BEWLEY KEVIN (GB)
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"Health Protection Report", vol. 9, no. 14, 24 April 2015 (2015-04-24) - 24 April 2015 (2015-04-24), pages 1 - 19, XP002765045, Retrieved from the Internet
KEVIN BEWLEY: "The identification of immune-reactive proteins recognized in response to Coxiella burnetii infection", April 2015 (2015-04-01), XP002765046, Retrieved from the Internet
| CLAIMS 1. An immunogenic composition comprising one or more protein antigens selected from: (1) a CBU_0091 antigen; (2) a CBIM648 antigen; (3) a CBU_0532 antigen; (4) a CBU_0758 antigen; (5) a CBIM652 antigen; (6) a CBU_0510 antigen; and (7) a CBU_2009 antigen. 2. The immunogenic composition of claim 1 comprising a pharmaceutically acceptable carrier or excipient. 3. The immunogenic composition of claim 1 or claim 2 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 4, wherein the bacteriostatic agent is co-trimoxazole. 6. The immunogenic composition of claim 3, wherein the one or more additional therapeutic agents comprises a bactericidal agent. 7. The immunogenic composition of claim 6, wherein the bactericidal agent is doxycycline and/or hydroxychloroquine. 8. The immunogenic composition of any one of claims 1-7, further comprising an adjuvant. 9. The immunogenic composition of claim 8, wherein the adjuvant is alum or Freund's adjuvant. 10. The immunogenic composition of any one of claims 1-9 for use in raising an immune response in a patient. 11. The immunogenic composition of any one of claims 1-10 for use in preventing, treating or suppressing C. burnetii infection in a patient. 12. Use of the immunogenic composition of any one of claims 1-10, in preventing, treating or suppressing C. burnetii infection in a patient. 13. A method of preventing, treating or suppressing C. burnetii infection in a patient, said method comprising administering to the patient the immunogenic composition of any one of claims 1-10. 14. The immunogenic composition, use or method of any one of claims 11-13, wherein said preventing, treating or suppressing comprises intravenous administration of said immunogenic composition to said patient. 15. The immunogenic composition, use or method of any one of claims 11-13, wherein said preventing, treating or suppressing comprises intraperitoneal administration of said immunogenic composition to said patient. 16. The immunogenic composition, use or method of any one of claims 11-13, wherein said preventing, treating or suppressing comprises intramuscular administration of said immunogenic composition to said patient. 17. The immunogenic composition, use or method of any one of claims 11-16, wherein said preventing, treating or suppressing comprises administration of one or more additional therapeutic agent(s). 18. The immunogenic composition, use or method of claim 17, wherein the one or more additional therapeutic agent(s) comprises a bacteriostatic agent. 19. The immunogenic composition, use or method of claim 18, wherein the bacteriostatic agent is co-trimoxazole. 20. The immunogenic composition, use or method of claim 17, wherein the one or more additional therapeutic agent(s) comprises a bactericidal agent. 21. The immunogenic composition, use or method of claim 20, wherein the bactericidal agent is doxycycline and/or hydroxychloroquine. 22. The immunogenic composition, use or method of any one of claims 17-21 , wherein said preventing, treating or suppressing comprises administration of said one or more additional therapeutic agent(s) prior to administration of said protein antigen. 23. The immunogenic composition, use or method of any one of claims 17-22, wherein said preventing, treating or suppressing comprises administration of said one or more additional therapeutic agent(s) after to administration of said protein antigen. 24. The immunogenic composition, use or method of any one of claims 17-23, wherein said preventing, treating or suppressing comprises administration of said one or more additional therapeutic agent(s) concurrently with administration of said protein antigen. 25. A nucleic acid encoding one or more protein antigens according to claim 1 , wherein said nucleic acid has been optimised for expression in a host cell. 26. The nucleic acid according to claim 25, wherein said host cell is E. coli. 27. A vector comprising a promoter operatively linked to nucleic acid according encoding one or more protein antigens according to claim 1. 28. A vector according to claim 27, wherein said nucleic acid has been optimised for expression in a host cell. 29. A vector according to claim 28, wherein said host cell is E. coli. 30. The vector of any one of claims 27-29, wherein the vector is a pET vector. 31. A host cell capable of protein expression comprising the vector of any one of claims 27- 30. 32. The host cell of claim 31 , wherein the host cell is an E. coli cell. 33. A C. burnetii protein antigen obtainable from the host cell of claim 31 or claim 32. 34. A composition comprising one or more nucleic acid(s) according to any of claims 25-26, or one or more nucleic acid(s) complementary thereto; or a vector according to any one of claims 27-30 or one or more nucleic acid(s) complementary thereto. 35. A composition according to claim 34 comprising a pharmaceutically acceptable carrier or excipient. 36. A composition according to claim 34 or claim 35 for use in nucleic acid immunisation of a patient. 37. A C. burnetii protein antigen according to claim 1 , for use in a method of generating an antibody capable of binding to said C. burnetii protein antigen. 38. An antibody that binds to a protein antigen according to claim 1. 39. A method of producing an antibody, said method comprising the following steps: (i) administering to a host animal C. burnetii antigen according to claim 1 ; (ii) allowing sufficient time for the generation of antibodies in the host animal; and (iii) obtaining the antibodies from the host animal. 40. The method of claim 39, wherein the host animal is a mammal. 41. The method of claim 40, wherein the mammal is a sheep, goat or horse. 42. A method for producing an antibody, said method comprising the following steps: (a) contacting a B cell with an effective amount of at least one protein antigen according to claim 1 ; (b) fusing the B cell of step (a) with a myeloma cell to obtain a hybridoma cell; and (c) isolating the antibody produced by the cultivated hybridoma cell. 43. An in vitro method for isolating antibodies that bind to C. burnetii protein antigen according to claim 1 , said method comprising: a) immobilising on a surface (for example on a matrix within a column) one or more C. burnetii protein antigen(s) according to claim 1 ; b) contacting the immobilised protein(s) with a solution containing antibodies that bind to the C. burnetii protein antigen(s); c) allowing said antibodies to bind to said C. burnetii protein antigen(s), thereby forming a bound complex of antibody and protein antigen(s); d) washing away any unbound antibody or protein; and e) eluting the bound antibodies from the surface, thereby providing affinity-purified antibodies. 44. The method of any one of claims 39-43, wherein the antibody is additionally purified. 45. An antibody obtainable by the method of any one of claims 39-44. 46. An antibody according to claim 38 or claim 45 for use in the prevention, treatment or suppression of C. burnetii infection in a patient. 47. Use of an antibody according to claim 38 or claim 45 in an in vitro immunoassay for confirming the presence or absence of a C. burnetii infection in a patient sample, wherein the presence of a C. burnetii infection is confirmed by detecting the binding of said antibody to a C. burnetii antigen present in said sample, and wherein failure to detect the binding of said antibody to a C. burnetii antigen in said sample confirms the absence of a C. burnetii infection. 48. An in vitro method for confirming the presence or absence of C. burnetii in a patient sample, wherein the presence of a C. burnetii is confirmed by detecting the binding of an antibody according to claim 38 or claim 45 to a C. burnetii antigen present in said sample, and wherein failure to detect the binding of an antibody according to claim 38 or claim 45 to a C. burnetii antigen in said sample confirms the absence of C. burnetii. 49. The use of claim 47 or the method of claim 48 wherein, upon confirmation of C. burnetii in the patient sample, said patient is administered agent for treatment or suppression of C. burnetii infection. 50. The use or method of claim 49, wherein said agent is selected from: (a) composition according to any one of claims 1-1 1 , 14-24 or 34-37; (b) antibody according to any one of claims 38, 45 or 46; (c) bacteriostatic agent; and/or (d) bactericidal agent. 51. The immunogenic composition, use, method or antibody of any one of claims 10-24, 34- 36, 38 or 46, wherein said patient is a mammal. 52. The immunogenic composition, use, method or antibody of claim 51 , wherein said mammal is a human. 53. The immunogenic composition, use, method or antibody of claim 51 , wherein said mammal is a non-human mammal. 54. The immunogenic composition, use, method or antibody of claim 53, wherein said non- human mammal is an ungulate, typically cow, sheep or goat. |
The present invention relates to antigens for the prevention, treatment or suppression of Coxiella burnetii infection. Also provided are methods for generating said antigens, methods for generating antibodies that bind to said antigens, and the use of said antibodies for the prevention, treatment, or suppression of C. burnetii infection.
Query (Q fever) is a bacterial infection affecting a variety of mammals, including humans. The causative agent of Q fever is the bacterium, Coxiella burnetii. C. burnetii has two phase variants: highly virulent phase I and avirulent phase II. Disease caused by C. burnetii can take several forms and has been described as clinically polymorphic.
Q fever is usually subclinical in livestock, although C. burnetii infection is associated with abortion in goats, cattle and sheep. Following initial abortions or infections with C. burnetii, animals no longer experience abortion, but typically remain sub-clinically infected, and females can carry C. burnetii indefinitely, exhibiting sporadic bacterial shedding in milk and at parturition.
Current evidence suggests that human infection by C. burnetii can occur after inhalation of as few as a single organism. This, coupled with the ability of C. burnetii to cause debilitating disease and the organism's extraordinarily high level of resistance to various means of inactivation have resulted in it being listed as a category B biological warfare and bioterrorism agent by the Centers for Disease Control (CDC).
In humans, presentation of C. burnetii infection ranges from asymptomatic, through acute disease, up to life-threatening chronic illness. In the majority of cases, acute disease presents as a self-limiting febrile illness with half of cases also suffering from severe headaches. In more severe cases of acute disease, atypical pneumonia is often reported. A proportion of those suffering from symptomatic acute Q fever will be admitted to hospital (2-4 %). The largest reported human outbreak of Q fever occurred in the Netherlands between 2007 and 2010, and was associated with a much higher hospitalisation rate, closer to 20%. This outbreak gave rise to over 4,000 reported human cases and led to the culling of 50,000 pregnant goats on 88 farms in an attempt to interrupt disease transmission.
Progression to chronic disease develops in approximately 5 % of those infected, and the vast majority of these cases present as a bacterial culture negative endocarditis, often in patients with predisposing heart-damage or immunosuppression. Without effective treatment, Q fever endocarditis is generally fatal, however earlier diagnosis of cases coupled with more effective treatment strategies has brought the death rate down to below 5 % in some cases.
l In addition to acute and chronic disease in humans, two other clinical manifestations of Q fever are of note due to their less than satisfactory outcomes with current treatment strategies. These are Q fever during pregnancy and post-Q fever fatigue syndrome (QFS). Infection with C. burnetii during pregnancy leads to premature delivery in almost half of those affected and spontaneous abortion in over a quarter. There are indications that in those infected during the first trimester and treated with suboptimal drug regimes, the abortion rate is 100 %. This is compounded by the fact that the frontline bactericidal drugs for treatment (doxycycline and hydroxychloroquine) are contraindicated for use during pregnancy. A bacteriostatic regimen (co-trimoxazole) has therefore been proposed for use until delivery. Without satisfactory treatment during and after pregnancy there is also a high probability for infection to lead to chronic Q fever in the mother (70 % was reported in a group of pregnant women in France). QFS was first reported in 1996, but an association between Q fever and chronic fatigue had been observed as early as 1982. Between 10 % and 15 % of those who have acute Q fever will develop a chronic fatigue syndrome that typically lasts between five and ten years, and in some cases even longer.
The current Q fever vaccine licensed for human use (in Australia only, named "Q-VAX") is a formalin-killed whole-cell vaccine (WCV) produced by cultivating virulent C. burnetii in live embryonated chicken eggs. Yolk sacs are removed and homogenised, and the preparation is inactivated with formaldehyde. This method of production is labour-intensive, poorly defined and difficult to reproduce, and is high-risk (requiring high biological containment). Moreover, the removal of fats and egg proteins is not 100% effective, leading to contamination of the final product with host cell components.
The current Q fever vaccine is also associated with severe local reactions in those with pre-existing immunity. The severity is such that pre-screening of potential vaccination candidates for skin reactions (to a small quantity of vaccine) and blood antibody levels must be performed prior to vaccination. Such screening is time-consuming and expensive. Moreover, despite pre-screening efforts, severe reactions against this vaccine are still reported in some individuals. Q-VAX is therefore deemed unsuitable for general vaccination strategies, and its use is limited to individuals in defined occupational risk groups, such as abattoir workers.
Alternative vaccines based on more highly purified whole-cell preparations such as chloroform:methanol residues (CMR) of the formalin-inactivated whole-cell material have been developed and shown to have similar levels of protection in some animal models with potentially fewer side-effects. However, the production of CMR vaccines suffers the same production issues as the current Q fever vaccine. Also, although vaccination with CMR appeared to protect against disease upon challenge, it did not protect against splenomegaly, hepatomegaly, or lesions in the liver. Indeed, animals vaccinated with CMR showed significant splenomegaly and hepatomegaly when either challenged with live C. burnetii or injected with killed WCV vaccine. This indicates that inadequate immunity against C. burnetii may lead to enhanced disease rather than partial protection. In addition, the production of CMR is hampered by the same limitations as WCV, particularly the requirement to produce large quantities of virulent C. burnetii at high containment during their manufacture. Vaccines based on the phase II, avirulent form of the organism (due to a 30 kb genomic deletion leading to a surface lipopolysaccharide lacking in O-antigen) were briefly investigated but found to be non-protective. Attempts to produce a vaccine based on a small number of recombinantly expressed immunodominant C. burnetii proteins also failed to induce protection in a mouse model.
