<i>Coxiella burnetii </i>antigens

Abstract

The present invention provides antigens, for use in the treatment or prevention of C. burnetii infection. Also provided are nucleic acids encoding such antigens, and antibodies raised against such antigens.

Claims

1. An immunogenic composition comprising one or more isolated protein antigens selected from the group consisting of: (1) a CBU_0091 antigen; (2) a CBU_1648 antigen; (3) a CBU_0532 antigen; (4) a CBU_0758 antigen; (5) a CBU_1652 antigen; and (6) a CBU_2009 antigen; and further comprising an immune-effective amount of an adjuvant.

2. The immunogenic composition of claim 1 comprising a pharmaceutically acceptable carrier or excipient.

3. The immunogenic composition of claim 1 comprising one or more additional therapeutic agents.

4. The immunogenic composition of claim 3, wherein the one or more additional therapeutic agents comprises a bacteriostatic drug.

5. The immunogenic composition of claim 3, wherein the one or more additional therapeutic agents comprises a bactericidal agent.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) There now follows a brief description of the Figures, which illustrate aspects and/or embodiments of the present invention.

(2) FIG. 1One-dimensional SDS-PAGE image of extracted C. burnetii protein run on a 12 Bis-Tris gel in MOPS-SDS running buffer at 200 V for 50 m. The three lanes are 10 l, 5 l, and 2.5 l of protein boiled in Laemmli buffer to ensure good band clarity. The figure shows that the extracted protein contained a wide range of proteins of different masses present in distinct species with little evidence of degradation (bands were sharply delineated with little smearing).

(3) FIG. 2Western blot of C. burnetii proteins (on the membrane) probed with the aerosol-exposed guinea pig sera (containing immune-reactive antibodies), that binding detected with anti-guinea pig IgG conjugate, and visualised with ECL prime substrate. Lane 1 is the transferred SeeBlue Plus2 Molecular Weight standard and lane 2 contains the MagicMark XP Standard. Figure shows that the sera from all of the exposed guinea pigs reacted to some degree with the proteins present in the C. burnetii extract whereas the unexposed guinea pig (A1-7) did not react. The image also suggests that individuals A1-4 and A1-6 reacted with a more proteins over a wider mass range than the other exposed animals.

(4) FIG. 3Linear Regression of protein quantitation standards and the resulting line equation used to calculate the protein quantities present in the C. burnetii extract. Line equation OD480 nm=0.856-0.003 g BSA; S=0.013; R.sup.2=94.8%; R2 (adj)=94.5%; P<0.001. The derived line equation was used to determine the loading quantities of proteins for the first-dimension (isoelectric focussing) of the 2D SDS-PAGE process to reduce the risk of under or overloading the gels.

(5) FIG. 4Coomassie-stained 2D PAGE separation of C. burnetii proteins across the wide pl range 3.0-11.0 (non-linear). Isoelectric point ranges from pl=3 at the left side of the image to pl=11 at the right side of the image.

(6) FIG. 5Coomassie-stained 2D PAGE separation of C. burnetii proteins across the narrower (acidic) pl range 3.0-5.6 (non-linear). Isoelectric point ranges from pl=3 at the left side of the image to pl=5.6 at the right side of the image.

(7) FIG. 6Coomassie-stained 2D PAGE separation of C. burnetii proteins across the narrower (basic) pl range 7.0-11.0 (non-linear). Isoelectric point ranges from pl=7 at the left side of the image to pl=11 at the right side of the image.

(8) FIG. 7Western Blue (BCIP/NBT)-stained antibody-probed Western blot of C. burnetii proteins detected in wide pl range 3-11 (non-linear). Immunoreactive proteins probed with antibody (IgG) present in guinea pig sera (A1-4), the bound antibody was detected with anti-Guinea Pig IgG/alkaline phosphatase conjugate. The areas marked C1-C9 indicate the locations from where spots of the corresponding protein gel were excised for protein identification.

(9) FIG. 8Western Blue (BCIP/NBT)-stained antibody-probed Western blot of C. burnetii proteins detected in lower, narrow pl range 3-5.6 (non-linear). Immunoreactive proteins probed with antibody (IgG) present in guinea pig sera (A1-4), the bound antibody was detected with anti-Guinea Pig IgG/alkaline phosphatase conjugate. The areas marked L1-L20 indicate the location from where spots of the corresponding protein gel were excised for protein identification.

(10) FIG. 9Western Blue (BCIP/NBT)-stained antibody-probed Western blot of C. burnetii proteins detected in higher, narrow pl range 7-11 (non-linear). Immunoreactive proteins probed with antibody (IgG) present in guinea pig sera (A1-4), the bound antibody was detected with anti-Guinea Pig IgG/alkaline phosphatase conjugate. The areas marked H1-H7 indicate the location from where spots of the corresponding protein gel were excised for protein identification.

(11) FIG. 10Output trace of the AKTA FPLC instrument during affinity purification of the guinea pig antisera. The blue trace shows protein concentration (arbitrary units) measured by UV, the two sample injection points are indicated by the pink vertical bars, the switch of the instrument from binding buffer to elution buffer is indicated by the change in conductivity shortly after 25 ml (green trace) and the eluted protein fraction identities are in red text from this point.

(12) FIG. 11Left-hand pane1D PAGE analysis of affinity purified guinea pig IgG. Lanes 1-3 non-reduced protein. Lanes 5-7 reduced protein. Lanes 1 and 5 are antisera, lanes 2 and 6 are purification column wash through, lanes 3 and 7 are the eluted, dialysed and concentrated IgG. In lane 7 the heavy (50 kDa) and light (23 kDa) chains of IgG can be clearly seen, in lane 3 the 150 kDa single band of the un-reduced IgG is not easily visualised, this is likely due to lane-to-lane bleed of the reducing agents partly reducing the IgG. Right-hand paneAntibody-probed Western Blots of C. burnetii protein detected with Western Blue substrate showing no difference in activity between the un-treated antisera and the affinity-purified antibody.

(13) FIG. 12The left-hand pane is the Coomassie-stained 1D PAGE of the immunoprecipitation (IP) experiment protein samples. Lanes 1 and 2 are the Native C. burnetii protein material pre- and post-IP. Lanes 3 and 4 are the Denatured material pre- and post-IP. Lane 7 is the post-IP (Convalescent guinea pig IgG-captured) eluted native proteins. Lane 10 is the post-IP eluted denatured proteins. The right-hand panel is the residual protein remaining in the gel after Western blotting the same material with the exception that lanes 5 and 6 are the eluted native and denatured protein materials. The greater sensitivity of the silver staining procedure shows that a good range of protein species were eluted from the IP experiment.

(14) FIG. 13Western-blot of the immunoprecipitation (IP) experiment protein samples probed with guinea pig convalescent sera (4427 1-4). Lanes 1 and 2 are the Native C. burnetii protein material pre- and post-IP. Lanes 3 and 4 are the Denatured material pre- and post-IP. Lane 5 is the post-IP (Convalescent guinea pig IgG-captured) eluted native proteins. Lane 6 is the post-IP eluted denatured proteins.

EXAMPLES

(15) In Vivo Experiments

(16) To generate representative immune-reactive sera, the inventors modelled the symptoms seen in acute Q fever disease in humans in small laboratory animal species. The key features of human disease are that the major mode of infection is by the inhalation of contaminated aerosols; that the organism needs to be in the wild-type virulent phase I form and; the disease is characterised by a fever that may reach a plateau of 40 C. before returning to normal. Further, relatively common disease features include pneumonia and hepatitis.

(17) While the mouse model for Q fever has been reported more frequently than guinea pig model, the mouse model lacks several critical features of human infection. In view of the lack of fever, fewer overt signs of disease and the lower susceptibility to infection in the mouse model, the inventors identified guinea pig, infected via an inhaled aerosol, as the preferred model for this study.

(18) Aerosol Infection of Guinea Pigs and Two Strains of Mouse

(19) An aerosol infection experiment (Collison nebulizer) was performed to assess the signs of clinical infection in guinea pig and two strains of mouse that the literature had suggested may show some signs of infection. This experiment was also performed to generate convalescent anti-sera and antibodies for subsequent use later in this study, to identify C. burnetii proteins that are immune-reactive.

(20) Two C. burnetii stocks, one spleen homogenate from an infected guinea pig and one egg yolk sack grown stock, were assessed for clinical signs of virulence after intraperitoneal infection with 1.510.sup.7 copies/ml. Both stocks proved virulent in this experiment but the egg-yolk sack-grown stock to a substantially greater degree. Animals were therefore challenged with egg yolk sack grown Coxiella burnetii, Nile Mile strain, designated batch EP2 GP1 EP1. Seven Dunkin-Hartley guinea pigs and two strains of mice (7BALB/c and 7NJ), all male, were used in this experiment. Each animal was implanted with a subcutaneous identification and temperature monitoring chip (Animalcare identichip with Bio-Thermo # XID050). The animals' weight, temperature, and general health were recorded twice daily for two days prior to aerosol challenge and four times each day post-challenge. Any animals with a bodyweight loss of greater than 20% or with severe disease symptoms were euthanized in accordance with Home Office project license 30/2423.

(21) The inventors designed, developed and implemented a real-time quantitative PCR assay to detect and enumerate C. burnetii in tissue, blood and culture samples. The PCR assay amplifies and detects the C. burnetii isocitrate-dehydrogenase (icd) gene.

(22) TABLE-US-00001 TABLE 1 qPCR titres of the bacterial culture material in the Collison nebuliser (generator 1 - C.sub.nebuliser) samples taken prior to challenge of each group as well as the impinger samples (C.sub.impinger) and the derived C.sub.aero concentrations in copies/ml (derived from C.sub.impinger 20 ml (the sample volume)/l/min (sample flow) 10 min(sampling time)/1000). Copies/ml Copies/ml Copies/ml Sample C.sub.nebuliser C.sub.impinger C.sub.aero Guinea Pig - Group 1 1.10 10.sup.7 1.30 10.sup.4 4.54 10.sup.0 Mouse (BALB/c) - Group 2 1.10 10.sup.7 3.20 10.sup.4 1.68 10.sup.1 Mouse (A/J) - Group 3 6.30 10.sup.6 1.10 10.sup.4 5.02 10.sup.0

(23) TABLE-US-00002 TABLE 2 Volume of infectious aerosol inspired by the animals in each group and the calculated inhaled dose (copies) of C. burnetii calculated from C.sub.aero Inspired volume. The inhaled dose is an estimate of the number of organisms to which each animal was exposed. Inspired Inhaled volume Dose Animal Group (mean weight) (ml) (copies) Guinea Pig (356.8 g) - Group 1 1,724 7.8 10.sup.3 Mouse (BALB/c) (20.7 g) - Group 2 204.1 3.4 10.sup.3 Mouse (A/J) (21.0 g) - Group 3 206.3 1.0 10.sup.3

(24) To estimate the dose of organism inhaled by each animal (Table 2) the C.sub.aero was multiplied by the inspired volume. Due to their smaller size, the mice received less inoculum but overall a dose of 1,000-7,800 organisms (copies) was predicted by the literature to be sufficient to infect laboratory animals. This was supported by the clinical data obtained by the inventors (data not shown).

(25) At the end of the study, (days 15 and 21 post-exposure respectively) two blood samples of 2 ml volume were taken under terminal anaesthesia by cardiac puncture from guinea pigs and placed into EDTA and SST blood tubes. Any remaining blood was placed into a Heparin tube. For mice, the blood samples were taken as for guinea pig but were divided equally between paediatric EDTA and SST blood tubes.

(26) In addition, the following organs were harvested for qPCR analysis; spleen, kidney, liver, heart, lung and testicle. For the qPCR, approximately of the total organ mass was removed from each organ. DNA was extracted and assessed by qPCR (data not shown).

(27) ResultsIn Vivo Experiments

(28) All guinea pigs in this study demonstrated overt signs of disease as evidenced by a lower rate of bodyweight gain and concurrent increase in body temperature. Therefore, the inoculum was virulent by the aerosol route at the dose used. Neither mouse strain demonstrated a measurable febrile response.

(29) No significant bacterial blood load of C. burnetii was found in any of the study animals. This is consistent with natural infections in humans with C. burnetii where bacteraemia is transient and confined to the febrile portion of disease. The bacterial load in the tissues of guinea pigs was undetectable in all but the lungs at day 21 post-infection. In contrast, both strains of mice had detectable C. burnetii in all tissues tested at day 21. This indicates that in guinea pig the infection is being cleared, consistent with an acute infection; whereas in mice the infection may be of a more chronic nature.

(30) The egg-yolk sack grown C. burnetii is clearly infectious in both guinea pig and mouse by the aerosol route. Clinical measurements were more pronounced in guinea pig than in mice. Disease after aerosol infection of guinea pigs appears to have more features consistent with human acute Q fever than mouse.

(31) Terminal blood was harvested from all animals and the anti-sera harvested and stored.

(32) Protein Isolation

(33) Proteomic comparisons between virulent phase I and avirulent phase II organisms have identified that, in addition to a truncated LPS, phase II organisms have a restricted proteome. Comparisons have also found differences in the proteomes of the two morphological forms, the large- (LCV) and small-cell variants (SCV). Therefore, the inventors performed immune-reactive protein isolation using phase I organisms that are present in a mixture of the two morphological forms.

(34) Immune reactive proteins are specific proteins, present in the pathogen, that have been recognised by the host during infection. This is evidenced by the presence of antibodies in the sera that bind to those proteins.

(35) The inventors also repost isolation of the proteins that are immunoreactive with the antibody (IgG fraction) present in the serum from the aerosol infected guinea pigs. These proteins are isolated to permit downstream mass spectrometric identification.

(36) Growth of C. burnetii

(37) The inventors grew a stock of C. burnetii, free of host proteins, in axenic media. This stock was produced from a low passage stock of phase I material and contained a mixture of the two morphological forms (SCV and LCV) to maximise the probability that a comprehensive representation of the organism's proteins are present in the preparation.

(38) Optimisation of Inoculation Concentration

(39) To axenically culture C. burnetii, acidified citrate cysteine media (ACCM-2) was prepared as described in the literature. Bacterial inoculum stock was C. burnetii, Nine Mile strain (EP2, GP1 EP1)30% (w/v) egg yolk sack in PBS homogenate. DNA was extracted from this material and its average titre estimated by three separate determinations in qPCR as described above. This titre was 2.910.sup.7 copies/ml. As this material was the first chick egg passage after a guinea pig passage, and was demonstrated by the inventors to be pathogenic in animals, it was concluded that it was in the virulent phase I form of the organism.

(40) Into the wells of six-well cell-culture plates (ThermoScientific/Nunc #140675) 2 ml of ACCM-2 media was pipetted. The wells were then inoculated with C. burnetii such that there were five concentrations of organisms, from 1.010.sup.2 copies/ml to 1.010.sup.6 copies/ml in 10.sup.1 increments. The plates were sealed into a 2.5 l gas-tight box with a microaerophilic atmosphere generating pack (Biomerieux GENbox microaer #96125) and the box placed in an incubator set at 37 C. At intervals throughout the ten day experiment, the box was opened, samples taken for DNA extraction/qPCR analysis, a fresh gas pack added, and the box re-sealed and returned to the incubator.

(41) The qPCR analysis data was plotted graphically to determine which seeding concentration yielded the best growth of the organism. Optimal growth was determined to be that which, given the smallest inoculum concentration, gave the greatest increase over the ten day incubation. Seeding concentration is an important consideration due to the proportion of egg yolk sac-associated protein contaminating the final material.

(42) The growth curves for different C. burnetii seeding concentrations show that for inoculation concentrations in the range 110.sup.2 to 110.sup.4 copies/ml, growth was exponential (results not shown). For the two highest titres (110.sup.5 and 110.sup.6 copies/ml), growth was initially exponential until day five post-inoculation, where the titre reaches a plateau and begins to fall. The optimal inoculation concentration was determined to be 110.sup.4 copies/ml because the endpoint titre at day ten post-inoculation is almost as high as the peak titre observed for higher inoculation concentrations but without the associated fall in titre at the end.

(43) Larger Scale Growth

(44) Ten 250 ml plastic sterile conical flasks were filled with 100 ml each of ACCM-2 media. Each flask was inoculated with C. burnetii (Nine Mile strain described above) such that the estimated titre in each flask was 110.sup.4 copies/ml. This consisted of 35 l of yolk sac homogenate per 100 ml flask. Each flask was sealed into an O-ring sealed, screw-capped BioJar containing a microaerophilic atmosphere generating pack. The BioJars were then incubated for nine days in a shaking incubator set at 37 C. and 75 rpm.

(45) Prior to harvest, a sample of the pooled cultured organisms was taken for DNA extraction/qPCR titration and other quality assessment measures. To harvest the bacteria the cultures were combined and centrifuged at 12,000g for 30 min to pellet the cells. The pellets were re-suspended in a small amount of the spent ACCM-2 media, combined and re-pelleted. All media was aspirated from the pellets and they were stored at 80 C. until lysis and protein extraction. The estimated titre of the culture at harvest was 1.110.sup.9 copies/ml (using icd real-time qPCR).

(46) Quality Assessments

(47) Electron Microscopy

(48) To assess the quality of the cultured C. burnetii prior to cell lysis and protein extraction, agarose-embedding followed by transmission electron microscopy (TEM) of a sample of the material was undertaken. The electron micrographs of the ACCM-2 grown C. burnetii (data not shown) show that the material (larger scale growth) consists almost exclusively of bacterial material with little to no contaminating matter. The micrographs show a mixture of morphological forms, the smaller electron-dense particles are likely to be the SCV and the larger more diffuse particles with visible structural detail (electron dense chromatin in the core and a double-walled plasma membrane) the LCV.

