FUSION POLYPEPTIDES DERIVED FROM STAPHYLOCOCCUS AUREUS ANTIGENS

Abstract

The present invention relates to fusion polypeptides comprising polypeptides derived from Staphylococcus aureus antigens, as well as vectors constructs comprising nucleic acid molecules encoding the fusion polypeptides. More particularly, the fusion polypeptides comprise: (i) a first polypeptide, wherein the first polypeptide is an EapH1 polypeptide, or a derivative or variant thereof; and (ii) a second polypeptide, wherein the second polypeptide is an EapH2 polypeptide, or a derivative or variant thereof. The invention also relates to the use of these fusion polypeptides and vectors, inter alia, as immunogenic compositions, particularly as vaccine compositions.

Claims

1. A fusion polypeptide comprising: (i) a first polypeptide, wherein the first polypeptide is an EapH1 polypeptide, or a derivative or variant thereof; and (ii) a second polypeptide, wherein the second polypeptide is an EapH2 polypeptide, or a derivative or variant thereof.

2. A fusion polypeptide as claimed in claim 1, wherein the first polypeptide: (a) is a polypeptide which has an amino acid sequence having at least 80%, 85%, 90%, 93%, 95%, 99% or 100% identity to SEQ ID NO: 2 or SEQ ID NO: 4, or (b) is a fragment of polypeptide (a), wherein the first polypeptide has the ability to induce antibodies which bind to a Staphylococcus EapH1 polypeptide.

3. A fusion polypeptide as claimed in claim 2, wherein the first polypeptide is a polypeptide which has an amino acid sequence having at least 93% amino acid sequence identity to SEQ ID NO: 4.

4. A fusion polypeptide as claimed in any one of the preceding claims, wherein the second polypeptide: (c) is a polypeptide which has an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 99% or 100% identity to SEQ ID NO: 6 or SEQ ID NO: 8, or (d) is a fragment of polypeptide (c), wherein the second polypeptide has the ability to induce antibodies which bind to a Staphylococcus EapH2 polypeptide.

5. A fusion polypeptide as claimed in claim 4, wherein the second polypeptide is a polypeptide which has an amino acid sequence having at least 97% amino acid sequence identity to SEQ ID NO: 8.

6. A fusion polypeptide as claimed in any one of the preceding claims, wherein the fusion polypeptide additionally comprises: (iii) a third polypeptide, wherein the third polypeptide comprises at least one Eap MAP domain, or a derivative or variant thereof.

7. A fusion polypeptide as claimed in claim 6, wherein the third polypeptide: (e) is a polypeptide which has an amino acid sequence having at least 80%, 85%, 90% or 95% identity to SEQ ID NO: 9, or (f) is a fragment of polypeptide (e), wherein the third polypeptide has the ability to induce antibodies which bind to a Staphylococcus Eap polypeptide.

8. A fusion polypeptide as claimed in any one of the preceding claims, wherein the fusion polypeptide additionally comprises a BitC, EsxA, ClfB, Spa and/or Hla polypeptide.

9. A virus-like particle comprising a fusion polypeptide as claimed in any one of claims 1 to 8.

10. An antibody against a fusion polypeptide as claimed in any one of claims 1 to 8, wherein the antibody: (a) disrupts or prevents binding of EapH1 to EapH2 or to Eap; or (b) disrupts or prevents binding of EapH2 to EapH1 or to Eap.

11. A nucleic acid molecule which codes for a fusion polypeptide or a virus-like particle as claimed in any one of claims 1 to 9.

12. A vector or plasmid comprising a nucleic acid molecule as claimed in claim 11, preferably wherein the vector is an adenoviral vector or a Modified Vaccinia Ankara viral vector, most preferably a non-replicating adenoviral vector or a non-replicating Modified Vaccinia Ankara viral vector.

13. A host cell comprising a vector or plasmid as claimed in claim 11, preferably wherein the host cell is a mammalian host cell, more preferably a yeast host cell.

14. A pharmaceutical composition comprising a fusion polypeptide or a virus-like particle as claimed in any one of claims 1 to 9, an antibody as claimed in claim 10, a nucleic acid molecule as claimed in claim 11, or a vector or plasmid as claimed in claim 12, optionally together with one or more pharmaceutically-acceptable carriers, excipients or diluents.

15. A vaccine composition comprising a fusion polypeptide or virus-like particle as claimed in any one of claims 1 to 9, a nucleic acid molecule as claimed in claim 11, or a vector or plasmid as claimed in claim 12, together with a pharmaceutically-acceptable adjuvant.

16. A fusion polypeptide or virus-like particle as claimed in any one of claims 1 to 9, an antibody as claimed in claim 10, a nucleic acid molecule as claimed in claim 11, or a vector or plasmid as claimed in claim 11, for use in therapy or for use as a medicament.

17. A fusion polypeptide or virus-like particle as claimed in any one of claims 1 to 9, an antibody as claimed in claim 10, a nucleic acid as claimed in claim 11, or a vector or plasmid as claimed in claim 11, for use in a method of preventing or treating a Staphylococcus aureus infection in a subject, for use in a method of preventing kidney abscesses due to a Staphylococcus aureus infection in a subject, for use in a method of reducing carriage of Staphylococcus aureus bacteria in a subject or for use in a method of inducing a T-cell response or a B-cell response to a Staphylococcus aureus antigen in a subject.

18. Use of a fusion polypeptide or virus-like particle as claimed in any one of claims 1 to 9, an antibody as claimed in claim 10, a nucleic acid molecule as claimed in claim 11, or a vector or plasmid as claimed in claim 11, in the manufacture of a medicament for use in a method of preventing or treating a Staphylococcus aureus infection in a subject, for use in a method of preventing kidney abscesses due to a Staphylococcus aureus infection in a subject, for use in a method of reducing carriage of Staphylococcus aureus bacteria in a subject, or for use in a method of inducing a T-cell response or a B-cell response to a Staphylococcus aureus antigen in a subject.

19. A method of treating a subject susceptible to Staphylococcus aureus infection comprising administering an effective amount of a fusion polypeptide or virus-like particle as claimed in any one of claims 1 to 9, an antibody as claimed in claim 10, a nucleic acid molecule as claimed in claim 11, a vector or plasmid as claimed in claim 11, or a composition as claimed in claim 14, to the subject.

20. A method of preventing kidney abscesses due to a Staphylococcus aureus infection in a subject comprising administering an effective amount of a fusion polypeptide or virus-like particle as claimed in any one of claims 1 to 9, an antibody as claimed in claim 10, a nucleic acid molecule as claimed in claim 11, a vector or plasmid as claimed in claim 11, or a composition as claimed in claim 14, to the subject.

21. A method of reducing carriage of Staphylococcus aureus bacteria in a subject comprising administering an effective amount of a fusion polypeptide or virus-like particle as claimed in any one of claims 1 to 9, an antibody as claimed in claim 10, a nucleic acid molecule as claimed in claim 11, a vector or plasmid as claimed in claim 11, or a composition as claimed in claim 14, to the subject.