Accordingly, there exists an urgent need for further vaccines against C. burnetii infection. In particular, there exists a need for vaccines with an improved safety profile. An improved safety profile would increase the number of individuals that may enjoy the benefits of vaccination against C. burnetii infection, and would reduce the risk of patients suffering from adverse reactions, and would reduce or avoid the requirement for pre-screening. There also exists a need for vaccines against C. burnetii, whose production does not require the culturing of organisms at high levels of biological containment. Advantageously, such vaccines would be less expensive to produce than existing vaccines; more simple to produce than existing vaccines; less dangerous to produce than existing vaccines; and would avoid the current challenges in providing acceptable batch-to-batch variation.
It is an object of the invention to provide further antigens for use in preventing, treating or and/or suppressing C. burnetii infection. It is also an object of the invention to provide immunogenic compositions that are effective in raising an immune response against C. burnetii, and which avoid the above-mentioned limitations with existing vaccines against C. burnetii.
The inventors have identified a number of C. burnetii protein antigens which are involved in immunity and infection. The protein antigens of the invention avoid one or more of the above- mentioned limitations associated with existing vaccines against C. burnetii infection. The protein antigens of the present invention are capable of eliciting an immune response against C. burnetii, and are thus suitable for use in vaccines against C. burnetii infection. The provision of specific protein antigens that stimulate the immune system avoids the requirement to administer poorly defined complex mixtures of microbial antigens (as is the case for WCV and CMR vaccines), and so the chances of adverse reactions to the vaccines of the invention are significantly reduced. Moreover, the protein antigens of the invention can be produced recombinantly, without the requirement for high-level containment, and at lower cost than the current vaccines.
The inventors have identified 71 immune-reactive proteins from C. burnetii. Despite previous efforts to identify immune-reactive proteins from C. burnetii, the inventors believe that none of these 71 immune-reactive proteins have been previously identified as such. Thus, in one aspect, the invention provides a protein antigen selected from: (1) a CBU_0091 antigen; (2) a CBIM648 antigen; (3) a CBIM652 antigen; (4) a CBU_0510 antigen; (5) a CBU_2009 antigen; (6) a CBIM579 antigen; (7) a CBIM 193 antigen; (8) a CBIM400 antigen; (9) a CBIM401 antigen; (10) a CBU_0962 antigen; (11) a CBU_0094 antigen; (12) a CBU_0338 antigen; (13) a CBIM352 antigen; (14) a CBU_0532 antigen; (15) a CBU_0758 antigen; (16) a CBU_0631 antigen; (17) a CBU_0075 antigen; (18) a CBIM 136 antigen; (19) a CBIM708 antigen; (20) a CBIM337 antigen; (21) a CBU_0232 antigen; (22) a CBU_0852 antigen; (23) a CBU_0326 antigen; (24) a CBU_0897 antigen; (25) a CBIM384 antigen; (26) a CBIM475 antigen; (27) a CBU_0517 antigen; (28) a CBU_0270 antigen; (29) a CBU_0629 antigen; (30) a CBU_0974 antigen; (31) a CBIM088 antigen; (32) a CBIM 116 antigen; (33) a CBIM296 antigen; (34) a CBU_1397 antigen; (35) a CBIM720 antigen; (36) a CBU_0638 antigen; (37) a CBU_0640 antigen; (38) a CBU_0073 antigen; (39) a CBU_1275 antigen; (40) a CBU_0297 antigen; (41) a CBU_0916 antigen; (42) a CBIM 183 antigen; (43) a CBIM235 antigen; (44) a CBU_2086 antigen; (45) a CBU_0043 antigen; (46) a CBU_0296 antigen; (47) a CBU_0531 antigen; (48) a CBU_0796 antigen; (49) a CBU_1830 antigen; (50) a CBU_0234 antigen; (51) a CBU_0445 antigen; (52) a CBU_0808 antigen; (53) a CBU_0851 antigen; (54) a CBU_1325 antigen; (55) a CBIM383 antigen; (56) a CBU_1473 antigen; (57) a CBIM594 antigen; (58) a CBIM841 antigen; (59) a CBU_1970 antigen; (60) a CBU_2087 antigen; (61) a CBU_0502 antigen; (62) a CBU_0288 antigen; (63) a CBU_0928 antigen; (64) a CBU_0738 antigen; (65) a CBU_2012 antigen; (66) a CBU_2092 antigen; (67) a CBU_0155 antigen; (68) a CBU_0943 antigen; (69) a CBU_1916 antigen; (70) a CBU_0114 antigen; and (71) a CBU_0656 antigen.
In one embodiment, the invention provides a protein antigen selected from: (1) a CBU_0091 antigen; (2) a CBIM648 antigen; (3) a CBIM652 antigen; (4) a CBU_0510 antigen; (5) a CBU_2009 antigen; (6) a CBIM579 antigen; (7) a CBIM 193 antigen; (8) a CBIM400 antigen; (9) a CBIM401 antigen; (10) a CBU_0962 antigen; (11) a CBU_0094 antigen; (12) a CBU_0338 antigen; (13) a CBIM352 antigen; (14) a CBU_0532 antigen; (15) a CBU_0758 antigen; (16) a CBU_0631 antigen; (17) a CBU_0075 antigen; (18) a CBIM 136 antigen; and (19) a CBIM708 antigen. These antigens are highly suitable for eliciting a humoral immune response to C. burnetii in a patient. CBU_0091 and CBIM648 antigens have also been demonstrated herein to elicit a cell-mediated immune response. The inventors have identified CBU_0532 and CBU_0758 as candidates for eliciting a cell-mediated immune response.
In one embodiment, the invention provides a protein antigen selected from: (1) a CBU_0091 antigen; (2) a CBIM648 antigen; (3) a CBIM652 antigen; (4) a CBU_0510 antigen; and (5) a and CBU_2009 antigen. These antigens are particularly suitable for eliciting a humoral immune response to C. burnetii in a patient. CBU_0091 and CBIM648 antigens have also been demonstrated herein to elicit a cell-mediated immune response. In one embodiment, the invention provides a protein antigen selected from: (1) a CBU_0091 antigen; (2) a CBIM648 antigen; (3) a CBU_0510 antigen; and (4) a CBU_2009 antigen; and optionally (5) a CBIM652 antigen. These antigens are particularly suitable for eliciting a humoral immune response to C. burnetii in a patient. CBU_0091 and CBIM648 antigens have also been demonstrated herein to elicit a cell-mediated immune response.
In one embodiment, the invention provides a protein antigen selected from: (1) a CBU_0091 antigen; (2) a CBIM648 antigen; (3) a CBU_0532 antigen; (4) a CBU_0758 antigen; (5) a CBIM652 antigen; (6) a CBU_0510 antigen; and (7) a CBU_2009 antigen. These antigens are particularly suitable for eliciting a humoral immune response to C. burnetii in a patient. CBU_0091 and CBIM648 antigens have also been demonstrated herein to elicit a cell- mediated immune response. The inventors have identified CBU_0532 and CBU_0758 as candidates for eliciting a cell-mediated immune response.
In one embodiment, the invention provides a protein antigen selected from: (1) a CBU_0091 antigen; (2) a CBIM648 antigen; (3) a CBU_0532 antigen; (4) a CBU_0758 antigen; (5) a CBU_0510 antigen; and (6) a CBU_2009 antigen; and optionally (7) a CBIM652. These antigens are particularly suitable for eliciting a humoral immune response to C. burnetii in a patient. CBU_0091 and CBIM648 antigens have also been demonstrated herein to elicit a cell-mediated immune response. The inventors have identified CBU_0532 and CBU_0758 as candidates for eliciting a cell-mediated immune response.
In one embodiment, the invention provides a protein antigen selected from: (1) a CBU_0091 antigen; and (2) a CBIM648 antigen. These antigens are particularly suitable for eliciting a humoral immune response and also a cell-mediated immune response to C. burnetii in a patient.
In one embodiment, the invention provides a protein antigen selected from: (1) a CBU_0091 antigen; (2) a CBIM648 antigen; (3) a CBU_0532 antigen; and (4) a CBU_0758 antigen.
In one embodiment, the invention provides a protein antigen selected from: (1) a CBU_0091 antigen; (2) a CBIM648 antigen; and (3) a CBU_0532 antigen.
In one embodiment, the invention provides a protein antigen selected from: (1) a CBU_0091 antigen; (2) a CBIM648 antigen; and (3) a CBU_0758 antigen.
In one embodiment, the invention provides a protein antigen selected from: (1) a CBIM 337 antigen; (2) a CBU_0232 antigen; (3) a CBU_0852 antigen; (4) a CBU_0326 antigen; (5) a CBU_0897 antigen; (6) a CBIM 384 antigen; (7) a CBIM475 antigen; (8) a CBU_0517 antigen; (9) a CBU_0270 antigen; (10) a CBU_0629 antigen; (11) a CBU_0974 antigen; (12) a CBIM088 antigen; (13) a CBIM 1 16 antigen; (14) a CBIM296 antigen; (15) a CBIM 397 antigen; (16) a CBIM 720 antigen; (17) a CBU_0638 antigen; (18) a CBU_0640 antigen; (19) a CBU_0073 antigen; (20) a CBIM275 antigen; (21) a CBU_0297 antigen; (22) a CBU_0916 antigen; (23) a CBIM 183 antigen; (24) a CBIM235 antigen; (25) a CBU_2086 antigen; (26) a CBU_0043 antigen; (27) a CBU_0296 antigen; (28) a CBU_0531 antigen; (29) a CBU_0796 antigen; (30) a CBIM830 antigen; (31) a CBU_0234 antigen; (32) a CBU_0445 antigen; (33) a CBU_0808 antigen; (34) a CBU_0851 antigen; (35) a CBIM 325 antigen; (36) a CBU_1383 antigen; (37) a CBIM473 antigen; (38) a CBIM594 antigen; (39) a CBIM841 antigen; (40) a CBIM970 antigen; (41) a CBU_2087 antigen; (42) a CBU_0502 antigen; (43) a CBU_0288 antigen; (44) a CBU_0928 antigen; (45) a CBU_0738 antigen; (46) a CBU_2012 antigen; (47) a CBU_2092 antigen; (48) a CBU_0155 antigen; (49) a CBU_0943 antigen; (50) a CBIM916 antigen; (51) a CBU_01 14 antigen; and (52) a CBU_0656 antigen. These antigens are highly suitable for eliciting a cell- mediated immune response to C. burnetii in a patient. Highly advantageously, CBU_0091 and CBIM648 antigens were demonstrated herein to be capable of eliciting a cell-mediated immune response (in addition to being capable of eliciting a humoral immune response, as demonstrated herein). CBU_0532 and CBU_0758 have also been identified as candidates for eliciting a cell-mediated immune response (in addition to being capable of eliciting a humoral immune response, as demonstrated herein).
Typically, the CBU_0091 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 62; the CBIM648 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 28; the CBIM652 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 29; the CBU_0510 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 69; the CBU_2009 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 71 ; the CBIM579 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 7; the CBIM 193 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 15; the CBIM400 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 18; the CBIM401 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 19; the CBU_0962 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 23; the CBU_0094 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 25; the CBU_0338 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 26; the CBIM352 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 27; the CBU_0532 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 35; the CBU_0758 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 36; the CBU_0631 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 41 ; the CBU_0075 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 54; the CBIM 136 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 64; the CBIM708 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 66; the CBIM337 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 1 ; the CBU_0232 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 2; the CBU_0852 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 3; the CBU_0326 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 4; the CBU_0897 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 5; the CBIM384 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 6; the CBIM475 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 8; the CBU_0517 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 9; the CBU_0270 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 10; the CBU_0629 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 11 ; the CBU_0974 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 12; the CBIM088 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 13; the CBIM 116 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 14; the CBIM296 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 16; the CBIM397 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 17; the CBIM720 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 20; the CBU_0638 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 21 ; the CBU_0640 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 22; the CBU_0073 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 24; the CBIM275 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 30; the CBU_0297 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 31; the CBU_0916 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 32; the CBIM 183 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 33; the CBIM235 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 34; the CBU_2086 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 37; the CBU_0043 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 38; the CBU_0296 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 39; the CBU_0531 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 40; the CBU_0796 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 42; the CBIM830 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 43; the CBU_0234 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 44; the CBU_0445 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 45; the CBU_0808 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 46; the CBU_0851 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 47; the CBIM325 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 48; the CBIM383 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 49; the CBIM473 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 50; the CBIM594 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 51 ; the CBIM841 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 52; the CBIM970 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 53; the CBU_2087 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 55; the CBU_0502 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 56; the CBU_0288 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 57; the CBU_0928 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 58; the CBU_0738 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 59; the CBU_2012 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 60; the CBU_2092 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 61 ; the CBU_0155 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 63; the CBU_0943 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 65; the CBIM916 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 67; the CBU_0114 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 68; and/or the CBU_0656 antigen comprises an amino acid sequence having 70% or more identity to SEQ ID NO: 70.