(49) Immunofluorescent Microscopy

(50) Quality assessment of the cultured C. burnetii was also performed using immunofluorescent microscopy. The immunofluorescent microscopy image of the ACCM-2 grown C. burnetii (data not shown) shows that the material consists almost exclusively of bacterial material (stained as bright green coccobacilli) with little to no contaminating matter. The Evans blue counterstain has stained very little material and this material is likely to be the dried proteinaceous residue from the ACCM-2 media (bacterial cells were not washed before drying onto the slides as it was found that without some quantity of salt and protein in the buffer, Coxiellae did not adhere to the glass).

(51) Extraction of Proteins from ACCM-2 Grown C. Burnetii

(52) The ACCM-2 grown C. burnetii pelleted organisms from the larger scale growth were weighed and found to contain approximately 165 mg (wet-pellet), this was re-suspended in PBS and divided equally between four 2 ml microcentrifuge tubes to yield approximately 41 mg/tube. This material was re-pelleted at 16,000g and the PBS discarded.

(53) The pellets were re-suspended in 1.0 ml each of Bug Buster master mix (containing Benzonase Nuclease and rLysozyme; Novagen #71456-3) containing 1 protease inhibitor cocktail (Roche cOmplete ULTRA, EDTA-free #05892953001) to lyse the bacterial cells, solubilise proteins and break down the nucleic acids. The lysis was allowed to continue for 2 h at room temperature before clarification by centrifugation at 16,000g for 20 min. The soluble protein fraction was then filtered through a 0.1 m PVDF syringe filter (Millipore # SLVV033RS) to remove any residual infectious particles.

(54) Soluble C. burnetii protein was stored at 80 C. in small aliquots until required.

(55) One Dimensional Protein Separation and Detection

(56) One Dimensional Protein Separation

(57) To produce a denatured, reduced protein preparation suitable for one-dimensional (1D) PAGE, 50 l of soluble protein was mixed with 50 l of 2 concentrated Laemmli sample buffer (Sigma 53401-10VL). This mixture was heated in a heating block at 95 C. for 10 min.

(58) The denatured, reduced proteins were loaded directly into an 1 mm thick, 8 cm square 12 Bis-Tris Protein Gel (Life Technologies NuPage Novex NP0343BOX) submerged in MOPS SDS running buffer (Life Technologies NuPage Novex NP0001) at the anode and MOPS SDS running buffer containing antioxidant (Life Technologies NuPage NP0005) at the cathode. The Bis-Tris gel system is a modification of the original SDS-PAGE that runs at a neutral pH rather than the basic pH of the original method. Into the first lane was loaded a pre-stained protein standard marker (Life Technologies SeeBlue Plus2 LC5925). The gel was then electrophoresed at 200 V for 50 min.

(59) Coomassie Staining of Gels

(60) For general protein visualisation, where sensitivity to protein bands or spots containing more than 10 ng was sufficient, or where downstream mass spectrometry analysis was desired, Coomassie staining was used. After electrophoresis, gels were removed from their plastic cassettes and subjected to three, 5 min washes with deionised water on a rocking platform. The gels were then covered with 20 ml of Coomassie G-250 stain (Life Technologies SimplyBlue SafeStain LC6060) and rocked on the platform for 1 h at room temperature. The stain was discarded and the gel de-stained using deionised water for two, 1 h washes.

(61) Gel images were captured using a PC running the Bio-Rad QuantityOne software package attached to a self-contained dark-room gel-documentation system containing a digital camera (BioRad XR System #170-8170).

(62) Silver Staining of Gels

(63) For greater sensitivity staining of protein gels (spot or band abundance as low as 0.25 ng), silver staining was used. A mass spectrometry compatible protocol, included in the reagent kit, was used (Pierce silver stain kit; ThermoScientific #24612). After electrophoresis, gels were removed from their plastic cassettes and subjected to two, 5 min washes with deionised water on a rocking platform. The gels were then fixed with two, 15 min washes in 30% (v/v) ethanol: 10% (v/v) acetic acid. Gels were washed with two; 5 min washes in 10% (v/v) ethanol followed by two, 5 min washes in deionised water.

(64) Gels were sensitised for 1 min in sensitiser working solution and washed for two, 1 min washes in deionised water. The gel was stained for 5 min in stain working solution followed by two, 20 s washes in deionised water. The stain was developed in developer working solution until the bands had a good intensity (less than 3 min) and the developing stopped by two, 10 min washes in 5% (v/v) acetic acid.

(65) Western Blotting

(66) To produce membranes with bound C. burnetii proteins, a Western Blot was performed. This was achieved using an iBlot semi-dry blotting system (Life Technologies IB1001UK) using a PVDF mini transfer stack (Life Technologies iBlot 164010-02). The pre-run PAGE gel was removed from the cassette, trimmed and floated off into deionised water. The iBlot anode stack was inserted into the iBlot device. The gel was carefully laid onto the PVDF membrane without introducing air-bubbles. An iBlot filter paper, wetted with deionised water, was carefully laid on top of the gel and the de-bubbling roller used to remove any residual air bubbles. The cathode stack was then place on the stack and the lid of the iBlot (fitted with a cathode sponge) closed. The transfer itself was performed by running programme preset P3 (20 V for 7 min). After transfer, the membranes were used either immediately for antibody probing or stored.

(67) For storage, the membranes were air-dried and placed into 50 ml screw-capped Falcon tubes and kept at 20 C. Before use, frozen membranes were warmed to room temperature and re-wetted with methanol for 10 s, thoroughly rinsed with deionised water and used as freshly-transferred membranes.

(68) Antibody Probing of Transferred Proteins

(69) Blocking of unoccupied protein binding sites on the membranes was achieved by incubating them in polypropylene tubes on a roller containing 30 ml of 5% (w/v) non-fat milk (Milk; Sigma M7049-1BTL) diluted in PBS containing 0.05% (v/v) Tween-20 buffer (PBS-T; Thermo Scientific Pierce 20PBS Tween 20 PI-28352) (5% Milk/PBS-T) at room temperature for 1-2 h, or overnight at 8 C.

(70) After blocking, the membranes were incubated with 5 ml of primary antibody diluted (generally at 1:1000) in 5% Milk/PBS-T for 1 h at room temperature on a roller. Membranes were washed three times for 5 min in PBS-T (without milk).

(71) Membranes were probed with secondary antibody diluted 1:3000 in 5% Milk/PBS-T for 1 h at room temperature. The conjugate used was dependent upon the detection method used downstream (see below). For ECL detection, anti-guinea pig IgG (whole molecule)-peroxidase produced in goat (Sigma A7289) was used and for BCIP/NBT detection and anti-guinea pig IgG (whole molecule)-alkaline phosphatase produced in goat (Sigma A5062) was used. After incubation, membranes were washed three times for 5 min in PBS-T.

(72) Horseradish peroxidase-conjugated secondary antibody was detected using enhanced chemiluminescence (Amersham ECL Prime Western Blotting Detection Reagent RPN 2232); reagents A and B were mixed 50:50 v/v, applied to the membranes and incubated in the dark at room temperature for 5 min. The ECL mixture was aspirated from the membranes and the membranes blotted dry. Visualisation and imaging was performed using a gel documentation system (BioRad XRS System #170-8071).

(73) Alkaline phosphatase-conjugated secondary antibodies were detected using 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) and nitro blue tetrazolium (NBT) (Western Blue substrate; Promega S3841). Approximately 5 ml were applied to the membranes and the reaction allowed to proceed at room temperature until protein spots/bands were clearly visible and the background had just began to take up stain. The reaction was stopped by washing the substrate away with deionised water. Visualisation and imaging was performed using a gel documentation system (BioRad XR System #170-8170).

(74) Confirmation of Antibody Activity in Convalescent Sera

(75) To confirm the reactivity of the antibodies present in the convalescent guinea pig sera from the aerosol-exposure experiment with the C. burnetii proteins, a 1D PAGE was performed with the C. burnetii proteins described above and the proteins blotted onto a membrane as described above. The membrane was cut into strips and the sera from each guinea pig used to probe a single strip.

(76) ResultsOne Dimensional Protein Separation

(77) Basic Assessment of Protein Extraction

(78) The initial assessment of the C. burnetii protein extraction process was performed by running three lanes of the reduced, denatured protein; 10 l, 5 l, 2.5 l. The gel image (FIG. 1) shows a range of many well-defined bands from approximately 100 kDa down to smaller than 19 kDa. This is an indication that the BugBuster extraction process is successfully extracting a mixture of proteins from the organism.

(79) Confirmation of Antibody Activity in Convalescent Sera

(80) The Western blots of C. burnetii proteins (on the membranes) probed with guinea pig sera (IgG fraction only detected by the conjugate) from the aerosol-exposure experiment show that the negative animal (4427 V1-7) had no response to the proteins present in the protein preparation (FIG. 2). The faint band seen at 40 kDa is likely to be bleed-through from the MW marker in the adjacent lane. Guinea pigs A1-1 to A1-6 all showed good responses to a wide range of proteins present in the preparation. The quality of the response in animals 1-4 and 1-6 is subjectively better in that the bands detected are sharper and more well-defined with more bands detected in the 19-60 kDa range; these two animals were autopsied at day 15 post-exposure whereas the others were autopsied at day 21 post-exposure.

(81) The one-dimensional protein separations and Western blots showed that the extracted proteins contained a satisfactory range of protein species and that the guinea pig sera from the aerosol-infection experiments reacted strongly with a restricted subset of the protein bands. This demonstrated that the antisera could be used to select only those proteins recognised by the guinea pig immune-system during infection, organism clearance and recovery.

(82) Two-Dimensional Protein Separation and Immune-Reactive Protein Isolation

(83) Prior to two-dimensional (2D) PAGE, protein samples require relatively accurate quantification as well as a more thorough clean-up procedure to ensure no substances (ionic detergents or salts) that will interfere with the isoelectric focusing (IEF) process are present.

(84) Protein Quantitation

(85) The C. burnetii protein prepared above was quantitated using the 2-D Quant Kit (GE Healthcare 80-6483-56) by the following procedure. A standard curve was prepared in 1.5 ml microcentrifuge tubes from the 2 mg/ml bovine sera albumin (BSA) supplied with the kit; 0, 10, 20, 30, 40, 50 g. Two microcentrifuge tubes were also set up with 10 l each of the C. burnetii protein. To all tubes, 500 l of precipitant (containing trichloroacetic acid) was added, the tubes vortex-mixed and incubated at room temperature for 3 min. To all tubes, 500 l of co-precipitant (containing deoxycholic acid) was added, the tubes vortex-mixed and the proteins pelleted by centrifugation at 10,000g for 5 min.

(86) The supernatants were decanted to waste and the tubes re-centrifuged at 10,000g for a brief pulse. Any residual supernatant was removed with a micropipette. To all tubes, 100 l of copper solution and 400 l of deionised water were added and the tubes vortex-mixed to dissolve the precipitated protein. To all tubes, 1 ml of working colour reagent (100 parts of colour reagent A mixed with 1 part colour reagent B) was added and mixed by inversion, the tubes were then incubated for 15 min at room temperature.

(87) The absorbance of all samples and the standard curve were read in triplicate on a NanoDrop spectrophotometer (ND-2000) using 10 mm disposable plastic cuvettes at wavelength 480 nm using deionised water as a reference. The standard curve data were subjected to linear regression analysis in MiniTab v16, the regression equation re-arranged and used to calculate the quantity of protein in the extracted C. burnetii protein preparation.

(88) Protein Purification

(89) Prior to first-dimension separation of proteins according to charge by isoelectric focussing, the proteins from the C. burnetii lysis material prepared above were subjected to a precipitation-based purification to remove contaminants such as salt that could give the material a high conductivity or charged detergents that could interfere with the separation. This purification was performed using the 2-D Clean-Up Kit (GE Healthcare 80-6484-51). For 7 cm pl=3-11 NL IEF separations 17 l (41 g) and pl=3-5.6 NL and pl=7-11 NL separations 25 l (60 g) per strip were used.

(90) The required number of g of lysed C. burnetii material was pipetted into 1.5 ml microcentrifuge tubes. To each tube, 300 l precipitant was added and the tubes vortex-mixed, the samples were then incubated on ice (4 C.) for 30 min. To each tube, 300 l of co-precipitant was added and the contents vortex-mixed. The tubes were centrifuged at 12,000g at 4 C. for 10 min. The supernatant was removed and discarded, the tubes were then pulse centrifuged at the same speed and temperature as above. Residual supernatant was removed and discarded using a fine micropipette. Onto the pellets, 40 l of co-precipitant was carefully layered so as to not disturb the pellet. The tubes were then incubated on ice for 5 min. The tubes were centrifuged at 12,000g at 4 C. for 10 min and the supernatant discarded using a micropipette.

(91) Onto each pellet was pipetted 25 l of deionised water and the tubes vortex-mixed for 30-60 s to disperse the pellet. Pre-chilled wash buffer (20 C.) was added to each tube, 1 ml per tube containing 5 l of wash additive and vortex-mixed. The tubes were then incubated in a freezer at 20 C. for 1 h with vortex-mixing every 15 min. Protein was pelleted by centrifuging the tubes at 16,000g at 4 C. for 10 min and the supernatant discarded. The pellets were allowed to air-dry for 5 min in a rack with the lids open at room temperature. Pellets were re-suspended in 125 l of IPG strip rehydration solution (GE Healthcare 17-6003-19) by repeated pipetting. The re-suspended, purified proteins were then stored at 80 C. until just prior to IEF strip rehydration.

(92) Isoelectric Focusing of C. burnetii Proteins

(93) For first dimension separation of proteins according to their charge or isoelectric point (pi), isoelectric focusing (IEF) was performed. An Ettan IPGPhor II instrument using 7 cm Immobiline DryStrip gels which consist of a pre-formed pH gradient immobilized into a polyacrylamide gel on a stiff plastic backing was used. To the thawed purified proteins suspended in rehydration solution, the immobilised pH gradient (IPG) buffer containing carrier ampholytes (GE Healthcare pl 3-11NL 17-6004-40, pl 7-11NL 17-6004-39, pl 3-5.6NL 17-6002-02) with the appropriate pl interval to the strip being run, was added to a final concentration of 1 (v/v). Into the IPG strip holder of the IPGPhor, 125 l of the protein preparation was pipetted. The protective backing was removed from the IPGstrip (GE Healthcare pl 3-11NL 17-6003-73, pl 7-11NL 17-6003-68, pl 3-5.6NL 17-6003-53), and the strip placed, gel side down, onto the protein solution. The strip was then overlaid with cover fluid (GE Healthcare 17-1335-01) and the lid placed on top. The rehydration was left overnight in the IPGPhor at room temperature.

(94) After rehydration, the IPG strips were removed from the strip holders and thoroughly washed with deionised water. The strip holders were washed and dried to remove all traces of cover fluid. Two filter paper electrode bridges were cut and placed into the strip holders and either both soaked with deionised water for acidic pl range (pl 3-3.5NL) or the anode with deionised water and the cathode with rehydration solution for basic pl ranges (pl 3-11NL and pl 7-11NL). The rehydrated IPG strip was carefully placed, gel side down, such that the electrode bridges made contact with each end of the gel, the strip holder filled with cover fluid and the lid placed on top. The lid of the IPGPhor was closed and the IEF programme, appropriate to the strip, run. The parameters used are presented below (Table 3). After IEF, the strips were washed with deionised water and either used immediately for second dimension separation or stored in petri dishes sealed with Parafilm M (Sigma-Aldrich P7793) at 20 C.

(95) TABLE-US-00003 TABLE 3 For first dimension separation of C. burnetii proteins by isoelectric point the IPG Strip running conditions here were used for focussing the 7 cm Immobiline DryStrips on the Ettan IPGPhor II Isoelectric focussing instrument. Temperature was held at 20 C. and current was capped at 50 A/strip. pI Interval Step voltage mode Voltage (V) Time (h:min) 3.0-11.0 NL 1. Step and Hold 300 1:00 2. Gradient 1,000 0:30 3. Gradient 5,000 1:20 4. Step and Hold 5,000 0:25 3.0-5.6 NL 1. Step and Hold 300 1:00 2. Gradient 1,000 0:30 3. Gradient 5,000 1:30 4. Step and Hold 5,000 0:36 7.0-11.0 NL 1. Step and Hold 300 1:00 2. Gradient 1,000 1:00 3. Gradient 5,000 1:30 4. Step and Hold 5,000 0:55

(96) Second Dimension Protein Separation

(97) For second dimension separation of the pl-separated proteins according to their size, polyacrylamide gel electrophoresis (PAGE) was performed. Lithium dodecyl sulphate buffer (4 NuPAGE LDS sample buffer; Life Technologies NP0008) was diluted to 1 with deionised water. The proteins in the IPG strips were reduced in immunoassay reagent troughs in 1LDS sample buffer containing 1:10 sample reducing agent (NuPAGE sample reducing agent 10; Life Technologies NP0009) at room temperature for 15 min with gentle rocking. The reducing buffer was decanted off and replaced with alkylating buffer consisting of 1LDS sample buffer containing 125 mM iodoacetamide (Sigma 11149) at room temperature for 15 min with gentle rocking. The alkylating buffer was decanted off and discarded.