22. A method of inducing a T-cell response or a B-cell response to a Staphylococcus aureus antigen in a subject comprising administering an effective amount of a fusion polypeptide or virus-like particle as claimed in any one of claims 1 to 9, an antibody as claimed in claim 10, a nucleic acid molecule as claimed in claim 11, a vector or plasmid as claimed in claim 11, or a composition as claimed in claim 14, to the subject.

23. A method of preventing or treating a Staphylococcus aureus infection in a subject, of inducing a T-cell response or a B-cell response to a Staphylococcus aureus antigen in a subject, the method comprising the steps of: (i) administering a first priming amount of a fusion polypeptide or virus-like particle as claimed in any one of claims 1 to 9, an antibody as claimed in claim 10, a nucleic acid molecule as claimed in claim 11, a vector or plasmid as claimed in claim 11, or a composition as claimed in claim 14, to the subject; and then (ii) administering a second boosting amount of a fusion polypeptide as claimed in any one of claims 1 to 9, an antibody as claimed in claim 10, a nucleic acid molecule as claimed in claim 11, a vector or plasmid as claimed in claim 11, or a composition as claimed in claim 14, to the subject.

24. A method as claimed in claim 23, the method comprising: (i) administering a vector as claimed in claim 11, wherein the vector is an adenovirus vector (e.g. AdHu5) to the subject; and then (ii) administering a vector as claimed in claim 11, wherein the vector is a non-replicating poxvirus vector to the subject (e.g. MVA).

25. A process for the production of a fusion polypeptide or virus-like particle as claimed in any one of claims 1 to 9, which process comprises expressing a nucleic acid molecule coding for said fusion polypeptide or virus-like particle in a suitable host and recovering the produced fusion polypeptide or virus-like particle.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0247] FIG. 1: Sequence diversity of EapH proteins in 104 S. aureus isolates

[0248] The presence and conservation of EapH1, EapH2, and comparator proteins in a set of 104 sequenced clinical S. aureus isolates representative of major S. aureus clones was determined by tblastn against de novo assemblies of Illumina sequencing of the clones. N=x/104 indicates that a homologue matching the query over 95% of the protein length was found in x sequences.

[0249] FIG. 2: Interaction between EapH proteins and Eap

[0250] Recombinant EapH and Eap proteins were produced from E. coli, and a C-terminal 6-HIS tag was either cleaved off or retained. Cleaved 6-HIS tags were removed by dialysis (A). Untagged proteins were immobilised on ELISA plates and His-Tagged partners applied at a range of concentrations (B-D). Following washing, detection of binding of a His-tagged ligand was detected by anti-His alkaline phosphatase polyclonal antibody (B). The extracellular domains of SAUSA300_2132 and SAUSA300_1795, two cell surface S. aureus proteins, were immobilised on the plate as negative controls. A model of the complex predicted is shown in E.

[0251] FIG. 3: Production of recombinant EapH proteins from viral vectors

[0252] Domains from the physiological Eap, EapH1, and EapH2 proteins (A) were expressed from adenovirus and modified Vaccinia Ankara (MVA) vectors behind a mammalian signal sequence, and fused to V5 epitope tag and IMX313 multimerising domain (B). V5 and IMX313 tagged proteins were detected from both supernatant (C) and cell lysates (D) following infection of HeLa cells with adenoviruses expressing these constructs.

[0253] FIG. 4: Immunogenicity of Adenovirus-MVA regimes expressing EapH proteins

[0254] Balb/c mice were vaccinated with adenovirus Hu5 expressing no antigen (control) or expressing EapH1_2, Eap domain 5, or EapH1_2_Eap constructs. Specific antibody against (B) EapH1 and (C) EapH2 were determined by LIPS on day 70. Anti-Eap end-point titres were determined on a random subset of animals from each group by ELISA (D). 3 days following i.v. challenge, the number of abscesses in the right kidney was determined by post-mortem MRI (E), and bacterial recovery from the left kidney determined (F).

[0255] FIG. 5: Protection following Adenovirus-MVA regimes expressing EapH1 and H2

[0256] Balb/c mice were vaccinated with adenovirus Hu5 expressing no antigen (control) or EapH1_2 antigen. The regime is identical to that shown in FIG. 4, and the experiment shown in FIG. 4 is summarised as experiment 1 here, along with two further larger experiments. Bacterial recovery from left kidney (A). Associations between renal bacterial load and peripheral blood anti-EapH1/H2 -IFN ELISPOT (B), anti-EapH1 antibody (C) and anti-EapH2 antibody (D) on day 70.

[0257] FIG. 6: Intranasal vaccination with vectors expressing EapH1 and H2

[0258] CD1 mice were vaccinated intranasally with a single dose of a viral vector (AdHu5 or MVA) vector expressing EapH1 and EapH2, or a control. Serological responses were monitored in tail bleeds. (A). Antibody responses against EapH1 and EapH2 were measured (B, C). 26 days after vaccination, mice were exposed to S. aureus by environmental contamination, and carriage of S. aureus monitored. Carriage levels at 1 day (D) and 28 days (E) after S. aureus exposure.

[0259] FIG. 7: Human serological responses to EapH1 and EapH2

[0260] IgG recognising EapH1, EapH2 and four control antigens (the cell surface proteins IsdA, IsdB, ClfB and the nuclease Nuc1) was quantified in a cohort of 42 humans, randomly selected from cohort studies, including 19 carriers (identified by having two S. aureus nasal swabs positive) and 23 non-carriers (A). Antibodies against EapH1 and EapH2 were also quantified (B). Correlations between antibody titres from the 4 control antigens, EapH1 and EapH2 with significant correlations (Spearman's rho differs from 0, p<0.01) indicated by plotting of a regression line (C). Carrier and non-carrier human EapH1 (D) and EapH2 (E) antibody titres relative to responses to control antigens (IsdA, IsdB, ClfB, Nuc1) were measured.

[0261] FIG. 8: Interaction between EapH proteins and Eap

[0262] Recombinant EapH and Eap proteins were produced from HEK293 cells, fused to either a C-terminal V5 tag or to Renilla luciferase. V5 tagged proteins were captured onto anti-V5 coated plate, and Renilla luciferase-ligands incubated at a range of concentrations.

[0263] FIG. 9: Stool carriage following intramuscular viral vector vaccination

[0264] Balb/c mice were vaccinated with adenovirus H5 expressing no antigen (control) or EapH1_2 antigen. Data on immunogenicity and impact on bacterial load is shown in FIG. 5. The regime is identical to that shown in FIG. 4, and the experiment shown in FIG. 4 is summarised as experiment 1 here, along with two further larger experiments. Effect of vaccination on proportion of mice with positive S. aureus bacterial recovery from stool is shown for the three experiments.

