COMBINATION VACCINE

Abstract

The disclosure relates to a composition comprising two or more immunogenic staphylococcal polypeptides and a multivalent vaccine composition comprising the immunogenic staphylococcal polypeptides.

Description

STATEMENT REGARDING SEQUENCE LISTING

[0001] The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is 100193_401D1_SEQUENCE_LISTING.txt. The text file is 40.1 KB, was created on Dec. 14, 2015, and is being submitted electronically via EFS-Web.

TECHNICAL FIELD

[0002] The disclosure relates to a composition comprising two or more immunogenic staphylococcal polypeptides and a multivalent vaccine composition comprising the immunogenic staphylococcal polypeptides in the prevention or treatment of staphylococcal infections in humans and animals.

BACKGROUND

[0003] Vaccines protect against a wide variety of infectious diseases. Many modern vaccines are therefore made from protective antigens of the pathogen, which are isolated by molecular cloning and purified. These vaccines are known as `subunit vaccines`. The development of subunit vaccines has been the focus of considerable research in recent years. The emergence of new pathogens and the growth of antibiotic resistance have created a need to develop new vaccines and to identify further candidate molecules useful in the development of subunit vaccines. Likewise the discovery of novel vaccine antigens from genomic and proteomic studies is enabling the development of new subunit vaccine candidates, particularly against bacterial pathogens. However, although subunit vaccines tend to avoid the side effects of killed or attenuated pathogen vaccines, their `pure` status means that subunit vaccines do not always have adequate immunogenicity to confer protection.

[0004] An approach to improve the efficacy of vaccine compositions is to provide multivalent vaccines comprising dominant antigens that provoke both a B cell and T cell response thereby mounting a more rigorous immune response in the subject receiving the vaccine. A typical multivalent vaccine might be a whole cell vaccine comprising multiple antigenic molecules. For example the Bacillus Calmette Guerin ["BCG"] vaccine includes an attenuated Mycobacterium bovis strain that provokes protective immunity in humans. For many pathogens chemical or heat inactivation while it may give rise to vaccine immunogens that confer protective immunity also gives rise to side effects such as fever and injection site reactions. In the case of bacteria, inactivated organisms tend to be so toxic that side effects have limited the application of such crude vaccine immunogens and therefore vaccine development has lagged behind drug-development. Moreover, effective vaccine development using whole cell inactivated organisms suffers from problems of epitope masking, immunodominance, low antigen concentration and antigen redundancy.

SUMMARY

[0005] Currently there is no effective vaccination procedure to prevent or treat Staphylococcus aureus infection. S. aureus is a bacterium whose normal habitat is the epithelial lining of the nose in about 20-40% of normal healthy people and is also commonly found on people's skin usually without causing harm. However, in certain circumstances, particularly when skin is damaged, this pathogen can cause infection. This is a particular problem in hospitals where patients may have surgical procedures and/or be taking immunosuppressive drugs. These patients are much more vulnerable to infection with S. aureus because of the treatment they have received. Antibiotic resistant strains of S. aureus have arisen since their wide spread use in controlling microbial infection. Methicillin resistant strains are prevalent and many of these resistant strains are also resistant to several other antibiotics.

[0006] S. aureus is therefore a major human pathogen capable of causing a wide range of diseases some of which are life threatening diseases including septicaemia, endocarditis, arthritis and toxic shock. This ability is determined by the versatility of the organism and its arsenal of components involved in virulence. At the onset of infection, and as it progresses, the needs and environment of the organism changes and this is mirrored by a corresponding alteration in the virulence determinants which S. aureus produces. At the beginning of infection it is important for the pathogen to adhere to host tissues and so a large repertoire of cell surface associated attachment proteins are made. The pathogen also has the ability to evade host defences by the production of factors that reduce phagocytosis or interfere with the ability of the cells to be recognised by circulating antibodies.

[0007] There is therefore a continuing need to identify staphylococcal antigens that are protective and can be used in multivalent vaccines. The combinations may be used in combination with non-protein immunogenic molecules such as polysaccharide antigens and anti-bacterial agents to provide a treatment regimen for control of staphylococcal infection. It is also within the scope of this disclosure to modify the treatment regimen to immunize subjects with a series of temporally separated administrations as an alternative to the administration of a single vaccine comprising multiple antigens.

[0008] This disclosure therefore relates to combination or multivalent immunogenic compositions and vaccines and their use in the prophylaxis and treatment of staphylococcal infections. We disclose polypeptides that individually are protective and are typically membrane spanning proteins that include an extracellular domain and are essential for staphylococcal cell growth. For example DivIB is an integral membrane protein comprising an intracellular domain, an intermembrane domain and an extracellular domain. DivIB and fragments thereof, provide protection from at least an S. aureus challenge in an animal model. The related gene DivIC is also an integral membrane protein the extracellular domain of which provokes protective immunity to staphylococcal infection. This disclosure also relates to antigens encoded by the genes PheP, YdiE and FtsL, each of which has an extramembranous domain.

[0009] According to an aspect of the invention there is provided an immunogenic composition comprising two or more different polypeptides wherein said polypeptides are encoded by different staphylococcal genes selected from the group consisting of:

[0010] i) a polypeptide, or immunogenic fragment thereof, comprising or consisting of the amino acid sequence as represented in SEQ ID NO.: 20;

[0011] ii) a polypeptide, or immunogenic fragment thereof, comprising or consisting of the amino acid sequence as represented in SEQ ID NO.: 21

[0012] iii) a polypeptide, or immunogenic fragment thereof, comprising or consisting of the amino acid sequence as represented in SEQ ID NO.: 22;

[0013] iv) a polypeptide, or immunogenic fragment thereof, comprising or consisting of the amino acid sequence as represented in SEQ ID NO.: 23;

[0014] v) a polypeptide, or immunogenic fragment thereof, comprising or consisting of the amino acid sequence as represented in SEQ ID NO.: 24; or

[0015] vi) a modified staphylococcal polypeptide wherein said polypeptide is a staphylococcal polypeptide variant of the amino acid sequences presented in SEQ ID NO.: 20, 21, 22, 23 or 24, wherein said sequences are modified by addition, deletion or substitution of one or more amino acid residues which modified polypeptides have retained or enhanced immunogenicity when compared to the polypeptide as represented in SEQ ID NO.: 20, 21, 22, 23 or 24.

[0016] A modified staphylococcal polypeptide or variant staphylococcal polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions, truncations that may be present in any combination. Among preferred variants are those that vary from a reference polypeptide by conservative amino acid substitutions. Such substitutions are those that substitute a given amino acid by another amino acid of like characteristics. The following non-limiting list of amino acids are considered conservative replacements (similar): a) alanine, serine, and threonine; b) glutamic acid and aspartic acid; c) asparagine and glutamine d) arginine and lysine; e) isoleucine, leucine, methionine and valine and f) phenylalanine, tyrosine and tryptophan. Most highly preferred are variants that retain or enhance the immunogenicity and/or activity as the reference polypeptide from which it varies.

[0017] In one embodiment, the variant polypeptides have at least 80-89% sequence identity, more preferably at least 90% identity, even more preferably at least 95% identity, still more preferably at least 97% identity, and most preferably at least 99% identity with the full length amino acid sequences illustrated herein.

[0018] In a preferred embodiment of the invention said immunogenic composition comprises or consists essentially of 2, 3, 4 or 5 staphylococcal polypeptides.

[0019] In a preferred embodiment of the invention there is provided an immunogenic composition comprising:

[0020] i) a polypeptide comprising SEQ ID NO.: 20, or an antigenic fragment thereof; and

[0021] ii) a polypeptide comprising SEQ ID NO.: 21, or antigenic fragment thereof.

[0022] In a preferred embodiment of the invention there is provided an immunogenic composition comprising:

[0023] i) a polypeptide comprising SEQ ID NO.: 20, or an antigenic fragment thereof; and

[0024] ii) a polypeptide comprising SEQ ID NO.: 22, or an antigenic fragment thereof.

[0025] In a preferred embodiment of the invention there is provided an immunogenic composition comprising:

[0026] i) a polypeptide comprising SEQ ID NO.:20, or an antigenic fragment thereof; and

[0027] ii) a polypeptide comprising SEQ ID NO.:23, or an antigenic fragment thereof.

[0028] In a preferred embodiment of the invention there is provided a composition comprising:

[0029] i) a polypeptide comprising SEQ ID NO.: 20, or an antigenic fragment thereof; and

[0030] ii) a polypeptide comprising SEQ ID NO.: 24, or an antigenic fragment thereof.

[0031] In a preferred embodiment of the invention there is provided an immunogenic composition comprising:

[0032] i) a polypeptide comprising SEQ ID NO.: 20, or an antigenic fragment thereof; and

[0033] ii) a polypeptide comprising SEQ ID NO.: 23 and 24, or an antigenic fragment thereof.

[0034] In a preferred embodiment of the invention there is provided an immunogenic composition comprising:

[0035] i) a polypeptide comprising SEQ ID NO.: 20, or an antigenic fragment thereof; and

[0036] ii) a polypeptide comprising SEQ ID NO.: 21 and 22, or an antigenic fragment thereof.

[0037] In a preferred embodiment of the invention there is provided an immunogenic composition comprising:

[0038] i) a polypeptide comprising SEQ ID NO.: 20, or an antigenic fragment thereof; and

[0039] ii) a polypeptide comprising SEQ ID NO.: 22 and 23, or an antigenic fragment thereof.

[0040] In a preferred embodiment of the invention there is provided an immunogenic composition comprising:

[0041] i) a polypeptide comprising SEQ ID NO.: 20, or an antigenic fragment thereof; and

[0042] ii) a polypeptide comprising SEQ ID NO.: 21 and 23, or an antigenic fragment thereof.

[0043] In a preferred embodiment of the invention there is provided an immunogenic composition comprising:

[0044] i) a polypeptide comprising SEQ ID NO.: 20, or an antigenic fragment thereof; and

[0045] ii) a polypeptide comprising SEQ ID NO.: 22 and 24, or an antigenic fragment thereof.

[0046] In a preferred embodiment of the invention there is provided an immunogenic composition comprising:

[0047] i) a polypeptide comprising SEQ ID NO.: 20, or an antigenic fragment thereof; and

[0048] ii) a polypeptide comprising SEQ ID NO.: 21 and 24, or an antigenic fragment thereof.

[0049] In a preferred embodiment of the invention there is provided an immunogenic composition comprising:

[0050] i) a polypeptide comprising SEQ ID NO.: 20, or an antigenic fragment thereof; and

[0051] ii) a polypeptide comprising SEQ ID NO.: 21, 23 and 24, or an antigenic fragment thereof.

