Lys Pro Glu Pro Lys Pro Glu Pro Lys Pro 245 250 255Glu Pro Lys Pro Glu Pro Lys Pro Gly Pro Lys Pro Glu Pro Lys Pro 260 265 270Glu Pro Lys Pro Gly Pro Lys Pro Glu Pro Lys Pro Glu Pro Lys Pro 275 280 285Gly Pro Lys Pro Gly Pro Lys Pro Glu Pro Lys Pro Gly Pro Lys Pro 290 295 300Glu Pro Lys Pro Glu Pro Lys Pro Glu Pro Lys Pro Glu Ala Lys Lys305 310 315 320Pro Glu Gln Pro Lys Pro Met Thr Lys Pro Gly Ala Lys Lys Pro Glu 325 330 335Gln Ser Leu Pro Ser Thr Gly Asp Ile Arg Asn Pro Phe Phe Thr Pro 340 345 350Ala Ala Ile Ala Ile Met Ile Ala Ala Gly Thr Ile Ala Ile Pro Lys 355 360 365Arg Lys Glu Glu Asp 3706201PRTArtificial SequenceChemically synthesized Eq5 protein having one N-terminal and four C-terminal amino acids originating from the pTYB4 vector 6Met Ala Thr Thr Leu Ala Gly Gln Thr Glu Val Arg Ala Asp Asn Ile1 5 10 15Leu Arg Leu Asp Met Thr Asp Lys Glu Ala Val Glu Lys Phe Ala Asn 20 25 30Glu Leu Lys Asn Glu Val His Lys Asn Tyr Arg Gly Ser Asn Thr Trp 35 40 45Gln Lys Leu Thr Leu Ile Leu Asn Gly Tyr Gln Asn Leu Arg Glu Gln 50 55 60Ile Glu Thr Glu Leu Lys Asn Ser Glu Gln Lys Val Lys Glu Leu Asn65 70 75 80Asp Lys Val Asn Ser Glu Thr Gln Gly Lys Gln Glu Leu Gln Asn Gln 85 90 95Leu Glu Lys Glu Lys Glu Glu Leu Glu Thr Leu Lys Lys Glu Leu Glu 100 105 110Ala Glu Lys Ala Lys Gly Thr Gly Glu Thr Glu Lys Leu Gln Lys Glu 115 120 125Ile Glu Ala Lys Asn Ala Met Ile Ser Asp Leu Gln Lys Gln Leu Glu 130 135 140Glu Thr Lys Gln Arg Val Gln Glu Phe Glu Ala Glu Val Gly Lys Leu145 150 155 160Met Ala Glu Lys Ala Asp Leu Gln Thr Lys Leu Asn Glu Gln Glu Gln 165 170 175Leu Asn Ala Lys Leu Gln Lys Glu Ile Glu Asp Leu Lys Ala Gln Ile 180 185 190Glu Lys Leu Lys His Leu Glu Pro Gly 195 2007392PRTStreptococcus zooepidemicus 7Met Met Lys Lys Gln Ser Phe Thr His Ser Arg Lys Pro Lys Phe Gly1 5 10 15Met Arg Lys Leu Ser Ile Gly Leu Ala Ser Cys Met Leu Gly Met Met 20 25 30Phe Leu