(98) The plastic backing strip of the IPG strip was trimmed to 7 cm and the strip carefully inserted into the IPG well of the PAGE gel (NuPAGE Novex 4-12% Bis-Tris ZOOM protein gel, 1 mm thick; Life Technologies NP0330BOX). Approximately 400 l of molten 0.5% (w/v) agarose (VWR Electran 438795A) in MOPS-SDS running buffer (NuPAGE MOPS SDS running buffer; Life Technologies NP0001) were pipetted into the well containing the IPG strip and allowed to set.

(99) The gel was loaded into the electrophoresis unit with MOPS SDS running buffer at the anode and MOPS SDS running buffer containing antioxidant (Life Technologies NuPage NP0005) at the cathode. Into the molecular weight lane, a pre-stained protein standard marker (Life Technologies SeeBlue Plus2 LC5925) was loaded. The gel was then electrophoresed at 200 V for 45 min.

(100) Gels were stained and visualised using either Coomassie or silver protocols as described above. Western blots and antibody probing of membranes were performed as described above.

(101) Isolation of Immune-Reactive Protein Spots

(102) Parallel 2D PAGE gels were prepared of the C. burnetii proteins for three pl ranges; 3-11, 3-5.6, and 7-11. One gel from each pair was used to produce a Western Blot, probed with guinea pig sera from the aerosol-exposure experiment group one, subject four (4427 1-4), and detected with Western Blue (NCIP/NBT) substrate as described above. The other gel was stained with Coomassie (as above); despite the use of a mass spectrometric-compatible protocol, silver staining was avoided because it is less compatible with down-stream mass spectrometry analysis than Coomassie.

(103) The stained Western blot membrane was placed on a lightbox and the Coomassie-stained protein gel placed in a petri dish on top of this. The protein gel was moved until the two gels were aligned using the molecular weight markers and the larger spot-features as a guide. Protein spots that visibly corresponded to spots on the Western blot membrane were carefully excised using sterile, trimmed, 1 ml micropipette tips or, for larger regions of interest, with a disposable scalpel. In some cases it could not be ascertained which protein spot corresponded to an individual spot on the Western blot membrane, in those cases no spot was collected.

(104) The spots and regions that were cut were collected into labelled 500 l microcentrifuge tubes and stored at 80 C. prior to further processing.

(105) ResultsTwo-Dimensional Protein Separation and Immune-Reactive Protein Isolation

(106) Protein Quantitation

(107) The standard curve generated during the protein assay (FIG. 3) yielded the line equation:
OD480 nm=0.8560.003g BSA

(108) To calculate the quantity of protein present in the C. burnetii protein preparation this was re-arranged to the form:

(109) $g\mathrm{BSA}=\frac{\mathrm{OD}480\mathrm{nm}-0.856}{0.003}$

(110) The two samples of the C. burnetii extract gave protein quantities of 21 and 26 g in the 10 l tested. For the purposes of the 2D PAGE the mean value of 2.4 g/l was used.

(111) Two-Dimensional Protein Separation

(112) Protein separations in two-dimensions over the three pl ranges chosen showed a satisfactory range of spots in terms of both molecular weight and pl (FIGS. 4-7). There appears to be a slight bias towards the number of spots in the low pl (3-5.6) range compared to the higher (7-11) range.

(113) Immune-Probing of 2D PAGE Western Blots and Protein Spot Excision

(114) Western blot membranes that were probed with guinea pig sera (4427 A1-4) were used to identify spots or regions on the Coomassie stained 2D PAGE gels that contained immune-reactive proteins. The locations and assigned identities of the cut proteins are indicated on the images of the blots below (FIGS. 7-9).

(115) Overall, the two-dimensional protein separations further supported the observations from the one-dimensional work by yielding a wide range of protein species in terms of both size and charge (pl). The Western blot analysis of the 2D-separated proteins again demonstrated that only a subset of the proteins present reacted with the sera from the guinea pigs. Sections of the 2D PAGE gels corresponding to reactive areas on the immunoblots were excised and stored for further analysis.

(116) Immunoprecipitation of Immune-Reactive Proteins

(117) A single spot picked from a 2D PAGE gel may well contain numerous protein species and there is no convenient method for identifying which of the identities corresponds to the immune-reactive protein species detected on the sera-probed Western blot. Accordingly, the inventors carried out immunoprecipitation (IP) to produce a second dataset of protein identities generated by using the guinea pig sera to capture the immune-reactive proteins. The captured proteins were then eluted, and identified by tandem mass spectrometry discussed below. The inventors were the first to use IP for the detection of immune-reactive proteins in C. burnetii.

(118) Antibody Affinity Purification

(119) Immunoprecipitation (IP) requires an affinity purified class G immunoglobulin (IgG) antibody to use as the capture antibody. This was prepared from 500 l of guinea pig sera as follows.

(120) A 1 ml recombinant Protein A (1 ml) column (HiTrap rProtein A FF GE Healthcare #17-5079-02) was fitted to an AKTA fast protein liquid chromatography (FPLC) instrument. The flow rate of the instrument was set to 1 ml/min. The column was flushed to remove any residual storage buffer with 5 ml of binding buffer (Appendix 1). The column was regenerated with 5 ml of Elution Buffer (Appendix 1) and finally equilibrated with 10 ml of binding buffer.

(121) The guinea pig antisera (4427 1-4) was made up to 5 ml with binding buffer and filtered through a 0.45 m syringe filter. This was then injected into a 5 ml loading loop on the FPLC instrument. Due to the precious nature of the antisera, the syringe was washed with binding buffer and the contents injected onto the column. The column was then washed with 15 ml of binding buffer.

(122) Finally, the bound IgG was eluted by washing 5 ml of elution buffer through the column. Fractions (71 ml) were collected into tubes each containing 200 l of 1 M Tris-HCl, pH 9.0 to rapidly neutralise the low pH of the elution buffer and minimise damage to the purified antibodies.

(123) Dialysis and Concentration of Affinity Purified Antibody

(124) To buffer-exchange the antibodies into a buffer compatible with IP, the pooled fractions (four and five) containing the eluted, affinity-purified IgG (approximately 2.5 ml) were dialysed against phosphate buffered saline (PBS) in a Slide-A-Lyzer dialysis cassette (Thermo #66380-10,000 MWCO). Three dialyses were performed, two stirred 500 ml volumes at room temp for 2 h each, followed by a third overnight at 8 C.

(125) The dialysed antibody was concentrated using a centrifugal concentrator (VivaSpin500 Sartorious # VS0121; 30K MWCO) by first washing the storage buffer off the column with 500 l PBS for 10 min at 15,000g. The purified antibody was loaded onto the column 500 l at a time and concentrated for 10 min at 15,000g until all of the antibody had been loaded and concentrated, and the final volume was approximately 50 l. The concentrated antibody was quantitated by measuring the absorbance at 280 nm (NanoDrop ND-2000 spectrophotometer) using PBS as a blank.

(126) Purity and Activity Check of Affinity Purified Antibody

(127) Non-reducing and reducing 1D PAGE was performed on the neat guinea pig sera, the column wash through, and the eluted, dialysed and concentrated IgG. To reduce the samples they were heated to 95 C. for 10 min in the presence of 50 mM dithiothreitol (DTT; Life Technologies P2325).

(128) In addition, to assess there was no activity loss during processing of the IgG, two Western blot strips of C. burnetii protein were probed as described above, one with the pre-treated guinea pig sera (4427 1-4) and the other with the purified, concentrated IgG.

(129) Preparation of Proteins for Immunoprecipitation

(130) For capture of the proteins recognised by the antibodies by their conformation, the native epitopes, 0.5 mg of C. burnetii protein was buffer-exchanged into the IP lysis/wash buffer supplied with the Pierce Crosslink Magnetic IP (Thermo #8805) kit. The buffer-exchange was carried out by performing three, 1 ml concentrations into IP lysis/wash buffer in a 5 kDa MWCO centrifugal concentrator (Sartorius Vivaspin 2; VS0211) at 20 C. and 4,000g.

(131) The majority of proteins isolated so far using the 2D PAGE spot picking method, were all denatured and reduced and consisted of predominantly linear epitopes. To produce a denatured protein preparation, 0.5 mg of C. burnetii protein was made to 1% (v/v) sodium dodecyl sulphate with 10% (v/v) stock solution (Life Technologies 24730-020) and 10 mM dithiothreitol (DTT; Life Technologies P2325) and heated to 95 C. for 15 min. This mixture was then alkylated by the addition of iodoacetamide (IAA; Sigma 11149) to a final concentration of 50 mM and incubated in the dark at room temperature for 45 min. To remove the substances that would interfere with the IP reaction, the denatured, reduced, and alkylated proteins were buffer-exchanged into IP lysis/wash buffer as described above for the native proteins.

(132) Immunoprecipitation of C. burnetii Immune-Reactive Proteins

(133) Immunoprecipitation was performed using a Pierce Crosslink Magnetic IP (Thermo #8805) kit. For each immunoprecipitation experiment, antibody was bound to protein A/G magnetic beads by pipetting 50 L of the magnetic beads into a 2.0 ml microcentrifuge tube. The tubes were placed in a magnetic stand and the storage buffer discarded. The beads were twice washed for 1 min in 500 l of modified coupling buffer (MCBconsisting of 100 l of 20 coupling buffer and 100 l mL of IP lysis/wash buffer made up to 2 ml with deionised water) on a rotating platform followed by magnetic collection and discarding of the supernatant. The affinity-purified guinea pig IgG was diluted to a final volume of 200 l of MCB containing 20 g of antibody. To bind antibody to the beads, the diluted antibody solution was added to the beads and the tubes incubated on a rocking platform for 30 min at room temperaturevortexing every 10 min during incubation. The beads were collected with a magnetic stand and the supernatant discarded.

(134) The beads were washed with 100 l of MCB, vortexed, magnetically collected and the supernatant discarded. This was repeated with a 300 l volume of MCB twice more to remove any unbound antibody.

(135) Antibody was cross-linked to the magnetic beads using disuccinimidyl suberate (DSS). The DSS was diluted to 0.25 mM in dimethylformamide (DMF). The cross-linking buffer consisted of 100 l coupling buffer containing 20 M DSS. The cross-linking buffer was added to the beads, vortexed and incubated for 30 min at room temperature on a rocking platform. The beads were collected in a magnetic stand and the cross-linking solution discarded. To remove any non-crosslinked antibody and stop the cross-linking reaction, 100 l volumes of elution buffer were added to the beads and mixed for 5 min at room temperature before magnetically collection and discarding the supernatant. This was performed twice. The beads were finally given two, 200 l washes in 8 C. IP lysis/wash buffer.

(136) The native or denatured protein preparations (see preparation of proteins for immunoprecipitation) were added to tubes containing cross-linked magnetic beads and incubated for 1 h at room temperature followed by overnight incubation at 8 C. on a rotating mixer. The beads were collected and the unbound proteins removed and retained. The beads were washed twice with 500 l of IP lysis/wash buffer and once with 500 l of deionised water. Bound proteins were eluted by adding 100 L of elution buffer to each tube followed by incubate for 5 min at room temperature on a rotating platform. The beads were magnetically collected, the supernatant containing the eluted proteins removed, and the pH neutralised with 10 l of neutralisation buffer. The elution step was repeated twice and the supernatants pooled.

(137) The eluted proteins were immediately purified and concentrated by precipitation using a 2-D Clean-Up Kit (GE Healthcare 80-6484-51) described above. The precipitated protein was denatured and reduced by re-suspended in 1 Laemmli buffer, heating to 95 C. for 10 min and subjecting to 1D PAGE as described above. Western blots were also performed and the blots probed with guinea pig antisera.

(138) The eluted protein lanes from the Coomassie-stained gel were cut into 7 sections (each approximately 1 cm0.5 cm) and stored at 20 C. for mass spectrometric analysis.

(139) ResultsImmunoprecipitation of Immune-Reactive Proteins

(140) Antibody Affinity Purification

(141) The output of the FPLC instrument is shown below (FIG. 10). It can be seen that fractions four and five contain the bulk of the eluted IgG. The concentrated antibody preparation produced yielded a mean quantity of 2.9 g/l (A280 nm=3.945).

(142) The purity and activity assessment (FIG. 11) show that the majority of the non-antibody serum proteins were washed from the column in the flow-through (lanes 2 and 6) and the affinity purified antibody contained almost pure IgG (heavy and light IgG chains in the reduced gel image). In addition, the antibody-probed Western blots show the activity of the purified IgG to be indiscernible from the untreated antisera from which it was derived.

(143) Immunoprecipitation

(144) The protein results of the immunoprecipitation (IP) experiment (FIG. 12) do not show any obvious loss of protein in the post-IP lanes. However, the eluted proteins from both the native and denatured protein samples contain a good range of sizes of protein species. Some of these proteins are present at low abundance, evidenced by the fact that they are only visible on the more sensitive silver-stained gels.

(145) Immunoprobing the Western-blot membrane with the IgG present in guinea pig sera (4427 A1-4) shows reactions with proteins present in all lanes (FIG. 13). This is further confirmation that the IP experiment did not remove all of the immune-reactive proteins from the post-IP samples. Reactions with the eluted protein lanes demonstrate that the proteins that were immunoprecipitated were immune-reactive species. Many of the protein species bands from the highly-sensitive silver-stained gel were not detected on the immunoblot; this is likely due to the low abundance of these proteins rather than their lack of immunogenicity. However, it is possible that some of the native proteins, due to their denatured and reduced state here, would not be recognised by their corresponding antibody.

(146) Protein Identification

(147) The inventors sought to identify the proteins contained in the 2D-PAGE spots that corresponded to reactive areas on the immunoblots, and identify the proteins contained in the gel slices of immunoreactive proteins captured by the convalescent guinea pig IgG by immunoprecipitation. The inventors also employed in silico predictive tools to obtain more information about the newly-identified identified proteins.

(148) In Gel Tryptic Digestion of Proteins

(149) Excised gel spots or regions from Coomassie-stained polyacrylamide gels were de-stained and the proteins contained in the gel, reduced and alkylated. These proteins were then digested or cleaved into peptides with trypsin followed by passive elution using the procedure described below based on a published protocol.

(150) Destaining buffer, consisting of 25 mM ammonium bicarbonate (Fisher Scientific #10207183) in 50% (v/v) aqueous acetonitrile (Fisher Scientific #10080000) and digestion buffer, consisting of 25 mM aqueous ammonium bicarbonate were prepared in advance of the procedure and stored at 8 C. Trypsin enzyme (Trypsin Gold, Mass spectrometry grade; Promega V5280) was re-suspended in 50 mM acetic acid to produce a 1 g/l stock. This stock was divided into 10 l aliquots and stored at 80 C. For each digestion, a fresh vial of trypsin was thawed. To minimise missed-cleavage artefacts during analysis of the mass spectrometric data downstream, freeze-thawed enzyme was never used for this work.

(151) Each gel piece was incubated with 500 l of destaining buffer for 30 min at 37 C. in a shaking incubator set at 300 rpm. The destaining solution was carefully aspirated from around the gel piece and discarded. This step was repeated until all of the blue colouring from the gel had been removed. The gel pieces were then dehydrated by incubating twice with 500 l of acetonitrile for 10 min, after the second incubation the acetonitrile was removed and the gel pieces allowed to air-dry (caps open) at room temperature for 10 min.

(152) The proteins were reduced by adding 50 l of 10 mM dithiothreitol (DTT; Life Technologies P2325) diluted in 25 mM ammonium bicarbonate to each gel piece and heating to 60 C. for 30 min. After incubation, the excess solution was discarded. To alkylate the proteins, the gel pieces were suspended in 50 l of 55 mM of iodoacetamine (Sigma 11149) diluted in 25 mM ammonium bicarbonate and incubated at room temperature in the dark for 45 min. The alkylation buffer was removed and the gel pieces subjected to three, 5 min washes with 500 l digestion buffer. The gel pieces were then dehydrated by incubating twice with 500 l of acetonitrile for 10 min, after the second incubation the acetonitrile was removed and the gel pieces allowed to air-dry (caps open) at room temperature for 10 min.

(153) Trypsin was thawed and diluted in 25 mM ammonium bicarbonate to a working concentration of 10 ng/l. To each gel piece, 75 l of working trypsin solution was added and the tubes incubated overnight at 37 C. at 300 rpm in a shaking incubator.

(154) Peptides were extracted by centrifuging the gel pieces at 10,000g for 5 min and aspirating off and storing the digested peptide/trypsin solution in individual tubes. The tubes were then incubated at 37 C. for 1 h with 100 l of 0.1% (v/v) aqueous trifluoroacetic acid (Fisher Scientific #10311725). The gel pieces were again centrifuged at 10,000g for 5 min, the trifluoroacetic acid aspirated off and combined and mixed with the retained trypsinised peptide solution.

(155) The extracted peptides were frozen at 80 C. until required for mass spectrometric analysis.

(156) Mass Spectrometry Analysis

(157) Tryptic peptide mixtures from the in-gel trypsin digestion were separated using nanoflow reversed phase liquid chromatography (RPLC) and analysed using a tandem mass spectrometer (nLC-MS/MS). Online chromatography was performed with the Thermo Easy nLC 1000 ultra-high pressure HPLC system (Thermo Fisher Scientific Ltd.) coupled to the Q Exactive mass spectrometer (Thermo Fisher Scientific Ltd.). The instrument was controlled by the Xcalibur software (Q Exactive Plus 2.3, ThermoFisher Scientific Ltd.).