[0052] In a preferred embodiment of the invention there is provided an immunogenic composition comprising:

[0053] i) a polypeptide comprising SEQ ID NO.: 20, or an antigenic fragment thereof; and

[0054] ii) a polypeptide comprising SEQ ID NO.: 22, 23 and 24, or an antigenic fragment thereof.

[0055] In a preferred embodiment of the invention there is provided an immunogenic composition comprising:

[0056] i) a polypeptide comprising SEQ ID NO.: 20, or an antigenic fragment thereof; and

[0057] ii) a polypeptide comprising SEQ ID NO.: 21, 22 and 24, or an antigenic fragment thereof.

[0058] In a preferred embodiment of the invention there is provided an immunogenic composition comprising:

[0059] i) a polypeptide comprising SEQ ID NO.: 20, or an antigenic fragment thereof; and

[0060] ii) a polypeptide comprising SEQ ID NO.: 21, 22 and 23, or an antigenic fragment thereof.

[0061] In a preferred embodiment of the invention there is provided an immunogenic composition comprising:

[0062] i) a polypeptide comprising SEQ ID NO.: 20, or an antigenic fragment thereof; and

[0063] ii) a polypeptide comprising SEQ ID NO.: 21, 22, 23 and 24, or an antigenic fragment thereof.

[0064] In an alternative preferred embodiment of the invention there is provided an immunogenic composition comprising:

[0065] i) a polypeptide comprising SEQ ID NO.: 21, or an antigenic fragment thereof; and

[0066] ii) a polypeptide comprising SEQ ID NO.: 22, or an antigenic fragment thereof.

[0067] In a preferred embodiment of the invention there is provided an immunogenic composition comprising:

[0068] i) a polypeptide comprising SEQ ID NO.: 21, or an antigenic fragment thereof; and

[0069] ii) a polypeptide comprising SEQ ID NO.: 23, or an antigenic fragment thereof.

[0070] In a preferred embodiment of the invention there is provided an immunogenic composition comprising:

[0071] i) a polypeptide comprising SEQ ID NO.: 21, or an antigenic fragment thereof; and

[0072] ii) a polypeptide comprising SEQ ID NO.: 24, or an antigenic fragment thereof.

[0073] In a preferred embodiment of the invention there is provided an immunogenic composition comprising:

[0074] i) a polypeptide comprising SEQ ID NO.: 21, or an antigenic fragment thereof; and

[0075] ii) a polypeptide comprising 23 and 24, or an antigenic fragment thereof.

[0076] In a preferred embodiment of the invention there is provided an immunogenic composition comprising:

[0077] i) a polypeptide comprising SEQ ID NO.: 21, or an antigenic fragment thereof; and

[0078] ii) a polypeptide comprising SEQ ID NO.: 22, and 23, or an antigenic fragment thereof.

[0079] In a preferred embodiment of the invention there is provided an immunogenic composition comprising:

[0080] i) a polypeptide comprising SEQ ID NO.: 21, or an antigenic fragment thereof; and

[0081] ii) a polypeptide comprising SEQ ID NO.: 22 and 24, or an antigenic fragment thereof.

[0082] In a preferred embodiment of the invention there is provided an immunogenic composition comprising:

[0083] i) a polypeptide comprising SEQ ID NO.: 21, or an antigenic fragment thereof; and

[0084] ii) a polypeptide comprising SEQ ID NO.: 22, 23 and 24, or an antigenic fragment thereof.

[0085] In an alternative preferred embodiment of the invention there is provided an immunogenic composition comprising:

[0086] i) a polypeptide comprising SEQ ID NO.: 22, or an antigenic fragment thereof; and

[0087] ii) a polypeptide comprising SEQ ID NO.: 23 and 24, or an antigenic fragment thereof.

[0088] In a preferred embodiment of the invention there is provided an immunogenic composition comprising:

[0089] i) a polypeptide comprising SEQ ID NO.: 22, or an antigenic fragment thereof; and

[0090] ii) a polypeptide comprising SEQ ID NO.: 23, or an antigenic fragment thereof.

[0091] In a preferred embodiment of the invention there is provided an immunogenic composition comprising:

[0092] i) a polypeptide comprising SEQ ID NO.: 22, or an antigenic fragment thereof; and

[0093] ii) a polypeptide comprising SEQ ID NO.: 24, or an antigenic fragment thereof.

[0094] In an alternative preferred embodiment of the invention there is provided an immunogenic composition comprising:

[0095] i) a polypeptide comprising SEQ ID NO.: 23, or an antigenic fragment thereof; and

[0096] ii) a polypeptide comprising SEQ ID NO.: 24, or an antigenic fragment thereof.

DETAILED DESCRIPTION

[0097] In a preferred embodiment of the invention said composition is a vaccine composition and includes at least one carrier and/or adjuvant.

[0098] Adjuvants (immune potentiators or immunomodulators) have been used for decades to improve the immune response to vaccine antigens. The incorporation of adjuvants into vaccine formulations is aimed at enhancing, accelerating and prolonging the specific immune response to vaccine antigens. Advantages of adjuvants include the enhancement of the immunogenicity of weaker antigens, the reduction of the antigen amount needed for a successful immunisation, the reduction of the frequency of booster immunisations needed and an improved immune response in elderly and immunocompromised vaccines. Selectively, adjuvants can also be employed to optimise a desired immune response, e.g. with respect to immunoglobulin classes and induction of cytotoxic or helper T lymphocyte responses. In addition, certain adjuvants can be used to promote antibody responses at mucosal surfaces. Aluminium hydroxide and aluminium or calcium phosphate has been used routinely in human vaccines.

[0099] Adjuvants can be classified according to their source, mechanism of action and physical or chemical properties. The most commonly described adjuvant classes are gel-type, microbial, oil-emulsion and emulsifier-based, particulate, synthetic and cytokines. More than one adjuvant may be present in the final vaccine product according to the invention. They may be combined together with a single antigen or all antigens present in the vaccine, or each adjuvant may be combined with one particular antigen. The origin and nature of the adjuvants currently being used or developed is highly diverse. For example, aluminium based adjuvants consist of simple inorganic compounds and PLG is a polymeric carbohydrate. MDP is derived from bacterial cell walls; saponins are of plant origin, squalene is derived from shark liver and recombinant endogenous immunomodulators are derived from recombinant bacterial, yeast or mammalian cells. There are several adjuvants licensed for veterinary vaccines, such as mineral oil emulsions that are too reactive for human use. Similarly, complete Freund's adjuvant, although being one of the most powerful adjuvants known, is not suitable for human use.

[0100] A carrier is an immunogenic molecule which, when bound to a second molecule augments immune responses to the latter. The term carrier is construed in the following manner. A carrier is an immunogenic molecule which, when bound to a second molecule augments immune responses to the latter. Some antigens are not intrinsically immunogenic yet may be capable of generating antibody responses when associated with a foreign protein molecule such as keyhole-limpet haemocyanin or tetanus toxoid. Such antigens contain B-cell epitopes but no T cell epitopes. The protein moiety of such a conjugate (the "carrier" protein) provides T-cell epitopes which stimulate helper T-cells that in turn stimulate antigen-specific B-cells to differentiate into plasma cells and produce antibody against the antigen.

[0101] The vaccine compositions of the invention can be administered by any conventional route, including injection, intranasal spray by inhalation of for example an aerosol or nasal drops. The administration may be, for example, intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous, or intradermally. The vaccine compositions of the invention are administered in effective amounts. An "effective amount" is that amount of a vaccine composition that alone or together with further doses, produces the desired response. In the case of treating a particular bacterial disease the desired response is providing protection when challenged by an infective agent.

[0102] In a preferred embodiment of the invention said vaccine composition is adapted for administration as a nasal spray.

[0103] In a preferred embodiment of the invention said vaccine composition is provided in an inhaler and delivered as an aerosol.

[0104] The amounts of vaccine will depend, of course, on the individual patient parameters including age, physical condition, size and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the individual components or combinations thereof be used sufficient to provoke immunity; that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art, however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons.

[0105] The doses of vaccine administered to a subject can be chosen in accordance with different parameters, in particular in accordance with the mode of administration used and the state of the subject. In the event that a response in a subject is insufficient at the initial doses applied, higher doses (or effectively higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits.

[0106] In general, doses of vaccine are formulated and administered in effective immunizing doses according to any standard procedure in the art. Other protocols for the administration of the vaccine compositions will be known to one of ordinary skill in the art, in which the dose amount, schedule of injections, sites of injections, mode of administration and the like vary from the foregoing. Administration of the vaccine compositions to mammals other than humans, (e.g. for testing purposes or veterinary therapeutic purposes), is carried out under substantially the same conditions as described above. A subject, as used herein, is a mammal, preferably a human, and including a non-human primate, cow, horse, pig, sheep or goat.

[0107] In a preferred embodiment of the invention there is provided a vaccine composition according to the invention that includes at least one additional anti-bacterial agent.

[0108] In a preferred embodiment of the invention said agent is a second different vaccine and/or immunogenic agent (for example a bacterial polypeptide and/or polysaccharide antigen).

[0109] According to a further aspect of the invention there is provided a composition comprising a nucleic acid molecule[s] comprising or consisting of nucleotide sequences of two or more different staphylococcal genes and encoding immunogenic polypeptides selected from the group consisting of:

[0110] i) a nucleic acid molecule comprising or consisting of the nucleotide sequence as represented in SEQ ID NO.: 1 or 6;

[0111] ii) a nucleic acid molecule comprising or consisting of the nucleotide sequence as represented in SEQ ID NO.: 2 or 7;

[0112] iii) a nucleic acid molecule comprising or consisting of the nucleotide sequence as represented in SEQ ID NO.: 3 or 8;

[0113] iv) a nucleic acid molecule comprising or consisting of the nucleotide sequence as represented in SEQ ID NO.: 4 or 9;

[0114] v) a nucleic acid molecule comprising or consisting of the nucleotide sequence as represented in SEQ ID NO.: 5 or 10;

[0115] vi) a nucleic acid molecule comprising or consisting of a nucleotide sequence wherein said sequence is degenerate as a result of the genetic code to the nucleotide sequence defined in i-v above; or

[0116] vii) a nucleic acid molecule the complementary strand of which hybridizes under stringent hybridization conditions to the sequence in SEQ ID NO.: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 and wherein said nucleic acid molecule encodes a staphylococcal antigenic polypeptide.