Typically, the CBU_0091 antigen comprises 7 or more consecutive amino acids of SEQ ID NO: 62; the CBIM648 antigen comprises 7 or more consecutive amino acids of SEQ ID NO: 28; the CBIM652 antigen comprises 7 or more consecutive amino acids of SEQ ID NO: 29; the
CBU. _0510 antigen comprises 7 or more consecutive amino acids of SEQ ID NO 69 the
CBU. _2009 antigen comprises 7 or more consecutive amino acids of SEQ ID NO 71 the
CBU. _1579 antigen comprises 7 or more consecutive amino acids of SEQ ID NO 7 the
CBU. _1193 antigen comprises 7 or more consecutive amino acids of SEQ ID NO 15 the
CBU. _1400 antigen comprises 7 or more consecutive amino acids of SEQ ID NO 18 the
CBU. _1401 antigen comprises 7 or more consecutive amino acids of SEQ ID NO 19 the
CBU. _0962 antigen comprises 7 or more consecutive amino acids of SEQ ID NO 23 the
CBU. _0094 antigen comprises 7 or more consecutive amino acids of SEQ ID NO 25 the
CBU. _0338 antigen comprises 7 or more consecutive amino acids of SEQ ID NO 26 the
CBU. _1352 antigen comprises 7 or more consecutive amino acids of SEQ ID NO 27 the
CBU. _0532 antigen comprises 7 or more consecutive amino acids of SEQ ID NO 35 the
CBU. _0758 antigen comprises 7 or more consecutive amino acids of SEQ ID NO 36 the
CBU. _0631 antigen comprises 7 or more consecutive amino acids of SEQ ID NO 41 the
CBU. _0075 antigen comprises 7 or more consecutive amino acids of SEQ ID NO 54 the
CBU. _1136 antigen comprises 7 or more consecutive amino acids of SEQ ID NO 64 the
CBU. _1708 antigen comprises 7 or more consecutive amino acids of SEQ ID NO 66 the
CBU. _1337 antigen comprises 7 or more consecutive amino acids of SEQ ID NO 1 the
CBU. _0232 antigen comprises 7 or more consecutive amino acids of SEQ ID NO 2 the
CBU. _0852 antigen comprises 7 or more consecutive amino acids of SEQ ID NO 3 the
CBU. _0326 antigen comprises 7 or more consecutive amino acids of SEQ ID NO 4 the
CBU. _0897 antigen comprises 7 or more consecutive amino acids of SEQ ID NO 5 the CBU. _1384 antigen comprises 7 or more consecutive amino acids of SEG ID NO : 6; the
CBU. _1475 antigen comprises 7 or more consecutive amino acids of SEG ID NO : 8; the
CBU. _0517 antigen comprises 7 or more consecutive amino acids of SEQ ID NO: 9; the
CBU. _0270 antigen comprises 7 or more consecutive amino acids of SEQ ID NO: 10; the
CBU. _0629 antigen comprises 7 or more consecutive amino acids of SEQ ID NO: 11 ; the
CBU. _0974 antigen comprises 7 or more consecutive amino acids of SEQ ID NO: 12; the
CBU. _1088 antigen comprises 7 or more consecutive amino acids of SEQ ID NO: 13; the
CBU. _1116 antigen comprises 7 or more consecutive amino acids of SEQ ID NO: 14; the
CBU. _1296 antigen comprises 7 or more consecutive amino acids of SEQ ID NO: 16; the
CBU. _1397 antigen comprises 7 or more consecutive amino acids of SEQ ID NO: 17; the
CBU. _1720 antigen comprises 7 or more consecutive amino acids of SEQ ID NO: 20; the
CBU. _0638 antigen comprises 7 or more consecutive amino acids of SEQ ID NO: 21 ; the
CBU. _0640 antigen comprises 7 or more consecutive amino acids of SEQ ID NO: 22; the
CBU. _0073 antigen comprises 7 or more consecutive amino acids of SEQ ID NO: 24; the
CBU. _1275 antigen comprises 7 or more consecutive amino acids of SEQ ID NO: 30; the
CBU. _0297 antigen comprises 7 or more consecutive amino acids of SEQ ID NO: 31 ; the
CBU. _0916 antigen comprises 7 or more consecutive amino acids of SEQ ID NO: 32; the
CBU. _1183 antigen comprises 7 or more consecutive amino acids of SEQ ID NO: 33; the
CBU. _1235 antigen comprises 7 or more consecutive amino acids of SEQ ID NO: 34; the
CBU. _2086 antigen comprises 7 or more consecutive amino acids of SEQ ID NO: 37; the
CBU. _0043 antigen comprises 7 or more consecutive amino acids of SEQ ID NO: 38; the
CBU. _0296 antigen comprises 7 or more consecutive amino acids of SEQ ID NO: 39; the
CBU. _0531 antigen comprises 7 or more consecutive amino acids of SEQ ID NO: 40; the
CBU. _0796 antigen comprises 7 or more consecutive amino acids of SEQ ID NO: 42; the
CBU. _1830 antigen comprises 7 or more consecutive amino acids of SEQ ID NO: 43; the
CBU. _0234 antigen comprises 7 or more consecutive amino acids of SEQ ID NO: 44; the
CBU. _0445 antigen comprises 7 or more consecutive amino acids of SEQ ID NO: 45; the
CBU. _0808 antigen comprises 7 or more consecutive amino acids of SEQ ID NO: 46; the
CBU. _0851 antigen comprises 7 or more consecutive amino acids of SEQ ID NO: 47; the
CBU. _1325 antigen comprises 7 or more consecutive amino acids of SEQ ID NO: 48; the
CBU. _1383 antigen comprises 7 or more consecutive amino acids of SEQ ID NO: 49; the
CBU. _1473 antigen comprises 7 or more consecutive amino acids of SEQ ID NO: 50; the
CBU. _1594 antigen comprises 7 or more consecutive amino acids of SEQ ID NO: 51 ; the
CBU. _1841 antigen comprises 7 or more consecutive amino acids of SEQ ID NO: 52; the
CBU. _1970 antigen comprises 7 or more consecutive amino acids of SEQ ID NO: 53; the
CBU. _2087 antigen comprises 7 or more consecutive amino acids of SEQ ID NO: 55; the
CBU. _0502 antigen comprises 7 or more consecutive amino acids of SEQ ID NO: 56; the
CBU. _0288 antigen comprises 7 or more consecutive amino acids of SEQ ID NO: 57; the
CBU. _0928 antigen comprises 7 or more consecutive amino acids of SEQ ID NO: 58; the
CBU. _0738 antigen comprises 7 or more consecutive amino acids of SEQ ID NO: 59; the
CBU. _2012 antigen comprises 7 or more consecutive amino acids of SEQ ID NO: 60; the
CBU. _2092 antigen comprises 7 or more consecutive amino acids of SEQ ID NO: 61 ; the CBU_0155 antigen comprises 7 or more consecutive amino acids of SEQ I D NO: 63; the CBU_0943 antigen comprises 7 or more consecutive amino acids of SEQ I D NO: 65; the CBIM 916 antigen comprises 7 or more consecutive amino acids of SEQ I D NO: 67; the CBU_01 14 antigen comprises 7 or more consecutive amino acids of SEQ ID NO: 68; and/or the CBU_0656 antigen comprises 7 or more consecutive amino acids of SEQ ID NO: 70.
The protein antigens of the invention are referred to by their "CBU" Locus Tag, and are described below:
1. CBLM337 antigens
The original "CBIM 337" sequence is annotated as "DNA polymerase I II alpha subunit", and is involved in DNA metabolism - replication, recombination and repair. For reference purposes, the amino acid sequence of the full length CBIM 337 sequence is provided in SEQ I D NO: 1 herein.
Preferred CBIM 337 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity (e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ I D NO: 1 ; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ ID NO: 1 , wherein "n" is 7 or more (e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ I D NO: 1 comprise an epitope of SEQ I D NO: 1 . Other preferred fragments lack one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ I D NO: 1 . Preferred fragments of SEQ I D NO: 1 comprise an epitope of SEQ I D NO: 1.
2. CBU_0232 antigens
The original "CBU_0232" sequence is annotated as "DNA-directed RNA polymerase beta' chain" and is involved in transcription. For reference purposes, the amino acid sequence of the full length CBU_0232 sequence is provided in SEQ ID NO: 2 herein.
Preferred CBU_0232 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity (e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ I D NO: 2; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ ID NO: 2, wherein "n" is 7 or more (e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ I D NO: 2 comprise an epitope of SEQ I D NO: 2. Other preferred fragments lack one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids {e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ ID NO: 2. Preferred fragments of SEQ ID NO: 2 comprise an epitope of SEQ ID NO: 2.
3. CBU_0852 antigens
The original "CBU_0852" sequence is annotated as "Polyribonucleotide nucleotidyltransferase/ Polynucleotide adenylyltransferase" and is involved in transcription. For reference purposes, the amino acid sequence of the full length CBU_0852 sequence is provided in SEQ ID NO: 3 herein.
Preferred CBU_0852 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity {e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ ID NO: 3; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ ID NO: 3, wherein "n" is 7 or more {e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ ID NO: 3 comprise an epitope of SEQ ID NO: 3. Other preferred fragments lack one or more amino acids {e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids {e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ ID NO: 3. Preferred fragments of SEQ ID NO: 3 comprise an epitope of SEQ ID NO: 3.
4. CBU_0326 antigens
The original "CBU_0326" sequence is annotated as "Phosphoribosylamine-glycine ligase" and is involved in nucleotide and nucleoside biosynthesis and metabolism. For reference purposes, the amino acid sequence of the full length CBU_0326 sequence is provided in SEQ ID NO: 4 herein.
Preferred CBU_0326 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity {e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ ID NO: 4; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ ID NO: 4, wherein "n" is 7 or more {e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 61 , 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ ID NO: 4 comprise an epitope of SEQ ID NO: 4. Other preferred fragments lack one or more amino acids {e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids {e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ ID NO: 4. Preferred fragments of SEQ ID NO: 4 comprise an epitope of SEQ ID NO: 4. 5. CBU_0897 antigens
The original "CBU_0897" sequence is annotated as "Amidophosphoribosyltransferase" and is involved in nucleotide and nucleoside biosynthesis and metabolism. For reference purposes, the amino acid sequence of the full length CBU_0897 sequence is provided in SEQ I D NO: 5 herein.
Preferred CBU_0897 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity (e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ I D NO: 5; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ ID NO: 5, wherein "n" is 7 or more (e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 61 , 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ I D NO: 5 comprise an epitope of SEQ I D NO: 5. Other preferred fragments lack one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ I D NO: 5. Preferred fragments of SEQ I D NO: 5 comprise an epitope of SEQ I D NO: 5.
6. CBLM384 antigens
The original "CBIM 384" sequence is annotated as "Uridylate kinase" and is involved in nucleotide and nucleoside biosynthesis and metabolism. For reference purposes, the amino acid sequence of the full length CBU_1384 sequence is provided in SEQ ID NO: 6 herein.
Preferred CBU_1384 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity (e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ I D NO: 6; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ ID NO: 6, wherein "n" is 7 or more (e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ I D NO: 6 comprise an epitope of SEQ I D NO: 6. Other preferred fragments lack one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ I D NO: 6. Preferred fragments of SEQ I D NO: 6 comprise an epitope of SEQ I D NO: 6.
7. CBLM579 antigens
The original "CBU_1579" sequence is annotated as "Trp repressor binding protein" and is involved in regulatory function. For reference purposes, the amino acid sequence of the full length CBU_1579 sequence is provided in SEQ I D NO: 7 herein. Preferred CBIM 579 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity (e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ I D NO: 7; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ ID NO: 7, wherein "n" is 7 or more (e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ I D NO: 7 comprise an epitope of SEQ I D NO: 7. Other preferred fragments lack one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ I D NO: 7. Preferred fragments of SEQ I D NO: 7 comprise an epitope of SEQ I D NO: 7.
8. CBLM475 antigens
The original "CBIM 475" sequence is annotated as "Aspartyl/glutamyl-tRNA(Asn/Gln) amidotransferase subunit B" and is involved in translation (protein biosynthesis). For reference purposes, the amino acid sequence of the full length CBIM475 sequence is provided in SEQ I D NO: 8 herein.
Preferred CBIM475 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity (e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ I D NO: 8; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ ID NO: 8, wherein "n" is 7 or more (e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ I D NO: 8 comprise an epitope of SEQ I D NO: 8. Other preferred fragments lack one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ I D NO: 8. Preferred fragments of SEQ I D NO: 8 comprise an epitope of SEQ I D NO: 8.
9. CBU_0517 antigens
The original "CBU_0517" sequence is annotated as "Aspartate aminotransferase/ Succinyldiaminopimelate aminotransferase" and is involved in amino acid biosynthesis and metabolism. For reference purposes, the amino acid sequence of the full length CBU_0517 sequence is provided in SEQ I D NO: 9 herein.
Preferred CBU_0517 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity (e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ I D NO: 9; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ ID NO: 9, wherein "n" is 7 or more {e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ I D NO: 9 comprise an epitope of SEQ I D NO: 9. Other preferred fragments lack one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ I D NO: 9. Preferred fragments of SEQ I D NO: 9 comprise an epitope of SEQ I D NO: 9.
10. CBU_0270 antigens
The original "CBU_0270" sequence is annotated as "Short-chain alcohol dehydrogenase" and is involved in energy metabolism - electron transport. For reference purposes, the amino acid sequence of the full length CBU_0270sequence is provided in SEQ I D NO: 10 herein.
Preferred CBU_0270 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity (e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ I D NO: 10; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ I D NO: 10, wherein "n" is 7 or more (e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ I D NO: 10 comprise an epitope of SEQ ID NO:
10. Other preferred fragments lack one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10,
1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ ID NO: 10. Preferred fragments of SEQ I D NO: 10 comprise an epitope of SEQ ID NO: 10.
11. CBU_0629 antigens
The original "CBU_0629" sequence is annotated as "Proline dehydrogenase/Delta-1- pyrroline-5-carboxylate dehydrogenase" and is involved in energy metabolism - electron transport. For reference purposes, the amino acid sequence of the full length CBU_0629 sequence is provided in SEQ I D NO: 1 herein.