(158) For chromatographic separation, buffer A (0.1% (v/v) aqueous formic acid) and buffer B (0.1 (v/v) formic acid in acetonitrile) were used as mobile phases for gradient separation. Each sample (10 l) was loaded onto a reversed phase Nano Trap Column (Acclaim PepMap 100, 100 m i.d.2 cm long, C.sub.18, 5 m, 100 ) and further separated on an C.sub.18 reversed-phase nanocolumn (Acclaim PepMap100, 75 m i.d.15 cm long, C.sub.18, 3 m, 100 ; ThermoFisher Scientific Ltd.) with a linear gradient of 4-75% buffer B at a flow rate of 300 nl/min over 30 min, then to 95% B over 1 min and held at 95% B for 7 min (see Table 4). Due to loading, lead-in and washing steps, the total time for the nLC-MS/MS runs was 53 min.

(159) TABLE-US-00004 TABLE 4 NanoLC parameters used to feed the electrospray ionisation (ESI) component of the tandem mass spectrometer. Buffer A (default) consisted of 0.1% (v/v) aqueous formic acid and buffer B of 0.1% (v/v) formic acid in acetonitrile. Time (min) Duration (min) Flow rate (nl/min) % buffer B (in A) 0.00 0.00 300 4 30.00 30.00 300 75 31.00 1.00 300 95 38.00 7.00 300 95

(160) General mass spectrometric conditions were set as follows: spray voltage at 1.6 kV, capillary temperature at 260 C., S-lens RE level at 50. Nitrogen was used as collision gas, but no sheath or auxiliary gases were applied.

(161) For data acquisition, the instrument was operated in positive ion mode and a data-dependent top 20 method was used. Full scans (300-2,000 amu) were acquired at a resolution of 70,000 at m/z=200 with maximum ion injection time (IIT) of 100 ms. MS/MS was performed by higher-energy collisional dissociation (HCD) fragmentation using collision-induced dissociation (CID). Resolution for HCD spectra was set to 17,500 at m/z=200 amu with maximum IIT of 50 ms. Normalized collision energy was set as 27%. The underfill ratio, specifying the minimum percentage of the target ion value likely to be reached at maximum fill time was defined as 1.0%. Default dynamic exclusion of 15.0 s was selected to prevent an ion from triggering a subsequent data-dependent scan after it has already triggered a data-dependent scan.

(162) In Silico Analyses and Protein Identification

(163) MS data were generated in the form of .RAW files (ThermoFinnigan file format), which contain all of the spectra detected from the LC-MS/MS analysis for each sample. Spectra acquired were searched against the non-redundant Uniprot protein database (http://www.uniprot.orgcontaining 8,955 C. burnetii protein sequences including randomly generated peptide decoys (TDA) to reduce the false discovery rate) using Proteome Discoverer (Version 1.4, Thermo Scientific). The search parameters used were: Enzyme: trypsin; Fixed (or static) Modifications: carbamidomethylation of cysteine; Variable Modifications: oxidation of methionine; Missed Cleavage Sites: 2; peptide mass tolerance10 ppm. The search results were filtered using Scaffold (Version 4, Proteome Software, USA) to minimise the number of false positives, as indicated by a false discovery rate (FDR) of <2%. Protein identifications were accepted with at least two unique, exclusive identified peptides.

(164) Organisation of Identified Proteins

(165) The lists of identified proteins were compared to the five published reports of proteins discovered by 2D-PAGE followed by immunoblotting, spot-picking and mass spectrometry and the four reports of proteins discovered using microarray and ELISA/ELISPOT and in vitro translated open-reading frames of the C. burnetii genome. This comparison yielded three groups of identified proteins; those that were present in both 2D-PAGE picked spots and in the immunoprecipitated proteins, those that were present only in the immunoprecipitated proteins, and those present only in 2D-PAGE picked spots. These were further sub-divided into two groups each; those previously described in one or more of the seven published reports and those that were unique to this work.

(166) Protein Functional Characterisation

(167) Functional annotation of all identified proteins was based on the cellular process information from the COG database, the UniProt server, and the InterPro domains and functional sites database. The proteins were then assigned to 20 functional categories based on the criteria used in two published C. burnetii proteomics articles.

(168) Newly-identified immune reactive proteins were further characterised using a range of tools to ascertain their size and isoelectric point using the Compute pl/Mw tool on the ExPASy server, their predicted subcellular localisation with PSORTb v3.0.2 and SOSUI.sub.GramN, their predicted non-classical secretion probability with SecretomeP v2.0, the presence of predicted signal peptides using SignalP v4.1, the presence of predicted integral beta-barrels using BOMP, the presence of predicted lipoproteins using Lipo and LipoP v1.0, and the presence of predicted transmembrane regions using TMHMM v2.0. Table 5 shows the internet locations of these tools.

(169) TABLE-US-00005 TABLE 5 Tools used to predict subcellular localisation and functionality of identified C. burnetii proteins. Tool name (and version where Uniform resource locator (URL) of server hosting applicable) the tool Compute pI/Mw tool web.expasy.org/compute_pi/ PSORTb v3.0.2 www.psortb.org/psortb/ SOSUI.sub.GramN harrier.nagahama-i- bio.ac.jp/sosui/sosuigramn_submit.html SecretomeP v2.0 www.cbs.dtu.dk/services/SecretomeP/ SignalP v4.1 www.cbs.dtu.dk/services/SignalP/ BOMP services.cbu.uib.no/tools/bomp Lipo services.cbu.uib.no/tools/lipo LipoP v1.0 www.cbs.dtu.dk/services/LipoP/ TMHMM v2.0 www.cbs.dtu.dk/services/TMHMM/

(170) ResultsProtein Identification

(171) Due to the fact that an immunoreactive spot picked from a 2D-PAGE gel can contain several proteins, proteins that were identified by spot picks alone have been excluded from the tables presented herein. Proteins identified in spot picks and captured by the guinea pig convalescent IgG during the immunoprecipitation (IP) experiments and proteins identified only by IP are presented.

(172) Novel proteins that have not been described as immune-reactive in the literature previously as isolated by 2D-PAGE spot picks and validated by immunoprecipitation (IP) are presented (Table 6) as well as those isolated by IP only (Table 7).

(173) TABLE-US-00006 TABLE 6 Novel proteins identified by 2D-PAGE spot picks and by immunoprecipitation methods that have not been previously reported as immunoreactive in the literature; the calculated molecular weight (MW) in Daltons (Da) and the estimated average isoelectric point (pI) are shown for each protein. SEQ ID Locus Functional Classification Spot MW NO: Tag Protein Name locations (Da) pI DNA metabolism - Replication, recombination and repair 1 CBU_1337 DNA polymerase III alpha subunit L20 128,481 5.7 Transcription 2 CBU_0232 DNA-directed RNA polymerase beta H2 157,104 7.6 chain 3 CBU_0852 Polyribonucleotide L6, L8, L9, 76,331 5.4 nucleotidyltransferase/Polynucleotide L21, L22, adenylyltransferase H2, Nucleotide and nucleoside biosynthesis and metabolism 4 CBU_0326 Phosphoribosylamine-glycine ligase L11, L13, 47,631 6.1 L19, H3, H4, H6, H7, C5 5 CBU_0897 Amidophosphoribosyltransferase H3, H4 55,936 6.0 6 CBU_1384 Uridylate kinase H1, C6 26,362 9.1 Regulatory function 7 CBU_1579 Trp represser binding protein H5, H6, 21,156 7.0 H7 Translation - Protein Biosynthesis 8 CBU_1475 Aspartyl/glutamyl-tRNA(Asn/Gln) H2, L16, 53,454 5.4 amidotransferase subunit B L17, L21, H3, H4, H7, C1, C2, C3 Amino acid biosynthesis and metabolism 9 CBU_0517 Aspartate aminotransferase/ L12, L13, H3, 46,419 6.4 Succinyldiaminopimelate H4, H6, H7, aminotransferase L19, C5 Energy metabolism - electron transport 10 CBU_0270 Short-chain alcohol L10, L11, L13, 44,875 5.8 dehydrogenase L19, H7, C5 11 CBU_0629 Proline dehydrogenase/Delta-1- H2 116,423 6.3 pyrroline-5-carboxylate dehydrogenase 12 CBU_0974 Acetyl-CoA acetyltransferase H6, H7 42,243 7.7 13 CBU_1088 Bifunctional NAD(P)H-hydrate L16, L17, H7 51,699 5.7 repair enzyme Nnr 14 CBU_1116 Alanine dehydrogenase H6 39,472 6.1 15 CBU_1193 Thioredoxin reductase H5, C2, C3, 34,620 5.9 C4 16 CBU_1296 ATP-NAD kinase L15, H5, C1, 32,892 5.3 C2, C4 17 CBU_1397 Succinyl-CoA synthetase beta L10, L11, L12, 42,333 5.5 chain L13, L19, H3, H4, H6, H7 18 CBU_1400 Succinate dehydrogenase iron- C6 27,792 8.2 sulfur protein 19 CBU_1401 Succinate dehydrogenase H2, L21, L22, 65,438 6.7 flavoprotein subunit H3, H4 20 CBU_1720 Aconitate hydratase H2, L22, H4 101,389 5.8 Intermediary metabolism and other metabolic pathways 21 CBU_0638 Dihydrolipoamide L2, L10, L11, 40,846 5.2 acetyltransferase component of L12, L13, L19, pyruvate dehydrogenase H3, H4, H6, complex H7, C5 22 CBU_0640 Pyruvate dehydrogenase E1 L10, L11, L12, 41,138 5.3 component alpha subunit L13, L19, H3, H4, H6, H7, C1 23 CBU_0962 Short chain dehydrogenase H1, H5, H6, 25,567 6.9 H7 Posttranslational modification, degradation, protein turnover, chaperones 24 CBU_0073 Xaa-Pro aminopeptidase H2, L21 68,185 5.6 25 CBU_0094 ClpB protein H2, L22 96,769 5.5 26 CBU_0338 Membrane alanine H2, L22, H4 103,023 6.1 aminopeptidase Cell division, chromosome partitioning 27 CBU_1352 Cell division protein ftsH L15 71,610 6.2 Protein and peptide secretion and trafficking 28 CBU_1648 DotA protein H4, C8 86,867 5.4 29 CBU_1652 IcmX protein H3, C3 41,352 6.0 Adaptation to atypical conditions - response to starvation 30 CBU_1275 Starvation sensing protein rspA L10, L19, H2, 45,431 5.7 H3, H4, H6, H7, C5

(174) TABLE-US-00007 TABLE 7 Novel proteins identified by immunoprecipitation method that have not been previously reported as immunoreactive in the literature; the calculated molecular weight (MW) in Daltons and the estimated average isoelectric point (pI) are shown for each. SEQ ID Locus Functional Classification MW NO: Tag Protein Name (Da) pI DNA metabolism - Replication, recombination and repair 31 CBU_0297 Exodeoxyribonuclease III 30,453 9.2 32 CBU_0916 Endonuclease/Exonuclease/phosphatase 29,568 9.2 family protein 33 CBU_1183 Glycine-rich RNA-binding protein 13,149 9.6 34 CBU_1235 Oligoribonuclease 21,012 5.7 DNA - medicated transformation (Competance) 35 CBU_0532 COME operon protein 1 13,493 10.5 36 CBU_0758 Lipoprotein, ComL family 30,899 9.6 Transcription 37 CBU_2086 Transcription termination factor rho 46,814 6.3 Nucleotide and nucleoside biosynthesis and metabolism 38 CBU_0043 Xanthosine triphosphate pyrophosphatase 21,777 4.7 39 CBU_0296 Orotate phosphoribosyltransferase 24,190 6.4 40 CBU_0531 Orotidine 5-phosphate decarboxylase 25,849 7.6 41 CBU_0631 Phosphoribosylformylglycinamidine synthase 146,552 6.3 42 CBU_0796 Adenosine 5-monophosphoramidase/ 12,481 6.3 Guanosine 5-monophosphoramidase 43 CBU_1830 Ribose-phosphate pyrophosphokinase 35,221 5.8 Translation - protein biosynthesis 44 CBU_0234 SSU ribosomal protein S7P 21,291 10.3 45 CBU_0445 SSU ribosomal protein S16P 20,726 9.9 46 CBU_0808 Valyl-tRNA synthetase 106,648 8.6 47 CBU_0851 SSU ribosomal protein S15P 10,316 10.4 48 CBU_1325 Bacterial Protein Translation Initiation Factor 19,456 9.9 3 (IF-3) 49 CBU_1383 Ribosome Recycling Factor (RRF) 20,945 6.4 50 CBU_1473 Aspartyl/glutamyl-tRNA(Asn/Gln) 11,102 4.7 amidotransferase subunit C 51 CBU_1594 GatB/Yqey domain protein 16,744 6.0 52 CBU_1841 Peptidyl-tRNA hydrolase 20,771 9.0 Amino acid biosynthesis and metabolism 53 CBU_1970 Diaminopimelate epimerase 30,071 6.1 Energy metabolism - electron transport 54 CBU_0075 2-polyprenyl-6-methoxyphenol hydroxylase 45,231 9.7 55 CBU_2087 Thioredoxin 12,613 4.9 Intermediary metabolism and other metabolic pathways 56 CBU_0502 DNase, TatD family 28,627 5.9 57 CBU_0288 Phosphopantetheine adenylyltransferase 17,967 6.2 58 CBU_0928 Pyridoxamine 5-phosphate oxidase 23,636 6.3 Posttranslational modification, degradation, protein turnover, chaperones 59 CBU_0738 ATP-dependent endopeptidase clp proteolytic 21,602 6.1 subunit clpP 60 CBU_2012 ATP-dependent endopeptidase hsl ATP- 52,123 5.5 binding subunit hslU Lipopolysaccharide biosynthesis and metabolism 61 CBU_2092 Phosphoenolpyruvate carboxykinase [ATP] 56,809 5.8 Protein and peptide secretion and trafficking 62 CBU_0091 Peptidoglycan-associated lipoprotein OmpA- 21,357 9.5 like 63 CBU_0155 Type 4 pili biogenesis protein pilB (nuleotide- 57,831 8.6 binding protein) Pathogenicity and pathogenesis 64 CBU_1136 Enhanced entry protein enhC, 117,740 9.3 tetratricopeptide repeat family Detoxication and Resistance 65 CBU_0943 Rhodanese-related sulfurtransferases 16,593 8.7 66 CBU_1708 Superoxide dismutase 22,274 6.2 Adaptation to atypical condition - response to starvation 67 CBU_1916 Universal stress protein A 15,779 6.6 Poorly characterised 68 CBU_0114 Protein yajQ 18,184 7.9 69 CBU_0510 Hypothetical protein 11,275 5.5 70 CBU_0656 Hypothetical transcriptional regulatory protein 12,103 4.9 71 CBU_2009 Hypothetical protein 50,169 9.1

(175) The inventors also identified 36 immunoreactive C. burnetii proteins that have previously been described in the literature, thereby further validating the processes and methods used herein.

(176) Further Characterisation of Unpublished Proteins

(177) Of the 71 novel immune reactive proteins identified, 19 were identified as having particularly advantageous features and/or a predicted non-cytoplasmic location.

(178) TABLE-US-00008 TABLE 8 Novel identified proteins (previously unpublished) that have predicted notable features and/or non-cytoplasmic locations. Predictions of transmembrane regions using TMHMM2.0, lipoproteins using Lipo and LipoP, secretion using SecretomeP2.0, signal peptides using SignalP4.1 and beta-barrel outer membrane regions using BOMP. Of particular interest for possible antibody-mediated vaccine targets are CBU_0091, CBU_1648, and CBU_1652 as inhibition of those features has been shown in the literature to inhibit bacteria replication. The two hypothetical proteins are also worthy of future study due to their, as yet, unknown functions. Predicted cellular location SEQ ID Locus Signal (program used) NO: Tag Protein Name Transmembrane Lipoprotein Secreted Peptide BOMP PSORTb SoSui.sub.GramN 54 CBU_0075 2-polyprenyl-6- + C C methoxyphenol hydroxylase 62 CBU_0091 Peptidoglycan- + + OM OM associated lipoprotein OmpA-like 25 CBU_0094 ClpB protein + C C 26 CBU_0338 Membrane alanine C OM aminopeptidase 69 CBU_0510 Hypothetical protein + ukn C 35 CBU_0532 COME operon protein 1 + + C OM 41 CBU_0631 Phosphoribosylformyl- IM C glycinamidine synthase 36 CBU_0758 Lipoprotein, ComL family + OM C 23 CBU_0962 Short chain E C dehydrogenase 64 CBU_1136 Enhanced entry protein + + 1 E ukn enhC, tetratricopeptide repeat family 15 CBU_1193 Thioredoxin reductase C P 27 CBU_1352 Cell division protein ftsH + + IM IM 18 CBU_1400 Succinate IM ukn dehydrogenase iron- sulfur protein 19 CBU_1401 Succinate IM ukn dehydrogenase flavoprotein subunit 7 CBU_1579 Trp repressor binding + ukn C protein 28 CBU_1648 DotA protein + + + 1 IM IM 29 CBU_1652 IcmX protein + + + ukn P 66 CBU_1708 Superoxide dismutase + P C 71 CBU_2009 Hypothetical protein + + ukn IM Key: C = Cytoplasmic; IM = Inner/Cytoplasmic Membrane; P = Periplasmic; OM = Outer Membrane; E = Extracellular; ukn = unknown

(179) The inventors have identified five particularly preferred proteins. CBU0091 (SEQ ID NO: 62) is described as OmpA-like, is predicted to be situated on the outer membrane and be secreted. In addition, another OmpA molecule (CBU1260) has been reported as the first C. burnetii invasin, antibodies against which were demonstrated to inhibit bacterial internalization into cells. CBU1648 (DotA, SEQ ID NO: 28) and CBU1652 (lcmX, SEQ ID NO: 29) are both constituents of the type IV secretion system of C. burnetii and are essential for replication within cells. It is possible, therefore, that antibodies binding to these proteins could inhibit the organism. Finally, both CBU0510 (SEQ ID NO: 69) and CBU2009 (SEQ ID NO: 71) are hypothetical proteins with predicted secretory functions. Although the function of these proteins is unknown, the inventors believe that these proteins play a key role in protective ability.