[0117] Hybridization of a nucleic acid molecule occurs when two complementary nucleic acid molecules undergo an amount of hydrogen bonding to each other. The stringency of hybridization can vary according to the environmental conditions surrounding the nucleic acids, the nature of the hybridization method, and the composition and length of the nucleic acid molecules used. Calculations regarding hybridization conditions required for attaining particular degrees of stringency are discussed in Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 2001); and Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Acid Probes Part I, Chapter 2 (Elsevier, New York, 1993). The T, is the temperature at which 50% of a given strand of a nucleic acid molecule is hybridized to its complementary strand. The following is an exemplary set of hybridization conditions and is not limiting:

Very High Stringency (Allows Sequences that Share at Least 90% Identity to Hybridize)

[0118] Hybridization: 5×SSC at 65° C. for 16 hours

[0119] Wash twice: 2×SSC at room temperature (RT) for 15 minutes each

[0120] Wash twice: 0.5×SSC at 65° C. for 20 minutes each

High Stringency (Allows Sequences that Share at Least 80% Identity to Hybridize)

[0121] Hybridization: 5×-6×SSC at 65° C.-70° C. for 16-20 hours

[0122] Wash twice: 2×SSC at RT for 5-20 minutes each

[0123] Wash twice: 1×SSC at 55° C.-70° C. for 30 minutes each

Low Stringency (Allows Sequences that Share at Least 50% Identity to Hybridize)

[0124] Hybridization: 6×SSC at RT to 55° C. for 16-20 hours

[0125] Wash at least twice: 2×-3×SSC at RT to 55° C. for 20-30 minutes each.

[0126] In a preferred embodiment of the invention said composition is a vaccine composition and includes at least one carrier and/or adjuvant.

[0127] The nucleic acid or DNA combination vaccines comprise nucleic acid molecules that encode antigenic polypeptides as herein disclosed. The specific combinations of polypeptide antigens as represented by amino acid SEQ ID can be substituted for the corresponding nucleotide SEQ ID as herein disclosed in the manufacture of DNA vaccines.

[0128] According to a further aspect of the invention there is provided a combination vaccine according to the invention for use in the protection or treatment of a subject animal to a staphylococcal infection or condition that results from a staphylococcal infection.

[0129] In a preferred embodiment of the invention said staphylococcal infection is caused by a staphylococcal species selected from the group consisting of: S. epidermidis, S. aureus, S. hominis, S. haemolyticus, S. wameri, S. capitis, S. saccharolyticus, S. auricularis, S. simulans, S. saprophyticus, S. cohnii, S. xylosus, S. hyicus, S. caprae, S. gallinarum, and S. intermedius.

[0130] In a further preferred embodiment of the invention said staphylococcal species is S. aureus or S. epidermidis.

[0131] In a preferred embodiment of the invention said subject is a human.

[0132] In an alternative preferred embodiment of the invention said subject is a non-human animal, preferably a livestock animal, for example cattle.

[0133] In a preferred embodiment of the invention said live stock animal is vaccinated against bacterial mastitis caused by staphylococcal bacterial cells.

[0134] In a preferred embodiment of the invention said life stock animal is a caprine animal (e.g. sheep, goat).

[0135] In a preferred embodiment of the invention said life stock animal is a bovine animal (e.g. a cow).

[0136] Staphylococcal mastitis is a serious condition that affects live stock and can result in considerable expense with respect to controlling the disease through administration of antibiotics and in terms of lost milk yield. The vaccine according to the invention provides cost effective control of bacterial, in particular staphylococcal mastitis.

[0137] Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", means "including but not limited to", and is not intended to (and does not) exclude other moieties, additives, components, integers or steps.

[0138] Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0139] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

[0140] An embodiment of the invention will now be described by example only, as described herein.

Materials and Methods

[0141] Construction of Plasmids for the Overexpression in E. coli of the Extramembranous Fragments of the S. aureus Proteins

[0142] The PheP selected peptide was synthesized and conjugated through a cysteine at its C terminal to the carrier protein KLH to undertake as a chimeric protein used in vaccinations. The extramembranous fragments of YdiE, DivIB, DivIC and FtsL were PCR amplified from the chromosome of strain S. aureus SH1000 (Horsburgh M J, Aish J L, White I J, Shaw L, Lithgow J K, Foster S J: sigmaB modulates virulence determinant expression and stress resistance: characterization of a functional rsbU strain derived from Staphylococcus aureus 8325-4. J. Bacteriol. 184:5457-5467, 2002) using oligonucleotide pairs indicated on Table 1 according to the following PCR reaction conditions: 1 initial denaturation cycle of 94° C. for 4 min; 30 amplification cycles of denaturation 94° C. for 30 seconds, annealing 45° C. for 30 seconds, and extension at 72° C. for up to 2.5 minutes; finally, ongoing amplification rounds were allow to complete at 72° C. for 4 min.

[0143] The restrictions sites engineered within the oligonucleotides are also indicated in Table 1 (underlined; Ncol or Xhol). The amplified fragments were digested with the corresponding restriction enzymes (Ncol for the 5' end, and Xhol for the 3' end) and cloned into the equivalent sites of the pET-21d(+) expression vector from Novagen (Cat. No. 69743-3) and resulting in the overexpression plasmids indicated in Table 1 generating a T7-tagged (partial, at the N-terminal) and 6×His-tagged (at the C-terminal end) form of the extramembranous fragments. In the SEQ IDs the T7- and His-tags are indicated in bold, and the extramembranous portion of the proteins of interest are underlined. The over expression plasmids were transferred into E. coli BL21 for over expression of the recombinant protein fragment.

[0144] The cloning of the PCR amplified fragment indicated above into the recipient pET21d(+) recipient plasmid vector at the Ncol and Xhol sites entailed the addition of hinge amino acids between the T7-tag and the extramembranous fragment, and between the latter and the His-tag. These amino acids are neither bold nor underlined in the SEQ IDs.

Over Expression of SEQ ID NOS.: 25-28

[0145] SEQ ID NOS.: 25 through 28 were over expressed from plasmids pGL597, pGL601, pALB26, and pALB27 in E. coli BL21 strain using Brain Heart Infusion Broth (CMO225, Oxoid, United Kingdom) in the presence of 100 μg/ml ampicillin and the Plac promoter gratuitous inducer IPTG (Isopropyl β-D-1-thiogalactopyranoside, 1 mM) for 4 to 6 hours at 37° C. and vigorous shaking. Following harvesting of the cells by centrifugation (5,000×g for 15 minutes at 4° C.) and subsequent lysis with 1 mg/ml lysozyme in phosphate buffer (Buffer A; 0.1M pH7.2) containing 0.5M NaCl) for one hour and subsequent sonication (3 cycles of 10 second pulses in sonicating water bath) the soluble and insoluble forms of the proteins of interest were separated by centrifugation at 13,000×g for 10 minutes. The precipitate was then resuspended in Buffer A containing 8M urea by freeze/thawing (3 cycles of freezing at -80° C. for 10 minutes and subsequent thawing to room temperature) and sonication (3 cycles of 10 second pulses in sonicating water bath), and subsequent centrifugation for 25 minutes at 18,000×g). The over expressed proteins of interest in the supernatant and the solubilised pellet were purified by initial specific binding (through their His-tag) to a nickel (NiSO4)-bound Sepharose chromatography column (Ni-Sepharose) and elution with an imidazole solution run through the column in the following stepwise manner: 5% for 5 minutes, 30% for 60 minutes, 35% for 60 minutes, 50% for 100 minutes and 55% for 100 minutes. Fractions from this stepwise elution were analysed in acrylamide denaturing gels with a 4% acrylamide/bis-acrilamide stacking layer and a 12% acrylamide/bis-acrylamide separating layer. The fractions containing the over expressed proteins of interest were pooled and dyalized against sterile phosphate buffer (8 g NaCl, 0.2 g KCl, 1.44 g Na₂HPO₄, 0.24 g KH₂PO₄, per liter of distilled H₂O, pH 7.4).

[0146] All the proteins of interest were successfully over expressed from the indicated strains and under the indicated conditions. They were also subsequently extracted from the total cellular protein content of the over expressing E. coli strains with more than 95% purity. Examples of the purification obtained for each of the proteins are indicated below.

Evaluation of Vaccination-Mediated Protection of Balb/C Mice Against Infection by S. aureus

[0147] One week after the second boost each animal was infected with an i.v. (tail vein) injection of 100 microliters of endotoxin-free PBS containing 1.1×10⁷ (±0.5×10⁷) cells of S. aureus strain Newman. The latter were prepared from cultures growing to early stationary phase in Brain Heart Infusion medium (BHI), which were then washed three times with the same volume of PBS.

[0148] After 10 to 14 days the animals were sacrificed according to Schedule 1 cervical dislocation. The pair of kidneys from each animal was extracted in aseptic conditions, and homogenized in sterile PBS. Serial dilutions of the kidney homogenates were carried out in PBS and plated on BHI agar plates. Plates containing between 10 to 150 staphylococcal colonies were counted and dilution corrected. The number of viable cells in the kidneys was inferred from the number of colony forming units (CFU) on the plates. Evaluation of the possible protection against infection conferred by vaccination with DivIB-2 was determined from difference in the number of S. aureus cells in the kidneys of animals vaccinated with KLH and those vaccinated with DivIB-2. The statistic significance of the difference was calculated using the Mann-Whitney test. A significantly higher (p<0.05) number of S. aureus in KLH vaccinated animals compared to the DivIB-2 vaccinated animals was concluded as protection.

Vaccination: Generic Protocol for Polyvalent Vaccines

[0149] Combination (or polyvalent) vaccines including variations of the antigens (conjugated selected PheP peptide, YdiE, DivIB, DivIC and FtsL) will follow an identical protocol with the following modifications. The vaccine priming and boost mixtures will contain rather than a single component 2 or more of the components. The total volume of mixed vaccine used for priming and boosting injections will fluctuate in a range of 50-100 microliters per animal. Similarly the total amount in each of those injections may vary between 50-100 micrograms. The amount of each antigen to contribute to the total amount of vaccine in the priming or boosting mix will vary between 20% to 80% of the total.

[0150] The various combinations of antigens to be evaluated as a vaccine mix will be undertaken according to the matrix in Table 2. The combinations are grouped in 3 tiers. Depending on results from the 1^st Tier of experiments the 2^nd Tier of experiments would be undertaken accordingly, and depending on the results from the latter the 3^rd Tier will be undertaken. In each Tier a vaccination experiment will contain an antigen in the Y axis, together with those ticked along the X axis, and labelled with the same colour. Each vaccination experiment is labelled with a different colour.