Thr Thr Ser His Val Ser Gly Glu Val Val Glu Val Trp Pro 35 40 45Tyr Gly Gln Asp Pro Asn Asp Lys Ile Glu Val Leu Ser Gln Ser Glu 50 55 60Tyr Ser Glu Tyr Leu Gln Arg Leu His Asp Val Glu Asp Phe Gln Ala65 70 75 80Glu Lys Lys Lys Glu Gly Val Val Arg Thr Gln Trp Leu Glu Gly Val 85 90 95Asn Val Thr Asp His Asp Phe Arg Lys Ile Thr Asp Gly Gly Ser Val 100 105 110Tyr Tyr Ala Thr Pro Leu Leu Asn Asp Arg Gly Tyr Tyr Asp Ile Asn 115 120 125Lys Asn Phe Asn Gln Asp Ser Asp Lys Cys Ala Ala Ala Val Ala Val 130 135 140Asn Met Phe His Tyr Trp Leu Asp Arg Asn Lys Asp Asn Val Ala Lys145 150 155 160Phe Leu Ser Gln Ser Pro Asp His Gly Phe Val Glu Gly Glu Pro Thr 165 170 175Phe Asn Leu Val Asp Phe Gln Tyr Thr Tyr Ala Ser Pro Tyr Glu Glu 180 185 190Gly Gly Tyr Arg Asp Asn Ser Lys Leu Phe Asp Phe Ile Ser Lys Ala 195 200 205Phe Asn Lys Pro Leu Trp Ala Asn Lys Leu Leu Asp Ala Tyr Ile Asn 210 215 220Gly Tyr Gly Tyr Ile Asp Arg Tyr Val Lys Asn Thr Pro His Ser Gly225 230 235 240Gln Asn Asn Ser Lys Phe Asn Phe Phe Lys Lys Val Phe Asp Gly Lys 245 250 255Leu Leu Thr Asp Ile Gln Gln Ile Phe Asp Tyr Tyr Thr Leu Ser Ser 260 265 270Glu Leu Arg Glu Ala Leu Asp Thr Gly Lys Ala Ile Gly Leu Ala Tyr 275 280 285Gly Pro Gly Asp Leu Arg Arg Ser Leu Gly His Ile Ile Ser Val Trp 290 295 300Gly Ala Asp Ile Asn Glu Asp Gly Asn Val Val Ala Ile Tyr Val Thr305 310 315 320Asp Ser Asp Asp Lys Lys Leu Thr Ile Gly Asn Lys Lys Asp Arg Ile 325 330 335Gly Leu Lys Arg Tyr Lys Leu Tyr Ser Asp Asn Val Gly Arg Ala Arg 340 345 350Leu Thr Ala Tyr Ala Thr Glu Asn Gln Gln Thr Gly Gly Glu Val Arg 355 360 365Gly Ile Glu Thr Leu Asp Met Ala Thr Gln Asp Trp Ala Asp Tyr Phe 370 375 380Ser Arg Thr Asp Glu Ala Glu Gln385 3908570PRTStreptococcus zooepidemicus 8Met Lys Lys Phe Thr Lys Arg Cys Leu Lys Gly Cys Gly Leu Val Gly1 5 10 15Leu Val Phe Ser Thr Gly Leu Val Ala Leu Ser Asp Asn Ile Asp Ser 20 25 30Ala Leu Thr Val Gly Ala Glu Thr Ala Thr Thr Ala Asn Ala Phe Glu 35 40 45Glu Ser Gly Asp Gln Gln His Lys Asn Trp His Ile Tyr Ile Pro Glu 50 55 60Val Tyr Thr Val Lys Val Gly Gln Pro Ile Thr Ile Glu Asp Ile Leu65 70 75 80Ser Gln Ile Thr Ile Thr Arg Lys Gly Glu Asp Ser Gln Gly Lys Thr 85 90 95Ser Pro Gly Met Ile Tyr Thr Tyr Glu Glu Tyr Pro Lys Val Arg Gly 100 105 110Ile Glu Val Ser Ala Gly Thr Ile Trp Phe Asp Phe Tyr Asn Ser Gly 115 120 125Asn Trp Val Asn Asn Asp Val Leu Ala Thr Phe Asn Glu Pro Gly Gly 130 135 140Thr Tyr Thr Leu Ser Ala Trp Ala Tyr Tyr Ala Asn Glu Asn Val Lys145 150 155 160Lys Gln Phe Val Phe Lys Leu Gln Val Glu Asn Ser Asp Lys Arg Ala 165 170 175Leu Glu Gln Ser Leu Ala Thr Ala Asn Glu Lys Leu Gln Ala Pro Glu 180 185 190Gly Thr Tyr Ser Asp Glu Ser Leu Gln Arg Leu Gln Glu Ser Val Phe 195 200 205Leu Gly Gln Thr Tyr Leu Asn Arg Asp Pro Glu Gln Gln Glu Val Asp 210 215 220Asp Met Lys Ala Thr Ile Asp Ser Ala Val Ser Gly Leu Val Asp Leu225 230 235 240Thr Val Leu Asn Thr Ala Val Glu Thr Ala Thr Pro Leu Leu Thr Asp 245 250 255Gly Lys Glu Tyr Pro Lys Glu Ala Tyr Asp Ser Leu Val Gln Lys Leu 260 265 270Ala Ala Ala Ala Lys Leu Gln Asn Ser Phe Asn Pro Ser Gln Glu Glu 275 280 285Val Asn Glu Ala Ala Thr Asp Leu Thr Gln Ala Leu Thr Thr Leu Lys 290 295 300Thr Ala Val Ala His Glu Ala Leu Asp Gln Ala Leu Ala Lys Leu Leu305 310 315 320Glu Leu Tyr Arg Glu Asn Pro Asn Leu Ala Leu Thr Ser Glu Pro Leu 325 330 335Lys Glu Leu Tyr Asn Lys Ala Ile Glu Ala Ala Gly Thr Phe Tyr Arg 340 345 350Thr Val Ser Lys Asp Lys Glu Arg Lys Gly Ile Ser Leu Tyr Glu Leu 355 360 365Glu Arg Tyr Thr Thr Glu Thr Asn Ser Val Val Asp Thr Ile Leu Lys 370 375 380Val Lys Ala Ala Ile Ala Glu Glu Gly Lys Ala Lys Leu Arg Ser Ala385 390 395 400Leu Asp Gln Leu Asn Ala Leu Ile Gly Glu Asn Leu Asp Leu Ser Pro 405 410 415Tyr Thr Ala Ala Ser Ala Gln Ala Tyr Thr Asp Gln Leu Ala Lys Ala 420 425 430Lys Glu Val Ala Ala Ala Gly Glu Thr Ala Tyr Ala Gln Glu Thr Glu 435 440 445Pro Thr Ala Ile Thr Asn Ser Leu Ile Lys Val Leu Asn Ala Lys Lys 450 455 460Ser Leu Ser Asp Ala Lys Ala Ala Leu Val Ala Lys Pro Val Asp Pro465 470 475 480Val Asp Pro Val Asp Pro Val Asp Pro Val Asp Pro Val Asp Pro Ile 485 490 495Asp Pro Val Asp Pro Val Lys Pro Val Asp Pro Glu Val Lys Pro Glu 500 505 510Pro Lys Pro Glu Ser Lys Pro Glu Ala Lys Lys Glu Asp Lys Lys Ala 515 520 525Ala Asp Lys Gln Gln Val Leu Pro Ala Thr Ala Asp Thr Ala Asn Pro 530 535 540Phe Phe Thr Ala Ala Ala Leu Ala Val Ile Ala Cys Ala Gly Gln Leu545 550 555 560Ala Ile Val Ser Arg Arg Lys Glu Ser Asn 565 5709411PRTStreptococcus zooepidemicus 9Met Asn Lys Lys Ser Ala Arg Arg Lys Arg Lys Asp Leu Ile Thr Lys1 5 10 15Leu Ala Met Thr Ser Ala Leu Thr Leu Gly Val Gly Ala Ala Ala Thr 20 25 30Ile Ala Gly Gln Thr Glu Val Arg Ala Glu Val Leu Thr Leu Asn Met 35 40 45Lys Asp Lys Ala Lys Val Glu Glu Phe Ala Asn Lys Leu Lys Asp Tyr 50 55 60Ala Lys Gln Lys Lys Ser Gly Gln Ile Thr Leu Gln Glu Leu Ser Leu65 70 75 80Ile Leu Asp Gly Tyr Arg Asn Ile Arg Glu Gln Ile Glu Gln Asp Leu 85 90 95Ala Thr Thr Glu Lys Thr Lys Asn Phe Tyr Gly Glu Gln Leu Ile Leu 100 105 110Thr Asp Lys Leu Tyr Gln Ser Glu Lys Glu Lys Lys Glu Lys Leu Glu 115 120 125Ala Glu Leu Gln Leu Ser Gln Gln Lys Ile His Asp Leu Asp Glu Lys 130 135 140His Gln Lys Glu Lys Leu Glu Leu Gln Glu Gln Leu Glu Ala Ser Asn145 150 155 160Gln Lys Ile Lys Glu Leu Glu Met Ala Lys Ser Thr Ala Glu Ala Glu 165 170 175Ile Asn Arg Leu Thr Ala Glu Lys Asn Gly Leu Gln Glu Lys Leu Asn 180 185 190Asn Gln Glu Lys Leu Asn Ala Glu Leu Gln Ala Lys Leu Ala Lys Gln 195 200 205Glu Glu Leu Asn Ala Lys Leu Gln Lys Glu Ile Asp Glu Leu Asn Ala 210 215 220Gln Leu Glu Lys Leu Lys His Cys Gln Asp Thr Pro Lys Pro Glu Pro225 230 235 240Lys Pro Glu Pro Lys Pro Glu Pro Lys Pro Glu Pro Lys Pro Glu Pro 245 250 255Lys Pro Glu Pro Lys Pro Glu Pro Lys Pro Glu Pro Lys Pro Glu Pro 260 265 270Lys Pro Glu Pro Lys Pro Glu Pro Lys Pro Glu Pro Lys Pro Glu Pro 275 280 285Lys Pro Glu Pro Lys Pro Glu Pro Lys Pro Glu Pro Lys Pro Glu Pro 290 295 300Lys Pro Glu Pro Lys Pro Glu Pro Lys Pro Glu Pro Lys Pro Glu Pro305 310 315 320Lys Pro Glu Pro Lys Pro Glu Pro Lys Pro Glu Pro Lys Pro Glu Pro 325 330 335Lys Pro Glu Pro Lys Pro Glu Pro Lys Pro Glu Pro Lys Pro Glu Pro 340 345 350Lys Pro Glu Ala Lys Lys Pro