Preferred CBU_0629 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity (e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ I D NO: 1 1 ; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ I D NO: 1 1 , wherein "n" is 7 or more (e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ I D NO: 1 1 comprise an epitope of SEQ ID NO: 1 1 . Other preferred fragments lack one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids {e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ ID NO: 11. Preferred fragments of SEQ ID NO: 1 1 comprise an epitope of SEQ ID NO: 1 1.
12. CBU_0974 antigens
The original "CBU_0974" sequence is annotated as "Acetyl-CoA acetyltransferase" and is involved in energy metabolism - electron transport. For reference purposes, the amino acid sequence of the full length CBU_0974 sequence is provided in SEQ ID NO: 12 herein.
Preferred CBU_0974 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity {e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ ID NO: 12;and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ ID NO: 12, wherein "n" is 7 or more {e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ ID NO: 12 comprise an epitope of SEQ ID NO: 12. Other preferred fragments lack one or more amino acids {e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 ,
12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids {e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ ID NO: 12. Preferred fragments of SEQ ID NO: 12 comprise an epitope of SEQ ID NO: 12.
13. CBLM088 antigens
The original "CBIM088" sequence is annotated as "Bifunctional NAD(P)H-hydrate repair enzyme Nnr" and is involved in energy metabolism - electron transport. For reference purposes, the amino acid sequence of the full length CBIM 088 sequence is provided in SEQ ID NO: 13 herein.
Preferred CBIM088 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity {e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ ID NO: 13; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ ID NO: 13, wherein "n" is 7 or more {e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ ID NO: 13 comprise an epitope of SEQ ID NO: 13. Other preferred fragments lack one or more amino acids {e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids {e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ ID NO: 13. Preferred fragments of SEQ ID NO: 13 comprise an epitope of SEQ ID NO: 13. 14. CBU_1116 antigens
The original "CBIM 1 16" sequence is annotated as "Alanine dehydrogenase" and is involved in energy metabolism - electron transport. For reference purposes, the amino acid sequence of the full length CBIM 1 16 sequence is provided in SEQ I D NO: 14 herein.
Preferred CBIM 1 16 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity (e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ I D NO: 14; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ I D NO: 14, wherein "n" is 7 or more (e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ I D NO: 14 comprise an epitope of SEQ ID NO:
14. Other preferred fragments lack one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ ID NO: 14. Preferred fragments of SEQ I D NO: 14 comprise an epitope of SEQ ID NO: 14.
15. CBLM 193 antigens
The original "CBIM 193" sequence is annotated as "Thioredoxin reductase" and is involved in energy metabolism - electron transport. For reference purposes, the amino acid sequence of the full length CBIM 193 sequence is provided in SEQ ID NO: 15 herein.
Preferred CBIM 193 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity (e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ I D NO: 15; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ I D NO: 15, wherein "n" is 7 or more (e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ I D NO: 15 comprise an epitope of SEQ ID NO:
15. Other preferred fragments lack one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ I D NO: 15. Preferred fragments of SEQ I D NO: 15 comprise an epitope of SEQ ID NO: 15.
16. CBU_1296 antigens
The original "CBIM296" sequence is annotated as "ATP-NAD kinase" and is involved in energy metabolism - electron transport. For reference purposes, the amino acid sequence of the full length CBIM 296 sequence is provided in SEQ ID NO: 16 herein. Preferred CBIM296 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity (e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ I D NO: 16; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ I D NO: 16, wherein "n" is 7 or more (e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ I D NO: 16 comprise an epitope of SEQ ID NO:
16. Other preferred fragments lack one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ ID NO: 16. Preferred fragments of SEQ I D NO: 16 comprise an epitope of SEQ ID NO: 16.
17. CBLM397 antigens
The original "CBIM 397" sequence is annotated as and is involved in energy metabolism - electron transport "Succinyl-CoA synthetase beta chain". For reference purposes, the amino acid sequence of the full length CBIM 397 sequence is provided in SEQ ID NO: 17 herein.
Preferred CBIM 397 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity (e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ I D NO: 17; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ I D NO: 17, wherein "n" is 7 or more (e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ I D NO: 17 comprise an epitope of SEQ ID NO:
17. Other preferred fragments lack one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ I D NO: 17. Preferred fragments of SEQ I D NO: 17 comprise an epitope of SEQ ID NO: 17.
18. CBLM400 antigens
The original "CBIM 400" sequence is annotated as and is involved in energy metabolism - electron transport "Succinate dehydrogenase iron-sulfur protein". For reference purposes, the amino acid sequence of the full length CBIM400 sequence is provided in SEQ I D NO: 18 herein.
Preferred CBIM400 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity (e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ I D NO: 18; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ I D NO: 18, wherein "n" is 7 or more {e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ I D NO: 18 comprise an epitope of SEQ ID NO:
18. Other preferred fragments lack one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ ID NO: 18. Preferred fragments of SEQ I D NO: 18 comprise an epitope of SEQ ID NO: 18.
19. CBLM401 antigens
The original "CBIM 401 " sequence is annotated as and is involved in energy metabolism - electron transport "Succinate dehydrogenase flavoprotein subunit". For reference purposes, the amino acid sequence of the full length CBIM 401 sequence is provided in SEQ ID NO: 19 herein.
Preferred CBIM401 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity (e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ I D NO: 19; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ I D NO: 19, wherein "n" is 7 or more (e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ I D NO: 19 comprise an epitope of SEQ ID NO:
19. Other preferred fragments lack one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ I D NO: 19. Preferred fragments of SEQ I D NO: 19 comprise an epitope of SEQ ID NO: 19.
20. CBLM720 antigens
The original "CBIM 720" sequence is annotated as "Aconitate hydratase" and is involved in energy metabolism - electron transport. For reference purposes, the amino acid sequence of the full length CBIM 720 sequence is provided in SEQ ID NO: 20 herein.
Preferred CBIM 720 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity (e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ I D NO: 20; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ I D NO: 20, wherein "n" is 7 or more (e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ I D NO: 20 comprise an epitope of SEQ ID NO: 20. Other preferred fragments lack one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids {e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ ID NO: 20. Preferred fragments of SEQ I D NO: 20 comprise an epitope of SEQ ID NO: 20.
21. CBU_0638 antigens
The original "CBU_0638" sequence is annotated as "Dihydrolipoamide acetyltransferase component of pyruvate dehydrogenase complex" and is involved in intermediary metabolism and other metabolic pathways. For reference purposes, the amino acid sequence of the full length CBU_0638 sequence is provided in SEQ I D NO: 21 herein.
Preferred CBU_0638 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity (e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ I D NO: 21 ; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ I D NO: 21 , wherein "n" is 7 or more (e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ I D NO: 21 comprise an epitope of SEQ ID NO:
21 . Other preferred fragments lack one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ ID NO: 21 . Preferred fragments of SEQ I D NO: 21 comprise an epitope of SEQ ID NO: 21.
22. CBU_0640 antigens
The original "CBU_0640" sequence is annotated as "Pyruvate dehydrogenase E1 component alpha subunit" and is involved in intermediary metabolism and other metabolic pathways. For reference purposes, the amino acid sequence of the full length CBU_0640 sequence is provided in SEQ I D NO: 22 herein.
Preferred CBU_0640 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity (e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ I D NO: 22; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ I D NO: 22, wherein "n" is 7 or more (e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ I D NO: 22 comprise an epitope of SEQ ID NO: 22. Other preferred fragments lack one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ I D NO: 22. Preferred fragments of SEQ I D NO: 22 comprise an epitope of SEQ ID NO: 22. 23. CBU_0962 antigens
The original "CBU_0962" sequence is annotated as "Short chain dehydrogenase" and is involved in intermediary metabolism and other metabolic pathways. For reference purposes, the amino acid sequence of the full length CBU_0962 sequence is provided in SEQ I D NO: 23 herein.
Preferred CBU_0962 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity (e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ I D NO: 23; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ I D NO: 23, wherein "n" is 7 or more (e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ I D NO: 23 comprise an epitope of SEQ ID NO:
23. Other preferred fragments lack one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ ID NO: 23. Preferred fragments of SEQ I D NO: 23 comprise an epitope of SEQ ID NO: 23.
24. CBU_0073 antigens
The original "CBU_0073" sequence is annotated as "Xaa-Pro aminopeptidase" and is involved in posttranslational modification, degradation, protein turnover, chaperones. For reference purposes, the amino acid sequence of the full length CBU_0073 sequence is provided in SEQ I D NO: 24 herein.
Preferred CBU_0073 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity (e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ I D NO: 24; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ I D NO: 24, wherein "n" is 7 or more (e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ I D NO: 24 comprise an epitope of SEQ ID NO:
24. Other preferred fragments lack one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ I D NO: 24. Preferred fragments of SEQ I D NO: 24 comprise an epitope of SEQ ID NO: 24.
25. CBU_0094 antigens
The original "CBU_0094" sequence is annotated as "ClpB protein" and is involved in posttranslational modification, degradation, protein turnover, chaperones. For reference purposes, the amino acid sequence of the full length CBU_0094 sequence is provided in SEQ I D NO: 25 herein.
Preferred CBU_0094 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity (e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ I D NO: 25; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ I D NO: 25, wherein "n" is 7 or more (e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ I D NO: 25 comprise an epitope of SEQ ID NO:
25. Other preferred fragments lack one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ ID NO: 25. Preferred fragments of SEQ I D NO: 25 comprise an epitope of SEQ ID NO: 25.
26. CBU_0338 antigens
The original "CBU_0338" sequence is annotated as "Membrane alanine aminopeptidase" and is involved in posttranslational modification, degradation, protein turnover, chaperones. For reference purposes, the amino acid sequence of the full length CBU_0338 sequence is provided in SEQ I D NO: 26 herein.
Preferred CBU_0338 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity (e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ I D NO: 26; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ I D NO: 26, wherein "n" is 7 or more (e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ I D NO: 26 comprise an epitope of SEQ ID NO:
26. Other preferred fragments lack one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ I D NO: 26. Preferred fragments of SEQ I D NO: 26 comprise an epitope of SEQ ID NO: 26.
27. CBLM352 antigens
The original "CBIM 352" sequence is annotated as "Cell division protein ftsH" and is involved in cell division, chromosome partitioning. For reference purposes, the amino acid sequence of the full length CBIM 352 sequence is provided in SEQ I D NO: 27 herein.
Preferred CBIM 352 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity (e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ I D NO: 27; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ I D NO: 27, wherein "n" is 7 or more (e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ I D NO: 27 comprise an epitope of SEQ ID NO:
27. Other preferred fragments lack one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ ID NO: 27. Preferred fragments of SEQ I D NO: 27 comprise an epitope of SEQ ID NO: 27.
28. CBLM648 antigens
The original "CBIM648" sequence is annotated as "DotA protein" and is involved in protein and peptide secretion and trafficking. For reference purposes, the amino acid sequence of the full length CBIM 648 sequence is provided in SEQ ID NO: 28 herein.
Preferred CBIM648 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity (e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ I D NO: 28; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ I D NO: 28, wherein "n" is 7 or more (e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ I D NO: 28 comprise an epitope of SEQ ID NO:
28. Other preferred fragments lack one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ I D NO: 28. Preferred fragments of SEQ I D NO: 28 comprise an epitope of SEQ ID NO: 28.
29. CBLM652 antigens
The original "CBIM652" sequence is annotated as "IcmX protein" and is involved in protein and peptide secretion and trafficking. For reference purposes, the amino acid sequence of the full length CBIM 652 sequence is provided in SEQ ID NO: 29 herein.
Preferred CBIM652 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity (e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ I D NO: 29; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ I D NO: 29, wherein "n" is 7 or more (e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ I D NO: 29 comprise an epitope of SEQ ID NO: 29. Other preferred fragments lack one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids {e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ ID NO: 29. Preferred fragments of SEQ ID NO: 29 comprise an epitope of SEQ ID NO: 29.
30. CBLM275 antigens
The original "CBIM275" sequence is annotated as "Starvation sensing protein rspA" and is involved in aptation to atypical conditions - response to starvation. For reference purposes, the amino acid sequence of the full length CBIM275 sequence is provided in SEQ ID NO: 30 herein.
Preferred CBIM275 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity {e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ ID NO: 30; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ ID NO: 30, wherein "n" is 7 or more {e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ ID NO: 30 comprise an epitope of SEQ ID NO:
30. Other preferred fragments lack one or more amino acids {e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids {e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ ID NO: 30. Preferred fragments of SEQ ID NO: 30 comprise an epitope of SEQ ID NO: 30.
31. CBU_0297 antigens
The original "CBU_0297" sequence is annotated as "Exodeoxyribonuclease III" and is involved in DNA metabolism - Replication, recombination and repair. For reference purposes, the amino acid sequence of the full length CBU_0297 sequence is provided in SEQ ID NO: 31 herein.
Preferred CBU_0297 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity {e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ ID NO: 31 ; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ ID NO: 31 , wherein "n" is 7 or more {e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ ID NO: 31 comprise an epitope of SEQ ID NO: 31. Other preferred fragments lack one or more amino acids {e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids {e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ ID NO: 31. Preferred fragments of SEQ ID NO: 31 comprise an epitope of SEQ ID NO: 31. 32. CBU_0916 antigens
The original "CBU_0916" sequence is annotated as
"Endonuclease/Exonuclease/phosphatase family protein" and is involved in DNA metabolism - Replication, recombination and repair. For reference purposes, the amino acid sequence of the full length CBU_0916 sequence is provided in SEQ ID NO: 32 herein.