Example 1

[0151] The experimental sample consisted in a combination antigen of the extracellular domains of YdiE and DivIB. The amount of antigen administered to each mouse (Female Balb/C, approx. 5-6 weeks old) was 5 ug of YdiE plus 50 ug of DivIB. Those amounts were contained within 100 ul of eluent consisting on a 50:50 v:v of PBS (Phosphate Buffer Saline) and Complete Freund's Adjuvant (used for the vaccination priming) or Incomplete Freund's Adjuvant (used for the vaccination boost). Priming was undertaken day 0, Boost 1 at 14 days, and Boost 2 at 21 days. Subsequently, 7 days later, i.e., at day 28 the animals were infected with Staphylococcus aureus strain Newman. Each test group (control and experimental) had 10 animals. The bacterial dose administered to the animals (both, control and experimental) contained 4×10⁶ bacteria in 100 ul of PBS. The infection period was run for 3 days, and the weight of the animals was monitored daily (we also extracted organs to evaluate bacterial loads in organs, results not shown). At that point the animals were sacrificed. The output of the experiment was calculated as the percentage body weight loss between day 3 and day 0 for every animal. See Tables 2-4.

TABLE-US-00001 TABLE 2 Multivalent vaccine experiments: Vaccinogen combinations Antigen Tier 1 FtsL SEQ 5 SEQ10 SEQ19 Tier 2 Tier 3 YdiE DivlB DivlC SEQ24 YdiE DivlB DivlC FtsL YdiE DivlB DivlC FtsL PheP SEQ 1 SEQ 6 SEQ 15 SEQ 20 YdiE SEQ 2 SEQ 7 SEQ 16 SEQ 21 DivlB SEQ 3 SEQ 8 SEQ 17 SEQ 22 DivlC SEQ 4 SEQ 9 SEQ 18 SEQ 23

TABLE-US-00002 TABLE 3 Body Weight of Immunized and Infected Mice % Body Weight Loss Control Sample Experimental Samples I IV Adjuvant II III Combination Animal alone Antigen: Antigen: Antigen: Number (Freunds) RYdiE rDivlB rYdiE rDivlB 1 11.1 11.3 13.1 2.3 2 12.7 4.3 0.0 2.0 3 9.7 9.0 3.5 0.0 4 7.6 12.6 0.5 -4.3 5 9.4 1.0 7.1 -0.5 6 9.9 6.2 3.3 1.8 7 2.0 3.8 2.1 1.5 8 4.2 5.3 1.9 1.4 9 1.0 3.4 1.0 1.6 10 3.1 7.0 6.8 3.9 11 7.7 4.7 -2.5 -0.5

TABLE-US-00003 TABLE 4 CFU in Kidneys of Immunized and Infected Mice Log10 CFUs in Kidneys per animal Control Sample Experimental Samples I IV Adjuvant II III Combination Animal alone Antigen: Antigen: Antigen: Number (Freunds) rYdiE rDivlB rYdiE rDivlB 1 7.82 6.98 5.6 6.04 2 7.73 6.5 6.5 6.12 3 7.7 5.61 4.64 5.97 4 6.98 7.61 5.99 5.61 5 6.74 5.09 6.54 5.38 6 7.32 6.38 6.03 5.88 7 6.5 6.6 6.41 6.74 8 6.2 6.75 6.66 5.96 9 5.61 6.72 7.88 6.98 10 6.06 6.63 6.63 6.68 11 7.04 6.45 4.74