[0265] FIG. 10: Stool carriage following intranasal viral vector vaccination

[0266] CD1 mice were vaccinated intranasally with a single dose of a viral vector (AdHu5 or MVA) vector expressing EapH1 and EapH2, or a control. The experiment consisted of eight cages of mice; one mouse per cage received a different treatment. Stool counts in mice receiving adenovirus expressing either Control or EapH1_2 are shown, following exposure to S. aureus by environmental contamination.

EXAMPLES

[0267] The present invention is further illustrated by the following Examples, in which parts and percentages are by weight and degrees are Celsius, unless otherwise stated. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

Example 1: Materials and Methods

Bioinformatic Analyses

[0268] Velvet-assembled contigs from Illumina next-generation sequencing of a collection of S. aureus strains [26] were interrogated by tblastn (NCBI Blast suite v. 2.4.0) using default parameters. Ungapped matches to more than 80% of the query with e<10.sup.10 were considered significant.

Bacterial Strains and Growth

[0269] Staphylococcus aureus strain Newman was kindly provided by Prof. T. Foster, Trinity College, Dublin (Ireland). For infection of mice, S. aureus Newman were grown on Horse Blood Agar (HBA, Oxoid, UK), three colonies were inoculated into 10 mL tryptic soy broth (TSB, Oxoid, UK) and grown overnight at 37 C., 130 rpm. The resulting culture was subcultured 1:100 into 10 mL of fresh TSB and incubated statically at 37 C. for 2.5 h. Bacteria were harvested by centrifugation, washed once and then resuspended in 10 mL Phosphate Buffered Saline (PBS, Sigma Aldrich, UK) at approximately 110.sup.8 cells/mL. The actual bacterial concentrations were determined by dilution plating.

Antigens and Viral Vectors

[0270] Vaccine Eap contained C-terminal MAP domain of S. aureus Eap protein with an adjacent C-terminal basic region (a. a. 481-584 of WP_001549158.1); vaccine EapH_1_2 contained MAP domains of EapH1 (a. a. 24-141 of WP_001549607.1) and EapH2 (a. a. 24-144 of WP_000769689.1); vaccine Eap_EapH1_EapH2 was composed of all three antigens (Table 1). DNA sequences encoding vaccine constructs were human codon optimized and combined with Human Tissue Plasminogen Activator leader signal sequence (TPA), V5 epitope tag and IMX313 oligomerization domain (Spencer et al, 2012). DNA strings were synthesised by Life Technologies Ltd. using GeneArt Gene Synthesis and subcloned by restriction digest into a pMono2 mammalian expression vector (WO2014/053861A2). For Adenovirus vaccines antigen constructs or pMono2 backbone (for control vaccine) were subcloned from pMono2 into pAdH5-PL-pDEST shuttle vector using Gateway technology (Life Technologies), linearized by PacI and transfected into replication-deficient adenovirus human serotype 5 (AdHu5) as described elsewhere (Draper et al., 2008; Gilbert et al., 2002). For Modified Vaccinia Ankara (MVA) vaccines, antigens or pMono2 backbone were transferred into an MVA shuttle vector by restriction digestion, linearized with AatII and transfected into MVA (Draper et al., 2008; Gilbert et al., 2002). All inserts were confirmed by sequencing.

TABLE-US-00001 TABLE 1 log2 fold change in protein concentration in Amino acids supernatant in USA300 used in rsp mutants of Protein NCBI Locus id vaccine USA300 Eap WP_001549158.1 Map 481 . . . 584 ND (pseudogene) EapH1 WP_001549607.1 SAUSA300_2614 24 . . . 141 3.53 EapH2 WP_000769689.1 SAUSA300_0883 24 . . . 144 3.83

Testing Antigen Production by Viral Vectors

[0271] HeLa cells we seeded at 10.sup.5 cells per well on the 24 well plate and allowed to grow overnight. The media in the wells was replaced by 210.sup.8, 210.sup.7 or 210.sup.6 I.U. of adenovirus preparation (in 250 l) and incubated for 2 h. Further 250 l of media was added to the wells and the plates were incubated overnight. All incubation steps were done at 37 C., 5% COO.sub.2. 96-well NUNC plate was coated with 1:500 dilution of mouse anti-V5 antibody (Abcam) in PBS, blocked with 1% BSA/PBS and incubated with 50 l of supernatant from adenovirus-infected HeLa cells. They were further incubated with 1:1000 dilution of serum from rabbits immunized with IMX313 protein (kindly provided by IMAXIO) and 1:500 dilution of the goat anti-rabbit IgG HRP conjugated antibody (Jackson ImmnoResearch). Plates were washed with PBS/0.05% Tween after each incubation, developed with TMB Substrate Solution (ThermoFisher), stopped with 2M H.sub.2SO.sub.4 and read at 450 nm.

Recombinant Protein Production

[0272] DNA strings encoding C-terminal MAP domain and adjacent basic region of Eap and MAP domains of EapH1 and EapH2 with N-terminal His-tag and 3C protease site were ordered from GeneArt Gene Synthesis (ThermoFisher) (Table 2). DNA strings were inserted into the pOPIN-F vector (kindly provided by the Dr Ray Owens, Oxford University, Oxford, UK) via their complimentary sites using In-Fusion reaction (Clontech). The resulting plasmids were transformed into DH5a Competent cells (Thermofisher). Plasmids were purified from transformed cells using QIAprep Spin Miniprep Kit (QIAGEN) and insert sequence was validated by Sanger sequencing using T7 forward and TriEx reverse primers. Purified plasmids were transformed into One Shot BL21 Star (DE3) Chemically Competent E. coli cells according to the manufacturer's protocol. Protein expression was done using Overnight Express Autoinduction System 1 (Novagen). Proteins were purified using BugBuster Ni-NTA His*Bind Purification Kit (Merck Millipore) and dialysed against PBS using Slide-A-Lyzer G2 Dialysis Cassettes, 7K MWCO (Thermofisher). On average 2 to 10 mg of protein was obtained from 50 ml culture.