Glu Gln Pro Lys Pro Met Thr Lys Pro 355 360 365Gly Ala Lys Lys Pro Glu Gln Ser Leu Pro Ser Thr Gly Asp Ile Arg 370 375 380Asn Pro Phe Phe Thr Pro Ala Ala Ile Ala Ile Met Ile Ala Ala Gly385 390 395 400Thr Ile Ala Ile Pro Lys Arg Lys Glu Glu Asp 405 41010349PRTStreptococcus equi 10Met Lys Thr Ile Ala Tyr Pro Asn Lys Pro His Ser Leu Ser Ala Gly1 5 10 15Leu Leu Thr Ala Ile Ala Ile Phe Ser Leu Ala Ser Ser Asn Ile Thr 20 25 30Tyr Ala Asp Asp Tyr Gln Arg Asn Ala Thr Glu Ala Tyr Ala Lys Glu 35 40 45Val Pro His Gln Ile Thr Ser Val Trp Thr Lys Gly Val Thr Pro Leu 50 55 60Thr Pro Glu Gln Phe Arg Tyr Asn Asn Glu Asp Val Ile His Ala Pro65 70 75 80Tyr Leu Ala His Gln Gly Trp Tyr Asp Ile Thr Lys Ala Phe Asp Gly 85 90 95Lys Asp Asn Leu Leu Cys Gly Ala Ala Thr Ala Gly Asn Met Leu His 100 105 110Trp Trp Phe Asp Gln Asn Lys Thr Glu Ile Glu Ala Tyr Leu Ser Lys 115 120 125His Pro Glu Lys Gln Lys Ile Ile Phe Asn Asn Gln Glu Leu Phe Asp 130 135 140Leu Lys Ala Ala Ile Asp Thr Lys Asp Ser Gln Thr Asn Ser Gln Leu145 150 155 160Phe Asn Tyr Phe Arg Asp Lys Ala Phe Pro Asn Leu Ser Ala Arg Gln 165 170 175Leu Gly Val Met Pro Asp Leu Val Leu Asp Met Phe Ile Asn Gly Tyr 180 185 190Tyr Leu Asn Val Phe Lys Thr Gln Ser Thr Asp Val Asn Arg Pro Tyr 195 200 205Gln Asp Lys Asp Lys Arg Gly Gly Ile Phe Asp Ala Val Phe Thr Arg 210 215 220Gly Asp Gln Thr Thr Leu Leu Thr Ala Arg His Asp Leu Lys Asn Lys225 230 235 240Gly Leu Asn Asp Ile Ser Thr Ile Ile Lys Gln Glu Leu Thr Glu Gly 245 250 255Arg Ala Leu Ala Leu Ser His Thr Tyr Ala Asn Val Ser Ile Ser His 260 265 270Val Ile Asn Leu Trp Gly Ala Asp Phe Asn Ala Glu Gly Asn Leu Glu 275 280 285Ala Ile Tyr Val Thr Asp Ser Asp Ala Asn Ala Ser Ile Gly Met Lys 290 295 300Lys Tyr Phe Val Gly Ile Asn Ala His Arg His Val Ala Ile Ser Ala305 310 315 320Lys Lys Ile Glu Gly Glu Asn Ile Gly Ala Gln Val Leu Gly Leu Phe 325 330 335Thr Leu Ser Ser Gly Lys Asp Ile Trp Gln Lys Leu Ser 340 34511349PRTStreptococcus zooepidemicus 11Met Lys Thr Ile Ala Tyr Pro Asn Lys Pro His Ser Leu Ser Ala Gly1 5 10 15Leu Leu Thr Ala Ile Ala Ile Phe Ser Leu Ala Ser Ser Asn Ile Thr 20 25 30Tyr Ala Asp Asp Tyr Gln Arg Asn Ala Ala Glu Val Tyr Ala Lys Glu 35 40 45Val Pro His Gln Ile Thr Ser Val Trp Thr Lys Gly Val Thr Pro Leu 50 55 60Thr Pro Glu Gln Phe Arg Tyr Asn Asn Glu Asp Val Ile His Ala Pro65 70 75 80Tyr Leu Ala His Gln Gly Trp Tyr Asp Ile Thr Lys Val Phe Asp Gly 85 90 95Lys Asp Asn Leu Leu Cys Gly Ala Ala Thr Ala Gly Asn Met Leu His 100 105 110Trp Trp Phe Asp Gln Asn Lys Thr Glu Ile Glu Ala Tyr Leu Ser Lys 115 120 125His Pro Glu Lys Gln Lys Ile Ile Phe Asn Asn Gln Glu Leu Phe Asp 130 135 140Leu Lys Ala Ala Ile Asp Thr Lys Asp Ser Gln Thr Asn Ser Gln Leu145 150 155 160Phe Asn Tyr Phe Arg Asp Lys Ala Phe Pro Asn Leu Ser Ala Arg Gln 165 170 175Leu Gly Val Met Pro Asp Leu Val Leu Asp Met Phe Ile Asn Gly Tyr 180 185 190Tyr Leu Asn Val Phe Lys Thr Gln Ser Thr Asp Val Asn Arg Pro Tyr 195 200 205Gln Asp Lys Asp Lys Arg Gly Gly Ile Phe Asp Ala Val Phe Thr Arg 210 215 220Gly Asp Gln Thr Thr Leu Leu Thr Ala Arg His Asp Leu Lys Asn Lys225 230 235 240Gly Leu Asn Asp Ile Ser Thr Ile Ile Lys Gln Glu Leu Thr Glu Gly 245 250 255Arg Ala Leu Ala Leu Ser His Thr Tyr Ala Asn Val Ser Ile Ser His 260 265 270Val Ile Asn Leu Trp Gly Ala Asp Phe Asn Ala Glu Gly Asn Leu Glu 275 280 285Ala Ile Tyr Val Thr Asp Ser Asp Ala Asn Ala Ser Ile Gly Met Lys 290 295 300Lys Tyr Phe Val Gly Ile Asn Ala His Gly His Val Ala Ile Ser Ala305 310 315 320Lys Lys Ile Glu Gly Glu Asn Ile Gly Ala Gln Val Leu Gly Leu Phe 325 330 335Thr Leu Ser Ser Gly Lys Asp Ile Trp Gln Lys Leu Ser 340 34512600DNAStreptococcus equi 12aaataatttt gtttaacttt aagaaggaga tataaccatg gctctagatg ctacaacggt 60gttagagcct acaacagcct tcattagaga agctgttagg gaaatcaatc agctgagtga 120tgactacgct gacaatcaag agcttcaggc tgttcttgct aatgctggag ttgaggcact 180tgctgcagat actgttgatc aggctaaagc agctcttgac aaagcaaagg cagctgttgc 240tggtgttcag cttgatgaag caagacgtga ggcttacaga acaatcaatg ccttaagtga 300tcagcacaaa agcgatcaaa aggttcagct agctctagtt gctgcagcag ctaaggtggc 360agatgctgct tcagttgatc aagtgaatgc agccattaat gatgctcata cagctattgc 420ggacattaca ggagcagcct tgttggaggc taaagaagct gctatcaatg aactaaagca 480gtatggcatt agtgattact atgtgacctt aatcaacaaa gccaaaactg ttgaaggtgt 540caatgcgctt aaggcaaaga ttttatcagc tctaccgtag ctcgagcccg ggtgctttgc 60013180PRTStreptococcus equi 13Met Ala Leu Asp Ala Thr Thr Val Leu Glu Pro Thr Thr Ala Phe Ile1 5 10 15Arg Glu Ala Val Arg Glu Ile Asn Gln Leu Ser Asp Asp Tyr Ala Asp 20 25 30Asn Gln Glu Leu Gln Ala Val Leu Ala Asn Ala Gly Val Glu Ala Leu 35 40 45Ala Ala Asp Thr Val Asp Gln Ala Lys Ala Ala Leu Asp Lys Ala Lys 50 55 60Ala Ala Val Ala Gly Val Gln Leu Asp Glu Ala Arg Arg Glu Ala Tyr65 70 75 80Arg Thr Ile Asn Ala Leu Ser Asp Gln His Lys Ser Asp Gln Lys Val 85 90 95Gln Leu Ala Leu Val Ala Ala Ala Ala Lys Val Ala Asp Ala Ala Ser 100 105 110Val Asp Gln Val Asn Ala Ala Ile Asn Asp Ala His Thr Ala Ile Ala 115 120 125Asp Ile Thr Gly Ala Ala Leu Leu Glu Ala Lys Glu Ala Ala Ile Asn 130 135 140Glu Leu Lys Gln Tyr Gly Ile Ser Asp Tyr Tyr Val Thr Leu Ile Asn145 150 155 160Lys Ala Lys Thr Val Glu Gly Val Asn Ala Leu Lys Ala Lys Ile Leu 165 170 175Ser Ala Leu Pro 180141158DNAStreptococcus equi 14atgatgaaaa aacaatcatt cacacactca cgtaaaccta aattcggtat gagaaaatta 60tctattggcc ttgcctcatg tatgctagga atgatgttcc taacaacagg acatgtttct 120ggtgaggtag ttgaagtttg gcctaatggg caaaatccta atggtaaaat agaaattcta 180agtcaaactg agcactctga gcatttacag aaattacgcg atattgaaga tttccaagct 240caaaagcaag ctgatcatgt tcgttacact aaatggttag atggggtaac tgttgatgag 300catgaattca gaaaaatcaa ggaatatgac acagaatatt atgtaacacc tcttttaagt 360ggtaaaggtt actatgatat caataaagat ttcaatcaag atagtgataa atgtgctgcc 420gctgtagcgg ctaatatgtt ccattattgg tttgatagaa atagagacag tattaatcgt 480ttcttaagtc aaagtccagg tgaaaatggt gttattaaac ttgaaaatga aaaaacaata 540gaagtatcaa aatttttaga aacttaccgt agtgatggtg attatcttga taaaagtccg 600ttttttgacc ttatcagtaa cagctttaaa ggtcctgttt gggcaaataa gctattggat 660gcttacatta acggctatgg ttatatccat aaatttgcta aaaatactcc acattctaaa 720aataataata gtaaatttaa tttctttaaa aaagtatttg atggtaatct cttgacagat 780attcaccaaa tttttgatta