Sequence CWU 1

1

3611455DNAStaphylococcus aureus 1atggaagata ataaaatgaa ccgtagtctt aactcaagac acatttccat gattgctata 60ggtggtgcaa ttgggactgg tctatttgta gctactggta atatcatttc tcaagctggt 120cctggaggcg ctatactcgc ttatcttgtt attggtgtca tgctatattt cttaatgtca 180tcaattggag agttggcaac attttatcca gtatcaggtt cattcagctc ttattcaaca 240cgctttattg actcatctct tggctttacc atgggatggt tgtattgggc attgtggtca 300ttagttacaa gtgttgatgt catagtagcg tcaaatgtgc tttatttctg ggacacattt 360aaatttttcc accccattac ttggagctta atctttatta caattttact attattaaac 420attttttctg taaaatcatt tggagaaact gagttttggt tatcattgat taaagtgtta 480acaattatcg tattcgttat ttttggcttt ttaatgattt tcggtatctt aggtggtcat 540acatatggat ttgaaaacta tacaaaaggc caagcaccgt ttgttggtgg tatctctggt 600ttcttaggcg tattattagt cgccggattt tcggttggtg gtacagaagt agtagcagta 660actgctggtg aatcagatga ccctaaaaag tctatgccta aggcaattaa acaagtattt 720tggcgtattc ttttattcta tgtcttatca attgcagtaa ttggtgcaat tattccgtac 780acagatccat cattattaag agcaagtagt tcaataagtc aaagcccatt tacaattgta 840ttcgatagag taggcatagc ctttgcagca tcagtaatca acgcggttat tttaacttca 900ttattatccg ctgcaaattc aggtgtttat acaacaggca gaatgttgta ttccttaagt 960tcagacaaaa aagcacccca atttttaagt aaattaaaca agacaactaa gttaccttta 1020agagcattat taactactta tgcagtcgtt gttattgtta ttatttatgc aaactttaat 1080tcaaatgccg tttttaattt acttgaaatt attggttcaa tgattatagt tgtttgggga 1140tcaagcattt ggtcacaaat acgattgcga caagctatta aaaaacaagg tcaagaccct 1200aataaggtcc taccatataa agcacctttt tatccattag gaccaatcat tgtcatcact 1260acactattat tcttgctatt tggtggctca gttgaatata ttttaaaaga tcaatggtta 1320aatgctttta aaaacttttt acctttaatc attctagcgt tgatttactt tattcataaa 1380atcattcaca aaacaaaatt tgtaaagcta gaaacaatta atttaaaacc acacgattat 1440gacaatcaaa aataa 145521026DNAStaphylococcus aureus 2atgactaaag atatattaat actagctgtt gaaacaagtt gtgatgaaac aagcgttagt 60gttataaaaa atggcagaga tattttatca aatacagttt taagtcagat tgaaagtcat 120aaacgatttg gcggtgtcgt tcccgaagtg gcaagtagac atcacgttga aggtataaca 180gcaacaataa acgaggctct aggggatgcc gatgtatcaa tagaagatat tgatgccata 240gcggttacag aaggccctgg actaattggt gcgttactaa taggtgttaa tgcagccaaa 300gcattggcat ttgcttacga taagccactt attcctgttc atcatattgc aggacatata 360tatgctaatc acatagaaga gccattaaca ttcccgctaa ttgcacttat tgtttcaggt 420ggacatactg aattagttta tatgaaggat catttatcat ttgaagtcat tggtgaaaca 480cgagatgacg cagtaggtga ggcttatgat aaagtggcac gaacaattgg tttaaattat 540ccaggtggtc cacaagttga tcggttggct gctgaaggtg aagatactta ttcattccct 600cgtgtttggt tggataaaga tagttatgat tttagtttta gtgggttgaa aagtgccgta 660atcaatcaac ttcacaatca acgacaaaaa aatattccaa tcattgaagc taacgtagca 720acgagctttc aaaacagtgt tgtagaggtg ctcacgttta aagctattca agcttgtaaa 780gaatatggtg ttcagcgatt aattgttgct ggtggcgtgg cgagtaataa aggattacgt 840caatctttag cggatcaatg caaagtcaat gacattcaat taactatccc aagtcctaaa 900ttatgcacag ataatgctgc aatgataggc gttgccggcc actatttgta tcagcaaggt 960cgatttgctg atttagcatt aaatgggcac agcaatatag atttagaaga gtattctgca 1020gaataa 102631320DNAStaphylococcus aureus 3atggatgata aaacgaagaa cgatcaacaa gaatcaaatg aagataaaga tgaattagaa 60ttatttacga ggaatacatc taagaaaaga cggcaaagaa aaagatcaaa ggctacacat 120ttttctaatc aaaataaaga tgatacatct caacaagctg attttgatga agaaatttac 180ttgataaata aagacttcaa aaaagaagaa agcaatgata aaaataatga ttctgcttct 240agtcatgcga atgataataa tatcgatgat tctacagact ctaatattga aaatgaggat 300tatagatata atcaagaaat tgacgaccaa aatgaatcga atgtaatttc agtcgacaac 360gaacaacctc aatcagctcc taaagaacaa aatagcgact cgattgatga ggaaacagta 420acgaaaaaag aacgaaaaag taaagtaaca caattaaagc cattaacact tgaagaaaag 480cggaagttaa gacgtaagcg acaaaagcga atccaataca gtgttattac aatattggta 540ttgttgattg ctgttatatt aatttacatg ttttcaccac ttagtaaaat tgcgcatgta 600aatataaatg gaaataatca cgttagtact tcaaagataa acaaagtttt aggtgttaaa 660aatgattcaa ggatgtatac gtttagtaaa aaaaatgcta ttaatgatct cgaagagaat 720ccattaatca aaagtgttga gatacacaag caattaccaa acacattaaa cgtagatatc 780acagaaaatg aaattattgc tttagtgaaa tataaaggta aatatttacc tttattagaa 840aatggtaaat tgcttaaagg ttcaaatgat gtcaaaatta atgatgcacc tgtcatggat 900ggtttcaaag gtacaaaaga agatgatatg attaaggcgt tatctgaaat gacacctgaa 960gttagacgat atattgccga agtgacatac gccccaagta aaaacaaaca aagcagaatt 1020gaattgttta cgacagatgg acttcaagta atcggtgata tttcgacgat atctaagaaa 1080atgaaatatt atccgcagat gtcacaatca ttatcaaggg atagttcggg taaactaaaa 1140acacgaggct atattgattt atcagtcggt gcttcattta tcccataccg tggaaacacg 1200tctagtcaat cagaaagcga taaaaatgtg actaaatcat ctcaagagga aaatcaagca 1260aaagaagaat tacaaagcgt tttaaacaaa attaacaaac aatcaagtaa gaataattaa 13204393DNAStaphylococcus aureus 4atgaaaaata aagtagaaca tatagaaaat cagtacacgt cgcaagagaa caagaaaaaa 60caacgtcaaa aaatgaaaat gcgtgttgtt cgtaggcgta ttacagtatt tgcgggcgta 120ttacttgcga taattgttgt tttatcaatc ttgcttgttg tccaaaaaca tcgcaatgat 180attgatgcac aggagcgaaa agcgaaagaa gcacagtttc aaaagcaaca aaatgaagaa 240attgcgttaa aagaaaagtt gaataatctg aatgacaaag attacattga aaaaattgcg 300cgtgatgatt attacttaag caacaaaggt gaagtgattt ttaggttgcc agaagacaaa 360gattcgtcta gctcaaaatc ttcgaaaaaa taa 3935402DNAStaphylococcus aureus 5atggctgtag aaaaagtgta ccaaccatat gacgaacaag tttataatag tataccgaag 60caacaaccac aaactaagcc cgaaaagaag actgtttcga gaaaagtggt tgtacaatta 120actaaatttg aaaaagtttt atacataact ttgattactg taattgctat gttaagtatt 180tatatgctat ctttaaaaat ggatgcgtat gatacgcgag gaaagattgc agatttagat 240tataaaatag ataaacaatc aagtgaaaac agtgctttac aatctgaaat caaaaagaat 300tcttcttatg aacgcatata cgaaaaggct aagaaacagg ggatgagcct tgagaacgat 360aatgtaaagg tagtgcgtag taatggcgaa gcaaaaaatt aa 402642DNAArtificial Sequenceselected pheP peptide DNA sequence 6ctttatttct gggacacatt taaatttttc caccccatta ct 427687DNAArtificial Sequenceextramembranous ydiE/gcp sequence 7gttcatcata ttgcaggaca tatatatgct aatcacatag aagagccatt aacattcccg 60ctaattgcac ttattgtttc aggtggacat actgaattag tttatatgaa ggatcattta 120tcatttgaag tcattggtga aacacgagat gacgcagtag gtgaggctta tgataaagtg 180gcacgaacaa ttggtttaaa ttatccaggt ggtccacaag ttgatcggtt ggctgctgaa 240ggtgaagata cttattcatt ccctcgtgtt tggttggata aagatagtta tgattttagt 300tttagtgggt tgaaaagtgc cgtaatcaat caacttcaca atcaacgaca aaaaaatatt 360ccaatcattg aagctaacgt agcaacgagc tttcaaaaca gtgttgtaga ggtgctcacg 420tttaaagcta ttcaagcttg taaagaatat ggtgttcagc gattaattgt tgctggtggc 480gtggcgagta ataaaggatt acgtcaatct ttagcggatc aatgcaaagt caatgacatt 540caattaacta tcccaagtcc taaattatgc acagataatg ctgcaatgat aggcgttgcc 600ggccactatt tgtatcagca aggtcgattt gctgatttag cattaaatgg gcacagcaat 660atagatttag aagagtattc tgcagaa 6878741DNAArtificial Sequenceextramembranous divIB sequence, also named in preceding patent DivIB-1 8ccacttagta aaattgcgca tgtaaatata aatggaaata atcacgttag tacttcaaag 60ataaacaaag ttttaggtgt taaaaatgat tcgaggatgt atacgtttag taaaaaaaat 120gctattaatg atctcgaaga ggatccatta atcaaaagtg ttgagataca caagcaatta 180ccaaacacat taaacgtaga tatcacagaa aatgaaatta ttgctttagt gaaatataaa 240ggtaaatatt tacctttatt agaaaatggt aaattgctta aaggttcaaa tgatgtcaaa 300attaatgatg cacctgtcat ggatggtttc aaaggtacaa aagaagatga tatgattaag 360gcgttatctg aaatgacacc tgaagttaga cgatatattg ccgaagtgac atacgcccca 420agtaaaaaca aacaaagcag aattgaattg tttacgacag atggacttca agtaatcggt 480gatatttcga cgatatctaa gaaaatgaaa tattatccgc agatgtcaca atcattatca 540agggatagtt cgggtaaact aaaaacaaga ggctatattg atttatcagt cggtgcttca 600tttatcccat accgtggaaa cacgtctagt caatcagaaa gcgataaaaa tgtgactaaa 660tcatctcaag aggaaaatca agcaaaagaa gaattacaaa gcgttttaaa