(180) Further Characterisation of Mechanisms of Immunity

(181) Overlapping peptide pools representing the entire open reading frame (ORF) of four vaccine candidate proteins (CBU_0510, CBU_0091, CBU_2009 and CBU_1648) were synthesised. The peptides in each pool were 15 amino acids long and were overlapping such that each 15mer started at a five amino acid offset. These peptide pools (2 g/peptide) were used to stimulate splenocytes harvested from acutely infected and recovered (convalescent) mice in an interferon- ELISpot re-stimulation assay. Peptide pools inducing an increase in spot count relative to unstimulated controls provide strong evidence that the protein that the pool represents induces protective cell-mediated immunity against part of Coxiella burnetii. Data are provided in Table 9, below:

(182) TABLE-US-00009 TABLE 9 Further characterisation of mechanisms of immunity Peptide pool (Ag) Mean increase (SFU*) Statistically significant** CBU_0510 0 No - p = 0.7 CBU_0091 29 Yes - p = 0.0029 CBU_2009 2 No - p = 0.16 CBU_1648 22.5 Yes - p = 0.0029 *SFUSpot Forming Units **Analysis performed by Mann-Whitney test

(183) The inventors found that peptide pools corresponding to CBU_0091 and CBU_1648 gave highly statistically significant interferon- responses in re-stimulated splenocytes. This is strong evidence that these vaccine antigens promote protective cell-mediated immunity to Coxiella burnetii, and (combined with their ability to elicit a humoral immune response, as demonstrated herein) renders these antigens highly desirable for use in immunisation. Peptide pools corresponding to CBU_0510 and CBU_2009 did not show a cell-mediated response, and so the inventors believe that these antigens contribute to immunity through antibody-mediated means.

(184) Summary of Results

(185) Despite previous efforts in the literature to identify immune reactive proteins of C. burnetii, the inventors have surprisingly identified 71 new immune reactive proteins.

(186) The identified proteins fall into a diverse range of functional groups, only a small proportion of which are surface exposed. The inventors believe that the surface-located proteins are directly involved in the antibody-mediated humoral immune response, and propose that these surface-exposed proteins elicit antibodies that can neutralize or hinder bacterial attachment, entry into host cells and/or replication. Antibodies raised against these antigens are believed to provide neutralization of C. burnetii, and these antigens are highly desirable for use in immunogenic compositions, such as vaccines.

(187) The inventors believe that immune recognition of proteins that are not surface exposed is also a phenomenon in C. burnetii infection. This belief is based the intracellular lifecycle of C. burnetii whereupon, during processing in the host's antigen-presenting cells, any of the organism's proteins, not just those located on the surface of the organism, could be presented to the CD4.sup.+ T-cells during immune recognition. The inventors therefore believe that the non-surface located proteins described herein are processed and presented by the host immune system, and are thus also highly desirable for use in immunogenic compositions, particularly vaccines. Such antigens are particularly useful for eliciting a cell-mediated immune response to C. burnetii in a patient.