taacactttt tcagataaat taagtgaggc tctctatact 840ggtaaagcca ttggattggc ctacggacct ggagacttgc gtcgttcact aggtcatatt 900atttctgtct ggggagctga tcttgacgat cagaatcgcg tggtagctat ttatgtaact 960gattctgatg ataaaaagtt aactatagga aatgagagag ttggtttgaa gcgatataaa 1020gtatctagcg atgatcaagg tcgtgctcgt ctgacgactc gtgataaaga taacacaggt 1080ggtgaaattc gatctattga aacattagat atgggtacac aagagtgggc agattacttc 1140aacaagacag aaaaataa 1158151860DNAStreptococcus equi 15atgaagaaat tcacgaaacg gtgtcttaag ggctgtggtc ttgttggatt agttttcagc 60acaggattgg ttgccttgtc ggataatatt gatagcgctt taacagtagg ggcggaaacg 120actactgcta gtgcatttga aaataatggg acaggtcaac atctgaactg gcacatagat 180attccacaag aatatacagt tgaattagga gaaccaatta ctatctcaga tcttatgagt 240caaattacgg ttactcgtaa aggtagtaat gggactgtta atgatggaga tacttttgac 300tttatttcga atggagatgg ttcaagagga attgataccc ctggagtaaa aatatggttt 360gacttttaca atgctgcggg tacttccttt ttaactgatg aaatgttagc ttcgcctaca 420tatgctgtac cggggggatc ttatactatt aaagcttggg tattctatgg gaaaaatgat 480accaaaaagc tcttcacatt taaactaaaa aattccaaca gcaataaaac tgagttaagg 540aagtcgttag aggaggctaa gctaaaactc agccagcctg aaggaacgta ttctgatgaa 600tcactgcaag ccttgcaatc agcggttact cttggtaaga cctatttaaa cagtgaccct 660gatcaaaata cagtagatca atctgttact actattgatt ccgctattac tagtcttgtt 720aatcttaatg ctttaaatga agctattaat caagctacac cttttataac agatggcaaa 780gagtatccta aagaagcgta tgacggtctt gtgcaaaagc ttgcagcggc agctaagctt 840caaaattcat ttggtccttc acaaggagat gttgataagg ctgcgactga tttaacgcaa 900gctcttacga cgcttaagac tgctgtagcg catgaagcct tagatcaagc cttggctaag 960ctgttagagc tttaccgaga aaatccaaat cttgctttga catcagagtc tttgaaggaa 1020ttgtacaata aggccattga agcagcaggt accttctata gaactgttaa caaggataaa 1080gagagaaaag acatttccct ttatgagcta gagcgctaca ctacagaaac aaattcagtt 1140gttgatacta ttttaaaggt aaaggctgcg attgccgaag aaggaaaggc aaaattgcgt 1200tctgctttag accaattaaa tgctcttatc ggagaaaatc tagacctatc tccatataca 1260gcagcttctg ctcaagccta tacagaccag ctagctaagg ctaaggaggt cgcagcagcg 1320ggtgagacag cttatgctca ggagacagaa ccgacagcta ttactaacag cttggttaag 1380gtgttaaatg ctaagaaatc cctctcagat gccaaggcag ccttggttgc taaaccggtc 1440gatccagtag atccagtaga cccagtggat ccggtagacc cagtagatcc ggtagaccca 1500gtggatccgg tagacccagt ggatccagta gacccagtag acccagtaga cccagtggat 1560ccggtagacc cagtggatcc ggtagacccg gtcgatccaa tcgacccagc ggatccagta 1620aaaccatcag atcctgaggt taagccagag cctaaaccag aatctaagcc tgaagctaag 1680aaggaggaca agaaagcagc tgataagcag caagtgcttc cggcaactgc tgatacagct 1740aatccattct ttacagcagc agctcttgca gttattgctt gtgcaggcca gcttgctatt 1800gtgtcaagac gcaaagaatc aaattaactg taggcgatga ttttccccct ttaattaatt 1860161140DNAStreptococcus equi 16atgaacaaaa aatcagcaag acgcaggcgt aagaatctta ttacgaagct tgcgatgaca 60agtgccttaa ccctgggtgt aggcgcagcg actaccctag caggacaaac agaagtacgg 120gctgataata tcttacgctt agatatgaca gataaagaag cagttgaaaa attcgctaac 180gagcttaaaa atgaagtcca taaaaactat cgtggtagta atacttggca aaagcttacc 240cttatactta atggttatca aaaccttaga gaacaaatag agaccgagct aaaaaatagt 300gaacaaaaag taaaagagct taatgataag gttaatagtg aaactcaagg aaaacaagag 360ttacagaatc agcttgagaa agaaaaagaa gagttagaaa cactaaaaaa agagcttgaa 420gctgagaagg ctaaaggaac tggagaaaca gagaagcttc aaaaggaaat tgaagcaaaa 480aatgcaatga tttctgacct acaaaaacag cttgaggaaa ctaagcaaag ggttcaagag 540tttgaagctg aagtaggtaa attaatggcc gaaaaggcag acctacaaac aaaattaaat 600gaacaagagc agcttaacgc taagcttcaa aaagaaattg aagacttaaa ggctcagatt 660gaaaagctta agcactgtca agatacacct aagccagagc ctaagccaga gcctaagcca 720gagcctaagc cagagcctaa gccagagcct aagccagagc ctaagccaga gcctaagcca 780gagcctaagc cagggcctaa gccagagcct aagccagagc ctaagccagg gcctaagcca 840gagcctaagc cagagcctaa gccagggcct aagccagggc ctaagccaga gcctaagcca 900gggcctaagc cagagcctaa gccagagcct aagccagagc ctaagcctga agctaagaag 960cctgaacaac ctaaaccaat gactaaacca ggagctaaga agcctgagca atcacttcca 1020tcaactggtg acatcagaaa tccattcttc acgcctgcag ctattgctat tatgatcgca 1080gcaggtacca ttgccattcc aaaacgcaag gaagaagatt aaacaaatta acaatcccca 1140171179DNAStreptococcus zooepidemicus 17atgatgaaaa aacaatcatt cacacactca cgtaaaccta aattcggtat gagaaaatta 60tctattggcc ttgcctcatg tatgctagga atgatgttcc taacaacaag ccatgtttct 120ggtgaggtag ttgaagtttg gccttatggg caagatccta atgataaaat agaagtttta 180agtcaatctg agtattccga atatttacag agattacacg atgttgaaga tttccaagct 240gaaaagaaaa aagaaggagt tgtccgtaca caatggttag agggtgtgaa cgttactgac 300catgacttcc ggaaaatcac tgatggtggt agtgtttatt atgccacacc tcttttaaat 360gatagaggct attatgatat caacaagaat ttcaatcaag acagtgataa atgtgctgct 420gctgtggcag ttaatatgtt ccattattgg cttgatagga ataaagataa tgtagctaag 480tttcttagtc aaagtccaga ccatggtttt gttgaaggtg aacctacttt taacttagta 540gattttcaat atacatatgc atctccatat gaagaaggag gatataggga caatagtaaa 600ctcttcgact ttattagcaa ggcttttaat aagcctcttt gggcaaataa attgttagat 660gcttacatta atggctatgg ctatatcgac agatacgtta aaaatacccc gcattctgga 720caaaataata gtaaatttaa tttctttaaa aaagtatttg atggcaagct cttgacagat 780attcaacaaa tttttgatta ttatacttta tcgtctgagc tacgtgaagc tcttgatact 840ggcaaagcta ttggtttagc ctatggacct ggagatttac gccgttctct gggacatatt 900atctccgtct ggggagctga cattaatgaa gatggaaatg tcgtggctat ttatgtgact 960gattccgatg ataaaaaatt aactataggg aataaaaaag accgaattgg tttgaagcga 1020tacaaactgt attctgataa cgtgggacga gctcgcctaa cagcctatgc tacagaaaac 1080caacaaactg gtggtgaagt tcgagggatt gaaactttag atatggctac acaagattgg 1140gcagattatt ttagcaggac agacgaagca gaacaataa 1179181713DNAStreptococcus zooepidemicus 18atgaagaaat tcacgaaacg gtgtcttaag ggctgcggtc ttgttggatt agttttcagc 60acaggattgg ttgccttgtc ggataatatt gatagcgctt taacagtagg ggcggaaacg 120gctactactg ctaatgcatt tgaagaaagt ggtgaccaac aacataaaaa ttggcatatt 180tatattccag aggtttatac tgttaaagtc ggtcagccaa tcaccattga ggatatctta 240agtcagatta cgattactcg taagggagaa gattcgcaag gtaaaacatc tcccggaatg 300atctatactt atgaagaata ccctaaagta cgaggaattg aagtttcagc aggaactatt 360tggtttgatt tttataattc tggaaactgg gtaaataatg atgttttagc taccttcaac 420gaacctggag gaacttatac cttatctgct tgggcatact atgctaacga aaatgtaaaa 480aaacaatttg ttttcaaact tcaagttgaa aatagtgata agcgtgcatt agaacaatct 540cttgctactg ctaacgaaaa gttacaggct cctgaaggaa cgtattctga tgaatcactg 600caacgtttac aagaatcagt tttccttggt caaacttatt tgaacaggga tcctgagcaa 660caagaagtgg acgatatgaa ggcaactatt gattctgctg tttctggtct tgttgatctt 720actgtcttaa ataccgcagt tgaaacagca acaccattgt taacagatgg taaggagtat 780cctaaagaag cgtatgatag ccttgttcaa aagcttgcag cagcagctaa gcttcaaaat 840tcctttaacc catcacaaga agaagttaac gaggctgcga ctgatttaac gcaagctctt 900acgacgctta agactgctgt agcgcatgaa gccttagatc aagccttggc taagctgtta 960gagctttacc gagaaaatcc aaaccttgct ttgacatcag agcctttgaa ggaattgtac 1020aataaggcca ttgaagcagc aggcaccttc tatagaactg ttagcaagga taaagagaga 1080aaaggcattt ccctttatga gctagagcgt tacactacag aaacaaactc agttgttgat 1140actattttaa aggtaaaggc tgcaattgcc gaagaaggaa aggcaaaatt gcgttctgct 1200ttagaccaat taaatgctct tatcggagaa aatctagacc tatctccata tacagcagct 1260tctgctcaag cctatacaga ccagctagct aaggctaagg aggttgcagc agcgggtgag 1320acagcttatg ctcaggagac agaaccgaca gctattacta acagcttgat taaggtgcta 1380aatgctaaga aatccctctc agatgccaag gcagcattgg ttgctaaacc ggtagatccg 1440gtagacccag tagatccggt agacccagtg gatccggtag acccaattga tccagtagat 1500ccagtaaaac cagtcgatcc tgaggttaag ccagagccta aaccagaatc taagcctgaa 1560gctaagaagg aggacaagaa agcagctgat aagcagcaag tgcttccggc aactgctgat 1620acagctaacc cattctttac agcagcagct cttgcagtta ttgcttgtgc aggccagctt 1680gctattgtgt caagacgcaa agaatcaaat taa 1713191236DNAStreptococcus zooepidemicus 19atgaacaaaa aatcagcaag acgcaagcgt aaggatctta tcacgaagct tgcgatgaca 60agtgccttaa ccctgggtgt aggcgcagca gctaccatag caggacaaac agaagtacgg 120gctgaggttc taaccttaaa tatgaaagat aaagctaaag ttgaagaatt cgctaataag 180cttaaagatt acgcaaagca aaagaaatct ggccaaatta