TABLE-US-00002 TABLE2 Sequencessynthesised Gene Sequenceusedinviralvector Eap aagttctgtttcagggcccgGTTCCGTACACCATCGCGGTTAACGGTACCTCTACCCCGA SEQID TCCTGTCTAAACTGAAAATCTCTAACAAACAGCTGATCTCTTACAAATACCTGAACGACA NO:15 AAGTTAAATCTGTTCTGAAATCTGAACGTGGTATCTCTGACCTGGACCTGAAATTCGCGA AACAGGCGAAATACACCGTTTACTTCAAAAACGGTAAAAAACAGGTTGTTAACCTGAAAT CTGACATCTTCACCCCGAACCTGTTCTCTGCGAAAGACATCAAAAAAATCGACATCGACG TTAAACAGTACACCAAATCTAAAAAAAACAAAtaaagctttctagaccat EapH1 aagttctgtttcagggcccgCCGTCTCACGAAGCGTCTGCGGACTCTAACAACGGTTACA SEQID AAGAAATGACCGTTGACGGTTACCACACCGTTCCGTACACCATCTCTGTTGACGGTATCA NO:16 CCGCGCTGCACCGTACCTACTTCATCTTCCCGGAAAACAAAAACGTTCTGTACCAGGAAA TCGACTCTAAAGTTAAAAACGAACTGGCGTCTCAGCGTGGTGTTACCACCGAAAAAATCA ACAACGCGCAGACCGCGACCTACACCCTGACCCTGAACGACGGTAACAAAAAAGTTGTTA ACCTGAAAAAAAACGACGACGCGAAAAACTCTATCGACCCGTCTACCATCAAACAGATCC AGATCGTTGTTAAAtaaagctttctagaccat EapH2 aagttctgtttcagggcccgGCGGGTAACGAAGTTTCTGCGGCGGAAAAAGACAAACTGC SEQID CGGCGACCCAGAAAGCGAAAGAAATGCAGAACGTTCCGTACACCATCGCGGTTGACGGTA NO:17 TCATGGCGTTCAACCAGTCTTACCTGAACCTGCCGAAAGACTCTCAGCTGTCTTACCTGG ACCTGGGTAACAAAGTTAAAGCGCTGCTGTACGACGAACGTGGTGTTACCCCGGAAAAAA TCCGTAACGCGAAATCTGCGGTTTACACCATCACCTGGAAAGACGGTTCTAAAAAAGAAG TTGACCTGAAAAAAGACTCTTACACCGCGAACCTGTTCGACTCTAACTCTATCAAACAGA TCGACATCAACGTTAAAACCAAAtaaagctttctagaccat

Murine Renal Abscess Model

[0273] For the experiments in this study, a total of 127 weight-matched female BALB/C mice, aged 6 weeks were obtained from Harlan Laboratories (Bicester, UK). Mice were housed randomly in cages of 3, 4 or 6. To determine the protective effect of the vaccines we used a murine intravenous challenge model. Treatment groups were allocated such that each cage contained at least one animal receiving each treatment. Mice received an intramuscular injection with 10.sup.9 IU AdHu5 expressing vaccine antigen or no antigen, followed 10 weeks later with a boost vaccination comprising 10.sup.7 PFU MVA expressing either the vaccine antigen or GFP. Two weeks after the boost vaccination mice were challenged with 10.sup.7 CFU S. aureus Newman bacterial suspension in 0.1 ml PBS injected into the lateral tail vein. Mice were weighed and monitored daily for signs of illness. Three days post infection mice were sacrificed and kidneys and spleens were harvested. The left kidney was homogenised in PBS, plated and viable bacteria per gram tissue were counted. Viable S. aureus per gram of tissue/ml of blood were enumerated by spreading serial diluted aliquots of homogenized tissue (GentleMACS, M-tubes, Miltenyi Biotec, Bisley, UK) for colony formation using an Autoplate machine (Quadrachem, UK) on horse blood agar (HBA) (Oxoid). Plates were incubated for 24 hours at 37 C. in air, and colonies were counted using an automated counter (QCount, Quadrachem, UK).

Abscess Formation

[0274] In addition to enumerating bacterial recovered from kidneys, abscess formation was examined using Magnetic Resonance Imaging (MRI) of post-mortem material. Briefly, after mice were sacrificed the right kidney was fixed and stored in 4% paraformaldehyde (Alfa Aesar, UK). MRI was performed as described [27]. Image analysis was performed using Amira software version 5.6 (FEI Visualisation Sciences Group). Kidneys were analysed for total abscess volume, individual abscess volumes and the number of abscesses detected in each kidney, and compared across treatment groups.

Intranasal Vaccination and Experimental Colonisation of Mice

[0275] 40 female CD1 mice, aged 6 weeks were obtained from Envigo (Netherlands). Mice were housed randomly in cages of 5. Treatment groups were allocated such that each cage contained at one animal receiving each treatment. Mice received 100 l intranasal dose of PBS, 10.sup.9 IU AdHu5 expressing vaccine antigen or no antigen, or 10.sup.7 PFU MVA expressing either the vaccine antigen or GFP. To assess efficacy of vaccines in reducing S aureus carriage, we experimentally colonised mice 26 days after vaccination. S. aureus was prepared by overnight culture in TSB (Oxoid) at 37 C., 130 rpm, washed and resuspended in PBS (Sigma). Contamination of cages was performed by spraying this inoculum onto bedding using a 100 ml plastic spray bottle with a hand-pumped vaporiser (product 215-3092, VWR International). Each cage received 5-10 ml of S. aureus culture at 510.sup.9 cfu/ml. Mice were not sprayed directly, and cages were not cleaned for 7 days after spraying.

Evaluation of S. aureus Carriage in Mice

[0276] Naturally and experimentally colonised mice were screened for gut carriage of S. aureus during the course of the experiments. Stool samples were taken on arrival, at various time points during the experiment. Stools were weighed and homogenised in sterile PBS (Sigma Aldrich, UK) before plating on Brilliance Staph 24 agar (Oxoid, UK) to determine CFU/g stool. Negative samples were enriched in 5% salt meat broth (Oxoid, UK) at 37 C., 130 rpm, for at least 24 h before plating on Brilliance Staph 24 agar. Samples negative upon enrichment were considered as negative in the analysis.

Luciferase Immunoprecipitation System (LIPS) Assay

[0277] The antibody response to vaccination in mice was assessed by the LIPS assay as previously described (van Diemen et al., 2013; Burbelo et al., 2005). The fusion of the target antigen and Renilla luciferase was expressed in HEK293 cells. Antigen-Renilla luciferase fusion proteins were combined with serially diluted mouse sera obtained one day before challenge. After 1 h incubation at room temperature the mixture was transferred into MultiScreen.sub.HTS HV opaque 0.45 m filter plates (EMD Millipore) containing 3% Protein A/G UltraLink Resin (ThermoFisher). After 1 h incubation and subsequent washings with Buffer A (10 mM Tris, 100 mM NaCl, 5 mM MgCl.sub.20.6 H.sub.2O, 1% Triton X-100) and PBS (Sigma Aldrich, UK) Renilla luciferase assay reagent (Renilla luciferase assay system, Promega, UK) was added and chemoluminiscence was measured using CLARIOstar microplate reader (BMG LABTECH). Log transformation was applied to luminescence data before subtracting the assay background.

IFN- Enzyme-Linked ImmunoSpot (ELISPOT) Assay

[0278] Individual mouse peripheral blood samples were treated with ACK lysis buffer to remove RBCs prior to stimulation with relevant peptides spanning the Eap, EapH1 or EapH2 portions of the immunogen (final concentration of 2 g/ml) in the presence of homologeous splenocytes (510.sup.6 cells/ml), on High Protein Binding Immobilon-P membrane plates (MAIPS4510, Millipore) coated with 5 mg/ml anti-mouse IFN- (AN18, Mabtech). After 18-20 hours, IFN- spot forming cells (SFC) were visualised by staining membranes with anti mouse IFN- biotin (1 g/ml, R4-6A2, Mabtech) followed by streptavidin-Alkaline Phosphatase (1 g/ml, Mabtech) and development with AP conjugate substrate kit (BioRad, UK). The number of SFC were counted with an ELISpot reader (AID, Germany). Log(SFC) was used in statistical analysis because of the approximate log-normal distribution of ELISpot counts in the animals [28].