(188) TABLE-US-00010 SEQUENCES: SEQIDNO:1 MTISFVHLKIHSEYSIVDSVVRIDQLLQRAVDLKMPAVALTDEVNLFALVKFYRQAINKGIKPIIGSELLLAEGDDVFRF TALCQNQIGFRHLIQLLSRAYVEGRQRDHVLIQWEWLVQANEGLIILSGARRGNVGQALLQRRSPLAEERLTRWIN HFPGRFYLELQRTRRDQEEEYIHSVIELALKHRVPVVATNEVCFLSQGDFEAHEARVCIHQGYLLQDVNRPREYSDQ QYFKSAEEMTALFSDIPEALENTVEIAKRCSVPLSLDEVFLPKFPVPANLKVEDYFRAQAKQGLTRRLVGLEMKNNLT HKDYEERLETEITVITKMGFASYFLIVADFIAWAKQHHIPVGPGRGSGAGSLVAYSLGITELDPLEHDLLFERFLNLER VSMPDFDIDFCMEGRDRVIDYVAERYGQEAVAQIITYGTMAARAVLRDVGRVLGLPYGYVDKIAKLVPFELGVTLE KALEQEEILAKRYAEDEEVKNLIDLAMKLEGLTRNAGKHAGGVVIAPTKLTDFVPLYSEPGSDHVVTQFDKDDVEAV GLVKFDFLGLRTLTIINWAVQNINAKRKIQNETELDIGTIPLDDPKTYALLKSCATTAVFQLESRGMKELIRRLQPDNF ADIMALVALFRPGPLQSGMVETFIACKHGEQSVHFLHPALEPILRTTYGVILYQEQVMQIAQVLAGYSLGAADVLR HAMGKKKPEEMAKQRAVFLEGTKARGLKEALANQIFDLMEKFSGYGFNKSHSAAYALIAYQTAWLKAHYPAEFM AAVLSSDMDNTDKVVGFINECRDMNLELLPPNINWSHYPFTVNTKGQIVYGLGAIKGVGEAAAMNIVAYREAEGE FKGLFNFCSRVDLRKVNRRAVEPLIRSGAMDTFGVSRASLFESLTKAFQAAEQRNRDMILGQHDLFGEEVKGIDED YTEVPEWNDSDRLRGEKETLGLYVSGHPLQACIKEMKAVGAVPINHLSLSEKNSVVVAGMMMGMRTITTRSGKR MAILSLEDQTGKIDVTLFNDLYQQVAADLTDHAILVIRGTVGRDDYTGGQKMVADMLLTLDKVREQMVKRLLIRV AGQDGVDQLLTELPPLIKPYVGGRCPVAIAYQSETAIAELLLGETWRVKLDDKLLSELSKLYGKDQVELEY SEQIDNO:2 MRDLVKQLKSEKHTAEFDALRIKLASPEEVRSWSYGEVKKPETINYRTFKPEREGLFCAKIFGPIKDYECLCGKYKRLK HRGVICEKCGVEVTLAKVRRERMGHIELASPVAHIWYLKSLPSRIGLLLDVTLRDIERILYFEAYVVVDPGMTDLEPR QLLSEEAYLDALEEYGDDFTALMGAEAIQRLLRDIDVEAEVEALRTELQTTTSETKTKKLTKRLKVLSAFLESGNKPEW MILTVLPVLPPDLRPLVPLDGGRFATSDLNDLYRRVINRNNRLKRLLDLNAPDIIVRNEKRMLQEAVDALLDNGRRG RAILGSNRRQLKSLADMIKGKSGRFRQNLLGKRVDYSGRSVIVVGPTLKLHQAGLPKKMALELFKPFIFSKLQLRGLA TTVKAAKKLVENEGPEVWDILEEVIREHPILLNRAPTLHRLGIQAFEPVLVEGKAIQLHPLVCTAYNADFDGDQMAV HVPLTLEAQLEARSLMMSTNNVLHPANGEPIIVPSQDVVLGLYYITRDRVNAKGEGMRFADAQEVVRAYENDQV DLHARITVRIKEGILNEAGEIEESDRLVNTAAGRILLWQIVPKGLPFALVDQPMTKKAVTKLLDFCYRNLGLKTTVIFA DKLMYMGFHYATHSGVSIGINDLVVPDQKEAIISRAEDEVREIEKQYASGLVTHGERRNKVIDIWSRTNDQVAKA MMEKIAVEKVKDAEGKEVAQSSFNSIYMMSDSGARGSAAQTRQLAGMRGLMARPDGTIIETPITANFREGLNVL QYFISTHGARKGLADTALKTANSGYLTRRLVDVAQDLVVTEHDCGTEASIEMMPHIEGGDVVEPLRERVLGRILAEP VMDPKSRKELLAKDTFLDERRVDILEEHSIDRVRVRSAITCEARYGICSMCYGRDLARGHVVNVGEAIGVVAAQSIG EPGTQLTMRTFHIGGAASRATAANNIGVKSTGKIKLRNLKIVEQAQGNLVAVSRSGELVVQDLQGSEREHYKVPY GATISVRDGDSVKAGQIVAQWDPHTHPIITEVAGTLRFVDLVDGVTMNRQTDELTGLSSIVITSTKQRSASGKELRP MVKLVDKNDDDLFLPGGKVPAHYFLPEGTFLTKEDGTTVNIGDVLARIPQETSKTRDITGGLPRVADLFEARRPKDA AILAEISGVVSFGKDTKDKGRLIITAPDGTTHEELIPKWRHVSVFEGETVEKGEVIADGPRDPHDILRLLGVNALANYI VNEVQEVYRLQGVKINDKHIEVIVRQMLRKVKITQPGDTDLLQNEQVERTRVREENEKIIKKDGTVAKVEPILLGITK ASLATESFISAASFQETTRVLTAASVAGKRDDLRGLKENVIVGRLIPAGTGFSYHQQRRAVAGKSVEEKEIEEKRVTA SEAEQALSEALKSSAPQEAKAAQKDE SEQIDNO:3 MNKIRKTFQYGKHEVTFETGEMARQATGAVVVRMGDTVLLVSVVAKKEAEEGRDFFPLTVNYQEKTYAAGKIPG GYFKREGRPTEKETLTSRLIDRPLRPLFPKGFTNEVQVIATVLSVDSKVPTDIPAILGASAAIGLSGIPFNGSLGAARVG YRGGEYLLNPSLDELKDSALDLVVAGTRDAVLMVESEAQELPESVMLGAVLHGHQAMQVAIQAIAEFIQEAGGAK WEWEPPTVNTALEKWVVEKSEAPLKKAYQIQEKTARQAQIQAIRDQLLADRAAEREGEENAVNEHELAVIFHELE RRIVREQILTGQPRIDGRDTKTVRPITVKVGVLPRSHGSALFTRGETQALVVTTLGTERDAQSIDDLDGDRQEEFIFH YNFPPFCVGEVGFMSGPKRREIGHGRLAKRAVVPVVPTLDKFPYVIRVVSEILESNGSSSMASVCGSSLALMDAGV PTKAPVAGIAMGLIKENDKYAVLSDILGDEDHLGDMDFKVAGTSNGVTALQMDIKIEGITKEIMEQALDQAKEGRL HILSIMNKVLDKPRSQVSDLAPQYVTMKINPEKIRDVIGKGGVVIREITEATNCAIDISDDGTIKIAAHTTEEGEAAKR RIEELTAEVELGKVYEGTVVKITDFGAFVQILPNTQGLVHISQIAQERVENVRDYLEEGQVIRVKVIEIDRQGRVRLS MKQID SEQIDNO:4 MLGVAEKCYDLTIMNILIIGNGGREHALAWKVAQSPRVEKIWVAPGNAGTARELKTQNVPIGVTDIKSLIAFAKKN QINLTLVGPEIPLAAGIVDHFQQENLIVFGPTQAAAQLETSKSFCKTFMRRHGIPTARFEAFRNTSDAFSYLEQQSFPI VIKASGLAAGKGVVIAQSLQEAKETVIAMMEEKQFGNAGAEIVIEEFLAGEELSFIAMVDGEHILPLAGSQDHKRRD DGDRGPNTGGMGAYSPVPQLSDALQEKIMTTIMQPTVTALKSEGILYRGFLYAGIMITLNNEPKVLEFNVRLGDPE TQPLMMRLRSDLIELILSALSGRLNQTQSAWDSRAALTVVLAAGGYPAHYQKGDIIQGLDQLSLPDVKVFHAGTQE INHQVVTDGGRVLGVTALGKDLREAQQKAYQAAQLITWPNCYYRHDIGHRAIS SEQIDNO:5 MCGIVG1IANGIVNQALYDALTILQHRGQDAAGIMTSDGERVFLRKSNGLVRDAIREPHMLHLVGNMGIGHVRYP TAGSESPAESQPFYVNSPYGLSLVHNGNLVNVKELTNDLIRSDLRHLNTTSDSEILLNVVAHELQHYGGVQLSPKQL FKAMTKVYERVEGAFAAVMIITGYGVIGFRDPHAIRPLVYGRRDNGNGPEYMLASESIALDALGFELIDDVGPGEVI YFDREGSVHRERCAKQVSHSPCIFEYIYLARPDSIIDGVPVYQARSGMGESLAQKILRERPDHGIDVVIPIPDTSRNAA QALARALDVPYSEGFVKNRYIGRTFIMPGQAKRRSSVRLKLNAIKAEFANKTVLLVDDSIVRGTTSKEIIQMARDVG AKKVYFASAAPEVRYPNVYGIDMPTADELIAHNKSTEEVMHSIGADWLVYQNLEDVYQAINDAMGSRKPKIERFE DSVFTGDYIAGNITKEYLAELAESRNDAAKMKKRALNEQEEANGLL SEQIDNO:6 MTNGPQPLYRRVLLKMSGEALMGKGLHAIDPNVLDRMAKDVTQVYQLGVQIAIVIGGGNFFRGAALQAAGINRI TGDYMGMLATLMNALALRDAFERSNLPVRILSAIPMTGVADAFHRRKAIHHLQQGRVVIFAAGTGNPLVTTDSAA SLRGIEINADVVLKATNVDGVYSDDPAKNPQAKLYKHLSYQEALKKELAVMDLAAFCQCRDYNMPLRVFNINKPG ALLSVIMNQEEGTLVDQGQ SEQIDNO:7 MPFILVLYYSRYGATAEMAEQVARGVERVNKIEARIRTVPSVSPKTEATEPDVPKDGPPYVTHDDLKNCVGLALGSP TRFGNMAAPLKYFLDTTSALWQSGSLIGKPAGFFTSTASLHGGQETTLLSMMMPLIHHGAIIVGVPYSETELFTTTA GGTPYGPSHMAGADSNWPLTQTEKNLCQALGKRLAEISLKLKA SEQIDNO:8 MEWEPVIGLEVHVQLRTQSKIFSGAATAYGAEPNTQACAIDLGLPGVLPVLNKEAVKLAVCFGLSVNASIPPYSIFA RKNYFYPDLPKGYQISQYNFPIVQNGHLDIENEDGTTKRIGITRAHLEEDAGKSFHEGMQGYSGIDFNRAGTPLLEIV SEPDIRSAQEAVAYLKALHSLVRYIGVSDANMQEGAFRCDVNISLRPKSEEKFGTRAEIKNVNSFRFVERAILFEINRQ KEILENGGTIVQETRLYDAVQDETRSMRTKEEAHDYRYFPDPDLLPVEIGPEFIEAVKNQLPELPWEKRKRFAASYQL SNYDVKLLTTQIEIANYFETVLKIDKTIPPKLAANWINGDLAAALNKNNLSITQSPINAEQLAGLLHRIADNTLSGSMG KQVFETMWGGEGDADTIIERHGLKQITDTEALEKIIDEVIENNPTQVEQYRSGKDKLIAFFVGQVMKATKGKANPQ QVNELFKKKL SEQIDNO:9 MTFQKPCFPHCLPVYFPLLYHSNHKELRKMNDVLSVRAQQLEPSVTLAVSDLARELLNKGHDVISLSAGEPDFDTP DFIKQSAIKAIQEGFTKYTNVDGTPALKAAIVHKLKRDNHLNYEPSEILVSGGAKQSIYNVLMGTLNAGDEAIIPAPY WVSYPPMVQLAEAKPIIISATIDQNFKLTPGQLSQAITPQSRLLILNSPNNPSGVAYTESELKALADVLMEHPQILILS DEIYEYILWGQNRFVNILNVCPELRDRTIIINGASKAYAMTGWRIGYAAGPKSIIQAMKKIQSQSTSSPNSIAQVAAT TALGAQRGDFAYMYEAYKTRHDLVLKALNQMKGVHCIPADGAFYLFPDVSAAIQQLGLEDDIKLGTYLLDKTKVAV VPGSAFGSPGHVRLSCATSTEKLQEALERLASVLDY SEQIDNO:10 MIVQPKVRGFICTTAHPEGCARHVGEWINYAKQEPSLTGGPQKVLIIGASTGFGLASRIVAAFGAGAKTIGVFFERP ASGKRTASPGWYNTAAFEKTALAAGLYAKSINGDAFSDEIKQQTIDLIQKDWQGGVDLVIYSIASPRRVHPRTGEIF NSVLKPIGQTYHNKTVDVMTGEVSPVSIEPATEKEIRDTEAVMGGDDWALWINALFKYNCLAEGVKTVAFTYIGPE LTHAVYRNGTIGRAKLHLEKTARELDTQLESALSGQALISVNKALVTQASAAIPVVPLYISLLYKIMKEKNIHEGCIEQ MWRLFKERLYSNQNIPTDSEGRIRIDDWEMREDVQAEIKRLWESINTGNVETVSDIAGYREDFYKLFGFGLNGIDY ERGVEIEKAIPSITVTPENPE SEQIDNO:11 MTDTHLLFFEKAIAQNAIRPSLNKTYRMDETTCVNHLLKTIAFTPRLEAAVSRLAKELVTAVREQESEKGGIEGFMM QYDLSTEEGILLMCLAEALLRVPDKETENLLIRDKLTSAEWNKYVGASESSFVNFATWGLALSGKILKKEKDGQFKNV WRNLVRRSGEPVIRKAVREAMKLMSEHFVLGRTIEEAVKRSQSAIKEGFRHSYDMLGEVARTQEDADRYYDSYHR AISVLGKSHPTKSVHEAPGISVKLSALYPRYDFKKRELAVPFLIERVKELALHAKEQKIGMTIDAEEADRLDISLDIFEAL FTDEAFENWQGLGLAVQAYQKRAFYLIDWLIDLAQRQKRRIPVRLVKGAYWDTEIKLAQMEGLSGYPVFTRKVNT DISYIACAQKMLNAQDAIYPQFATHNAYSVAAILNLMDHHYDNYEFEFQQLQGMGKALHHYIVTKLKLPCRVYAP VGYHEDLLPYLVRRLLENGANSSFVNRIADKTVPVDQLIESPVKKIEAFGDIPNPKIPLPKGIFKTRTNSSGIDLSNFAE LMPLNEEIHHALEKEWEAAPFLQEIKNGKPVFDPTDNRRQIGVIELANESDVEKAIQAGHSAFPNWDQKGISARAT ILRKMADLLEKHKAELMAVVVREGGRTLQNALSEVREATDFCRYYAEQAEQHLSDKALPGYTGESNTLRMNGRGII LCISPWNFPIAIFTGQIAAALVTGNAVIAKPSGQTPLTGALVTRLFHEAGVPKEILQLMPGSGKTVGQALIEDTKISGV IFTGSDATARHIQKTLAARPGPIVPFVAETSGINAMIADSTALPEQLVNDVIVSAFDSAGQRCSALRILYIQEDIADDV IKMLKGAMAEIKMGDPLLLSTDVGPVIDANAQKTLQKHQALMQKEAKLIYKVDLPRETDFGTFVAPQAYELPNLGL ITEEVFGPILHVIRYKRENLNKVIEEINGLGYGLTFGIQSRIDETVDYIQQRINAGNIYVNRNTVGAVVGVQPFGGSW LSGTGPKAGGPHYLPRFCIESTLTINTTAAGGNASLMAMED SEQIDNO:12 MENPIVIVSAARTPMGHYGGYFKEMPAPELGAAVIKAVVERAGLQPAEIDEVIMGCVLPAGQGQAPARQAALKA GLPVSTPCTTINKMCGSGMKAIMLAHDEILADSYPHIIAGGMENMSRAPYLMMKARFGYRLGHDRIYDHMMLD GLEDAYDKGKAMGVFAEKCVDKYQFTREALDKFAIESLLRAKKANENGSFAPEIVPITITHQRETLTVDHDENAMK ANPEKIPQLKPVFKADGAVTAANSSSISDGAAAVTLMRLSEAKRLNIQPLAKIIGHFTYAEDPSWFTTAPIGAIRGLLK KISWKKEAVDLFEINEAFAAVTMAAMKEIGLAHNKVNIHGGACALGHPIGASGARILVTLLYALQKNNLQRGIASLC IGGGEATAIAIERGF SEQIDNO:13 MTVLYQNRQIRELERLAVESGISEYELMCRAGEAAFKALLARWPEAQEITVCCGKGNNGGDGLVLARLAYENGLKV TVYLAGQRHQLKGAAAQAANACEASNLPILPFPEPLLFKGEVIVDALLGSGLSGEVKAPYDHLIAAINQAGQYVLAL DVPSGINVDSGEVQGTAVKANLTVTFIAPKRGLYTDKAPAYCGELIVDRLGLSESFFRAVFTDTRLLEWKGVFPLLPK RARDAHKGSYGHVLVIGGDYGMGGAVRMAAEAAARVGAGLVTVATRPEHVPIVSGPRPELMCHQVAAADDLK PLLTAATVVVIGPGLGKSDWAKSLLNKVLETDLPKVLDADSLNLLAESPSQREDWILTPHPGEASRLLGISCNEVQRD RFQAINDLQEKYQGVLVLKGVGTLIKDESQAYYVCPAGNPGMATGGMGDILSGIIGGLVAQRLSLASAAQAGVFIH SMAADRAAEEGGERGLLATDLFPHLRVLVNP SEQIDNO:14 MLIGVPKEVKIEEYRVGLTPYSVRELVLHGHQVIMERDAGNAINFTDEAYLAAGAKIVDTPVEVYQAEMIVKVKEP QSSEYALIREGQILFTYLHLAPDPQQAQALIKSGCIAIAYETVTDNEGGLPLLSPMSQVAGRLAIQAGAHCLEKPEGG SGILLGGVPGVYAGKVTVIGGGVVGSNAVRMAMGKKAQVTVLDKSLRRLQELDFQFGGRLNTAYSTESSIEHYVID ADLVVGAVLVPGHSAPKLVGQDVLKKMRPGSVMVDVAIDQGGCFETSKPTTHKKPTYVIDGIVHYCVANMPGAV PRTSTLALNNATLPYVIALADKGYRQAFLDDPHFLNGLNVYCGQITHKGVAQGLQQEFNPPLALL SEQIDNO:15 MNKPQHHSLIILGSGPAGYTAAIYAARANLKPIMITGMEQGGQLMTTTDVDNWPGEAPGLQGPQLMERMQKH AERLDTQFIFDHINEADLNQRPFLLKGDNATYSCDALIIATGASARYLGLPSEKAYMGKGVSACATCDGFFYRGKKV AVVGGGNTAVEEALYLSHIASHVTLIHRRDKLRAEKMLSAQLIKKVEEGKVAIVWSHVIEEVLGDDQGVTGVHLKH VKEEKTQDLTIDGLFIAIGHDPNTKIFKEQLEMDEAGYLRAKSGLQGNATATNIPGVFAAGDVTDHVYRQAITAAG MGCMAALDAERYLDSLNQA SEQIDNO:16 MLKIVSKPSFNRIALMGREGVEGVPETLAALKDYLVSLNREVILEENAAHMIDGSRLLTVPANDLKKKADLLIVVGG DGSLLNAAHIAVPQQLPVLGINRGRLGFLTDIPPNELTQISDILDGHYREEVRFLLEGTVEEGDEIVAQGIALNDIVLLP GNAPKMIEFDIFINDEFVCNQRADGLIITTPTGSTAYALSGGGPILHPQLNAMALVPMFPHTLSSRPIVVDAESQIKI TISPENDVSPYVSNDGQERVSIKPGGNVYTRKYHYPLHLIHPTDYNYYDTLRRKLDWEKRAAKV SEQIDNO:17 MNLHEYQSKHLLKKYNIPVPASEVVFNPDAAVDAAAKIGGDRWVVKAQVHAGGRGKAGGVRLVKNKEELKSAVK ALLGTRLVTYQTDERGQPVNQILVEQTSDIARELYLGAVIDRASQRIVFMASTEGGVEIEKVAEKSPEKILKVTIDPAI GLQPFQCRQLFFGLGLQDLKQMRSFTDIVMGLYRLFTERDLSLLEINPLVITGSGELICLDAKINIDDSALYRQSELRE MRDTTQEDEHETMAQQWELNYIKLDGNIGCMVNGAGLAMATMDLIKLSGGDPANFLDVGGSATKERVTEAFKI IVSDKNVKGILVNIFGGIVRCDLIADGIISAVKEVGIDVPVVVRLEGNNAQLGAKKLADSGMNIIAAKGFADAAEQIV KQVGVIA SEQIDNO:18 MNSKKSRIMTFSIMRFNPETDKKPYMQDFELDVSAIQGKMLLNALEALREKHPDIGLRRSCAEGVCGSDGMNING KNALACVTQLKDLPDRVVVRPLPGFPIIRDLIVDMEQFYAQYKKVKPYLLNDQEAPQKERLQSPEERAKLDGLYECIL CACCSSSCPSYWWNPDKFIGPAGLLWSYRFIADSRDSKEKERLDAMKDPYSVFRCRTIMDCATVCPKNLNPAKAIR KIRTEMLQETESGE SEQIDNO:19 