ctttgcaaga actttccctt 240atacttgatg ggtacagaaa tattagggag cagatagaac aagacttagc tactacagaa 300aaaactaaaa atttctatgg agaacagtta attcttactg ataaacttta tcagtctgaa 360aaagaaaaga aagaaaagct agaagctgag ctacaactaa gccaacaaaa aattcatgac 420cttgatgaaa aacatcaaaa agagaaatta gagctacaag aacaacttga ggcttcaaat 480caaaagatta aagagcttga aatggcaaag agcacagctg aagctgaaat aaatagacta 540acagctgaaa aaaatggatt acaagaaaaa ttaaataatc aagaaaagct taatgctgag 600ttacaagcaa aattagctaa gcaagaagag cttaacgcta agcttcaaaa ggaaattgac 660gaattaaatg ctcagcttga aaagcttaag cattgtcaag atacacctaa gccagagcct 720aagccagagc ctaagccaga gcctaagcca gagcctaagc cagagcctaa gccagagcct 780aagccagagc ctaagccaga gcctaagcca gagcctaagc cagagcctaa gccagagcct 840aagccagagc ctaagccaga gcctaagcca gagcctaagc cagagcctaa gccagagcct 900aagccagagc ctaagccaga gcctaagcca gagcctaagc cagagcctaa gccagagcct 960aagccagagc ctaagccaga gcctaagcca gagcctaagc cagagcctaa gccagagcct 1020aagccagagc ctaagccaga gcctaagcca gagcctaagc ctgaagctaa aaagcctgaa 1080caacctaaac caatgactaa accaggggct aagaagcctg agcaatcact tccatcaact 1140ggtgacatca gaaatccatt cttcacacct gcagctattg ctattatgat cgcagcaggt 1200accattgcaa ttccaaaacg caaggaagaa gactaa 1236201050DNAStreptococcus equi 20atgaaaacaa tagcttatcc aaataaacct cactccttat cagctggtct cttaactgct 60atagctattt ttagcctggc gagttcaaac attacttatg ctgacgatta ccaaaggaat 120gctacggaag cttatgccaa agaagtacca catcagatca cttctgtatg gaccaaaggt 180gttacaccac taacacccga gcagtttcga tataataacg aagatgtgat ccatgcgcca 240tatcttgctc atcaaggctg gtacgatatc accaaggcct tcgatgggaa ggataatctc 300ttgtgtggcg cagcaacggc aggtaatatg ctgcattggt ggtttgatca aaataaaaca 360gagattgaag cctatttaag taaacaccct gaaaagcaaa aaatcatttt taacaaccaa 420gagctatttg atttgaaagc tgctatcgat accaaggaca gtcaaaccaa tagtcagctt 480tttaattatt ttagagataa agcctttcca aatctatcag cacgtcaact cggggttatg 540cctgatcttg ttctagacat gtttatcaat ggttactact taaatgtgtt taaaacacag 600tctactgatg tcaatcgacc ttatcaggac aaggacaaac gaggtggtat tttcgatgct 660gttttcacca gaggagatca gacaacgctc ttgacagctc gtcatgattt aaaaaataaa 720ggactaaatg acatcagcac cattatcaag caagaactga ctgaaggaag agcccttgct 780ttatcacata cctacgccaa tgttagcatt agccatgtga ttaacttgtg gggagctgat 840tttaatgctg aaggaaacct tgaggccatc tatgtcacag actcagatgc taatgcgtct 900attggtatga aaaaatattt tgtcggcatt aatgctcata gacatgtcgc catttctgcc 960aagaaaatag aaggagaaaa cattggcgct caagtattag gcttatttac gctttccagt 1020ggcaaggaca tatggcagaa actgagctaa 1050211050DNAStreptococcus zooepidemicus 21atgaaaacaa tagcttatcc aaataaacct cactccttat cagctggtct cttaactgct 60atagctattt ttagcctggc gagttcaaac attacttatg ctgacgatta ccaaaggaat 120gctgcggaag tttatgccaa agaagtacca catcagatca cttctgtatg gaccaaaggt 180gttacaccac taacacccga gcagtttcga tataataacg aagatgtgat ccatgcgcca 240tatcttgctc atcaaggctg gtacgatatc accaaggtct tcgatgggaa ggataatctc 300ttgtgtggcg cagcaacggc aggtaatatg ctgcattggt ggtttgatca aaataaaaca 360gagattgaag cctatttaag taaacaccct gaaaagcaaa aaatcatttt taacaaccaa 420gagctatttg atttgaaagc tgctatcgat accaaggaca gtcaaaccaa tagtcagctt 480tttaattatt ttagagataa agcctttcca aatctatcag cacgtcaact cggggttatg 540cctgatcttg ttctagacat gtttatcaat ggttactact taaatgtgtt taaaacacag 600tctactgatg tcaatcgacc ttatcaggac aaggacaaac gaggtggtat tttcgatgct 660gttttcacca gaggagatca gacaacgctc ttgacagctc gtcatgattt aaaaaataaa 720ggactaaatg acatcagcac cattatcaag caggaactga ctgaaggaag agcccttgct 780ttatcacata cctacgccaa tgttagcatt agccatgtga ttaacttgtg gggagctgat 840tttaatgctg aaggaaacct tgaggccatc tatgtcacag actcagatgc taatgcgtct 900attggtatga aaaaatattt tgtcggcatt aatgctcatg gacatgtcgc catttctgcc 960aagaaaatag aaggagaaaa cattggcgct caagtattag gcttatttac gctttccagt 1020ggcaaggaca tatggcagaa actgagctaa 10502233DNAArtificial SequenceSynthetic oligonucleotide as PCR primer 22catgccatgg aggtagttga agtttggcct aat 332336DNAArtificial SequenceSynthetic oligonucleotide as PCR primer 23ccgctcgagt ttttctgtct tgttgaagta atctgc 362430DNAArtificial SequenceSynthetic oligonucleotide as PCR primer 24gtagccatgg aaacgactac tgctagtgca 302529DNAArtificial SequenceSynthetic oligonucleotide as PCR primer 25ctggctcgag cggtttagca accaaggct 292631DNAArtificial SequenceSynthetic oligonucleotide as PCR primer 26catgccatgg cgactaccct agcaggacaa a 312731DNAArtificial SequenceSynthetic oligonucleotide as PCR primer 27ctagctcgag gtgcttaagc ttttcaatct g 3128594PRTStreptococcus equi 28Met Ala Thr Asn Leu Ser Asp Asn Ile Thr Ser Leu Thr Val Ala Ser1 5 10 15Ser Ser Leu Arg Asp Gly Glu Arg Thr Thr Val Lys Val Ala Phe Asp 20 25 30Asp Lys Lys Gln Lys Ile Lys Ala Gly Asp Thr Ile Glu Val Thr Trp 35 40 45Pro Thr Ser Gly Asn Val Tyr Ile Gln Gly Phe Asn Lys Thr Ile Pro 50 55 60Leu Asn Ile Arg Gly Val Asp Val Gly Thr Leu Glu Val Thr Leu Asp65 70 75 80Lys Ala Val Phe Thr Phe Asn Gln Asn Ile Glu Thr Met His Asp Val 85 90 95Ser Gly Trp Gly Glu Phe Asp Ile Thr Val Arg Asn Val Thr Gln Thr 100 105 110Thr Ala Glu Thr Ser Gly Thr Thr Thr Val Lys Val Gly Asn Arg Thr 115 120 125Ala Thr Ile Thr Val Thr Lys Pro Glu Ala Gly Thr Gly Thr Ser Ser 130 135 140Phe Tyr Tyr Lys Thr Gly Asp Ile Gln Pro Asn Asp Thr Glu Arg Val145 150 155 160Arg Trp Phe Leu Leu Ile Asn Asn Asn Lys Glu Trp Val Ala Asn Thr 165 170 175Val Thr Val Glu Asp Asp Ile Gln Gly Gly Gln Thr Leu Asp Met Ser 180 185 190Ser Phe Asp Ile Thr Val Ser Gly Tyr Arg Asn Glu Arg Phe Val Gly 195 200 205Glu Asn Ala Leu Thr Glu Phe His Thr Thr Phe Pro Asn Ser Val Ile 210 215 220Thr Ala Thr Asp Asn His Ile Ser Val Arg Leu Asp Gln Tyr Asp Ala225 230 235 240Ser Gln Asn Thr Val Asn Ile Ala Tyr Lys Thr Lys Ile Thr Asp Phe 245 250 255Asp Gln Lys Glu Phe Ala Asn Asn Ser Lys Ile Trp Tyr Gln Ile Leu 260 265 270Tyr Lys Asp Gln Val Ser Gly Gln Glu Ser Asn His Gln Val Ala Asn 275 280 285Ile Asn Ala Asn Gly Gly Val Asp Gly Ser Arg Tyr Thr Ser Phe Thr 290 295 300Val Lys Lys Ile Trp Asn Asp Lys Glu Asn Gln Asp Gly Lys Arg Pro305 310 315 320Lys Thr Ile Thr Val Gln Leu Tyr Ala Asn Asp Gln Lys Val Asn Asp 325 330 335Lys Thr Ile Glu Leu Ser Asp Thr Asn Ser Trp Gln Ala Ser Phe Gly 340 345 350Lys Leu Asp Lys Tyr Asp Ser Gln Asn Gln Lys Ile Thr Tyr Ser Val 355 360 365Lys Glu Val Met Val Pro Val Gly Tyr Gln Ser Gln Val Glu Gly Asp 370 375 380Ser Gly Val Gly Phe Thr Ile Thr Asn Thr Tyr Thr Pro Glu Val Ile385 390 395 400Ser Ile Thr Gly Gln Lys Thr Trp Asp Asp Arg Glu Asn Gln Asp Gly 405 410 415Lys Arg Pro Lys Glu Ile Thr Val Arg Leu Leu Ala Asn Asp Ala Ala 420 425 430Thr Asp Lys Val Ala Thr Ala Ser Glu Gln Thr Gly Trp Lys Tyr Thr 435 440 445Phe Thr Asn Leu Pro Lys Tyr Lys Asp Gly Lys Gln Ile Thr Tyr Thr 450 455 460Ile Gln Glu Asp Pro Val Ala Asp Tyr Thr Thr Thr Ile Gln Gly Phe465 470 475 480Asp Ile Thr Asn His His Glu Val Ala Leu Thr Ser Leu Lys Val Ile 485 490 495Lys Val Trp Asn Asp Lys Asp Asp Tyr Tyr His Lys Arg Pro Lys Glu 500 505 510Ile Thr Ile Leu Leu Lys Ala Asp Gly Lys Val Ile Arg Glu His Gln 515 520 525Met Thr Pro Asp Gln Gln Gly Lys Trp Glu Tyr Thr Phe Asp Gln Leu 530 535 540Pro Val Tyr Gln Ala Gly Lys Lys Ile Ser Tyr Ser Ile Glu Glu Lys545 550 555 560Gln Val Ala Gly Tyr Gln Ala Pro Val Tyr Glu Val Asp Glu Gly Leu 565 570 575Lys Gln Val Thr Val Thr Asn Thr Leu Asn Pro Ser Tyr Lys Leu Glu 580 585 590Pro Gly29302PRTStreptococcus equi 29Met Thr Asn Lys Thr Lys Arg Thr Gly Leu Val Arg Lys Tyr Gly Ala1 5 10 15Cys Ser Ala Ala Ile Ala Leu Ala Ala Leu Ala Ser Leu Gly Ala Gly 20 25 30Lys Ala Val Lys Ala Asp Gln Pro Ala Ala Leu Lys Tyr Pro Glu Pro 35 40 45Arg Asp Tyr Phe Leu His Thr Arg Glu Gly Asp Val Ile Tyr Asp Glu 50 55 60Asp Ile Lys Arg Tyr Phe Glu Asp Leu Glu Ala Tyr Leu Thr Ala Arg65 70 75 80Leu Gly Gly Ile Asp Lys Lys Val Glu Glu Ala Ala Gln Lys Pro Gly 85 90 95Ile Pro Gly Pro Thr Gly Pro Gln Gly Pro Lys Gly Asp Lys Gly Asp 100 105 110Pro Gly