caaaattaac 720aaacaatcaa gtaagaataa t 7419225DNAArtificial Sequenceextramembranous divIC sequence 9aaacatcgca atgatattga tgcacaggag cgaaaagcga aagaagcaca gtttcaaaag 60caacaaaatg aagaaattgc gttaaaagaa aagttgaata atctgaatga caaagattac 120attgaaaaaa ttgcgcgtga tgattattac ttaagcaaca aaggtgaagt gatttttagg 180ttgccagaag acaaagattc gtctagctca aaatcttcga aaaaa 22510204DNAArtificial Sequenceextramembranous ftsL sequence 10aaaatggatg cgtatgatac gcgaggaaag attgcagatt tagattataa aatagataaa 60caatcaagtg aaaacagtgc tttacaatct gaaatcaaaa agaattcttc ttatgaacgc 120atatacgaaa aggctaagaa acaggggatg agccttgaga acgataatgt aaaggtagtg 180cgtagtaatg gcgaagcaaa aaat 20411720DNAArtificial Sequence(T7-tag / intermediate bases / ydiE extramembranous / intermediate bases / His-tag sequence 11atggctgttc atcatattgc aggacatata tatgctaatc acatagaaga gccattaaca 60ttcccgctaa ttgcacttat tgtttcaggt ggacatactg aattagttta tatgaaggat 120catttatcat ttgaagtcat tggtgaaaca cgagatgacg cagtaggtga ggcttatgat 180aaagtggcac gaacaattgg tttaaattat ccaggtggtc cacaagttga tcggttggct 240gctgaaggtg aagatactta ttcattccct cgtgtttggt tggataaaga tagttatgat 300tttagtttta gtgggttgaa aagtgccgta atcaatcaac ttcacaatca acgacaaaaa 360aatattccaa tcattgaagc taacgtagca acgagctttc aaaacagtgt tgtagaggtg 420ctcacgttta aagctattca agcttgtaaa gaatatggtg ttcagcgatt aattgttgct 480ggtggcgtgg cgagtaataa aggattacgt caatctttag cggatcaatg caaagtcaat 540gacattcaat taactatccc aagtcctaaa ttatgcacag ataatgctgc aatgataggc 600gttgccggcc actatttgta tcagcaaggt cgatttgctg atttagcatt aaatgggcac 660agcaatatag atttagaaga gtattctgca gaactcgagc accaccacca ccaccactga 72012774DNAArtificial SequenceT7-tag / intermediate bases / divIB extramembranous / intermediate bases / His-tag sequence 12atggctccac ttagtaaaat tgcgcatgta aatataaatg gaaataatca cgttagtact 60tcaaagataa acaaagtttt aggtgttaaa aatgattcga ggatgtatac gtttagtaaa 120aaaaatgcta ttaatgatct cgaagaggat ccattaatca aaagtgttga gatacacaag 180caattaccaa acacattaaa cgtagatatc acagaaaatg aaattattgc tttagtgaaa 240tataaaggta aatatttacc tttattagaa aatggtaaat tgcttaaagg ttcaaatgat 300gtcaaaatta atgatgcacc tgtcatggat ggtttcaaag gtacaaaaga agatgatatg 360attaaggcgt tatctgaaat gacacctgaa gttagacgat atattgccga agtgacatac 420gccccaagta aaaacaaaca aagcagaatt gaattgttta cgacagatgg acttcaagta 480atcggtgata tttcgacgat atctaagaaa atgaaatatt atccgcagat gtcacaatca 540ttatcaaggg atagttcggg taaactaaaa acaagaggct atattgattt atcagtcggt 600gcttcattta tcccataccg tggaaacacg tctagtcaat cagaaagcga taaaaatgtg 660actaaatcat ctcaagagga aaatcaagca aaagaagaat tacaaagcgt tttaaacaaa 720attaacaaac aatcaagtaa gaataatctc gagcaccacc accaccacca ctga 77413255DNAArtificial SequenceT7-tag / intermediate bases / divIC extramembranous / intermediate bases / His-tag sequence 13atggctaaac atcgcaatga tattgatgca caggagcgaa aagcgaaaga agcacagttt 60caaaagcaac aaaatgaaga aattgcgtta aaagaaaagt tgaataatct gaatgacaaa 120gattacattg aaaaaattgc gcgtgatgat tattacttaa gcaacaaagg tgaagtgatt 180tttaggttgc cagaagacaa agattcgtct agctcaaaat cttcgaaaaa actcgagcac 240caccaccacc accac 25514234DNAArtificial SequenceT7-tag / intermediate bases / ftsL extramembranous / intermediate bases / His-tag sequence 14atggctaaaa tggatgcgta tgatacgcga ggaaagattg cagatttaga ttataaaata 60gataaacaat caagtgaaaa cagtgcttta caatctgaaa tcaaaaagaa ttcttcttat 120gaacgcatat acgaaaaggc taagaaacag gggatgagcc ttgagaacga taatgtaaag 180gtagtgcgta gtaatggcga agcaaaaaat ctcgagcacc accaccacca ccac 23415484PRTStaphylococcus aureus 15Met Glu Asp Asn Lys Met Asn Arg Ser Leu Asn Ser Arg His Ile Ser 1 5 10 15 Met Ile Ala Ile Gly Gly Ala Ile Gly Thr Gly Leu Phe Val Ala Thr 20 25 30 Gly Asn Ile Ile Ser Gln Ala Gly Pro Gly Gly Ala Ile Leu Ala Tyr 35 40 45 Leu Val Ile Gly Val Met Leu Tyr Phe Leu Met Ser Ser Ile Gly Glu 50 55 60 Leu Ala Thr Phe Tyr Pro Val Ser Gly Ser Phe Ser Ser Tyr Ser Thr 65 70 75 80 Arg Phe Ile Asp Ser Ser Leu Gly Phe Thr Met Gly Trp Leu Tyr Trp 85 90 95 Ala Leu Trp Ser Leu Val Thr Ser Val Asp Val Ile Val Ala Ser Asn 100 105 110 Val Leu Tyr Phe Trp Asp Thr Phe Lys Phe Phe His Pro Ile Thr Trp 115 120 125 Ser Leu Ile Phe Ile Thr Ile Leu Leu Leu Leu Asn Ile Phe Ser Val 130 135 140 Lys Ser Phe Gly Glu Thr Glu Phe Trp Leu Ser Leu Ile Lys Val Leu 145 150 155 160 Thr Ile Ile Val Phe Val Ile Phe Gly Phe Leu Met Ile Phe Gly Ile 165 170 175 Leu Gly Gly His Thr Tyr Gly Phe Glu Asn Tyr Thr Lys Gly Gln Ala 180 185 190 Pro Phe Val Gly Gly Ile Ser Gly Phe Leu Gly Val Leu Leu Val Ala 195 200 205 Gly Phe Ser Val Gly Gly Thr Glu Val Val Ala Val Thr Ala Gly Glu 210 215 220 Ser Asp Asp Pro Lys Lys Ser Met Pro Lys Ala Ile Lys Gln Val Phe 225 230 235 240 Trp Arg Ile Leu Leu Phe Tyr Val Leu Ser Ile Ala Val Ile Gly Ala 245 250 255 Ile Ile Pro Tyr Thr Asp Pro Ser Leu Leu Arg Ala Ser Ser Ser Ile 260 265 270 Ser Gln Ser Pro Phe Thr Ile Val Phe Asp Arg Val Gly Ile Ala Phe 275 280 285 Ala Ala Ser Val Ile Asn Ala Val Ile Leu Thr Ser Leu Leu Ser Ala 290 295 300 Ala Asn Ser Gly Val Tyr Thr Thr Gly Arg Met Leu Tyr Ser Leu Ser 305 310 315 320 Ser Asp Lys Lys Ala Pro Gln Phe Leu Ser Lys Leu Asn Lys Thr Thr 325 330 335 Lys Leu Pro Leu Arg Ala Leu Leu Thr Thr Tyr Ala Val Val Val Ile 340 345 350 Val Ile Ile Tyr Ala Asn Phe Asn Ser Asn Ala Val Phe Asn Leu Leu 355 360 365 Glu Ile Ile Gly Ser Met Ile Ile Val Val Trp Gly Ser Ser Ile Trp 370 375 380 Ser Gln Ile Arg Leu Arg Gln Ala Ile Lys Lys Gln Gly Gln Asp Pro 385 390 395 400 Asn Lys Val Leu Pro Tyr Lys Ala Pro Phe Tyr Pro Leu Gly Pro Ile 405 410 415 Ile Val Ile Thr Thr Leu Leu Phe Leu Leu Phe Gly Gly Ser Val Glu 420 425 430 Tyr Ile Leu Lys Asp Gln Trp Leu Asn Ala Phe Lys Asn Phe Leu Pro 435 440 445 Leu Ile Ile Leu Ala Leu Ile Tyr Phe Ile His Lys Ile Ile His Lys 450 455 460 Thr Lys Phe Val Lys Leu Glu Thr Ile Asn Leu Lys Pro His Asp Tyr 465 470 475 480 Asp Asn Gln Lys 16341PRTStaphylococcus aureus 16Met Thr Lys Asp Ile Leu Ile Leu Ala Val Glu Thr Ser Cys Asp Glu 1 5 10 15 Thr Ser Val Ser Val Ile Lys Asn Gly Arg Asp Ile Leu Ser Asn Thr 20 25 30 Val Leu Ser Gln Ile Glu Ser His Lys Arg Phe Gly Gly Val Val Pro 35 40 45 Glu Val Ala Ser Arg His His Val Glu Gly Ile Thr Ala Thr Ile Asn 50 55 60 Glu Ala Leu Gly Asp Ala Asp Val Ser Ile Glu Asp Ile Asp Ala Ile 65 70 75 80 Ala Val Thr Glu Gly Pro Gly Leu Ile Gly Ala Leu Leu Ile Gly Val 85 90 95 Asn Ala Ala Lys Ala Leu Ala Phe Ala Tyr Asp Lys Pro Leu Ile Pro 100 105 110 Val His His Ile Ala Gly His Ile Tyr Ala Asn His Ile Glu Glu Pro 115 120 125 Leu Thr Phe Pro Leu Ile Ala Leu Ile Val Ser Gly Gly His Thr Glu 130 135 140 Leu Val Tyr Met Lys Asp His Leu Ser Phe Glu Val Ile Gly Glu Thr 145 150 155 160 Arg Asp Asp Ala Val Gly Glu Ala Tyr Asp Lys Val Ala Arg Thr Ile 165 170 175 Gly Leu Asn Tyr Pro Gly Gly Pro Gln Val Asp Arg Leu Ala Ala Glu 180 185 190 Gly Glu Asp Thr Tyr Ser Phe Pro Arg Val Trp Leu Asp Lys Asp Ser 195 200 205 Tyr Asp Phe Ser Phe Ser Gly Leu Lys Ser Ala Val Ile Asn Gln Leu 210 215 220 His Asn Gln Arg Gln Lys Asn Ile Pro Ile Ile Glu Ala Asn Val Ala 225 230 235 240 Thr Ser Phe Gln Asn Ser Val Val Glu Val Leu Thr Phe Lys Ala Ile 245 250 255 Gln Ala Cys Lys Glu Tyr Gly Val Gln Arg Leu Ile Val Ala Gly Gly 260 265 270 Val Ala Ser Asn Lys Gly Leu Arg Gln Ser Leu Ala Asp Gln Cys Lys 275 280 285 Val Asn Asp Ile Gln Leu Thr Ile Pro Ser Pro Lys Leu Cys Thr Asp 290 295 300 Asn Ala Ala Met Ile Gly Val Ala Gly His Tyr Leu Tyr Gln Gln Gly 305 310 315 320 Arg Phe Ala Asp Leu Ala Leu Asn Gly His Ser Asn