MSSIRVKQYDALIVGAGGAGLRAALEMAQSRQYKVAVVSKVFPTRSHTVSAQGGIAAALGNVVPDKPIWHMFDT VKGSDYLGDQDAIQYMCEQAPPSVYELEHYGLPFSRLDDGRIYQRAFGGHTRDFGKEMARRTCACADRTGHAML HTLYQKNVEAGTHFYYEWYGIDLVRGAQGGIAGMIAMNMETSELVFFKSRATIFATGGAGRIYETTSNAYTNTGD GIGMVLRAGLPVQDMEFWQFHPTGIYGVGCLITEGARGEGGYLINKDGERFMERYSPHLKDLDCRDVVARSILQE VMAGGGVGPKKDHVLLKLDHLGEKVLRERLPGIIELSEKFANVDITKEPIPILPTCHYMMGGIPTNIHGQALTVDEN GKDQIIEGLFAAGECACVSVHGANRLGTNSLLDLVVFGRAIGLHLEEALKTELKHRSENPDDIDAAIARLKRWEKPN NVENPALLRQEMRKAMSEDFGVFREEQKMKQGLERLQKLNERLQRAKLTDTSRTFNNARIEALELDNLMEVSYAT AVSAQQRTESRGAHSRYDYKERDDANWLKHTVYFRDGHIAYRPVNMKPKGMDPFPPKSRD SEQIDNO:20 MAGCGLTDFCRTFECVKLKRKIGCEVTMADSLKTRRELTAGGKTYHYHSLKAAEDAGLSNIHRLPYSLKILLENQLRH EDGETVTQTHIEAFAHWLKDKHSDREIAYRPARVLMQDFTGVPAVVDLAAMRDAMARMKGDPTKINPHCPVDL IIDHSVQVDEFGNEEAFRDNVRIEMERNHERYTFLKWGQQAFRHFQLVPPGTGICHQVNLEYLGRGVWSSQQDG EWLAYPDTLVGTDSHTTMINGLGVLGWGVGGIEAEAAMLGQPISMLIPEVIGFYLSGQLCEGITATDLVLTVTQML RQKGVVGKFVEFYGPGLAELPLADRATIGNMAPEYGATCGLFPIDAETIKYLELTGRDAEAIELVKAYSKAQGTWHD ENTPEPIFSDTLSLDLSTVEPSLAGPKRPQDRVPLAKLKKTIEGVIATAERDQELDHSFQSTGDFDLHHGDVVIAAITS CTNTSNPSVMLAAGLLAKNAVEKGLQRKPWVKSSLAPGSKVVTDYLHKTGLIDYLEKIGFYLVGYGCTTCIGNSGPL PETVAKTVTENDLIVSSVLSGNRNFEGRIHPLVKTNWLASPPLVVAFALAGTTRIDLTKDPLGHNDRGEPIFLNDIWP SNAEIAKTVMQVRNDMFRKEYADVFEGDEEWQRIHVSAGDTFSWQTNSTYVKNPPFFENMSAKPEPLKNIIDARI LAILGDSVTTDHISPAGAIKADSPAGKYLIEHGIDIKDFNSYGSRRGNHEVLMRGTFANIRIRNEMLSKVEGGFTKHF PDGEQLPIYDAAMKYHSENIPLVVIAGKEYGTGSSRDWAAKGPRLLGVKAVVAESFERIHRSNLVGMGVLPLEFKN DDNRHSLKLEGNEVIDITGLENDLQPGGDVIMTVKRKDGTIEKIPLHCRIDTQNELAYYQHGGILQFVLRQMLRSS SEQIDNO:21 MKVFKLPDLGEGLPDATIREWYIAVGDEVKIDQPLVAMETAKALVDVPSPLAGKIEKLFGEVGDVIETGSPLIGFEGE AETEEPKDTGTVVGAIETSDIVLEESGAGIPVKKAAEKKNFKATPAVRMLAKQLGVDLTKITPKSSLISAEEVKQAAQ ITKTGKTQKIEGELTPLSPVRRAMAQSMSQSHREVVPVSLMDDGDLSAWKGEQDITLRIIRAIEAACQAVPIMNAH FDGETLGYKLNETINIGIAVDTPQGLYVPVLKDVSHQDDTALRNQINRFKELAQSRSFPPEDLRDATIMLSNFGAFA GRYANPILLPPMVTIIGVGRTRDEIVPVDGKPAVHRILPLSVISDHRVITGGEIARFLKQLIDSLEKAS SEQIDNO:22 MTPKTTTVANFTIRYLQFLDANSNPTQPFPDFADPDMLLYLYRRMALIRQLDNKAINLQRTGKMGTYPSSRGQEA VGIGMGSAMQKEDIFCPYYRDQGALFEHGIKLSEILAYWGGDERGSRYANPDVKDDFPNCVPIAGQLLHAAGVAY AVKYRKQARAVLTICGDGGTSKGDFYEAINLAGCWQLPLVFIINNNQWAISVARGEQTHCQTLAQKAIAGGFEGW QVDGNDVIAVRYAVSKALEKARDGGGPTLIEALSYRLCDHTTADDATRYIPQEEWKVAWQKEPIARLGYYLESQGL WSREKEAVLQKELAQEVDQVVEEFLTMPPPKATDMFDYLYAELPVSLEKQREELADNKPSHPSGREG SEQIDNO:23 MKRILITGANRGIGLELVKQYLAAGWHVDGCYRDKKASNSLFELAAEKKQSLTLHELDVLDEKAIQALGEHLKNQPI DILFNNAGVSAKNLREFGSIHDTENACEVFKINTIAPLLMVQALLESVEKSEKKLIINMSSEMGSIAQNVNGNYYVYR ASKSALNAITKSLAIDLKRRGITVISMNPGWVRTDMGGEQAPLDVISSVRGMREVIERVDIKSTGGFLGYDGGEMPW SEQIDNO:24 MRTLQLREGNMTNLIADRLAALRRLMHEIGVDYYYVPSSDPHKNEYVPSCWQRRAWISGFTGSAGDVVVGIDKA FLWTDPRYFLQAEQQLDDSLYHLMKMGQGETPAIDQWLTQQRNGIVFAVDPRLINLQQSEKIQRALEKQNGKLL ALDENLIDRVWKDQPPLPQSAIQLQPLQYAGLSAEDKLAALRQTLQKESADAIVLNTLDAIAWLFNIRGNDVAYNP LVISYAVITQNEASLFVDPHKITEGDRSYFKKIPVHIEPYEGIGKLLESLSGSVWLDPGATNLWLRDQLKNTASLILKPS PITLAKALKNPVEQKGAREAHIIDAIAMIQFLHWLENHWQSGVSEISAAEKLEFFRRGDSRCLDLSFPSISGFGPHGA IVHYSATTDTDATINDSAPYLIDSGGQYHYGTTDITRTIHLGTPTEEEKRLYTLVLKGHLAIRQAVFPKGTCGEHLNAL AHQFLWREALDYGHGTGHGVGSYLCVHEGPQAITSRYTGIPLQPGMIVSNEPGVYLTHKYGIRIENLCLVTEKFTVD DSLTGDGPFYSFEDLTLVPYCRKLINPNLLTSEEIQQINDYHQRVDQTLRDLLPANELNDWLHEATAPL SEQIDNO:25 MRIDKFTTAFQTALADAQSLAVGRDHQFIEPAHVMKVLLEQTQGTVAPLLEQSKVNLSRLIDGVNKAIDSYPQVEG TGGEVHVSRELSKILTLMDKFAQQNKDQYISSEWFIPAALEAKGQLRDVLIEAGADKKAIEKNIMNLRKGERVTEQS AEDQRQALAKYTIDLTEKAETGKLDPVIGRDEEIRRTVQVLQRRTKNNPVLIGEPGVGKTAIVEGLAQRIVNGEVPE GLKQKRLLALDMGALIAGAKFRGEFEERLKAVLKDIAKEEGRVILFIDELHTMVGAGKAEGAMDAGNMLKPALAR GELHCVGATTLDEYRKYIEKDAALERRFQKVLVEEPSTEDAIAILRGLKERYEVHHGVEITDPAIIAAATLSQRYITDRN LPDKAIDLIDEAASQIRMEMDSKPVELDRLERRLIQLKIEREALKKETDEASKKRLSDLETEIKNVEKEYSDLEEVWKSE KASLHGTQQIKEELEQARIELEAAGRAGDLARMSELQYGIIPELDKKLKAASQKEEQFHDHKLLRSRVTEEEVAEVVS KWTHIPVSKMLEGEREKLLHMETELHKRVIGQDEAVNAVANAIRRSRAGLSDPNRPVGSFLFLGPTGVGKTELCKA LAVFLFDTEDAMVRIDMSEFMEKHSVARLIGAPPGYVGYEEGGYLTEAIRRRPYSVILLDEIEKAHNDVFNVLLQVLD DGRLTDGQGRTVDFRNTVIVMTSNLGSDLIREFSGENYDKMKDAVMEVVAQHFRPEFINRIDEAVVFHSLKKEQIR NIAIIQIDRIKKRLKEKDYQLTISDDALDYLSELGYDPVYGARPLKRVLQQQLENPLSQKILEGKFVPGSLINIEKKGEQL EFKEA SEQIDNO:26 MGLEAFCLSSLQCQISFETAEPKMSNQKPRTVYLKDYRPSDFLVDTVHLYFDLHEEETHVKTILNLQRNPEGNATAP LALTGEAMTLKKVALDGQTLASSDYTLDASSLTIANVPNEFTLETEVVIKPQENTQLMGLYKSRGNFCTQCESHGFR RITYFLDRPDVMARYTTTITADKNKYPFLLSNGNLIETKILSDNRHWAHWEDPSKKPCYLFALVAGDFDLLEDTFVT QSGREIALRLYLEKGFKDQGPFSLAALKKAMRWDEKRFGREYDLDIYMIVAVSDFNMGAMENKGLNIFNTKYILAN PQSATDDNYVAIESVIGHEYFHNWSGNRVTCRDWFQITLKEGLTVFREQLFTEDTTSKGVARIGTVNILRNSQFPED AGPMAHPIRPRSYIEVNNFYTTTVYNKGSEVIRMVQTLLGEALFRKAMDLYFSRYDGQAVTTENFIQAMEDASGK NLEQFKRWYDQAGTPVLDLNSEYNANDKTLTLTVKQSCPPTPGQSEKLPFHLPLTLGFVGPECQDMPTQLAGEKK AIPGTRVLEIKDAETEFKFVNVNHKPTLSLLRGFSAPVRLNYPYSDEELVWLFQCDSDPFARYEAGQIFAQRLIFKLID DSYQGKPLKIDERFIDAHRKIIAGPHRDHWYEAALLQLPSINYLMQLMKKMDVEALHTIRQFVKKALSNALVDDLKI QYEHHQLPLYEYTPADIGKRKLKNICLAYLTESDDTQFRQVAYQQFKKSDNMTDTVGALSALLNHDCKERHQALDE FYQQWKDQPLVVNKWLMLHASSTLPSTLEAVRKLTKHPAFDVKNPNNVYSLLGTFGANAVCFHEGSGEGYRLIAD YVLAIDPANPQVAARVLQPLTRWQMMDKKRQELMKAELNRIAKAERLSSDVYEIVTKSLL SEQIDNO:27 MNSMIKNLLLWLVIAVVLITVFSNFGSRQSDVQPYSYSQFVQAVNNDKVSSVVIQGHEIKGVTKDNKHFTTYLPME DQALLNQLMAKGVSVKGEPPKQQSMFLHILISWLPFLILIFVWILFMRQMQGGGRGGGPMSFGRSKARLLSQDQ VKVTFDDVAGVDEAKEEVKELVEFLRDPGKFQRLGGKMPCGVLLVGPPGTGKTLLAKAVAGEAKVPFFTISGSDFV EMFVGVGASRVRDMFDQAKKQAPCIIFIDEIDAVGRHRGAGLGGGHDEREQTLNQLLVEMDGFEGKEGIIVMAA TNRPDVLDPALLRPGRFDRQVVVPLPDIKGREYILKVHMNKLPLAKDVKASVIARGTPGFSGADLANIVNEAALFAA RENKKDVSMSEFERAKDKIMMGAERRSMVMSDDEKKLTAYHEAGHAIVGLHMLEHDPVYKVTIIPRGRALGVTM FLPEHDRYSMTKRRLECQLAGLFGGRIAEEIIFGPDLVTTGASNDIEKATEIARNMVTKWGLSQKLGPLTYREEEGEV FLGRSVTQRKDISDATNKEIDSEVRRIVDTAYTTAKQTLEEHIEQLHLMAKALIKYETIGEAQIKEILAGKEPSPPPDW KEENGSASAHKENSEKELSEEKGEEKTVNPSRPRPAEDG SEQIDNO:28 MKKLVSSLLASISLFLISAAAWADNLPTDFTDNTAMNTHHDLSVTYLSQVFGTVGNVLHGMSGQMLGHLFYRLNE GIIVVAGMWLVYTVFTIVLRAAQDGSFMGPNKNVALVFLKIAFGFSLLVPNPATGYSLLQDVVMKVVVEGVGLAD QTWEYGLTYINNGGSLWRRPETNGAGKDIISQSTVNSVLGGNSQNKEGPGQKIFASAVCMYSSDDNQSPLKSNN NNIGPAVNGGPTVKYTYDVITDDSAHQFEFPGSGDTPPFKPGDDSCGAVTWDINNACTGAGSNSTKCTMAKEAV SELVTSLLPAAKKYYCSQHSSSDLCLGVTHNDAFAENETSFFGALLNYVNTIVPLVQFNSGKSADEAKRFIDEAQNEG WLSAGRYYWDLSQIQSHYDNVSNVDSYYPRTVDPTVNGNPEDDYQAALKQSLGYIYGVIDTANPHPIPVKGSVLY QLAQYAQSQHSGDTGGGEENWGHGGLDAGIALIGGIFSETIYDIYKLIHTFTTGSDGAMGPDPILFLHKIGIRAISVA ADIWFGFLGIMAIALFATGVCTATYNAQTPVQALLGWIKPLLMVVAVGLWGTGFVLAYYVPLYPYMLYTFGVIGW IIVVIEAMVAAPLIAFGLTHPEGHDFLGEAKQGGMLLLGVFLRPVLMVVGLIAGMILSYVALRIVVYTFSGLAVDLFA NTPSSGPASGSILHAATALMSNSMATAGSVTGAIVSLMVFPLVLIIFTILVYVVTTQSFSLIFALPDNVMRWIGIPGQ RSEYDRMATQLESKVGGFASSTGRSGGLQASERIGKGAANANLGKQLHLGPSKK SEQIDNO:29 MKNFRVLGIASFLALGVASTSALADIDPMSGVIKAIKEVGLEVQALAIASKKSVSNMKYQLDKNLDLALQADVEKNN ALQTVKNNAGTNTQNQISGTLLQFPEQVINASQLNDAQMAATIKNRKNLIPNLTTAIPASDTLYLTDAEDPLANTY GVAKPDSLYDNYFNFDSLFAPSAYNSDQQQAATTYLQYLTKPYQSLTDNIHFSELKDNLNKLSAEKRADKLKSFLNN PAYQKFQLAVRSLIATKSLAIDNFNTLLNERVPVKGLGAKVGMPDDPHLPKGYASPLQVENYIANQRINSPDWFKQ MKTASPAVVAREQVLILAEIESQLERNHLDNERLLATLSLMALQGTKNSEMELQTNTAADLNKLIDQIGK SEQIDNO:30 MKITDAKVFVCSPGRNFVTVKIYTDEGIYGLGDGTLNGRELAVASYLEDHLLPCLIGKDPSQIEDIWQYFYKGAYWR RGPVTMSAIGAIDMALWDIKGKALKTPVYNLLGGRSRKGVMVYGHANGKDVEETVDEVGKYIEKGYLAIRAQTGV PGLPSTYGVSPDKLFYEPAEKGLPPENVWSTEKYLNHVPKLFKKLRDVYGDDPHLLHDCHHRLTPIEAGRLGKELEP YHLFWLEDTVPAELQEGFRIIRNHTTTPLAVGEVFNVIYDCTTLITEQLIDYIRMSIVHGGGLTPMMKIASFADIYHVR TGCHGPTDVSPVTMAAALHFETAINNFGIQEFMRHTPETDEVFPHHYYFENGYLNVKDEPGLGVDFDEKLAAKYP YERAYLPINRKLDGTMYNW SEQIDNO:31 MRIITLNLNGIRAAARRGFFDWLKRQKADIVCLQETKACLEITNGDQFHPKGYHCYYHDAEKSGYSGVGIYCREKPD RVTTRLGWEHADKEGRYIQADFGSLSVASLYMPSGTTGEHRQKIKFDFMDRYMKRLKNIVHSKRSFIICGDWNIVH KEIDIKNFKSNQKYSGCLPEERAWLDEVFTKVGLVDAFRVVNQKPDQYTWWSSRGRAWEKNVGWRIDYQVITSD LKNSVKSERIYKDKRFSDHAPLIIDYEREISD SEQIDNO:32 MESLTPKRDAFTVLSYNIHKGFSARYRRFVLPDIREALRAIDADIVLLQEVQGKHHKSRLKKFAHADLPQTEFIAESK WPHYMYGKNAVYGSAHHGNALLSNFPFKMVENINVSLSQRASRSILHAIIDYEPTVELHVICIHLGLFRAERDYQLIT LSKRIEAHVPSHAPLIIAGDFNDWRRGAFNYMEKELELKEVYKVLEGKHAKTYPASRPTLEVDRIYYRGLKLLSGEIFN ESYWKKLSDHLPLHAKFAIE SEQIDNO:33 MPKHFYFYFLRKMTMSQNKIYVGSLSYDVTADELQSFFGQYGEIEEAKLIMDRETGRSKGFAFITYGTQDAAQEAV SKANGIDLQGRKIRVNIARENTGDRRRDGGSGGRGGRGGRF SEQIDNO:34 MDFSDDNLIWLDLEMTGLDPERDRIIEIATIVTNSHLDILAEGPAFAIHQPDKLLTAMDNWNTSHHTASGLLERVK NSSVDEVEAETLTLAFLEKYVSAGKSPLCGNSVCQDRRFLSRYMPRLNQFFHYRHLDVTTLKILAQRWAPQ1AAAH1 KESQHLALQDIRDSIEELRYYRAHLLNLSK SEQIDNO:35 MMFELFKEIFMKKIIQLISAVLITSLVFSAQAKPASEVIKNKLHRHAAVSTQKTGPVDINTADATLLTTLKGIGVKKAK AIIAYRKKEGNFKSIEALSSVPGISQKTVARLIRNNPHRLVVNP SEQIDNO:36 MFYNGRICLALNPEEGPMKKILFLATLLLILSGCVRKDVDPYQAYRGKTSAELFTSGERALAKKDYSEAVKNFEALDAI YPFGPHAEQAQLDIIYAYYKNNDTSSAIAAADRYIRLYPRGRNVDYAYYMRGVISFDLGLSWLQKLARVSPVSRDVS TLQQSFTSFATLAEVFPHSRYTPDALTRMRYIRNLMAQREIMIAEFYMKRRAYVAAANRGSYVVQHFQGSPQVAK ALAIMVQAYRALGLPKMADASNHLLQTNYPHTLEARKLRKA SEQIDNO:37 MNLTDLKQKSVPELMQIAQEMNLEYVSRTRKQDIIFAVLKAHAKKGEDIFGDGVLEILQDGFGFLRSADSSYLAGPD DIYVSPSQIRRFNLRTGDTVSGKIRPPKESERYFALLQVNEINLEKPEASKGKILFENLTPLFPNEQIRMETGNGSTEDI TARIIDLISPIGKGQRGLIVSPPKAGKTMMLQNIAHSITTNHPECVLIVLLIDERPEEVTEMDRSVKGEVVASTFDEPA SRHVQVAEMVIEKAKRLVEHKKDVVILLDSITRLARAYNTVIPASGKVLTGGVDANALQRPKRFFGAARNVEEGGSL TIIATALVETGSKMDDVIYEEFKGTGNMEIHLDRRIAEKRTFPAININRSGTRREELMMPQDVLQKVWILRKILHPM DELAASEFLIDRLKLTKTNNDFFDSMKG SEQIDNO:38 MLEIVLASQNSSKLAEMQELLRDLEIKFIPQTEFSVPDIEETGSTFVENAIIKARHAAKQTGLPALADDSGLTIAALNSA PGVFSSRYAGKNATDAERIQKVLEALEAADDSDRSASFHCVIALMENENDPAPLICHGVWEGEIAREPRGKNGFGY DPIFYVPSHQRTAAELDPQEKNAISHRGQALEQLSTVLTEAFLV SEQIDNO:39 MCNNDFMNQATEIAKLLLNIKAVTLNLHEPYRYTSGILSPIYCDNRLIISYPEKRKMIlEAFLQLIEKNHLSFDIVAGTAT AGIPHAAWIADRLDLPMIYVRAKAKTHGKQNQIEGRIRKGQRALIVEDLISTGKSALAAGLALREKGVTVTDCIAIFS YQLPQAQQNFSDANINCHALSHFDTLIEMAVDEGYIDEIEKQKALAWNKDPEHWQP SEQIDNO:40 MEKPDPKVIVAIDAGTVEQARAQINPLTPELCHLKIGSILFTRYGPAFVEELMQKGYRIFLDLKFYDIPQTVAGACRA VAELGVWMMNIHISGGRTMMETVVNALQSITLKEKPLLIGVTILTSLDGSDLKTLGIQEKVPDIVCRMATLAKSAGL DGVVCSAQEAALLRKQFDRNFLLVTPGIRLETDEKGDQKRVMTPRAAIQAGSDYLVIGRPITQSTDPLKALEAIDKDI KTR SEQIDNO:41 MYSIISCIPLRSIRATPILLKHDDLGSRMLFLQGSHVYTPFRHQQILFRLKQKQNTVRSVEAIYGYFVDGEKLLSRAEQE RLERLLPKAYFSDYPKSAENFSVWVTPRLGTISPWSSKATDIAHNCEIPINRIERGIYFIIDGIAKRDKKAIEKVASELYD