Ala Pro Gly Glu Arg Gly Pro Ala Gly Pro Lys Gly Asp Thr 115 120 125Gly Glu Ala Gly Pro Arg Gly Glu Gln Gly Pro Ala Gly Gln Ala Gly 130 135 140Glu Arg Gly Pro Lys Gly Asp Pro Gly Ala Pro Gly Pro Lys Gly Glu145 150 155 160Lys Gly Asp Thr Gly Ala Val Gly Pro Lys Gly Glu Lys Gly Asp Thr 165 170 175Gly Ala Thr Gly Pro Lys Gly Asp Lys Gly Glu Arg Gly Glu Lys Gly 180 185 190Glu Gln Gly Gln Arg Gly Glu Lys Gly Glu Gln Gly Gln Arg Gly Glu 195 200 205Lys Gly Glu Gln Lys Pro Lys Gly Asp Gln Gly Lys Asp Thr Lys Pro 210 215 220Ser Ala Pro Lys Ala Pro Glu Lys Ala Pro Ala Pro Lys Ala Pro Lys225 230 235 240Ala Ser Glu Gln Ser Ser Asn Pro Lys Ala Pro Ala Pro Lys Ser Ala 245 250 255Pro Ser Lys Ser Ala Ala Pro Thr Gly Gln Lys Ala Ala Leu Pro Ala 260 265 270Thr Gly Glu Ile Asn His Pro Phe Phe Thr Leu Ala Ala Leu Ser Val 275 280 285Ile Ala Ser Val Gly Val Leu Thr Leu Lys Gly Lys Lys Asp 290 295 30030320PRTArtificial SequenceChemically synthesized recombinant protein IdeE 30Gly Pro Leu Gly Ser Asp Asp Tyr Gln Arg Asn Ala Thr Glu Ala Tyr1 5 10 15Ala Lys Glu Val Pro His Gln Ile Thr Ser Val Trp Thr Lys Gly Val 20 25 30Thr Pro Leu Thr Pro Glu Gln Phe Arg Tyr Asn Asn Glu Asp Val Ile 35 40 45His Ala Pro Tyr Leu Ala His Gln Gly Trp Tyr Asp Ile Thr Lys Ala 50 55 60Phe Asp Gly Lys Asp Asn Leu Leu Cys Gly Ala Ala Thr Ala Gly Asn65 70 75 80Met Leu His Trp Trp Phe Asp Gln Asn Lys Thr Glu Ile Glu Ala Tyr 85 90 95Leu Ser Lys His Pro Glu Lys Gln Lys Ile Ile Phe Asn Asn Gln Glu 100 105 110Leu Phe Asp Leu Lys Ala Ala Ile Asp Thr Lys Asp Ser Gln Thr Asn 115 120 125Ser Gln Leu Phe Asn Tyr Phe Arg Asp Lys Ala Phe Pro Asn Leu Ser 130 135 140Ala Arg Gln Leu Gly Val Met Pro Asp Leu Val Leu Asp Met Phe Ile145 150 155 160Asn Gly Tyr Tyr Leu Asn Val Phe Lys Thr Gln Ser Thr Asp Val Asn 165 170 175Arg Pro Tyr Gln Asp Lys Asp Lys Arg Gly Gly Ile Phe Asp Ala Val 180 185 190Phe Thr Arg Gly Asp Gln Thr Thr Leu Leu Thr Ala Arg His Asp Leu 195 200 205Lys Asn Lys Gly Leu Asn Asp Ile Ser Thr Ile Ile Lys Gln Glu Leu 210 215 220Thr Glu Gly Arg Ala Leu Ala Leu Ser His Thr Tyr Ala Asn Val Ser225 230 235 240Ile Ser His Val Ile Asn Leu Trp Gly Ala Asp Phe Asn Ala Glu Gly 245 250 255Asn Leu Glu Ala Ile Tyr Val Thr Asp Ser Asp Ala Asn Ala Ser Ile 260 265 270Gly Met Lys Lys Tyr Phe Val Gly Ile Asn Ala His Arg His Val Ala 275 280 285Ile Ser Ala Lys Lys Ile Glu Gly Glu Asn Ile Gly Ala Gln Val Leu 290 295 300Gly Leu Phe Thr Leu Ser Ser Gly Lys Asp Ile Trp Gln Lys Leu Ser305 310 315 3203133DNAArtificial SequenceSynthetic primer 31tactggatcc gacgattacc aaaggaatgc tac 333232DNAArtificial SequenceSynthetic primer 32tgatctcgag ttagctcagt ttctgccata tg 32