Ile Asp Leu Glu 325 330 335 Glu Tyr Ser Ala Glu 340 17439PRTStaphylococcus aureus 17Met Asp Asp Lys Thr Lys Asn Asp Gln Gln Glu Ser Asn Glu Asp Lys 1 5 10 15 Asp Glu Leu Glu Leu Phe Thr Arg Asn Thr Ser Lys Lys Arg Arg Gln 20 25 30 Arg Lys Arg Ser Lys Ala Thr His Phe Ser Asn Gln Asn Lys Asp Asp 35 40 45 Thr Ser Gln Gln Ala Asp Phe Asp Glu Glu Ile Tyr Leu Ile Asn Lys 50 55 60 Asp Phe Lys Lys Glu Glu Ser Asn Asp Lys Asn Asn Asp Ser Ala Ser 65 70 75 80 Ser His Ala Asn Asp Asn Asn Ile Asp Asp Ser Thr Asp Ser Asn Ile 85 90 95 Glu Asn Glu Asp Tyr Arg Tyr Asn Gln Glu Ile Asp Asp Gln Asn Glu 100 105 110 Ser Asn Val Ile Ser Val Asp Asn Glu Gln Pro Gln Ser Ala Pro Lys 115 120 125 Glu Gln Asn Ser Asp Ser Ile Asp Glu Glu Thr Val Thr Lys Lys Glu 130 135 140 Arg Lys Ser Lys Val Thr Gln Leu Lys Pro Leu Thr Leu Glu Glu Lys 145 150 155 160 Arg Lys Leu Arg Arg Lys Arg Gln Lys Arg Ile Gln Tyr Ser Val Ile 165 170 175 Thr Ile Leu Val Leu Leu Ile Ala Val Ile Leu Ile Tyr Met Phe Ser 180 185 190 Pro Leu Ser Lys Ile Ala His Val Asn Ile Asn Gly Asn Asn His Val 195 200 205 Ser Thr Ser Lys Ile Asn Lys Val Leu Gly Val Lys Asn Asp Ser Arg 210 215 220 Met Tyr Thr Phe Ser Lys Lys Asn Ala Ile Asn Asp Leu Glu Glu Asn 225 230 235 240 Pro Leu Ile Lys Ser Val Glu Ile His Lys Gln Leu Pro Asn Thr Leu 245 250 255 Asn Val Asp Ile Thr Glu Asn Glu Ile Ile Ala Leu Val Lys Tyr Lys 260 265 270 Gly Lys Tyr Leu Pro Leu Leu Glu Asn Gly Lys Leu Leu Lys Gly Ser 275 280 285 Asn Asp Val Lys Ile Asn Asp Ala Pro Val Met Asp Gly Phe Lys Gly 290 295 300 Thr Lys Glu Asp Asp Met Ile Lys Ala Leu Ser Glu Met Thr Pro Glu 305 310 315 320 Val Arg Arg Tyr Ile Ala Glu Val Thr Tyr Ala Pro Ser Lys Asn Lys 325 330 335 Gln Ser Arg Ile Glu Leu Phe Thr Thr Asp Gly Leu Gln Val Ile Gly 340 345 350 Asp Ile Ser Thr Ile Ser Lys Lys Met Lys Tyr Tyr Pro Gln Met Ser 355 360 365 Gln Ser Leu Ser Arg Asp Ser Ser Gly Lys Leu Lys Thr Arg Gly Tyr 370 375 380 Ile Asp Leu Ser Val Gly Ala Ser Phe Ile Pro Tyr Arg Gly Asn Thr 385 390 395 400 Ser Ser Gln Ser Glu Ser Asp Lys Asn Val Thr Lys Ser Ser Gln Glu 405 410 415 Glu Asn Gln Ala Lys Glu Glu Leu Gln Ser Val Leu Asn Lys Ile Asn 420 425 430 Lys Gln Ser Ser Lys Asn Asn 435 18130PRTStaphylococcus aureus 18Met Lys Asn Lys Val Glu His Ile Glu Asn Gln Tyr Thr Ser Gln Glu 1 5 10 15 Asn Lys Lys Lys Gln Arg Gln Lys Met Lys Met Arg Val Val Arg Arg 20 25 30 Arg Ile Thr Val Phe Ala Gly Val Leu Leu Ala Ile Ile Val Val Leu 35 40 45 Ser Ile Leu Leu Val Val Gln Lys His Arg Asn Asp Ile Asp Ala Gln 50 55 60 Glu Arg Lys Ala Lys Glu Ala Gln Phe Gln Lys Gln Gln Asn Glu Glu 65 70 75 80 Ile Ala Leu Lys Glu Lys Leu Asn Asn Leu Asn Asp Lys Asp Tyr Ile 85 90 95 Glu Lys Ile Ala Arg Asp Asp Tyr Tyr Leu Ser Asn Lys Gly Glu Val 100 105 110 Ile Phe Arg Leu Pro Glu Asp Lys Asp Ser Ser Ser Ser Lys Ser Ser 115 120 125 Lys Lys 130 19133PRTStaphylococcus aureus 19Met Ala Val Glu Lys Val Tyr Gln Pro Tyr Asp Glu Gln Val Tyr Asn 1 5 10 15 Ser Ile Pro Lys Gln Gln Pro Gln Thr Lys Pro Glu Lys Lys Thr Val 20 25 30 Ser Arg Lys Val Val Val Gln Leu Thr Lys Phe Glu Lys Val Leu Tyr 35 40 45 Ile Thr Leu Ile Thr Val Ile Ala Met Leu Ser Ile Tyr Met Leu Ser 50 55 60 Leu Lys Met Asp Ala Tyr Asp Thr Arg Gly Lys Ile Ala Asp Leu Asp 65 70 75 80 Tyr Lys Ile Asp Lys Gln Ser Ser Glu Asn Ser Ala Leu Gln Ser Glu 85 90 95 Ile Lys Lys Asn Ser Ser Tyr Glu Arg Ile Tyr Glu Lys Ala Lys Lys 100 105 110 Gln Gly Met Ser Leu Glu Asn Asp Asn Val Lys Val Val Arg Ser Asn 115 120 125 Gly Glu Ala Lys Asn 130 2014PRTArtificial Sequenceselected extramembranous PheP sequence 20Leu Tyr Phe Trp Asp Thr Phe Lys Phe Phe His Pro Ile Thr 1 5 10 21229PRTArtificial Sequenceextramembranous YdiE sequence 21Val His His Ile Ala Gly His Ile Tyr Ala Asn His Ile Glu Glu Pro 1 5 10 15 Leu Thr Phe Pro Leu Ile Ala Leu Ile Val Ser Gly Gly His Thr Glu 20 25 30 Leu Val Tyr Met Lys Asp His Leu Ser Phe Glu Val Ile Gly Glu Thr 35 40 45 Arg Asp Asp Ala Val Gly Glu Ala Tyr Asp Lys Val Ala Arg Thr Ile 50 55 60 Gly Leu Asn Tyr Pro Gly Gly Pro Gln Val Asp Arg Leu Ala Ala Glu 65 70 75 80 Gly Glu Asp Thr Tyr Ser Phe Pro Arg Val Trp Leu Asp Lys Asp Ser 85 90 95 Tyr Asp Phe Ser Phe Ser Gly Leu Lys Ser Ala Val Ile Asn Gln Leu 100 105 110 His Asn Gln Arg Gln Lys Asn Ile Pro Ile Ile Glu Ala Asn Val Ala 115 120 125 Thr Ser Phe Gln Asn Ser Val Val Glu Val Leu Thr Phe Lys Ala Ile 130 135 140 Gln Ala Cys Lys Glu Tyr Gly Val Gln Arg Leu Ile Val Ala Gly Gly 145 150 155 160 Val Ala Ser Asn Lys Gly Leu Arg Gln Ser Leu Ala Asp Gln Cys Lys 165 170 175 Val Asn Asp Ile Gln Leu Thr Ile Pro Ser Pro Lys Leu Cys Thr Asp 180 185 190 Asn Ala Ala Met Ile Gly Val Ala Gly His Tyr Leu Tyr Gln Gln Gly 195 200 205 Arg Phe Ala Asp Leu Ala Leu Asn Gly His Ser Asn Ile Asp Leu Glu 210 215 220 Glu Tyr Ser Ala Glu 225 22247PRTArtificial Sequenceextramembranous DivIB sequence, also named in preceding patent DivIB-1 22Pro Leu Ser Lys Ile Ala His Val Asn Ile Asn Gly Asn Asn His Val 1 5 10 15 Ser Thr Ser Lys Ile Asn Lys Val Leu Gly Val Lys Asn Asp Ser Arg 20 25 30 Met Tyr Thr Phe Ser Lys Lys Asn Ala Ile Asn Asp Leu Glu Glu Asp 35 40 45 Pro Leu Ile Lys Ser Val Glu Ile His Lys Gln Leu Pro Asn Thr Leu 50 55 60 Asn Val Asp Ile Thr Glu Asn Glu Ile Ile Ala Leu Val Lys Tyr Lys 65 70 75 80 Gly Lys Tyr Leu Pro Leu Leu Glu Asn Gly Lys Leu Leu Lys Gly Ser 85 90 95 Asn Asp Val Lys Ile Asn Asp Ala Pro Val Met Asp Gly Phe Lys Gly 100 105 110 Thr Lys Glu Asp Asp Met Ile Lys Ala Leu Ser Glu Met Thr Pro Glu 115 120 125 Val Arg Arg Tyr Ile Ala Glu Val Thr Tyr Ala Pro Ser Lys Asn Lys 130 135 140 Gln Ser Arg Ile Glu Leu Phe Thr Thr Asp Gly Leu Gln Val Ile Gly 145 150 155 160 Asp Ile Ser Thr Ile Ser Lys Lys Met Lys Tyr Tyr Pro Gln Met Ser 165 170 175 Gln Ser Leu Ser Arg Asp Ser Ser Gly Lys Leu Lys Thr Arg Gly Tyr 180 185 190 Ile Asp Leu Ser Val Gly Ala Ser Phe Ile Pro Tyr Arg Gly Asn Thr 195 200 205 Ser Ser Gln Ser Glu Ser Asp Lys Asn Val Thr Lys Ser Ser Gln Glu 210 215 220 Glu Asn Gln Ala Lys Glu Glu Leu Gln Ser Val Leu Asn Lys Ile Asn 225 230 235 240 Lys Gln Ser Ser Lys Asn Asn 245 2375PRTArtificial Sequenceextramembranous DivIC sequence 23Lys His Arg Asn Asp Ile Asp Ala Gln Glu Arg Lys Ala Lys Glu Ala 1 5 10 15 Gln Phe Gln Lys Gln Gln Asn Glu Glu Ile Ala Leu Lys Glu Lys Leu 20 25 30 Asn Asn Leu Asn Asp Lys Asp Tyr Ile Glu Lys Ile Ala Arg Asp Asp 35 40 45 Tyr Tyr Leu Ser Asn Lys Gly Glu Val Ile Phe Arg Leu Pro Glu Asp 50 55 60 Lys Asp Ser Ser Ser Ser Lys Ser Ser Lys Lys 65 70 75 2468PRTArtificial Sequenceextramembranous FtsL sequence 24Lys Met Asp Ala Tyr Asp Thr Arg Gly Lys Ile Ala Asp Leu Asp Tyr 1 5 10 15 Lys Ile Asp Lys Gln Ser Ser Glu Asn Ser Ala Leu Gln Ser Glu Ile 20 25 30 Lys Lys Asn Ser Ser Tyr Glu Arg Ile Tyr Glu Lys Ala Lys Lys Gln 35 40 45 Gly Met Ser Leu Glu Asn Asp Asn Val Lys Val Val Arg Ser Asn Gly 50 55 60 Glu Ala Lys Asn 65 25239PRTArtificial SequenceT7-tag / intermediate bases / YdiE extramembranous / intermediate bases / His-tag sequence 25Met Ala Val His His Ile Ala Gly His Ile Tyr Ala Asn His Ile Glu 1 5 10 15 Glu Pro Leu Thr Phe Pro Leu Ile Ala Leu Ile Val Ser Gly Gly His 20 25 30 Thr Glu Leu Val Tyr Met Lys Asp His Leu Ser Phe Glu Val Ile Gly 35 40 45 Glu Thr Arg Asp Asp Ala Val Gly Glu Ala Tyr Asp Lys Val Ala Arg 50 55 60 Thr Ile Gly Leu Asn Tyr Pro Gly Gly Pro Gln Val Asp Arg Leu Ala 65 70 75 80 Ala Glu Gly Glu Asp Thr Tyr Ser Phe Pro Arg Val Trp Leu Asp Lys 85 90 95 Asp Ser Tyr Asp Phe Ser Phe Ser Gly Leu Lys Ser Ala Val Ile Asn 100 105 110 Gln Leu His Asn Gln Arg Gln Lys Asn Ile Pro Ile Ile Glu Ala Asn 115 120 125 Val Ala Thr Ser Phe Gln Asn Ser Val Val Glu Val Leu Thr Phe Lys 130 135 140 Ala Ile Gln Ala Cys Lys Glu Tyr Gly Val Gln Arg Leu Ile Val Ala 145 150 155 160 Gly Gly Val Ala Ser Asn Lys Gly Leu Arg Gln Ser Leu Ala Asp Gln 165 170 175 Cys Lys Val Asn Asp Ile Gln Leu Thr Ile Pro Ser Pro Lys Leu Cys 180 185 190 Thr Asp Asn Ala Ala Met Ile Gly Val Ala Gly His Tyr Leu Tyr Gln 195 200 205 Gln Gly Arg Phe Ala Asp Leu Ala Leu Asn Gly His Ser Asn Ile Asp 210 215 220 Leu Glu Glu Tyr Ser Ala Glu Leu Glu His His His His His His 225 230 235 26257PRTArtificial SequenceT7-tag / intermediate bases / DivIB extramembranous / intermediate bases / His-tag sequence; also termed DivB-2 in a preceding patent 26Met Ala Pro Leu Ser Lys Ile Ala His Val Asn Ile Asn Gly Asn Asn 1 5 10 15 His Val Ser Thr Ser Lys Ile Asn Lys Val Leu Gly Val Lys Asn Asp 20 25 30 Ser Arg Met Tyr Thr Phe Ser Lys Lys Asn Ala Ile Asn Asp Leu Glu 35 40 45 Glu Asp Pro Leu Ile Lys Ser Val Glu Ile His Lys Gln Leu Pro Asn 50 55 60 Thr Leu Asn Val Asp Ile Thr Glu Asn Glu Ile Ile Ala Leu Val Lys 65 70 75 80 Tyr Lys Gly Lys Tyr Leu Pro Leu Leu Glu Asn Gly Lys Leu Leu Lys 85 90 95 Gly Ser Asn Asp Val Lys Ile Asn Asp Ala Pro Val Met Asp Gly Phe 100 105 110 Lys Gly Thr Lys Glu Asp Asp Met Ile Lys Ala Leu Ser Glu Met Thr 115 120 125 Pro Glu Val Arg Arg Tyr Ile Ala Glu Val Thr Tyr Ala Pro Ser Lys 130 135 140 Asn Lys Gln Ser Arg Ile Glu Leu Phe Thr Thr Asp Gly Leu Gln Val 145 150 155 160 Ile Gly Asp Ile Ser Thr Ile Ser Lys Lys Met Lys Tyr Tyr Pro Gln 165 170 175 Met Ser Gln Ser Leu Ser Arg Asp Ser Ser Gly Lys Leu Lys Thr Arg 180 185 190 Gly Tyr Ile Asp Leu Ser Val Gly Ala Ser Phe Ile Pro Tyr Arg Gly 195 200 205 Asn Thr Ser Ser Gln Ser Glu Ser Asp Lys Asn Val Thr Lys Ser Ser 210 215 220 Gln Glu Glu Asn Gln Ala Lys Glu Glu Leu Gln Ser Val Leu Asn Lys 225 230 235 240 Ile Asn Lys Gln Ser Ser Lys Asn Asn Leu Glu His His His His His 245 250 255 His 2785PRTArtificial Sequence(T7-tag / intermediate bases / DivIC extramembranous / intermediate bases / His-tag sequence 27Met Ala Lys His Arg Asn Asp Ile Asp Ala Gln Glu Arg Lys Ala Lys 1 5 10 15 Glu Ala Gln Phe Gln Lys Gln Gln Asn Glu Glu Ile Ala Leu Lys Glu 20 25 30 Lys Leu Asn Asn Leu Asn Asp Lys Asp Tyr Ile Glu Lys Ile Ala Arg 35 40 45 Asp Asp Tyr Tyr Leu Ser Asn Lys Gly Glu Val Ile Phe Arg Leu Pro 50 55 60 Glu Asp Lys Asp Ser Ser Ser Ser Lys Ser Ser Lys Lys Leu Glu His 65 70 75 80 His His His His His 85 2878PRTArtificial SequenceT7-tag / intermediate bases / ftsL extramembranous / intermediate bases / His-tag sequence 28Met Ala Lys Met Asp Ala Tyr Asp Thr Arg Gly Lys Ile Ala Asp Leu 1 5 10 15 Asp Tyr Lys Ile Asp Lys Gln Ser Ser Glu Asn Ser Ala Leu Gln Ser 20 25 30 Glu Ile Lys Lys Asn Ser Ser Tyr Glu Arg Ile Tyr Glu Lys Ala Lys 35 40 45 Lys Gln Gly Met Ser Leu Glu Asn Asp Asn Val Lys Val Val Arg Ser 50 55 60 Asn Gly Glu Ala Lys Asn Leu Glu His His His His His His 65 70 75 2933DNAArtificial Sequencenucleotide primer 29ataataccat ggctgttcat catattgcag gac 333036DNAArtificial Sequencenucleotide primer 30ataatactcg agttctgcag aatactcttc taaatc 363136DNAArtificial Sequencenucleotide primer 31ataataccat ggctccactt agtaaaattg cgcatg 363234DNAArtificial Sequencenucleotide primer 32ataatactcg agattattct tacttgattg tttg 343335DNAArtificial Sequencenucleotide primer 33ataataccat ggctaaacat cgcaatgata ttgat 353433DNAArtificial Sequencenucleotide primer 34ataattctcg agttttttcg aagattttga gct 333535DNAArtificial Sequencenucleotide primer 35ataataccat ggctaaaatg gatgcgtatg atacg 353633DNAArtificial Sequencenucleotide primer 36ataatactcg agattttttg cttcgccatt act 33