End-Point Titre ELISA

[0279] 96-well NUNC ELISA plate was coated with 0.05 g/well of recombinant Eap protein diluted in PBS; and blocked with 1% BSA/PBS/0.05% Tween. Further wells were incubated with post-boost sera from 5 mice for each of the vaccination regimens, serially diluted 1:3 in blocking buffer with the lowest dilution of 1:100. Sera from nave SOPF Balb/C mice were used for background. Goat anti-mouse IgG Alkaline Phosphatase conjugated secondary antibody (Abcam) was used for detection, diluted 1:10,000 in blocking buffer. Plates were washed with PBS/0.05% Tween after each incubation, developed with SIGMAFAST p-Nitrophenyl phosphate tablets (Sigma) and read at 405 nm. After Background subtraction and log-transformation of the data, non-linear regression (dose-response stimulation; log(agonist) vs response-variable slope 4 parameters) was used to fit the curve and interpolate the end-point titre value using GraphPad Prism Software.

Interaction Assays

[0280] For this assay, His tag was excised from the Eap, EapH1 and EapH2 proteins using Pierce HRV 3C Protease Solution Kit (Thermofisher). 96-well NUNC plates were coated with 0.1 mM/well of Eap, EapH1 and EapH2 proteins with His tag excised, diluted in PBS. Plates were blocked with 2% BSA/PBS/0.05% Tween and incubated with PBS (as background) or His-tagged Eap, EapH1, EapH2, SAUSA300_2132 and SAUSA300_1795 at a range from 1,000 to 0.5 mM per well, diluted in PBS. The latter two staphylococcal proteins were used as negative controls. His-tagged proteins alone were coated on the plate as positive controls. Plate was incubated with mouse anti-Histidine tag antibody, Alkaline Phosphatase conjugated (Biorad) diluted 1:1000 in 1% BSA/PBS/0.05% Tween. As a positive control for successful coating some wells were incubated with post-boost sera from EapH1_EapH2 vaccinated mice and goat anti-mouse IgG, Alkaline Phosphatase conjugated (Abcam) secondary antibody. Plates were washed with PBS/0.05% Tween after each incubation, developed with SIGMAFAST p-Nitrophenyl phosphate tablets (Sigma) and read at 405 nm. After background subtraction, non-linear regression with least squares fit was used to create a fitting curve and interpolate K.sub.d using GraphPad Prism Software.

IgG Antibody to S. aureus Antigens in Human Cohorts

[0281] Forty-two serum samples randomly selected out of two cohorts of healthy adult volunteers (18 to 60 years old) were screened for antibodies against selected S. aureus antigens using LIPS, as described above. Linearity of the response was determined by including a standard curve comprising serial dilutions of human serum from a single, healthy donor. Sera were examined at 1:1000 dilution in PBS. Specific informed consent was obtained from both cohorts, as described by Whitehouse et al. [29]. Briefly, the Oxford Staphylococcus aureus carriage cohort (n=26) comprises healthy adult volunteers from 18 to 60 years old, declaring themselves to be of North European ancestry. Exclusion criteria were pregnancy, intake of immunomodulatory drugs, cancer, connective tissue disease, blood born viruses, or previous organ transplantation. The Submarine cohort (n=49) comprises healthy male submariners who gave serum at the start of a submarine patrol serum were taken, separated within 4 hours of sampling and stored at 80 C. For both cohorts two nasal swabs were taken at least 1 month apart, and cultured both directly on selective agar (BD Brilliance Staph 24) and by enrichment culture in Mannitol salt broth followed by plating on selective agar. Individuals who cultured positive at both time points were considered to be S. aureus carriers, and all others non-carriers. Specific informed consent was obtained from both cohorts, as described [29][30]. From these two studies, serum samples were selected randomly, having stratified for carriage status. Analysis was performed blinded on serum from 19 carriers and 23 non-carriers.

Statistical Analysis

[0282] Data on antibody response and IFN--specific cell numbers were statistically analysed for effect of vaccine by means of t-tests after a log.sub.10 transformation and correction for background. Specific antibody levels from the LIPS assay were expressed as fold increase over assay background by subtracting the log transformed assay luminescence background, which was considered to be the luminescence observed in the absence of any sera. The assay limit of detection was considered to be four standard deviations above the background. Post-hoc pairwise comparisons were performed using Dunnett's Multiple Comparison Test. For estimation of the association between S. aureus carriage and antibody titres, a general linear model fitting

y.sub.i=P.sub.i+C

where y.sub.i=log(fold increase in antibody concentration relative to background) [0283] P.sub.i=the i.sup.th protein examined and [0284] C=effect of carriage vs. non-carriage.

[0285] Differences were considered significant when p<0.05. The statistical packages used were R 2.15 (http://www.cran.org), and GraphPad Prism version 5.04 (GraphPad Software, Inc.).

Example 2: Prevalence of EapH1 and EapH2 Across S. aureus Lineages

[0286] We examined the presence of EapH1 and EapH2 proteins in a set of 104 sequenced clinical S. aureus isolates previously shown to be representative of major S. aureus clones [26]. In common with other recognised core S. aureus genes, such as IsdA and IsdB, we observed EapH1 and EapH2 homologues in all strains examined (FIG. 1). Genetic diversity relative to the query sequences used (WP_001549607.1, WP_000769689.1 respectively) was significantly greater (median 97%) for EapH1 than for EapH2 (median 99%). However, both genes are very highly conserved.

Example 3: Interactions Between EapH1, EapH2 and C-Terminus of Eap

[0287] Eap is a multimerising, cell surface associated protein [24]. We purified recombinant His-tagged Eap, EapH1 and EapH2 proteins from E. coli, and removed endotoxin using an affinity column. The His tag was cleaved from an aliquot of each protein, and an interaction assay established in which this material was immobilised on an EIA plate (FIG. 2A). Binding of a His-tagged ligand to the immobilised protein was detected using an anti-His antibody (FIG. 2B-D). We observed apparently saturable binding of Eap to itself with a K.sub.d estimate of 80 nM (95% Cl 29, 134), and with EapH2 (estimated K.sub.d 82 nM, 95% Cl 30, 132 nM), (FIG. 2 B-D). We also observed binding of EapH2 to EapH1 and to Eap (FIG. 2), but the affinity could not be reliably quantified as we were unable to obtain saturation of binding at the concentrations of protein available. Binding appeared specific, since similar-sized recombinant Staphylococcal cell surface proteins SAUSA300_2132 and SAUSA300_1795 did not show any binding to immobilized Eap, EapH1 or EapH2 proteins (FIG. 2 B-D).