Claims

1. An antigenic composition comprising at least one antigen, wherein said at least one antigen comprises at least part of a protein or polypeptide of Streptococcus equi subsp. equi or subsp. zooepidemicus and said at least part of said protein or polypeptide comprises at least one antigenic epitope or antigenic determinant of Streptococcus equi, and wherein said protein or polypeptide is selected from the group comprisinga protein or polypeptide which is designated EAG and has an amino acid sequence as shown in SEQ ID NO: 13;a protein or polypeptide which is designated IdeE and has an amino acid sequence as shown in SEQ ID NO: 10;a protein or polypeptide which is designated IdeE2 and has an amino acid sequence as shown in SEQ ID NO: 1;a protein or polypeptide which is designated Eq5 and has an amino acid sequence as shown in SEQ ID NO: 3;a protein or polypeptide which is designated Eq8 and has an amino acid sequence as shown in SEQ ID NO: 5;a protein or polypeptide which is designated IdeZ2 and has an amino acid sequence as shown in SEQ ID NO: 7;a protein or polypeptide which is designated Eqz5 and has an amino acid sequence as shown in SEQ ID NO: 8; anda protein or polypeptide which is designated Eqz8 and has an amino acid sequence as shown in SEQ ID NO: 9;or an analog or fragment thereof, and wherein a composition which comprises EAG comprises at least one further antigen, which is a protein or polypeptide which is selected from the group comprising IdeE, IdeE2, Eq5, Eq8, IdeZ2, Eqz5 and Eqz8.