Claims

1. A composition, comprising two or more different nucleic acid molecules each encoding an immunogenic staphylococcal extracellular domain polypeptide, wherein the nucleic acid molecules are selected from: (i) a nucleic acid molecule comprising or consisting of a nucleotide sequence as represented in SEQ ID NO.:7; (ii) a nucleic acid molecule comprising or consisting of a nucleotide sequence as represented in SEQ ID NO.:8; (iii) a nucleic acid molecule comprising or consisting of a nucleotide sequence as represented in SEQ ID NO.:9; (iv) a nucleic acid molecule comprising or consisting of a nucleotide sequence as represented in SEQ ID NO.:10; (v) any one of the nucleic acids of (i) to (iv), wherein the nucleotide sequence is degenerate as a result of the genetic code, provided that the degenerate nucleotide sequence encodes the same staphylococcal immunogenic extracellular domain polypeptide as the nucleic acid molecules of (i) to (iv), respectively; (vi) any one of the nucleic acid molecules of (i) to (iv), wherein the nucleic acid molecule comprises or consists of a nucleotide sequence having at least 90% identity to the nucleotide sequence of the nucleic acid molecules of (i) to (iv), respectively; and (vii) a nucleic acid molecule, wherein the complementary strand of the nucleic acid molecule hybridizes under stringent hybridization conditions to the nucleotide sequence as represented in SEQ ID NO.:7, 8, 9 or 10, and wherein the nucleic acid molecule encodes an immunogenic staphylococcal extracellular domain polypeptide.

2. An immunogenic composition, comprising: (i) an immunogenic extracellular domain polypeptide, or immunogenic fragment thereof, comprising or consisting of the amino acid sequence as represented in SEQ ID NO.:21, and an immunogenic extracellular domain polypeptide, or immunogenic fragment thereof, comprising or consisting of the amino acid sequence as represented in SEQ ID NO.:23; (ii) an immunogenic extracellular domain polypeptide, or immunogenic fragment thereof, comprising or consisting of the amino acid sequence as represented in SEQ ID NO.:21, and an immunogenic extracellular domain polypeptide, or immunogenic fragment thereof, comprising or consisting of the amino acid sequence as represented in SEQ ID NO.:24; (iii) an immunogenic extracellular domain polypeptide, or immunogenic fragment thereof, comprising or consisting of the amino acid sequence as represented in SEQ ID NO.:22, and an immunogenic extracellular domain polypeptide, or immunogenic fragment thereof, comprising or consisting of the amino acid sequence as represented in SEQ ID NO.:23; (iv) an immunogenic extracellular domain polypeptide, or immunogenic fragment thereof, comprising or consisting of the amino acid sequence as represented in SEQ ID NO.:22, and an immunogenic extracellular domain polypeptide, or immunogenic fragment thereof, comprising or consisting of the amino acid sequence as represented in SEQ ID NO.:24; (v) an immunogenic extracellular domain polypeptide, or immunogenic fragment thereof, comprising or consisting of the amino acid sequence as represented in SEQ ID NO.:23, and an immunogenic extracellular domain polypeptide, or immunogenic fragment thereof, comprising or consisting of the amino acid sequence as represented in SEQ ID NO.:24; (vi) an immunogenic extracellular domain polypeptide, or immunogenic fragment thereof, comprising or consisting of the amino acid sequence as represented in SEQ ID NO.:21, an immunogenic extracellular domain polypeptide, or immunogenic fragment thereof, comprising or consisting of the amino acid sequence as represented in SEQ ID NO.:23, and an immunogenic extracellular domain polypeptide, or immunogenic fragment thereof, comprising or consisting of the amino acid sequence as represented in SEQ ID NO.:24; (vii) an immunogenic extracellular domain polypeptide, or immunogenic fragment thereof, comprising or consisting of the amino acid sequence as represented in SEQ ID NO.:22, an immunogenic extracellular domain polypeptide, or immunogenic fragment thereof, comprising or consisting of the amino acid sequence as represented in SEQ ID NO.:23, and an immunogenic extracellular domain polypeptide, or immunogenic fragment thereof, comprising or consisting of the amino acid sequence as represented in SEQ ID NO.:24; or (viii) any one of the compositions of (i) to (vii), wherein the immunogenic extracellular domain polypeptide or immunogenic fragment thereof is an immunogenic extracellular domain polypeptide variant having at least 90% identity to the amino acid sequence presented in the corresponding SEQ ID NO.

3. The immunogenic composition according to claim 2, comprising: (i) an immunogenic extracellular domain polypeptide comprising SEQ ID NO.:21, or an antigenic fragment thereof; and (ii) an immunogenic extracellular domain polypeptide comprising SEQ ID NO.:23, or an antigenic fragment thereof.

4. The immunogenic composition according to claim 2, comprising: (i) an immunogenic extracellular domain polypeptide comprising SEQ ID NO.:21, or an antigenic fragment thereof; and (ii) an immunogenic extracellular domain polypeptide comprising SEQ ID NO.:24, or an antigenic fragment thereof.

5. The immunogenic composition according to claim 2, comprising: (i) an immunogenic extracellular domain polypeptide comprising SEQ ID NO.:21, or an antigenic fragment thereof; and (ii) an immunogenic extracellular domain polypeptide comprising SEQ ID NO.:23, or an antigenic fragment thereof, and (iii) an immunogenic extracellular domain polypeptide comprising SEQ ID NO.:24, or an antigenic fragment thereof.

6. The immunogenic composition according to claim 2, comprising (i) an immunogenic extracellular domain polypeptide comprising SEQ ID NO.:22, or an antigenic fragment thereof; and (ii) an immunogenic extracellular domain polypeptide comprising SEQ ID NO.:23, or an antigenic fragment thereof.

7. The immunogenic composition according to claim 2, comprising: (i) an immunogenic extracellular domain polypeptide comprising SEQ ID NO.:22, or an antigenic fragment thereof; and (ii) an immunogenic extracellular domain polypeptide comprising SEQ ID NO.:24, or an antigenic fragment thereof.

8. The immunogenic composition according to claim 2, comprising: (i) an immunogenic extracellular domain polypeptide comprising SEQ ID NO.:22, or an antigenic fragment thereof; and (ii) an immunogenic extracellular domain polypeptide comprising SEQ ID NO.:23, or an antigenic fragment thereof, and (iii) an immunogenic extracellular domain polypeptide comprising SEQ ID NO.:24, or an antigenic fragment thereof.

9. The immunogenic composition according to claim 2, comprising: (i) an immunogenic extracellular domain polypeptide comprising SEQ ID NO.:23, or an antigenic fragment thereof; and (ii) an immunogenic extracellular domain polypeptide comprising SEQ ID NO.:24, or an antigenic fragment thereof.

10. The immunogenic composition according to claim 2, further comprising a carrier, an adjuvant or both.

11. The immunogenic composition according to claim 2, wherein the composition is a vaccine.

12. The immunogenic composition according to claim 11, wherein the vaccine is adapted for administration as a nasal spray.

13. The immunogenic composition according to claim 12, wherein the vaccine is provided in an inhaler and delivered as an aerosol.

14. A method of treating a staphylococcal infection in an animal subject, comprising administering an effective amount of an immunogenic composition according to claim 1.

15. The method of claim 14, wherein the immunogenic composition further comprises a carrier, an adjuvant or both.

16. The method of claim 15, wherein the adjuvant is aluminium phosphate.

17. The method of claim 14, wherein the immunogenic composition further comprises an adjuvant and the composition is formulated as a gel.

18. The immunogenic composition according to claim 14, wherein the immunogenic composition is administered as an aerosol or nasal spray.

19. The immunogenic composition according to claim 14, wherein the staphylococcal infection is caused by a staphylococcal species selected from the group consisting of: S. epidermidis, S. aureus, S. hominis, S. haemolyticus, S. wameri, S. capitis, S. saccharolyticus, S. auricularis, S. simulans, S. saprophyticus, S. xylosus, S. hyicus, S. caprae, S. gallinarum, and S. intermedius.

20. The immunogenic composition according to claim 14, wherein the animal subject is human, caprine or bovine.

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