Preferred CBU_0916 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity (e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ I D NO: 32; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ I D NO: 32, wherein "n" is 7 or more (e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ I D NO: 32 comprise an epitope of SEQ ID NO:
32. Other preferred fragments lack one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ ID NO: 32. Preferred fragments of SEQ I D NO: 32 comprise an epitope of SEQ ID NO: 32.
33. CBLM 183 antigens
The original "CBIM 183" sequence is annotated as "Glycine-rich RNA-binding protein" and is involved in DNA metabolism - Replication, recombination and repair. For reference purposes, the amino acid sequence of the full length CBIM 183 sequence is provided in SEQ I D NO: 33 herein.
Preferred CBIM 183 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity (e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ I D NO: 33; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ I D NO: 33, wherein "n" is 7 or more (e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ I D NO: 33 comprise an epitope of SEQ ID NO: 33. Other preferred fragments lack one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ I D NO: 33. Preferred fragments of SEQ I D NO: 33 comprise an epitope of SEQ ID NO: 33. 34. CBLM235 antigens
The original "CBIM 235" sequence is annotated as Oligoribonuclease" and is involved in DNA metabolism - Replication, recombination and repair. For reference purposes, the amino acid sequence of the full length CBIM235 sequence is provided in SEQ ID NO: 34 herein.
Preferred CBIM235 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity (e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ I D NO: 34; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ I D NO: 34, wherein "n" is 7 or more (e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ I D NO: 34 comprise an epitope of SEQ ID NO:
34. Other preferred fragments lack one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ ID NO: 34. Preferred fragments of SEQ I D NO: 34 comprise an epitope of SEQ ID NO: 34.
35. CBU_0532 antigens
The original "CBU_0532" sequence is annotated as "COME operon protein 1" and is involved in DNA - medicated transformation (competence). For reference purposes, the amino acid sequence of the full length CBU_0532 sequence is provided in SEQ ID NO: 35 herein.
Preferred CBU_0532 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity (e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ I D NO: 35; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ I D NO: 35, wherein "n" is 7 or more (e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ I D NO: 35 comprise an epitope of SEQ ID NO:
35. Other preferred fragments lack one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ I D NO: 35. Preferred fragments of SEQ I D NO: 35 comprise an epitope of SEQ ID NO: 35.
36. CBU_0758 antigens
The original "CBU_0758" sequence is annotated as "Lipoprotein, ComL family" and is involved in DNA - medicated transformation (competence). For reference purposes, the amino acid sequence of the full length CBU_0758 sequence is provided in SEQ ID NO: 36 herein.
Preferred CBU_0758 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity (e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ I D NO: 36; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ I D NO: 36, wherein "n" is 7 or more (e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ I D NO: 36 comprise an epitope of SEQ ID NO:
36. Other preferred fragments lack one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ ID NO: 36. Preferred fragments of SEQ I D NO: 36 comprise an epitope of SEQ ID NO: 36.
37. CBU_2086 antigens
The original "CBU_2086" sequence is annotated as "Transcription termination factor rho" and is involved in transcription. For reference purposes, the amino acid sequence of the full length CBU_2086 sequence is provided in SEQ I D NO: 37 herein.
Preferred CBU_2086 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity (e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ I D NO: 37; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ I D NO: 37, wherein "n" is 7 or more (e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ I D NO: 37 comprise an epitope of SEQ ID NO:
37. Other preferred fragments lack one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ I D NO: 37. Preferred fragments of SEQ I D NO: 37 comprise an epitope of SEQ ID NO: 37.
38. CBU_0043 antigens
The original "CBU_0043" sequence is annotated as "Xanthosine triphosphate pyrophosphatase" and is involved in nucleotide and nucleoside biosynthesis and metabolism. For reference purposes, the amino acid sequence of the full length CBU_0043 sequence is provided in SEQ I D NO: 38 herein.
Preferred CBU_0043 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity (e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ I D NO: 38; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ I D NO: 38, wherein "n" is 7 or more (e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ I D NO: 38 comprise an epitope of SEQ ID NO:
38. Other preferred fragments lack one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ ID NO: 38. Preferred fragments of SEQ I D NO: 38 comprise an epitope of SEQ ID NO: 38.
39. CBU_0296 antigens
The original "CBU_0296" sequence is annotated as "Orotate phosphoribosyltransferase" and is involved in nucleotide and nucleoside biosynthesis and metabolism. For reference purposes, the amino acid sequence of the full length CBU_0296 sequence is provided in SEQ I D NO: 39 herein.
Preferred CBU_0296 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity (e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ I D NO: 39; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ I D NO: 39, wherein "n" is 7 or more (e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ I D NO: 39 comprise an epitope of SEQ ID NO:
39. Other preferred fragments lack one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ I D NO: 39. Preferred fragments of SEQ I D NO: 39 comprise an epitope of SEQ ID NO: 39.
40. CBU_0531 antigens
The original "CBU_0531" sequence is annotated as "Orotidine 5'-phosphate decarboxylase" and is involved in nucleotide and nucleoside biosynthesis and metabolism. For reference purposes, the amino acid sequence of the full length CBU_0531 sequence is provided in SEQ I D NO: 40 herein.
Preferred CBU_0531 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity (e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ I D NO: 40; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ I D NO: 40, wherein "n" is 7 or more (e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ ID NO: 40 comprise an epitope of SEQ ID NO:
40. Other preferred fragments lack one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids {e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ ID NO: 40. Preferred fragments of SEQ ID NO: 40 comprise an epitope of SEQ ID NO: 40.
41. CBU_0631 antigens
The original "CBU_0631" sequence is annotated as "Phosphoribosylformylglycinamidine synthase" and is involved in nucleotide and nucleoside biosynthesis and metabolism. For reference purposes, the amino acid sequence of the full length CBU_0631 sequence is provided in SEQ ID NO: 41 herein.
Preferred CBU_0631 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity {e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ ID NO: 41 ; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ ID NO: 41 , wherein "n" is 7 or more {e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ ID NO: 41 comprise an epitope of SEQ ID NO:
41. Other preferred fragments lack one or more amino acids {e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids {e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ ID NO: 41. Preferred fragments of SEQ ID NO: 41 comprise an epitope of SEQ ID NO: 41.
42. CBU_0796 antigens
The original "CBU_0796" sequence is annotated as "Adenosine 5'-monophosphoramidase / Guanosine 5'-monophosphoramidase" and is involved in nucleotide and nucleoside biosynthesis and metabolism. For reference purposes, the amino acid sequence of the full length CBU_0796 sequence is provided in SEQ ID NO: 42 herein.
Preferred CBU_0796 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity {e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ ID NO: 42; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ ID NO: 2, wherein "n" is 7 or more {e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ ID NO: 42 comprise an epitope of SEQ ID NO: 42. Other preferred fragments lack one or more amino acids {e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids {e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ I D NO: 42. Preferred fragments of SEQ I D NO: 42 comprise an epitope of SEQ I D NO: 42.
43. CBLM 830 antigens
The original "CBIM 830" sequence is annotated as "Ribose-phosphate pyrophosphokinase" and is involved in nucleotide and nucleoside biosynthesis and metabolism. For reference purposes, the amino acid sequence of the full length CBIM 830 sequence is provided in SEQ I D NO: 43 herein.
Preferred CBIM 830 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity (e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ I D NO: 43; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ I D NO: 43, wherein "n" is 7 or more (e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ I D NO: 43 comprise an epitope of SEQ ID NO:
43. Other preferred fragments lack one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ ID NO: 43. Preferred fragments of SEQ I D NO: 43 comprise an epitope of SEQ ID NO: 43.
44. CBU_0234 antigens
The original "CBU_0234" sequence is annotated as "SSU ribosomal protein S7P" and is involved in translation - protein biosynthesis. For reference purposes, the amino acid sequence of the full length CBU_0234 sequence is provided in SEQ I D NO: 44 herein.
Preferred CBU_0234 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity (e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ I D NO: 44; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ I D NO: 44, wherein "n" is 7 or more (e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ I D NO: 44 comprise an epitope of SEQ ID NO: 44. Other preferred fragments lack one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ I D NO: 44. Preferred fragments of SEQ I D NO: 44 comprise an epitope of SEQ ID NO: 44. 45. CBU_0445 antigens
The original "CBU_0445" sequence is annotated as "SSU ribosomal protein S16P" and is involved in translation - protein biosynthesis. For reference purposes, the amino acid sequence of the full length CBU_0445 sequence is provided in SEQ I D NO: 45 herein.
Preferred CBU_0445 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity (e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ I D NO: 45; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ I D NO: 45, wherein "n" is 7 or more (e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ I D NO: 45 comprise an epitope of SEQ ID NO:
45. Other preferred fragments lack one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ ID NO: 45. Preferred fragments of SEQ I D NO: 45 comprise an epitope of SEQ ID NO: 45.
46. CBU_0808 antigens
The original "CBU_0808" sequence is annotated as "Valyl-tRNA synthetase" and is involved in translation - protein biosynthesis. For reference purposes, the amino acid sequence of the full length CBU_0808 sequence is provided in SEQ I D NO: 46 herein.
Preferred CBU_0808 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity (e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ I D NO: 46; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ I D NO: 46, wherein "n" is 7 or more (e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ I D NO: 46 comprise an epitope of SEQ ID NO:
46. Other preferred fragments lack one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ I D NO: 46. Preferred fragments of SEQ I D NO: 46 comprise an epitope of SEQ ID NO: 46.
47. CBU_0851 antigens
The original "CBU_0851" sequence is annotated as "SSU ribosomal protein S15P" and is involved in translation - protein biosynthesis. For reference purposes, the amino acid sequence of the full length CBU_0851 sequence is provided in SEQ I D NO: 47 herein.
Preferred CBU_0851 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity (e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ I D NO: 47; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ I D NO: 47, wherein "n" is 7 or more (e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ I D NO: 47 comprise an epitope of SEQ ID NO:
47. Other preferred fragments lack one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ ID NO: 47. Preferred fragments of SEQ I D NO: 47 comprise an epitope of SEQ ID NO: 47.
48. CBLM325 antigens
The original "CBIM 325" sequence is annotated as "Bacterial Protein Translation Initiation Factor 3 (I F-3)" and is involved in translation - protein biosynthesis. For reference purposes, the amino acid sequence of the full length CBIM 325 sequence is provided in SEQ I D NO: 48 herein.
Preferred CBIM 325 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity (e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ I D NO: 48; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ I D NO: 48, wherein "n" is 7 or more (e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ I D NO: 48 comprise an epitope of SEQ ID NO:
48. Other preferred fragments lack one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ I D NO: 48. Preferred fragments of SEQ I D NO: 48 comprise an epitope of SEQ ID NO: 48.
49. CBLM383 antigens
The original "CBIM 383" sequence is annotated as "Ribosome Recycling Factor (RRF)" and is involved in translation - protein biosynthesis. For reference purposes, the amino acid sequence of the full length CBIM 383 sequence is provided in SEQ I D NO: 49 herein.
Preferred CBIM 383 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity (e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ I D NO: 49; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ I D NO: 49, wherein "n" is 7 or more (e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ I D NO: 49 comprise an epitope of SEQ ID NO: 49. Other preferred fragments lack one or more amino acids {e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids {e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ ID NO: 49. Preferred fragments of SEQ ID NO: 49 comprise an epitope of SEQ ID NO: 49.
50. CBLM473 antigens
The original "CBIM473" sequence is annotated as "Aspartyl/glutamyl-tRNA(Asn/Gln) amidotransferase subunit C" and is involved in translation - protein biosynthesis. For reference purposes, the amino acid sequence of the full length CBIM473 sequence is provided in SEQ ID NO: 50 herein.
Preferred CBIM473 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity {e.g. 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ ID NO: 50; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ ID NO: 50, wherein "n" is 7 or more {e.g. 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ ID NO: 50 comprise an epitope of SEQ ID NO:
50. Other preferred fragments lack one or more amino acids {e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids {e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ ID NO: 50. Preferred fragments of SEQ ID NO: 50 comprise an epitope of SEQ ID NO: 50.
51. CBLM594 antigens
The original "CBIM594" sequence is annotated as "GatB/Yqey domain protein" and is involved in translation - protein biosynthesis. For reference purposes, the amino acid sequence of the full length CBIM594 sequence is provided in SEQ ID NO: 51 herein.
Preferred CBIM594 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity {e.g. 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ ID NO: 51 ; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ ID NO: 51 , wherein "n" is 7 or more {e.g. 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ ID NO: 51 comprise an epitope of SEQ ID NO: 51. Other preferred fragments lack one or more amino acids {e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids {e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ ID NO: 51. Preferred fragments of SEQ ID NO: 51 comprise an epitope of SEQ ID NO: 51. 52. CBLM841 antigens
The original "CBIM 841 " sequence is annotated as "Peptidyl-tRNA hydrolase" and is involved in translation - protein biosynthesis. For reference purposes, the amino acid sequence of the full length CBIM 841 sequence is provided in SEQ I D NO: 52 herein.
Preferred CBIM 841 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity (e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ I D NO: 52; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ I D NO: 52, wherein "n" is 7 or more (e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ I D NO: 52 comprise an epitope of SEQ ID NO:
52. Other preferred fragments lack one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ ID NO: 52. Preferred fragments of SEQ I D NO: 52 comprise an epitope of SEQ ID NO: 52.
53. CBLM970 antigens
The original "CBIM 970" sequence is annotated as "Diaminopimelate epimerase" and is involved in amino acid biosynthesis and metabolism. For reference purposes, the amino acid sequence of the full length CBIM 970 sequence is provided in SEQ I D NO: 53 herein.