PLTESLLFDAEDLAQLFQHPAPKTFNDIPVLGKGEAALKEADQNLGLALSDPDIHYLLRAFHQLNRNPTDIELMMFA QVNSEHCRHKIFNAQWTIDGKEKKESLFDMIRYTYKTHPEKILVAYKDNAAVIEGFNCESFLINPSNHSYEKQKGRL HTVLKVETHNHPTAIAPFAGAATGSGGEIRDEAATGRGAQSLAGLAGFSVSHLRIPDFLQPWEKAPSKKSLHSDSKP KTLASALDIMLQGPIGAASFNNEFGRPTICGYFRTLEHLSSKTLKWGYHKPIMIAGGIGHIRESQIEKQSFTEGALLVV LGGPAMAIGLGGGSASSRTSGESTEALDFASVQRANPEMQRRAQEVINACLSLGDDNPILSLHDVGAGGLSNAFP ELVHATECGGEFELRHIPNAEPGMSPLEIWCNEAQERFVLAIKPESLKVFSGIAERERCPFAVVGRAKEEKKLILNDA HFHNRPIDLPLSFLFEDMPPMKREDKRVFSGETAWNISKINWADAVKRVLQYPCVADKSFLITIGDRTVGGMVAR DQMVGPWQIPVADVAVTAHSFTGYEGQALAMGERSPIAIVHPAASARMAVGEAITNIAAAPIKAISDIVLSANW MAAPDQPGEGAGLYEAVQTVAKELCPALGICIPVGKDSLSMQTSLEKEIVTAPLSLIITATAPVSDVRHALTPQLQTD VGETRLLLIDLGQGANFLGGSCLAQTYNLLGKQPPDVDDPLLLRRFFEAIQSLNQKNLLLAYHDRSDGGLLATLCEM AFTAHVGITIKLDSLGDDALASVFNEELGAVIQVKEKNIDIVFEILKSHKLQAHSHVIGELNQLDEIIFNFRGQTLYQET RTTLQRWWSETSYRLQSLRDNPECAKQQYDGLLDKKDTGLFTKITFDNNEDIALPYINSGKRPRVAILREQGTNGH REMAAAFHLAGFESVDVHMSDLLNERVNLMDFKGAVAGGGFSYGDVLGAGRGWAQVILMHPKIRDKFSLFFES KDRFALGVCNGCQLFSHLKSLIPGALHWPAFQRNVSEQFEARLSMVEIPQSPSLFFQGMAGSQLPVAVAHGEGRV VFEKNTQEFENEKLIALRYVNYAGQPTENYPANPNGSPKGITGLTTPDGRITILMPHPERVFRTVQFSWHPKQWSE MSPWMRIFKNARKWVG SEQIDNO:42 MTACVFCKIAKGEIGELIYEDKQVVAFNDAAPQAPIHILVIPHRHIETINDVTPGDEDLLGHMVVVATRLAHDKNM AADGYRLVMNCNRNGGQAVFHIHLHLLGGRQMHWPPG SEQIDNO:43 MPNVDDIRIFHGSANPSLAENVAKELNTTIGNALISRFSDGEIRFEIEENVRGRDIYLIQSTGHPTNEHVMELILMGD AFRRASAASITAVVPYFGYARQDRRVRSSRVPISAKVVADMMQKVGFSRLITVDLHADQIQGFFYMPVDNIYASIT ALEEYRLLDKLETPMIVSPDVGGVVRARAIAKRLNDSDLAIIDKRRPAPNQAEVMNVIGNVQNRHCVIVDDIVDTA GTLCHAASALKEKGALTVSSYCTHPVLSGNAVKNIMDSDIDELIVTDTIPLHEEAAKCRKITQISLSRLIAETISRINQKE SVSSMFLD SEQIDNO:44 MARRKAAPKRETLPDPLFHSELLAKFINAVMRNGKKSVAEKIVYGALDVVAKRVQNKSGEQGDGDGESGGKAGGI KKRSLGDIRTDENARALALETFKGALDKVMPNVEVKSRRVGGSTYQVPVEIRMARRQALARRWLVEYANKRNEKT MVLRLAHEILDAVEGRGGAIKKREDVHRMAKANQAFAHYKW SEQIDNO:45 MNDLNSDGLFLFHFQAHLRWTRLALACPHQFRIKYPTLTNTGTHMVVIRLARGGSKKNPFYHIVVADRRKPRDGR FIERVGYYNPMARGQDIRLQLEKERISHWLNQGAQTSLRVKHLIKKLEKSPEEAQKGGMRKGEFKRLQAEQAAKA QKKAVATEEPKAEEAKEAPPAESQAAEGKEE SEQIDNO:46 MEKTYDPKAIEKKWADYWEKRQLSKPTAQGSPYCIMLPPPNVTGTLHMGHGFQQTLMDTLIRYHRMKGERTLW QGGTDHAGIATQMVVEQQLAQEDLTREDLGRQAFIKRVWEWRERSGGKITHQMRRLGVSIDWSRERFSMDEG LSRATTEAFIRLHHEGLIYRGKRLVNWDPKLNTAISDLEVVTEEVEGHLWHIRYPLAEGSGHLIIATTRPETLLGDVAI AVHPQDERYQPFVGKKVRLPLTDRTIPVIADEAVDKEFGTGSLKITPGHDFNDYEIGQRHQLPLINILTSEGYLNENV PEPYRGLERFEARKKIIADLQRENLLEKTEPYRVPVPRGERSGVIIEPLLTDQWFIKMEALAKPAMEAVESGELKFIPK NWEKTYLQWLSNIQDWCISRQLWWGHRLPVWYDEEKNSYVGRSREEILKKYHLSPDVKLQQETDVLDTWFSASL WPFATLGWPEKTESFKTFYPTQVLVTGFDIIFFWVARMVMMGLKLTHKIPFHSVYIHGLIRDSQGRKMSKSKGNVI DPIDIIDGISLDALIEKRTHALLQPKMAKTIEKMTRKEFPNGIASFGTDALRFTFCALASRGRDINFDMGRIDGYRNFC NKIWNAARFVTMNTQEKDLNPEKPLSYSAADEWIRTRLQQTIKNAEEALSQYRFDLLAQTLYEFTWNEYCDWYVE FAKCILYDKQAKPAQLRGTRVALLEVLEILLRLLHPVMPFITEEIWQTVAPLAGKEGKSIMVEHWPQFNIHEMNYDA KVEIEWVKNVITAIRTLRAEIGISPAKRIPVIFGKGDEKDKKRIAKMKSYIKTLGKVSQLRFAKHDDCFSATATGIVERL EIHIPLAGVIDKQTEIARLKKEISKLQKEEEKSLKKLDNPNYLQRAPQEVVEKERLSLEKTQNALKKLQSQYASIESL SEQIDNO:47 MSLASAETAKIVKEYQLGKDDTGSPEVQVAILTAKIIKLTDHMKAHKHDHHSRRGLLRMVSQRRKLLNFLKRNDLQ RYLKLIERLGLRS SEQIDNO:48 MRLIDEKGEQVGVVRTDRALTMAEEAGLDLVEISPTAKPPVCRIMNFGKYQFEQSKRKAAQKKKQRLVHLKEVKF RPGTDVGDYQVKLRKIATFLDRGDKVKVSLRFRGREMQHRELGLELLGRVKRDLGNIVVEQEPRLEGRQMTMVV MKAKGEGNKTKREDHAEIKD SEQIDNO:49 MINDIINDSKSRMEKSLGSLKTELAKLRTCRAHPSLLEHIKVDYYNVETPLSQVASIAIENPRTLSITPWEKNMVGPIE KAIQKADLGLNPATVGMVIRVPLPPLTEERRKELARVVREEAEHARVAIRNIRREANNDLKELMKEKEISEDEERRA QTAIQKLTDAQIAEVDKMASQKEADLMAV SEQIDNO:50 MALLKSRDIDKIANLSKLIIPKNENDALLEALNKTFDLVIKMDKVDTSAVDPLAHPYNETQPLREDHVTESNQRDLFQ KSAPQVEAGLYMVPVVIDNEG SEQIDNO:51 MTESLKNRIQEDMKAAMRAQEKGRLGTIRLLLAAIKQREIDEQITLDDAGVMKVIEKMIKQRRDSITQYEAGNRPD LAEKEKQEIDVLQAYLPEALSDAEIDIAVKQAIEETGATSMKDMGQLMGVLKGKLQGRVDMSMVSKKVKEHLS SEQIDNO:52 MSGGVKLIAGLGNPGDQYARTRHNVGAWFLETLAQQRNQSLAKENKFHGFVAKCNDYWLLKPTTFMNESGQA VAALAHFYKIKPSEILIAHDELDFPAGDIRLKEGGGHGGHNGLRNIIQHLGSSDFYRLRIGINHPGHKDRVTPYVLSPP SENDRIAILAAIEKGLRLIPELVQGDFQKVMRELHS SEQIDNO:53 MKVNFTKMQGSGNDFVVIDATKTPFQLTTSQIQKMANRRFGVGFDQLLVIEPPKNNSVDFHFRIFNADGSEVGQ CGNGARCIARFIRAHQLSDREELRVSTLNEVLELKIQPDGKVSVKMGVPRFEPTEIPFIASGVANFYDIAVDNQIVKL GVVNIGNPHAIIPVERINAEEVGKLGARLSVHECFPEGANVGFMQVIDPQNIRLRVYERGTGETLACGSNACAAVA VGRRCGLLQERVVVSQPGGSLTIDWQGPLTPVTMTGPATTVFCGEWLD SEQIDNO:54 MNQTDIIIIGAGLVGTSVAVALQGHGIKIKILEHHLPSAAVTSSNDVRPLTLSFGSYQILKNLGVEADLANEACPISTV HVSDQGALGALRFRASEFNVPALGYVVSFAKLQQSLYQRAALQKNAEIVPISTIDDIQCNTNHAQVTFSTINGQQQ LQADLLIAADGTHSTARRLLKIPVEEENRNEVALIALLRLKQPHNHIAYERFTSQGTLALLPLFQANQCRLVWTLPKT KADEIEQLSDDEFRAVLHRVFKPYIGAIQSVERGKRFPLQMLIAQEQVRPSFVMLGNASHTLYPIAAQGFNLGLRDA AVLSEVLIDARRQLKPLGDIRFLQEYSRWRKTDQARITGLTRGLSQWFGVQLPLANQARGLGLLATGLLPPFKKRLA KRLMGLSGRLPQLMRGLKLDDAI SEQIDNO:55 MSEHVHTASDENFETEVLQADMPVLVDFWAEWCQPCKMISPVVEEIAKEYAGRVKVFKLNVDENAQTPTKYGV RGIPSLLIFREGEVVDRKVGALNKSQLAAFLDESLHFSS SEQIDNO:56 MFVDSHCHLNMLDLSPYEGDLGALIDKAKSMGVEHILCVGVDLTHAQTVIEIAARFENVSASVGLHPSEKVDHEPT VQELVEVANHPKVVAIGETGLDYYYNHSELGKMRDRFRCHVQAALKLKKPLIIHSRSAQTDTIQIMQEENAQSVGG VMHCFTESWEMAEQAMKLGFYISFSGIVTFKNAKNVAEVAKKVPLEKMLIETDAPYLAPVPYRGKKNEPQYIPYVA ERIAELKNIPLNEVARKTTENYYHLFG SEQIDNO:57 MKPIAIYPGTFDPLTNGHVDIIERALPLFNKIIVACAPTSRKDPHLKLEERVNLIADVLTDERVEVLPLTGLLVDFAKTH QANFILRGLRAVSDFDYEFQLAHMNYQLSPEIETIFLPAREGYSYVSGTMVREIVTLGGDVSPFVPPLVARHLQKRRE K SEQIDNO:58 MFRLDLLSDPLEQFKLWYDEAIRHETLHPDAMVLATADSKGKPSARNVLYKGISKGGFLIFTNYHSRKAHELDENPQ AAWVFYWPKTYKQVRGEGRVERLTQEESEAYFETRSYESQIAAWVSEQSQEIPDREYLITRYKKYREKFQDDVRCP EFWGGFRLIPDRMEFWVGQEHRLHDRFCYLKENQEWKIIRLAP SEQIDNO:59 MSVLVPMVVEQTSRGERAYDIYSRLLKDRVIFLVGQVEDHMANLAIAQMLFLESENPNKDINLYINSPGGAVTSA MAIYDTMQFVKPDVRTLCIGQAASAGALLLAGGAKGKRHCLPHSSVMIHQVLGGYQGQGTDIQIHAKQTQRVSD QLNQILAKHTGKDIERVEKDTNRDYFLTPEEAVEYGLIDSIFKERP SEQIDNO:60 MADLNHSYLTENAPLAAQMTMTPREIVAELDKFIIGQNDAKRAVAIALRNRWRRMQLGEELRREIFPKNILMIGPT GVGKTEIARRLSDLAGAPFLKIEATKFTEVGYVGRDVESIIRDLVDVAVKMTREKAIRQVKSLAEEAAEERVLDALIPP ARGGFQGEPTAEEKPTEKKESATRQLFRKKLRNGELDDKEIEVEVSAHPSFEIMGPPGMEEMVSQLQGIMSSMSS RRSKSRRLKVKDALRILGEEEAAKLVDEDQIKSTALASVEQNGIVFIDEIDKIVKREGAVGADVSREGVQRDLLPLVEG STVFTKYGMVKTDHILFIASGAFHIAKPSDLVPELQGRFPIRVELKALTADDFVRILTEPKASLTEQYTELLKTENFGLSF TKDGIKRLAEIAYQVNDRSENIGARRLHTIMERLLEEVSFEATDKQGESITIDADYVNKQLKKLAEDEDLSRYIL SEQIDNO:61 MEQIAARVTYINLSPDELIQHAVKNGEGVLSSTGALAVTTGKRTGRSPKDRFIVKDEQTADQVAWGNINQPVEQR TFDQLWERALRYLSERAVYISHLQVGADDNYFLPLKVVTEFAWHNLFACDLFIRPSGDHANGKPSWVILSAPGLKT DPERDGVNSDGAVMINLSQRRVLLVGMPYAGEMKKAMFSVLNYLLPPHDVLPMHCAANAGQSGDVALFFGLS GTGKTTLSADPHRFLIGDDEHGWSATSVFNFEGGCYAKCIDLSQEREPMIWNAIRHGAIMENVVLDENGVPDYAD ARLTQNSRAAYPREYIPLRVENNRGRPPDAVLFLTCDLDGVLPPVALLTKEQAAYYFLSGYTALVGSTEVGSVKGVTS TFSTCFGAPFFPRPPTVYAELLMKRIEATGCQVYLVNTGWTGGAYGEGGERFSIPTTRAIVNAVLSGKLKEGPTEVLS GFNLTIPKSALGVDDHLLNPRKTWEDVSAYDARAQRLIQKFRENFEKFKVLAAIREAGPSDVH SEQIDNO:62 MSRKFTDKIKGIVMNNLVKNSGLAVIALATLNLSGCKHHPAGANAATGLSDGTGAQAYALAEGKGYQGQLKKDSE GRIINPLVAPANQTYYFDFDSTQLRSLDLGAIRVQANYLATHSTAKVRLEGNTDNRGSREYNIGLGWRRDQAVARIL EQEGVAPKQIDMVSYGKERPAVMGNNENAWRLNRRVNLIYEAY SEQIDNO:63 MQTKVEGLAHILLQTNALTNSQIARAIEQAAGAQSPLLHYLVTEKIVSSEKIAEACATYFGLEAINLQTQPLNPSLCHE IPRKYLMRYAFIPLAVKSPTLAISDPLYFPLIEELQFQTNKQYKIVFAPYKSFAALINNFVSRQIYETVSQGEASIVELVN QVLTDAIYREASDVHFEPMQQHYRIRMCIDGILHTTTLLPNTQSPAMSSRLKVLAELDISEKRLPQDGRFYFTTLTHL KRDCRLSSCPTLFGEKIVIRLLNPVHHLLKFEELGLEEKPKQLIMKKIKQLQGLILVTGPTRSGKTVSLYAALNQINSTQ KNISTVEDPIEIQLAGVTQVNIRPKAGLNFAAVLRVFLRQDDVIMVGEIRDFETASIAVRAAHTGHLVLSTLHTNSA VECITRLIDMGIEPFNLASVLKLVVAQRLVRQLCAHCQATKISCPFCLNGYQGRTGIYEVLPITPSIIELILQKRSAQEIN ACAIQEGMQTLWQAALNKAKTGITNLNEIYRVIQSENNYA SEQIDNO:64 MKRIAVFILTLSFFSISYSDKNPVFQEYYEGNYRAAETGLKQLAEKNNGEATFYLATMYMNGFGVRRDFEKGFDYM TRAAELKYLPAQLYLGNYYFQQQKDLEKAVPWFKKAADAGDAGAQLFTGISYLNGYGVKKNIDIARKYFIRAAQNEI PMGQYELAKIFLASRHAGDRRMGRIWLTKAADKYNYPDAQYLLGTMLYTGNEAEKDPVKGVEWLEKAAANGSK EASKTLDKINRINTSDAKANSENRSEPTPWQIMVGLMQKAGVQLNNPITVTASINNFTKTPKSMALDKNSIIKLNLN LVNSKDIPPEKILSYMTQLNYKEEKFDLTVPAYPFEMPPGANNYKEAFQSLSRVANYGYAQSLFRLGQMYENGLGV QKDPETAFQLYMKAAEQNYLKAQYAIGTYYLQGKGVPQDYEKAISWFIRAALKGSLQAQFVLGNIYERGIKASNNK ILFKNFDRAKAMYSLAVGGNLPIAAYRLAELYVSGFLNPDNNVSLETQNWKKAYALYQKAAKSGLEKADVALGYFY LQQNQTTLAEKTFEIAQKAYQTNDPEAAMLLAILYDRGFGVNRNSRKSAEILEKLSKQNNAIAQFMLGNYYLKNKR KENIAISLLEKSANQGNGYAKYNLAILAKQNKYTKPGENFLSLLIRAANHYDKIKEILADYYLLDTPVPGSEKKAVAIYQ ELANKQDPAAELKLGFMNEHGLLFPKDYHKAEEWYQKSAEQGNPIAQYLLGNMYYLGRGVDRDVNKAIDWLKKS AAQNYVPAKVGLGFIYEMSKHNYPEAKKWYTLASKFHNPQALYNLGLMYEYGKGVKSDPQKAFRLYKDAAQNGL DLAAVQVAGMYLKGTGIGFDPNTALKMYSQAAQKNNSFATYQLGLMSESGVAQKIDLNKARLYYEKAAKEGSVE AQLALARFYEFGISVPADISKSINFYQAAAAEGNEFAKQQLTRLSNQGKSSSNAMPFQCVNQVALEKVKNSFWKK VTDWIAPVPNIDYMNAIDYLNSGKVEQATTALQKIIKVRPNFQPARETVSHYFCQKADRK SEQIDNO:65 MLETEKCTKIFLSFSLNSRRIIMNLSLTQDPQKAKEFFEKKMAFTTGPVEVSGMLKKNAKIQVVDVRAAEDYKKGHV PGAINLPSNEWEKAAEKLDKEKTNIIYCYSQVCHLAAKAAVKFAEQGFPVMEMEGGFKTWTEHKLETEK SEQIDNO:66 MAFELPDLPYKLNALEPHISQETLEYHHGKHHRAYVNKLNKLIEGTPFEKEPLEEIIRKSDGGIFNNAAQHWNHTFY WHCMSPDGGGDPSGELASAIDKTFGSLEKFKALFTDSANNHFGSGWAWLVKDNNGKLEVLSTVNARNPMTEGK KPLMTCDVWEHAYYIDTRNDRPKYVNNFWQVVNWDFVMKNFKS SEQIDNO:67 MDNYKKILVALALDPNSDRPLVEKAKELSANRDAQLYLIHAVEHLSSYGAAYGVAAGVDVEDMLLEEAKKRMNEIA SQLNISSDHQIVKVGPAKFLILEQAKNWGVDLIIVGSHGRHGIQLLLGSTSNAVLHGAKCDVLAVRIKGS SEQIDNO:68 MPSFDIQSELNKHEVSNAVDQANREVATRFDFKGSGATYKYEGNSITLQAETDFQLKQMIDILQNKFAKRQIDVAH MKLEDPIIQHKSAQQTVMLLEGIDQTAAKKIIKLIKDQKLKVQAAIQGEKVRVTGKKRDDLQSVIGLLKEQEIGLPLQ FDNFRD SEQIDNO:69 MSNSGKKFDFQGVLNNIKSMISPESNTPSPDPSDAIGMKIAELSVLAQQLTKSHEEQAKELANVNRLLNDLFKDLEA FRNPPENKTEEKQKDKKEETKKD SEQIDNO:70 MIGGKFNLGSLMKNAKKIQEMMQKAQDELAKIRVTGESGAGMVKLTMTAQHEVVEMNLDDELLKESKEVIEDLI KAALNDANQKILKITQEKMMSAGSLFGGNESDNEET SEQIDNO:71 MIRSGKMRKLINSIIGVALIVVIVLLVLPLGMSFWLKNNYPSILTRLSQAHNVSLKLINFDRGWFASKAVIQVIIPNSED KTTQPIKFTINQHIFNGPFIFSKNNHKVKLHCAKALVYTTSNDPNFTFHSSTLLRFNNSSKSSLYASNVNVANGQEQI VLKDTNLEILYNPLTQRLVLNAVIKSALISEQQKTILIMDNITWRNDLHHATPLWEGKRSLSLNKFTYYLTPEQLIEVK NFILENQQNAANDTTTFTFSSHADSIKDTSLNLAPLDIKFSLTQMNTAALVNLINTALNENHLKLNPQQLHQFHTPA INLLAQGLEVSLAHLTFGTEEGQVSVQGQLHLPAQNQSPDLSQIMVNAKGNLQAKMPMAWLKKELSRIYEDKKV ELDDQALTPEQIADQQIQYWINNKKLIPQNQDVELTINYDKGKLLVNNLPSHAPQQ