[0288] To exclude co-purification of other bacterial proteins or lipopolysaccharides with our recombinant proteins as an explanation for the interactions observed, we expressed Eap, EapH1 and EapH2 in 293 cells with either a C-terminal fusion to either a V5 tag or to Renilla luciferase epitope tags. V5-tagged proteins were captured onto anti-V5 coated plates, and capture of luciferase activity following addition of lysates containing luciferase fusion proteins quantified (FIG. 8). Absolute protein concentrations are not readily determined in this system, and saturation of binding not clearly achieved. Nevertheless, the pattern of binding observed is compatible with the data shown in FIG. 2. Taken together, these data support a model in which EapH1 and EapH2 may associate with each other, with Eap and perhaps other proteins near the cell surface (FIG. 2E). As such, they may be physically co-located, and with overlapping specificities contributing to potent, broad spectrum protease inhibition around the bacterium.

Example 4: EapH Protein Production from Viral Vectors

[0289] In view of this possible co-location of antigens, we investigated combinations of these three proteins as vaccine antigens since they may comprise complementary components of an immunomodulatory complex. Three constructs, designated Eap, EapH12Eap and EapH12 were constructed to contain different combinations of Map domains from the Eap, EapH1 or EapH2 proteins (FIGS. 3A, 3B). The MAP domains were expressed from both Adenovirus and modified Vaccinia Ankara (MVA) vectors as part of an expression cassette containing a eukaryotic leader sequence, and a 3 V5 tag (for antigen detection) together with an IMX313 multimerising sequence [31], which increases immunogenicity of some proteins. Cytosolic presence and secretion of antigens from viral vectors in vitro was readily detectable using a non-quantitative anti-V5 capture, anti-IMX313 detection sandwich EIA (FIGS. 3C, 3D). Identical adenovirus and MVA vectors, apart from the absence of the antigen expression cassette, were used as controls.

[0290] Balb/c mice were immunised with Adenovirus followed by MVA vaccine components (FIG. 4A). As judged by serological analysis on day 70, all components of all the vaccines were immunogenic, producing a strong antibody response against respective Map domains of Eap, EapH1 and EapH2 proteins (FIGS. 4B-D). Antibodies against EapH proteins were assessed by luciferase immunoprecipitation (LIPS) and a subset of sera were analysed by endpoint dilution against recombinant bacterial Eap. Weak immune responses against Eap were observed in 2/5 animals immunised with EapH12 alone, suggesting that some cross reactivity between Eap and EapH1/2 may occur in a subset of animals.

Example 5: EapH1 and H2 Reduce Abscess Numbers and Bacterial Recovery in i.v. Challenge Models

[0291] Following vaccination, mice were challenged i.v. with S. aureus strain Newman. All mice survived the challenge until day 3, when a post mortem was performed. The right kidney of each mouse was analysed using a sensitive post-mortem MRI technique [27], and abscess numbers quantified (FIG. 4E). The number of right kidney abscesses was significantly lower in the EapH12 vaccinated group in comparison to the control group (p=0.04). Bacterial recovery from the left kidney was also used to assess vaccine efficacy. The bacterial recovery in the EapH1H2 vaccine group differed from the control group by 0.7 log.sub.10 CFU/g, a difference which is not statistically significant (4F) (p=0.31, Mann-Whitney test).

[0292] Since power calculations using historical data from this infection model [32] indicated we are only powered to detect a 0.9 log decrease in bacterial numbers using this experimental design, we performed two additional similar experiments to investigate the impact of EapH1/H2 vaccination on bacterial recovery post i.v. infection. These indicated the effect observed was reproducible and consistent (FIG. 5A). A significant reduction in bacterial recovery was seen following Adenovirus/MVA vaccination with EapH1_2, across the 3 experiments. In the latter two experiments, blood lFN secreting cells were determined using tail bleeds on day 70, before i.v. challenge. These experiments showed induction of a geometric mean of 24,000 lFN secreting EapH1/H2 responsive T cells per l by the Adenovirus/MVA regime. However, neither blood ELISPOT nor serological responses against EapH1 or EapH2 were significantly associated with protection (FIGS. 5B-D). S aureus carriage levels in stools were also monitored and in 1 of the 3 experiments we found that a significantly higher proportion of mice were S. aureus negative in the Adenovirus-EapH1H2 group than the control adenovirus (p=0.006) vaccinated animals (FIG. 9). Thus, the protective mechanism(s) induced by the vaccine are unclear.

Example 6: Intranasal Vaccination with EapH Proteins Accelerates Loss of S. aureus Carriage

[0293] Adherence to mammalian cell surfaces has been proposed as a step in S. aureus carriage [33], as has generation of T cell responses against S. aureus [34]. Since EapH proteins may be exposed on the cell surface, and since viral vectors elicit potent T- and B-cell responses against encoded antigens [35], we tested whether eliciting mucosal responses against these proteins could alter S. aureus colonisation. CD1 mice without S. aureus colonisation were vaccinated intranasally with Adenovirus Hu5 expressing EapH1 and H2, a control Adenovirus expressing no antigen, MVA expressing EapH1 and H2, MVA expressing GFP, or PBS. One mouse from each vaccinated group was placed in each of eight cages (FIG. 6A). Vaccination induced systemic antibody responses against EapH1 and EapH2 (FIG. 6B,C). 26 days later, mice were exposed to S. aureus Newman by environmental contamination. Stool cultures 1 day later were positive in all mice (FIG. 6D). Carriage levels of S. aureus in stools were monitored. In all cages the carriage levels declined over time (FIG. 10), which has been observed previously in the animals in our facility. However, at the end of followup (day 54 after vaccination, 28 days after experimental colonisation), a significantly higher proportion of mice were S. aureus negative in the Adenovirus-EapH1H2 group than the control adenovirus (p=0.02) or PBS vaccinated animals (FIG. 6E). Thus, EapH1H2 vaccination may accelerate loss of carriage in murine models.

Example 7: Recognition of EapH1 and EapH2 by Human Populations

[0294] We examined a cohort of 42 humans, randomly selected from cohort studies, including 19 carriers (identified by having two S. aureus nasal swabs positive) and 23 non-carriers. We quantified IgG antibodies against the cell surface proteins IsdA, IsdB, ClfB and the nuclease Nuc1, antigens selected because antibodies against these proteins are known to be prevalent in humans [37, 38], using an immunoprecipitation assay. Antibodies against all four proteins were detected, with increased concentrations in carriers vs. non-carriers (fold increase 1.32, 95% Cl 1.07-1.64) (FIG. 7A). Antibodies against EapH1 and EapH2 were also detected (FIG. 7B). Significant correlations (Spearman's rho differs from 0, p<0.01) were seen between antibody quantities to Isd proteins and to Nuclease (FIG. 7C), with less significant associations (p<0.05) with ClfB antibodies. Antibody titres against EapH2, but not EapH1, were also associated with anti-Isd and Nuclease responses (FIG. 7C).