Preferred CBIM 970 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity (e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ I D NO: 53; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ I D NO: 53, wherein "n" is 7 or more (e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ I D NO: 53 comprise an epitope of SEQ ID NO:
53. Other preferred fragments lack one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ I D NO: 53. Preferred fragments of SEQ I D NO: 53 comprise an epitope of SEQ ID NO: 53.
54. CBU_0075 antigens
The original "CBU_0075" sequence is annotated as "2-polyprenyl-6-methoxyphenol hydroxylase" and is involved in energy metabolism - electron transport. For reference purposes, the amino acid sequence of the full length CBU_0075 sequence is provided in SEQ I D NO: 54 herein. Preferred CBU_0075 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity (e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ I D NO: 54; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ I D NO: 54, wherein "n" is 7 or more (e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ I D NO: 54 comprise an epitope of SEQ ID NO:
54. Other preferred fragments lack one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ ID NO: 54. Preferred fragments of SEQ I D NO: 54 comprise an epitope of SEQ ID NO: 54.
55. CBU_2087 antigens
The original "CBU_2087" sequence is annotated as "Thioredoxin" and is involved in energy metabolism - electron transport. For reference purposes, the amino acid sequence of the full length CBU_2087 sequence is provided in SEQ I D NO: 55 herein.
Preferred CBU_2087 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity (e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ I D NO: 55; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ I D NO: 55, wherein "n" is 7 or more (e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ I D NO: 55 comprise an epitope of SEQ ID NO:
55. Other preferred fragments lack one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ I D NO: 55. Preferred fragments of SEQ I D NO: 55 comprise an epitope of SEQ ID NO: 55.
56. CBU_0502 antigens
The original "CBU_0502" sequence is annotated as "DNase, TatD family" and is involved in intermediary metabolism and other metabolic pathways. For reference purposes, the amino acid sequence of the full length CBU_0502 sequence is provided in SEQ ID NO: 56 herein.
Preferred CBU_0502 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity (e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ I D NO: 6; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ I D NO: 56, wherein "n" is 7 or more (e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ ID NO: 56 comprise an epitope of SEQ ID NO: 56. Other preferred fragments lack one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids {e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ ID NO: 56. Preferred fragments of SEQ ID NO: 56 comprise an epitope of SEQ ID NO: 56.
57. CBU_0288 antigens
The original "CBU_0288" sequence is annotated as and is involved in intermediary metabolism and other metabolic pathways "Phosphopantetheine adenylyltransferase". For reference purposes, the amino acid sequence of the full length CBU_0288 sequence is provided in SEQ ID NO: 57 herein.
Preferred CBU_0288 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity {e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ ID NO: 57; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ ID NO: 57, wherein "n" is 7 or more {e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ ID NO: 57 comprise an epitope of SEQ ID NO:
57. Other preferred fragments lack one or more amino acids {e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids {e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ ID NO: 57. Preferred fragments of SEQ ID NO: 57 comprise an epitope of SEQ ID NO: 57.
58. CBU_0928 antigens
The original "CBU_0928" sequence is annotated as "Pyridoxamine 5'-phosphate oxidase" and is involved in intermediary metabolism and other metabolic pathways. For reference purposes, the amino acid sequence of the full length CBU_0928 sequence is provided in SEQ ID NO: 58 herein.
Preferred CBU_0928 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity {e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ ID NO: 58; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ ID NO: 58, wherein "n" is 7 or more {e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ ID NO: 58 comprise an epitope of SEQ ID NO: 58. Other preferred fragments lack one or more amino acids {e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids {e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ ID NO: 58. Preferred fragments of SEQ I D NO: 58 comprise an epitope of SEQ ID NO: 58.
59. CBU_0738 antigens
The original "CBU_0738" sequence is annotated as "ATP-dependent endopeptidase dp proteolytic subunit clpP" and is involved in posttranslational modification, degradation, protein turnover, chaperones. For reference purposes, the amino acid sequence of the full length CBU_0738 sequence is provided in SEQ I D NO: 59 herein.
Preferred CBU_0738 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity (e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ I D NO: 59; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ I D NO: 59, wherein "n" is 7 or more (e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ I D NO: 59 comprise an epitope of SEQ ID NO:
59. Other preferred fragments lack one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ ID NO: 59. Preferred fragments of SEQ I D NO: 59 comprise an epitope of SEQ ID NO: 59.
60. CBU_2012 antigens
The original "CBU_2012" sequence is annotated as "ATP-dependent endopeptidase hsl ATP-binding subunit hsIU" and is involved in posttranslational modification, degradation, protein turnover, chaperones. For reference purposes, the amino acid sequence of the full length CBU_2012 sequence is provided in SEQ I D NO: 60 herein.
Preferred CBU_2012 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity (e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ I D NO: 60; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ I D NO: 60, wherein "n" is 7 or more (e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ I D NO: 60 comprise an epitope of SEQ ID NO: 60. Other preferred fragments lack one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ I D NO: 60. Preferred fragments of SEQ I D NO: 60 comprise an epitope of SEQ ID NO: 60. 61. CBU_2092 antigens
The original "CBU_2092" sequence is annotated as "Phosphoenolpyruvate carboxykinase [ATP]" and is involved in lipopolysaccharide biosynthesis and metabolism. For reference purposes, the amino acid sequence of the full length CBU_2092 sequence is provided in SEQ I D NO: 61 herein.
Preferred CBU_2092 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity (e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ I D NO: 61 ; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ I D NO: 61 , wherein "n" is 7 or more (e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ I D NO: 61 comprise an epitope of SEQ ID NO:
61 . Other preferred fragments lack one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ ID NO: 61 . Preferred fragments of SEQ I D NO: 61 comprise an epitope of SEQ ID NO: 61.
62. CBU_0091 antigens
The original "CBU_0091 " sequence is annotated as "Peptidoglycan-associated lipoprotein OmpA-like" and is involved in protein and peptide secretion and trafficking. For reference purposes, the amino acid sequence of the full length CBU_0091 sequence is provided in SEQ I D NO: 62 herein.
Preferred CBU_0091 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity (e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ I D NO: 62; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ I D NO: 62, wherein "n" is 7 or more (e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ I D NO: 62 comprise an epitope of SEQ ID NO:
62. Other preferred fragments lack one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ I D NO: 62. Preferred fragments of SEQ I D NO: 62 comprise an epitope of SEQ ID NO: 62.
63. CBU_0155 antigens
The original "CBU_0155" sequence is annotated as "Type 4 pili biogenesis protein pilB (nuleotide-binding protein)" and is involved in protein and peptide secretion and trafficking. For reference purposes, the amino acid sequence of the full length CBU_0155 sequence is provided in SEQ I D NO: 63 herein.
Preferred CBU_0155 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity (e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ I D NO: 63; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ I D NO: 63, wherein "n" is 7 or more (e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ I D NO: 63 comprise an epitope of SEQ ID NO:
63. Other preferred fragments lack one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ ID NO: 63. Preferred fragments of SEQ I D NO: 63 comprise an epitope of SEQ ID NO: 63.
64. CBLM 136 antigens
The original "CBIM 136" sequence is annotated as "Enhanced entry protein enhC, tetratricopeptide repeat family" and is involved in pathogenicity and pathogenesis. For reference purposes, the amino acid sequence of the full length CBIM 136 sequence is provided in SEQ I D NO: 64 herein.
Preferred CBIM 136 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity (e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ I D NO: 64; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ I D NO: 64, wherein "n" is 7 or more (e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ I D NO: 64 comprise an epitope of SEQ ID NO:
64. Other preferred fragments lack one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ I D NO: 64. Preferred fragments of SEQ I D NO: 64 comprise an epitope of SEQ ID NO: 64.
65. CBU_0943 antigens
The original "CBU_0943" sequence is annotated as "Rhodanese-related sulfurtransferases" and is involved in detoxication and resistance. For reference purposes, the amino acid sequence of the full length CBU_0943 sequence is provided in SEQ I D NO: 65 herein.
Preferred CBU_0943 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity (e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ I D NO: 65; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ I D NO: 65, wherein "n" is 7 or more (e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ I D NO: 65 comprise an epitope of SEQ ID NO:
65. Other preferred fragments lack one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ ID NO: 65. Preferred fragments of SEQ I D NO: 65 comprise an epitope of SEQ ID NO: 65.
66. CBLM708 antigens
The original "CBIM 708" sequence is annotated as "Superoxide dismutase" and is involved in detoxication and resistance. For reference purposes, the amino acid sequence of the full length CBIM 708 sequence is provided in SEQ I D NO: 66 herein.
Preferred CBIM 708 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity (e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ I D NO: 66; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ I D NO: 66, wherein "n" is 7 or more (e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ I D NO: 66 comprise an epitope of SEQ ID NO:
66. Other preferred fragments lack one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ I D NO: 66. Preferred fragments of SEQ I D NO: 66 comprise an epitope of SEQ ID NO: 66.
67. CBU_1916 antigens
The original "CBIM 916" sequence is annotated as "Universal stress protein A" and is involved in adaptation to atypical condition - response to starvation. For reference purposes, the amino acid sequence of the full length CBIM 916 sequence is provided in SEQ I D NO: 67 herein.
Preferred CBIM 916 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity (e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ I D NO: 67; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ I D NO: 67, wherein "n" is 7 or more (e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ I D NO: 67 comprise an epitope of SEQ ID NO: 67. Other preferred fragments lack one or more amino acids {e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids {e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ ID NO: 67. Preferred fragments of SEQ ID NO: 67 comprise an epitope of SEQ ID NO: 67.
68. CBU_0114 antigens
The original "CBU_01 14" sequence is annotated as "Protein yajQ". For reference purposes, the amino acid sequence of the full length CBU_0114 sequence is provided in SEQ ID NO: 68 herein.
Preferred CBU_01 14 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity {e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ ID NO: 68; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ ID NO: 68, wherein "n" is 7 or more {e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ ID NO: 68 comprise an epitope of SEQ ID NO:
68. Other preferred fragments lack one or more amino acids {e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids {e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ ID NO: 68. Preferred fragments of SEQ ID NO: 68 comprise an epitope of SEQ ID NO: 68.
69. CBU_0510 antigens
The original "CBU_0510" sequence is annotated as "Hypothetical protein". For reference purposes, the amino acid sequence of the full length CBU_0510 sequence is provided in SEQ ID NO: 69 herein.
Preferred CBU_0510 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity {e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ ID NO: 69; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ ID NO: 69, wherein "n" is 7 or more {e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ ID NO: 69 comprise an epitope of SEQ ID NO: 69. Other preferred fragments lack one or more amino acids {e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids {e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ ID NO: 69. Preferred fragments of SEQ ID NO: 69 comprise an epitope of SEQ ID NO: 69. 70. CBU_0656 antigens
The original "CBU_0656" sequence is annotated as "Hypothetical transcriptional regulatory protein". For reference purposes, the amino acid sequence of the full length CBU_0656 sequence is provided in SEQ ID NO: 70 herein.
Preferred CBU_0656 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity (e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ ID NO: 70; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ ID NO: 70, wherein "n" is 7 or more (e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ ID NO: 70 comprise an epitope of SEQ ID NO:
70. Other preferred fragments lack one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ ID NO: 70. Preferred fragments of SEQ ID NO: 70 comprise an epitope of SEQ ID NO: 70.
71. CBU_2009 antigens
The original "CBU_2009" sequence is annotated as "Hypothetical protein". For reference purposes, the amino acid sequence of the full length CBU_2009 sequence is provided in SEQ ID NO: 71 herein.
Preferred CBU_2009 polypeptides for use with the invention comprise an amino acid sequence (a) having 70% or more identity (e.g. 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more) to SEQ ID NO: 71 ; and/or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ ID NO: 71 , wherein "n" is 7 or more (e.g. 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 or more). Preferred variants of SEQ ID NO: 71 comprise an epitope of SEQ ID NO: 71. Other preferred fragments lack one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 20, 25, 30 or more) from the N-terminus and/or one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 20, 25, 30 or more) from the C-terminus of SEQ ID NO: 71. Preferred fragments of SEQ ID NO: 71 comprise an epitope of SEQ ID NO: 71.
Protein antigens of the invention typically induce protective antibodies or stimulate an amnestic cytotoxic T-cell response against C. burnetii. In other words, protein antigens of the invention are typically protective antigens. In one embodiment of the invention, the protein antigen is involved in DNA metabolism - replication, recombination and repair. Thus, in one embodiment, the protein antigen is selected from: a CBIM 337 antigen, a CBU_ 0297 antigen, a CBU_0916 antigen, a CBIM 183 antigen, and a CBIM235 antigen.
In one embodiment of the invention, the protein antigen is involved in DNA - mediated transformation (competance). Thus, in one embodiment, the protein antigen is selected from: a CBU_0532 antigen and a CBU_0758 antigen.
In one embodiment of the invention, the protein antigen is involved in transcription. Thus, in one embodiment, the protein antigen is selected from: a CBU_0232 antigen, a CBU_0852 antigen, and a CBU_2086 antigen.
In one embodiment of the invention, the protein antigen is involved in nucleotide and nucleoside biosynthesis and metabolism. Thus, in one embodiment, the protein antigen is selected from: a CBU_0326 antigen, a CBU_0897 antigen, a CBIM 384 antigen, a CBU_0043 antigen, a CBU_0296 antigen, a CBU_0531 antigen, a CBU_0631 antigen, a CBU_0796 antigen, and a CBIM 830 antigen.
In one embodiment of the invention, the protein antigen is involved in regulatory function. Thus, in one embodiment, the protein antigen is a CBIM 579 antigen.