2. The antigenic composition of claim 1, wherein said at least one protein or polypeptide is selected from the group consisting of IdeE and IdeE2 and wherein a composition that comprises IdeE2 comprises at least one further antigen.

3. The antigenic composition of claim 1 or 2, wherein said at least one protein or polypeptide is selected from the group consisting of Eq5 and Eq8.

4. The antigenic composition of claim 1, wherein said at least one protein or polypeptide is selected from the group consisting of EAG, Eq5 and Eq8 and which composition further comprises at least one antigen, which is selected from the group comprising a protein or a polypeptide designated CNE (or SEC), which has an amino acid sequence as shown in SEQ ID NO: 28, and a protein or a polypeptide designated ScIC, which has an amino acid sequence as shown in SEQ ID NO: 29.

5. The antigenic composition of claim 4, wherein said at least one protein or polypeptide is selected from the group comprising IdeE and IdeE2.

6. The antigenic composition of claim 1, which comprises the antigens EAG, ScIC, CNE (or SEC), Eq5, Eq8, IdeE and IdeE2, or which composition comprises the antigens EAG, ScIC, CNE (or SEC), Eq5, and Eq8.

7. The antigenic composition of claim 1, wherein said at least one protein or polypeptide is selected from the group consisting of EAG, Eq8, and IdeE2 and which composition comprises at least one further antigen which is selected from the group comprising IdeE, Eq5, IdeZ2, Eqz5 and Eqz8.

8. The antigenic composition of claim 1, wherein at least one antigen is recombinantly produced.

9. The antigenic composition of claim 1, wherein at least one antigen is an isolated or purified antigen.

10. The antigenic composition of claim 1, which comprises at least one recombinant vector and at least one polynucleotide inserted therein that encodes said at least one protein or polypeptide, andwhich vector is able to express said polypeptide in vivo in a non-human mammal susceptible to infection with S. equi.

11. The antigenic composition of claim 10, wherein the vector is an expression vector which is a plasmid or a viral vector and wherein said polynucleotide has a nucleotide sequence as shown in SEQ ID NOS: 12 and 14-21.

12. The antigenic composition of claim 1, which is an immunogenic composition.

13. A vaccine composition for protecting non-human mammals against infection of Streptococcus equi, which comprises the antigenic composition of claim 1 as immunizing component, and a pharmaceutically acceptable carrier.

14. The vaccine composition of claim 13, which further comprises an adjuvant.

15. The vaccine composition of claim 13, which is a vaccine that protects susceptible mammals, suitably horses, against strangles caused by Streptococcus equi subsp. equi.

16. The vaccine composition of claim 13, which is provided in a physiologically administrable form, and suitably is administrable by subcutaneous or intranasal inoculation.

17. The vaccine composition of claim 15, which stimulates serum, mucosal and/or bronchial lavage antibody responses directed to Streptococcus equi antigens in mammals susceptible to Streptococcus equi, suitably horses.

18. A method for producing an antigen or an immunogen of an antigenic composition of claim 1, which method comprises(a) providing a DNA fragment encoding said antigen and introducing said fragment into an expression vector;(b) introducing said vector, which contains said DNA fragment, into a compatible host cell;(c) culturing said host cell provided in step (b) under conditions required for expression of the product encoded by said DNA fragment; and(d) isolating the expressed product from the cultured host cell, and, optionally,(e) purifying the isolated product from step (d) by affinity chromatography or other chromatographic methods known in the art.

19. A method for preparation of a vaccine composition according to claim 13, which vaccine composition contains as immunizing component, said antigenic or immunogenic composition, said method comprising mixing said antigenic or immunogenic composition and a pharmaceutically acceptable carrier.

20. Use of an antigenic or immunogenic composition of claim 1 in the preparation of a vaccine protecting against S. equi infection inclusive of strangles caused by subsp. equi infection in horses.

21. A method for the production of an antiserum, said method comprising administering an antigenic preparation of claim 1 to an animal host to produce antibodies in said animal host and recovering antiserum containing said antibodies produced in said animal host.

22. A method of prophylactic or therapeutic treatment of S. equi infection in non-human mammals, suitably horses, comprising administering to said mammal an immunologically effective amount of a vaccine composition of claim 13 or an antiserum produced by administering an antigenic composition to an animal host to produce antibodies in said animal host and recovering antiserum containing said antibodies produced in said animal host,wherein said antigenic composition comprises at least one antigen, wherein said at least one antigen comprises at least part of a protein or polypeptide of Streptococcus equi subsp. equi or subsp. zooepidemicus and said at least part of said protein or polypeptide comprises at least one antigenic epitope or antigenic determinant of Streptococcus equi, and wherein said protein or polypeptide is selected from the group comprisinga protein or polypeptide which is designated EAG and has an amino acid sequence as shown in SEQ ID NO: 13;a protein or polypeptide which is designated IdeE and has an amino acid sequence as shown in SEQ ID NO: 10;a protein or polypeptide which is designated IdeE2 and has an amino acid sequence as shown in SEQ ID NO: 1;a protein or polypeptide which is designated Eq5 and has an amino acid sequence as shown in SEQ ID NO: 3;a protein or polypeptide which is designated Eq8 and has an amino acid sequence as shown in SEQ ID NO: 5;a protein or polypeptide which is designated IdeZ2 and has an amino acid sequence as shown in SEQ ID NO: 7;a protein or polypeptide which is designated Eqz5 and has an amino acid sequence as shown in SEQ ID NO: 8; anda protein or polypeptide which is designated Eqz8 and has an amino acid sequence as shown in SEQ ID NO: 9;or an analog or fragment thereof, and wherein a composition which comprises EAG comprises at least one further antigen, which is a protein or polypeptide which is selected from the group comprising IdeE, IdeE2, Eq5, Eq8, IdeZ2, Eqz5 and Eqz8.

23. A method for protecting horses against Streptococcus equi infection, which comprises inoculating a horse subcutaneously or intranasally with a vaccine composition of claim 13 to induce an immune response against Streptococcus equi in said horse.

24. The method of claim 23, wherein an immune response in the form of IgG and/or IgA and/or IgM antibodies in the nasopharyngeal mucus is induced in said horse.

25. An antibody preparation comprising at least one, and suitably at least two, antibodies specific for a protein or polypeptide of the composition of claim 1, which antibody/antibodies is/are polyclonal or monoclonal; or which preparation comprises a fragment of said antibodies.

26. The antibody preparation of claim 25 which is used prophylactically or therapeutically against strangles and provides passive immunization when administered to a non-human mammal susceptible to infection by Streptococcus equi or infected by Streptococcus equi.

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