[0295] In view of this, and our finding that antibody generation against EapH proteins reduced carriage, we considered whether the relationship between antibody against well known S. aureus antigens (IsdA, IsdB, ClfB, Nuc1), and that against EapH proteins, were carriage-specific. Such an association might exist if in individuals with higher anti-EapH protein responses, carriage (and thence exposure to other S. aureus antigens) was inhibited. We computed a mean response to the reference antigens (IsdA, IsdB, ClfB, Nuc1) in each individual and compared with responses to EapH protein. In non-carriers, EapH1 (FIG. 7D), but non-EapH2 (FIG. 7E), responses, were significantly higher relative to the reference antigens. Thus, our data is compatible with the idea that the development of immune responses against EapH1 may be associated with loss of carriage.

TABLE-US-00003 SEQUENCES CompleteEapH1nucleotidesequencefromS.aureusNewman (fromWP001549607.1) SEQIDNO:1 ATGAAACTAAAATCATTTGTTACTGCCACTTTAGCATTGGGATTATTATCAACGGTCGGAGCTG CATTACCGAGTCACGAAGCATCTGCAGATAGTAATAACGGCTATAAAGAAATGACTGTGGATGG TTATCACACTGTTCCTTACACAATTTCAGTAGATGGTATTACTGCATTACATCGAACTTACTTT ATCTTCCCAGAAAATAAAAATGTTCTTTATCAAGAAATTGACAGTAAAGTAAAAAATGAATTAG CTTCTCAACGTGGTGTTACAACAGAAAAAATTAATAATGCCCAAACAGCAACTTATACGCTTAC TTTGAATGATGGTAATAAAAAAGTAGTGAATCTAAAGAAAAATGACGACGCTAAAAATTCAATT GATCCAAGTACAATCAAACAGATACAAATTGTAGTTAAATAA CompleteEapH1aminoacidsequencefromS.aureusNewman (fromWP001549607.1) SEQIDNO:2 MKLKSFVTATLALGLLSTVGAALPSHEASADSNNGYKEMTVDGYHTVPYTISVDGITALHRTYF IFPENKNVLYQEIDSKVKNELASQRGVTTEKINNAQTATYTLTLNDGNKKVVNLKKNDDAKNSI DPSTIKQIQIVVK CodonoptimisedEapH1antigennucleotidesequencefromS.aureus Newman SEQIDNO:3 CACGAGGCCTCTGCCGATAGCAACAACGGCTACAAAGAAATGACCGTGGATGGCTACCACACCG TGCCTTACACCATCTCTGTGGATGGAATCACCGCCCTGCACCGGACCTACTTCATCTTCCCCGA GAACAAGAACGTGCTGTACCAGGAAATCGACTCTAAAGTGAAGAACGAGCTGGCCTCCCAGAGA GGCGTGACAACCGAGAAGATTAACAACGCCCAGACCGCCACCTACACCCTGACCCTGAACGACG GCAACAAAAAGGTCGTGAATCTGAAGAAGAACGACGACGCCAAGAACAGCATCGACCCCAGCAC CATTAAGCAGATCCAGATCGTCGTGAAG EapH1antigenaminoacidsequencefromS.aureusNewman SEQIDNO:4 HEASADSNNGYKEMTVDGYHTVPYTISVDGITALHRTYFIFPENKNVLY QEIDSKVKNELASQRGVTTEKINNAQTATYTLTLNDGNKKVVNLKKNDDA KNSIDPSTIKQIQIVVK CompleteEapH2nucleotidesequencefromS.aureusNewman (fromWP000769689.1) SEQIDNO:5 ATGAAATTAAAATCATTTATAACTGTAACTTTGGCACTGGGCATGATCGCAACGACTGGCGCTA CTGTGGCAGGTAATGAGGTATCTGCAGCAGAAAAGGACAAACTACCGGCAACTCAAAAAGCTAA AGAAATGCAAAATGTTCCATATACAATTGCAGTAGATGGCATTATGGCTTTCAATCAATCTTAC TTAAATTTACCAAAAGATAGCCAATTATCATATTTAGATTTAGGAAATAAAGTTAAAGCTTTGT TATATGATGAACGCGGTGTAACACCTGAGAAGATTCGAAATGCAAAATCTGCCGTTTACACGAT TACTTGGAAAGATGGTAGTAAAAAAGAAGTGGATCTTAAGAAAGATAGCTACACAGCAAACTTG TTTGATTCAAATTCAATTAAACAAATTGATATTAATGTAAAAACTAAATAA CompleteEapH2aminoacidsequencefromS.aureusNewman (fromWP000769689.1) SEQIDNO:6 MKLKSFITVTLALGMIATTGATVAGNEVSAAEKDKLPATQKAKEMQNVPYTIAVDGIMAFNQSY LNLPKDSQLSYLDLGNKVKALLYDERGVTPEKIRNAKSAVYTITWKDGSKKEVDLKKDSYTANL FDSNSIKQIDINVKTK CodonoptimisedEapH2antigennucleotidesequencefromS.aureus Newman SEQIDNO:7 AACGAGGTGTCAGCCGCCGAGAAGGATAAGCTGCCCGCCACCCAGAAAGCCAAAGAAATGCAGA ACGTGCCCTACACAATCGCCGTGGACGGCATCATGGCCTTCAACCAGAGCTACCTGAACCTGCC CAAGGACAGCCAGCTGAGCTATCTGGACCTGGGCAACAAAGTGAAGGCCCTGCTGTACGACGAG CGGGGCGTGACCCCTGAGAAGATCAGAAACGCCAAGAGCGCCGTGTACACCATCACCTGGAAGG ACGGCAGCAAGAAAGAGGTGGACCTGAAGAAGGACAGCTACACCGCCAACCTGTTCGACAGCAA CAGCATCAAGCAGATCGACATCAACGTGAAAACAAAG EapH2MAPdomainaminoacidsequencefromS.aureusNewman SEQIDNO:8 NEVSAAEKDKLPATQKAKEMQNVPYTIAVDGIMAFNQSYLNLPKDSQL SYLDLGNKVKALLYDERGVTPEKIRNAKSAVYTITWKDGSKKEVDLKKDSYT ANLFDSNSIKQIDINVKTK EapMAPdomainaminoacidsequencefromS.aureusNewman (fromWP_001549158.1) SEQIDNO:9 VPYTIAVNGTSTPILSKLKISNKQLISYKYLNDKVKSVLKSERGISDLDLKFAK