In one embodiment of the invention, the protein antigen is involved in translation - protein biosynthesis. Thus, in one embodiment, the protein antigen is selected from: a CBIM475 antigen, a CBU_0234 antigen, a CBU_0445 antigen, a CBU_0808 antigen, a CBU_0851 antigen, a CBIM 325 antigen, a CBIM 383 antigen, a CBIM 473 antigen, a CBIM 594 antigen, and a CBIM 841 antigen.
In one embodiment of the invention, the protein antigen is involved in Amino acid biosynthesis and metabolism. Thus, in one embodiment, the protein antigen is selected from: a CBU_0517 antigen, and a CBIM 970 antigen.
In one embodiment of the invention, the protein antigen is involved in energy metabolism - electron transport. Thus, in one embodiment, the protein antigen is selected from: a CBU_0270 antigen, a CBU_0629 antigen, a CBU_0974 antigen, a CBIM 088 antigen, a CBIM 116 antigen, a CBIM 193 antigen, a CBIM296 antigen, a CBIM 397 antigen, a CBIM 400 antigen, a CBIM 401 antigen, a CBIM 720 antigen, a CBU_0075 antigen, and a CBU_2087 antigen.
In one embodiment of the invention, the protein antigen is involved in Intermediary metabolism and other metabolic pathways. Thus, in one embodiment, the protein antigen is selected from: a CBU_0638 antigen, a CBU_0640 antigen, a CBU_0962 antigen, a CBU_0502 antigen, a CBU_0288 antigen, and a CBU_0928 antigen. In one embodiment of the invention, the protein antigen is involved in posttranslational modification, degradation, protein turnover, chaperones. Thus, in one embodiment, the protein antigen is selected from: a CBU_0073 antigen, a CBU_0094 antigen, a CBU_0338 antigen, a CBU_0738 antigen, and a CBU_2012 antigen.
In one embodiment of the invention, the protein antigen is involved in cell division, chromosome partitioning. Thus, in one embodiment, the protein antigen is a CBIM352 antigen.
In one embodiment of the invention, the protein antigen is involved in Protein and peptide secretion and trafficking. Thus, in one embodiment, the protein antigen is selected from: a CBIM648 antigen, a CBIM652 antigen, a CBU_0091 antigen, and a CBU_0155 antigen.
In one embodiment of the invention, the protein antigen is involved in adaptation to atypical conditions - response to starvation. Thus, in one embodiment, the protein antigen is selected from: a CBIM275 antigen, and a CBIM916 antigen.
In one embodiment of the invention, the protein antigen is involved in lipopolysaccharide biosynthesis and metabolism. Thus, in one embodiment, the protein antigen is a CBU_2092 antigen.
In one embodiment of the invention, the protein antigen is involved in pathogenicity and pathogenesis. Thus, in one embodiment, the protein antigen is a CBIM 136 antigen.
In one embodiment of the invention, the protein antigen is involved in detoxication and Resistance. Thus, in one embodiment, the protein antigen is selected from: CBU_0943 antigen, and a CBIM 708 antigen.
In one embodiment of the invention, the protein antigen is selected from: a CBU_01 14 antigen, a CBU_0510 antigen, a CBU_0656 antigen, and a CBU_2009 antigen.
Proteins of the invention can take various forms (e.g. native, fusions, glycosylated, non- glycosylated, lipidated, non-lipidated, phosphorylated, non-phosphorylated, myristoylated, non-myristoylated, monomeric, multimeric, particulate, denatured, etc.).
Proteins of the invention can be prepared by various means (e.g. recombinant expression, purification from cell culture, chemical synthesis, etc.). Protein antigens of the invention are typically recombinant.
Polypeptides used with the invention are preferably provided in purified or substantially purified form i.e. substantially free from other polypeptides (e.g. free from naturally-occurring polypeptides), particularly from other C. burnetii or host cell polypeptides, and are generally at least about 50% pure (by weight), and usually at least about 90% pure, e.g. about 95% pure. In other words, typically, less than about 50%, and more preferably less than about 10%, e.g. 5% of a composition of the invention comprises other expressed proteins. Thus the antigens in the compositions of the invention are separated from the whole organism with which the molecule is expressed.
Protein antigens of the invention may be expressed as a fusion protein. Fusion proteins of the invention may comprise 1 , 2, 3, 4, 5 or more protein antigens of the invention.
In one embodiment, at least two of the protein antigens of the invention are in the form of a hybrid protein.
In one embodiment, a protein antigen of the invention comprises a sequence -P-A- or -A-P-, wherein: -P- is a protein antigen sequence as described above (i.e. a CBU antigen), and -A- is not a sequence as defined above. Examples of -A- include, purification tag(s), signal peptide(s), chaperonin(s), discussed below.
To facilitate removal of the -A- moiety during purification, a unique protease site may be inserted between the -A- moiety and the protein antigen per se. Such protease sites may include those for thrombin, factor Xa, enterokinase, PreScission™, Sumo™. Alternatively, removal of the -A- moiety may be achieved via inclusion of an intein sequence between the signal peptide and the protein antigen per se. Inteins are self-cleaving proteins and in response to a stimulus (e.g. lowered pH) are capable of self-splicing at the junction between the intein and the protein antigen construct thus eliminating the need for the addition of specific proteases. Examples of inteins include domains derived from Mycobacterium tuberculosis (RecA), and Pyrococcus horikoshii (Rad A).
In one embodiment, protein antigens of the invention comprise a fusion protein partner to facilitate soluble expression. Fusion protein partners may be attached at the N- or C- terminus of the protein antigen but are usually placed at the N-terminal end. Examples of fusion partners are: NusA, thyrodoxin, maltose-binding protein, small ubiquitin-like molecules (Sumo-tag).
To facilitate purification, protein antigens of the invention may include one or more purification tags to enable specific chromatography steps (e.g. metal ion chelating, affinity chromatography) to be included in the purification processes. Such purification tags may, for example, include: repeat histidine residues (e.g. 6-10 histidine residues), maltose binding protein, glutathione S-transferase; and strepavidin. These tags may be attached at the island/ or C-terminus of the protein antigens of the invention. To facilitate removal of such tags during purification, protease sites and/ or inteins (examples above) may be inserted between the protein antigen and the purification tag(s). Thus, a typical protein antigen of the invention (starting from the N-terminus) may comprise:
- a first purification tag
- a fusion protein partner (to facilitate expression)
- a first (preferably specific) protease sequence or intein sequence
- the protein antigen sequence
- an optional second (preferably specific) protease sequence or intein sequence
- an optional second purification tag
The first and second purification tags may be the same or different. Similarly, the first and second protease/ intein sequence may be the same or different. The first and second options are preferably different to enable selective and controllable cleavage/ purification.
In one embodiment spacers are introduced to distance the purification tag from the protein antigen - this can help to increase binding efficiency to affinity purification column media. The spacer may be placed (immediately) after the purification tag or between the fusion protein partner and the protein antigen per se. Typical spacer sequences may consist of between 10-40 amino acid residues to give either a linear or alpha-helical structure.
Accordingly, in one embodiment, a protein antigen of the invention may comprise (starting from the N-terminus):
- a first purification tag
- an optional first spacer sequence
- a fusion protein partner (to facilitate expression)
- an optional second spacer sequence
- a (preferably specific) protease sequence or intein sequence
- the protein antigen sequence
- an optional second (preferably specific) protease sequence or intein sequence
- an optional third spacer sequence
- an optional second purification tag
In one embodiment, protein antigens of the invention comprise a signal peptide to target them for secretion into the periplasm of the host cell. A signal peptide may be attached at the N- or C-terminus of the protein antigen but is usually placed at the N-terminal end. Examples of fusion partners are: PelB and ompT.
In one embodiment, protein antigens of the invention comprise a chaperonin protein to help enhance folding, presentation and immunogenicity of the protein antigen(s) per se. A chaperonin may be attached at the N- or C-terminus of the protein antigen but is usually placed at the N-terminal end. Examples of chaperonins are: GroEL and GroES.
In one embodiment, one or more protein antigens of the invention are used as carrier protein(s), typically for use in a conjugate vaccine. In one embodiment, protein antigen(s) of the invention are conjugated to one or more saccharide antigen(s). In one embodiment, protein antigen(s) of the invention are conjugated to one or more lipid antigen(s). In one embodiment, one or more protein antigen(s) of the invention are conjugated to one or more lipopolysaccharide antigen(s). Typically, conjugation of one or more protein antigen (s) of the invention to a polysaccharide, lipid or lipopolysaccharide provides an increased immune response, compared with the immune response elicited by un-conjugated polysaccharide, lipid or lipopolysaccharide. In one embodiment, the polysaccharide, lipid or lipopolysaccharide is from C. burnetii. Non-limiting methods of conjugating protein(s) to polysaccharide, lipid or lipopolysaccharide are known in the art; for example, via the use of heterobifunctional crosslinking agents or homobifunctional crosslinking agents.
For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences may be compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequent coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percentage sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
Optimal alignment of sequences for comparison may be conducted, for example, by the local homology alignment algorithm of Smith and Waterman [Adv. Appl. Math. 2: 484 (1981)], by the algorithm of Needleman & Wunsch [J. Mol. Biol. 48: 443 (1970)] by the search for similarity method of Pearson & Lipman [Proc. Nat'l. Acad. Sci. USA 85: 2444 (1988)], by computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA - Sequence Analysis Software Package of the Genetics Computer Group, University of Wsconsin Biotechnology Center, 1710 University Avenue, Madison, Ws. 53705), or by visual inspection [see Current Protocols in Molecular Biology, F.M. Ausbel et al, eds, Current Protocols, a joint venture between Greene Publishing Associates, In. And John Wiley & Sons, Inc. (1995 Supplement) Ausbubel].
Examples of algorithms suitable for determining percent sequence similarity are the BLAST and BLAST 2.0 algorithms [see Altschul (1990) J. Mol. Biol. 215: pp. 403-410; and "http://www.ncbi.nlm.nih.gov/" of the National Center for Biotechnology Information].
In one homology comparison, the identity exists over a region of the sequences that is at least 10 or 20 or 30 or 40 or 50 amino acid residues in length. In another homology comparison, the identity exists over a region of the sequences that is at least 60 or 70 or 80 or 90 or 100 amino acid residues in length.
Where the invention concerns an "epitope", this epitope may be a B-ce!l epitope and/or a T- cell epitope. Such epitopes can be identified empirically [e.g. using PEPSCAN or similar methods), or they can be predicted {e.g. using the Jameson- Wolf antigenic index, matrix- based approaches, MAPITOPE. TEP!TOPE, neural networks, Opti er & Epi er, ADEPT, Tsites, hydrophilicity, antigenic index or other methods known in the art. Epitopes are the parts of an immunogen that are recognised by and bind to the antigen binding sites of antibodies or T-celi receptors, and they may also be referred to as "antigenic determinants".
In one aspect, the invention provides an immunogenic composition comprising one or more protein antigens of the invention.
In one embodiment of the invention, the immunogenic composition comprises a single type of protein antigen of the invention. In one embodiment, immunogenic composition comprises two or more different types of protein antigen of the invention (e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different protein antigens).
In one embodiment, said one or more protein antigens of the invention are selected from: (1 ) a CBU_0091 antigen; (2) a CBIM648 antigen; (3) a CBIM 652 antigen; (4) a CBU_0510 antigen; (5) a CBU_2009 antigen; (6) a CBIM 579 antigen; (7) a CBIM 193 antigen; (8) a CBIM 400 antigen; (9) a CBIM 401 antigen; (10) a CBU_0962 antigen; (1 1 ) a CBU_0094 antigen; (12) a CBU_0338 antigen; (13) a CBIM 352 antigen; (14) a CBU_0532 antigen; (15) a CBU_0758 antigen; (16) a CBU_0631 antigen; (17) a CBU_0075 antigen; (18) a CBIM 136 antigen; and (19) a CBIM 708 antigen.
In one embodiment, said one or more protein antigens of the invention are selected from: (1 ) a CBU_0091 antigen; (2) a CBIM648; (3) a CBIM652 antigen; (4) a CBU_0510 antigen; and (5) a CBU_2009 antigen.
The immunogenic composition of the invention is typically a vaccine, preferably a subunit vaccine.
In one embodiment, the immunogenic composition comprises a pharmaceutically acceptable carrier or excipient. For the preparation of immunogenic compositions of the invention, the active immunogenic ingredients (whether these be protein antigen(s) of the present invention and/ or corresponding antibodies of the invention that bind thereto) may be mixed with carriers or excipients, which are pharmaceutically acceptable and compatible with the active ingredient. Suitable carriers and excipients include, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the immunogenic composition may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance the effectiveness of the immunogenic composition.
In one embodiment, the immunogenic composition comprises an adjuvant. A non-limiting example of an adjuvant with the scope of the invention is aluminium hydroxide. Other non- limiting examples of adjuvants include but are not limited to: N-acetyl-muramyl-L-threonyl-D- isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 1 1637, referred to as nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(T-2'- dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835A, referred to as MTP-PE), and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2 % squalene/ Tween 80 emulsion.
In one embodiment, immunogenic compositions of the invention comprise a chaperonin protein. In one embodiment, chaperonin is in the form of a fusion protein. In one embodiment, chaperonin is not in the form of a fusion protein.
In one embodiment of the invention, preventing, treating or suppressing C. burnetii infection comprises administration of one or more additional therapeutic agent(s). In one embodiment, the immunogenic composition comprises one or more additional therapeutic agent(s). Such additional therapeutic agent(s) include, for example, bacteriostatic agent(s), such as co-trimoxazole, and/or bactericidal agent(s), such as doxycycline and/or hydroxychloroquine.