QAKYTVYFKNGKKQVVNLKSDIFTPNLFSAKDIKKIDIDVKQYTKSKKNK Codon-optimisedEapMAPnucleotidesequence SEQIDNO:10 GTTCCGGAGTGCCCTATACCATTGCTGTGAACGGCACCAGCACCCCCATCCTGAGCAAGCTGAAGATCAG CAACAAGCAGCTGATCTCCTACAAGTACCTGAACGACAAAGTGAAAAGCGTGCTGAAGTCCGAGAGAGGC ATCAGCGACCTGGATCTGAAGTTCGCCAAGCAGGCCAAGTACACCGTGTATTTCAAGAACGGGAAGAAAC AGGTCGTGAACCTGAAAAGCGACATCTTCACCCCTAATCTGTTCAGCGCCAAGGACATCAAGAAAATTGA TATCGACGTGAAGCAGTACACCAAGAGCAAGAAGAACAAGGGATCCGGGCCCGGG EapproteinaminoacidsequencesfromStrainNewman (fromNCBIlocusCAB94853.1) SEQIDNO:11 MKFKSLITTTLALGVLASTGANFNNNEASAAAKPLDKSSSSLHHGYSKVHVPYAITVNGTSQNILSSLTF NKNQNISYKDLEDRVKSVLKSDRGISDIDLRLSKQAKYTVYFKNGTKKVIDLKVGIYTADLINTSEIKAI NINVDTKKQVEDKKKDKANYQVPYTITVNGTSQNILSNLTFNKNQNISYKDLEDKVKSVLESNRGITDVD LRLSKQAKYTVNFKNGTKKVIDLKSGIYTANLINSSDIKSININVDTKKHIENKAKRNYQVPYSINLNGT STNILSNLSFSNKPWTNYKNLTSQIKSVLKHDRGISEQDLKYAKKAYYTVYFKNGGKRILQLNSKNYTAN LVHAKDVKRIEITVKTGTKAKADRYVPYTIAVNGTSTPILSDLKFTGDPRVGYKDISKKVKSVLKHDRGI GERELKYAKKATYAVHFKNGTKKVININSNISQLNLLYVQDIKKIDIDVKTGTKAKADSYVPYTIAVNGT STPILSKLKISNKQLISYKYLNDKVKSVLKSERGISDLDLKFAKQAKYTVYFRNGKKQVVNLKSDIFTPN LFSAKDIKKIGIDVKQYTKSKKNK Staphylococcusaureusmap-ND2Cgene(AJ290973.2) SEQIDNO:12 AATTATCATAAAAAAGGAGTGATAATTTATGAAATTTAAGTCATTGATTACAACAACTTTAGCATTAGGT GTTTTAGCATCAACAGGTGCAAACTTTAATAATAATGAAGCGTCTGCCGCAGCTAAGCCATTAGATAAAT CATCAAGTTCGTTACACCATGGATATTCTAAAGTCCATGTTCCATATGCAATCACTGTGAACGGTACAAG CCAAAATATTTTATCAAGCTTAACATTTAATAAGAATCAAAATATTAGTTATAAAGATTTAGAGGATAGA GTTAAATCAGTTTTAAAATCAGACAGAGGTATTAGTGATATAGATTTAAGACTATCGAAGCAAGCGAAAT ATACTGTTTACTTTAAAAATGGAACAAAGAAAGTTATCGATTTGAAAGTAGGTATTTACACAGCTGATTT AATTAATACAAGTGAAATTAAAGCTATTAATATTAACGTAGATACTAAAAAGCAAGTTGAAGATAAAAAG AAAGATAAAGCAAATTACCAAGTTCCATACACAATCACTGTGAACGGTACAAGCCAAAATATTTTATCAA ACTTAACATTTAATAAGAATCAAAATATTAGTTACAAAGATTTAGAGGATAAAGTTAAATCAGTTTTAGA ATCAAATAGAGGTATTACCGATGTTGATTTAAGATTATCGAAGCAAGCGAAATATACAGTTAATTTTAAA AATGGAACGAAGAAAGTTATCGATTTGAAATCAGGTATTTACACAGCGAATTTAATAAATTCAAGTGATA TTAAAAGTATCAATATTAACGTAGATACAAAAAAACATATCGAAAATAAAGCTAAAAGAAACTATCAAGT TCCATATTCAATTAATTTAAATGGTACATCTACAAACATTTTATCGAATCTTTCATTTTCAAATAAACCT TGGACAAATTACAAAAATTTAACTAGTCAAATAAAATCAGTACTGAAGCATGATAGAGGTATTAGTGAAC AAGATTTAAAATATGCTAAGAAAGCTTATTATACTGTTTATTTTAAAAATGGTGGTAAAAGAATCTTACA GTTGAATTCAAAAAATTACACAGCAAACTTAGTTCATGCGAAAGATGTTAAGAGAATTGAAATTACTGTT AAAACAGGAACTAAAGCGAAAGCAGACAGATATGTACCATACACAATTGCAGTAAATGGCACATCAACAC CAATTTTATCAGATTTAAAATTTACAGGTGACCCACGTGTAGGCTACAAAGATATCTCTAAAAAAGTTAA ATCAGTACTGAAGCATGATAGAGGTATCGGGGAACGTGAATTAAAATATGCAAAAAAAGCTACTTACGCA GTACATTTTAAAAATGGAACGAAAAAAGTGATTAACATAAATTCAAATATTAGCCAACTGAATCTGCTTT ATGTTCAAGATATTAAAAAGATAGATATTGATGTTAAAACAGGAACTAAAGCGAAAGCGGATAGCTATGT ACCATATACAATTGCAGTAAATGGCACATCAACACCAATTTTATCAAAACTTAAAATTTCGAATAAACAA TTAATTAGTTACAAATATTTAAACGACAAAGTGAAATCTGTATTAAAAAGTGAAAGAGGCATCAGTGATC TTGACTTAAAATTTGCGAAACAAGCAAAATATACAGTATATTTCAGAAATGGAAAGAAACAAGTAGTGAA TTTAAAATCAGACATCTTTACACCTAATTTATTTAGTGCCAAAGATATTAAAAAGATTGGTATTGATGTA AAACAATACACTAAATCAAAAAAAAATAAATAAATTCTAATAATGTGAAATTCCCAGTAACAATAAATAA ATTTGAAAACATAGTTTCAAATGAATTTGTGTTCTATAATGCAAGCAAAATTACAATTAATGATTTAAGT ATAAAACTTAAATCAGCAATGGCAAATGATCAAGGGATAACTAAACATGACATAGGACTTGCTGAACGCG CAGTGTATAAAGTGTATTTTAAAAATGGTTCGTCAAAATATGTAGACTTAAAAACTGAGTATAAAGATGA AAGAGTATTTAAAGCAACTGACATTAAAAAAGTAGAAATTGAAATTAAATTTTAA TPAleader: SEQIDNO:13 ATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGAGCAGTCTTCGTTTCGCCCAGCCAGG AAATCCATGCCCGATTCAGAAGA BGHpolyA: SEQIDNO:14 ctgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctgga aggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcg Cattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggagg attgggaagacaatagcaggcatgctggggatgcggtgggctctatgg

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