Typically, the immunogenic compositions of the invention are prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. The preparation may also be emulsified, or the peptide encapsulated in liposomes or microcapsules. Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations or formulations suitable for distribution as aerosols. For suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of about 0.5 % to 10 %, including for instance, about 1 %-2 %.
Protein antigens of the invention may be formulated for human or animal use in a number of ways. For example, formulation may include treatment with an agent to introduce intramolecular cross-links. One example of such an agent is formaldehyde, which may be incubated, for example, with protein antigen of the invention for between 1-24 hours. Alternatively, longer incubation times of, for example, up to 2, 4, 6, 8 or 10 days may be employed. Following treatment with such an agent, protein antigens of the invention may be combined with a suitable adjuvant, which may differ depending on whether the protein antigen is intended for human or animal use.
An immunogenic composition of the invention vaccine may contain one or more protein antigen(s) of the present invention. Thus, in one embodiment, formulation of an immunogenic composition of the invention comprises the following steps:
providing one or more protein antigen(s) in suitable buffer system;
optionally treating said one or more protein antigen(s) of the invention with a toxoiding component such as formaldehyde; optionally transferring the one or more protein antigen(s) to a new buffer system; combining the one or more protein antigen(s) with one or more suitable adjuvants and optionally other excipients.
In one embodiment, the immunogenic composition is for use in raising an immune response in a patient.
In a preferred embodiment, the immune response is a protective immune response. A protective immune response confers immunological cellular memory upon the subject, with the effect that a secondary exposure to the same or a similar antigen is characterised by e.g. (a) shorter lag phase than after initial exposure to the antigen; (b) production of antibody which continues for a longer period than after initial exposure to the antigen; (c) a change in the type and quality of antibody produced in comparison to initial exposure to the antigen; (d) a shift in class response, with IgG antibodies appearing in higher concentrations and with greater persistence than IgM, than after initial exposure to the antigen; (e) increased average affinity (binding constant) of the antibodies for the antigen compared with initial exposure to the antigen; and/or (f) characteristics known in the art to characterize a secondary immune response.
In one embodiment, the immunogenic composition of the invention is for use in preventing, treating or suppressing C. burnetii infection in a patient. In one embodiment, the invention provides use of the immunogenic composition of the invention in preventing, treating or suppressing C. burnetii infection in a patient. In one embodiment, the invention provides a method of preventing, treating or suppressing C. burnetii infection in a patient, said method comprising administering to the patient the immunogenic composition of the invention.
The patient is typically a mammal. In one embodiment, the mammal is a human. In one embodiment, the mammal is non-human. Typical non-human patients include ungulates (typically cow, sheep or goat). Use of the invention with domesticated live stock is highly advantageous because it provides reduced abortion frequency (providing economic benefits, and reduced animal suffering), and decreased risk of secondary transmission of C. burnetii infection to humans.
Thus, the present invention provides an effective means for preventing, treating or suppressing C. burnetii infection (or a symptom thereof).
In one embodiment, immunogenic compositions of the invention are used prophylactically to prevent the onset of C. burnetii infection in a patient. In such embodiments, the patient is typically at increased risk of becoming infected with C. burnetii, e.g. a worker in an abattoir, resident in the close vicinity of an abattoir, laboratory workers, or military personnel. In one embodiment, the patient is pregnant. In one embodiment, the patient is likely to become pregnant. In one embodiment, the patient is immune-suppressed. In one embodiment, the patient has heart damage. Due to the reduced side effects associated with the protein antigens of the invention, immunogenic compositions of the invention may be used for widespread vaccination strategies. Immunogenic compositions for use in prophylaxis are administered at a prophylactically effective amount, i.e. they contain protein antigen(s) in any amount that, when administered alone or in combination to a patient, triggers an immune response against C. burnetii, and so inhibits or delays the onset or recurrence of at least one of the clinical symptoms of C. burnetii infection. In one embodiment, the prophylactically effective amount prevents the onset or reoccurrence of the C. burnetii infection. "Inhibiting" the onset means either lessening the likelihood of the infection's onset, or preventing the onset entirely.
In one embodiment, immunogenic compositions of the invention are used to treat or suppress C. burnetii infection in a patient. In such cases, the patient is infected with C.burnetii, or has a symptom of C.burnetii infection (e.g. symptoms from high fevers (up to 40-41 °C), severe headache, general malaise, myalgia, chills and/or sweats, non-productive cough, nausea, vomiting, diarrhoea, abdominal pain and chest pain). In one embodiment, treating or suppressing C. burnetii infection comprises administering a composition of the invention to the patient within 5 days of infection with C. burnetii. In one embodiment, the composition is administered to the patient within 2 days of infection, preferably within 1 day of infection with C. burnetii, more preferably within 12 hours of infection with C. burnetii, most preferably within 6 hours of infection with C. burnetii. Said "infection with C. burnetii' includes exposure to a sample suspected of containing, or known to contain C. burnetii.
In one embodiment, treating or suppressing C. burnetii infection comprises administering a composition of the invention to the patient within 5 days of displaying symptoms of C. burnetii infection. In one embodiment, the composition is administered to the patient within 2 days of displaying symptoms of C. burnetii infection, preferably within 1 day of displaying symptoms of C. burnetii infection, more preferably within 12 hours of displaying symptoms of C. burnetii infection, most preferably within 6 hours of displaying symptoms of C. burnetii infection.
In one embodiment, treating or suppressing C. burnetii infection comprises administering a composition of the invention to the patient 5 days or more after infection with C. burnetii. In one embodiment, the composition is administered to the patient between 5-10 days after infection with C. burnetii. In one embodiment, the composition is administered to the patient between 5- 10 days after infection with C. burnetii e.g. 5-9, 5-8 or 5-7 days after infection with C. burnetii. Said "infection with C. burnetii' includes exposure to a sample suspected of containing, or known to contain C. burnetii.
In one embodiment, treating or suppressing C. burnetii infection comprises administering a composition of the invention to the patient 5 days or more after displaying symptoms of C. burnetii infection. In one embodiment, the composition is administered to the patient between 5-10 days after displaying symptoms of C. burnetii infection. In one embodiment, the composition is administered to the patient between 5-10 days after displaying symptoms of C. burnetii infection e.g. 5-9, 5-8 or 5-7 days after displaying symptoms of C. burnetii infection.
In one embodiment, treating or suppressing C. burnetii infection in a patient comprises administering to the patient one or more protein antigens of the inveniton. In one embodiment, treating or suppressing C. burnetii infection in a patient comprises administering to the patient one or more antibodies of the invention. Typically, treating or suppressing C. burnetii infection in a patient comprises administering to the patient one or more antibodies of the invention.
Administration of immunogenic compositions of the invention is generally by conventional routes e.g. intravenous, intramuscular, subcutaneous, intraperitoneal, or mucosal routes. The administration may be by parenteral injection, for example, a subcutaneous or intramuscular injection. In one embodiment, administration is intravenous. In one embodiment, administration is intraperitoneal. In one embodiment, administration is intramuscular.
The immunogenic compositions are administered in a manner compatible with the dosage formulation, and in such amount as will be effective for treatment, prevention and/or suppression of C. burnetii infection. The quantity to be administered, which is generally in the range of 5 micrograms to 250 micrograms of antigen per dose, depends on the subject to be treated, capacity of the patient's immune system to synthesize antibodies, and the degree of protection desired. Precise amounts of active ingredient required to be administered may depend on the judgment of the practitioner and may be particular to each patient.
The immunogenic compositions of the invention may be given in a single dose schedule, or optionally in a multiple dose schedule. A multiple dose schedule is one in which a primary course of vaccination may be with 1-6 separate doses, followed by other doses given at subsequent time intervals required to maintain and or reinforce the immune response, for example, at 1-4 months for a second dose, and if needed, a subsequent dose(s) after several months. The dosage regimen will also, at least in part, be determined by the need of the individual and be dependent upon the judgment of the practitioner.
In one embodiment, the one or more additional therapeutic agent(s) is administered prior to administration of one or more protein antigen(s) of the invention. In one embodiment, the one or more additional therapeutic agent(s) is administered after administration of one or more protein antigen(s) of the invention. In one embodiment, the one or more additional therapeutic agent(s) is administered concurrently with one or more protein antigen(s) of the invention. In addition, the immunogenic compositions of the invention may be administered in conjunction with other immunoregulatory agents, for example, immunoglobulins, antibiotics, interleukins (e.g., IL-2, IL-12), and/or cytokines.
The invention provides nucleic acid encoding one or more of the protein antigens of the invention. The nucleic is typically an isolated nucleic acid.
Nucleic acids encoding protein antigens of the invention may be generated by PCR from C. burnetii genomic DNA. Amplification products may be sequenced by standard methods to ensure integrity. In a preferred embodiment, nucleic acids of the invention are codon optimised for expression in a host cell. Methods of optimising nucleic acids for expression in a host cell are known in the art. The host cell is preferably E. coli.
In one embodiment, the invention provides a vector comprising a promoter operatively linked to nucleic acid as defined above. In one embodiment, the vector is a pET vector. Other suitable vectors are readily identifiable.
The invention also provides a cell capable of protein expression comprising a vector as described above. Preferably, the cell capable of protein expression is not C. burnetii. In one embodiment, the cell is E. coli. In one embodiment, the cell is a human cell. In one embodiment, the human cell is a HEK293T cell.
The present invention also provides a method for expressing one or more of the aforementioned protein antigens of the invention, said method comprising:
1) providing a nucleic acid sequence that encodes one or more of said protein antigens in a host cell, wherein said nucleic acid sequence is operably linked to a promoter; and
2) expressing said nucleic acid sequence in the host cell.
The invention also provides a C. burnetii protein antigen obtainable from a host cell of the invention.
The invention also provides a composition comprising (i) one or more nucleic acid(s) of the invention, or one or more nucleic acid(s) complementary thereto. Optionally, said composition further comprises a pharmaceutically acceptable carrier or excipient. In one embodiment, said composition is for use in nucleic acid immunisation.
Antibodies of the present invention interact with epitopes of C. burnetii protein antigen(s) of the invention. An antibody that binds to a protein antigen of the invention is one capable of binding that antigen with sufficient affinity such that the antibody is useful as a therapeutic agent. An antibody that binds to a protein antigen of the invention is one that binds to said protein antigen with an affinity (K a ) of at least 10 4 M. In one embodiment, the antibody is a neutralising antibody. Neutralising activity of a substance may be measured by its ability to reduce or prevent the infection of mammalian cells grown in culture. Infection of cultured mammalian cells is associated with the presence of large vacuoles containing many C. burnetii.
Thus, the invention provides an antibody that binds to a protein antigen of the invention. Antibodies of the invention are typically protective antibodies.
The invention provides antibody of the invention for use in the prevention, treatment or suppression of C. burnetii infection in a patient.
The invention also provides a corresponding method prevention, treatment or suppression of C. burnetii infection in a patient, comprising administering to said patient antibody of the invention.
A therapeutically effective amount refers to the amount of the antibody, which when administered alone or in combination to a patient for treating, suppressing or preventing C. burnetii infection, or at least one of the clinical symptoms of C. burnetii infection, is sufficient to affect such treatment of the infection, or symptom. The therapeutically effective amount can vary depending, for example, on the antibody, the infection, and/or symptoms of the infection, severity of the infection, and/or symptoms of the infection, the age, weight, and/or health of the patient to be treated, and the judgment of the prescribing physician. An appropriate therapeutically effective amount in any given instance may be ascertained by those skilled in the art or capable of determination by routine experimentation. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody are outweighed by the beneficial effects.
In one embodiment, the method of treating C. burnetii infection comprises administering antibody of the invention systemically (e.g. once or twice per day, or once or twice or 3- or 4- times per every 3-4 days; for a short period of typically 1-2 weeks) followed by a more prolonged period of administration (e.g. once or twice or 3- or 4- or 5- or 6-times per day, or once or twice or 3- or 4- or 5- or 6-times per every 3-4 days, or once or twice or 3- or 4- or 5- or 6-times per week) of antibody. Administration routes include subcutaneous, intramuscular, intraperitoneal, and intravenous. The administration route is preferably intravenous, intramuscular or intraperitoneal.
When administered systemically, the antibodies are formulated accordingly (e.g. such formulations are typically provided as isotoxic aqueous formulations and do not require means for protection against stomach acid or stomach enzymes such as trypsin and/ or chymotrypsin). In one embodiment, preventing, treating or suppressing C. burnetii infection comprises administering antibody of the invention to the patient prior to infection with C. burnetii infection, and before presentation of symptoms of C. burnetii infection. In one embodiment, the composition is administered to the patient within 1 day of infection with C. burnetii. In one embodiment, the composition is administered to the patient within 2 days of infection with C. burnetii. In one embodiment, the composition is administered to the patient within 3 days of infection with C. burnetii. In one embodiment, the composition is administered to the patient within 4 days of infection with C. burnetii. In one embodiment, the composition is administered to the patient within 5 days of infection with C. burnetii. In one embodiment, the composition is administered to the patient within 6 days of infection with C. burnetii. In one embodiment, the composition is administered to the patient within 7 days of infection with C. burnetii.
In use, the present invention employs a composition, comprising antibody of the present invention in a form suitable administration. The purified intact antibodies, or their fragments, are formulated for such delivery. For example, antibody, or its fragment, at a concentration between 5-50 or 15-50 or 25-50 g/litre may be formulated in buffer. Examples of suitable buffer components include physiological salts such as sodium citrate and/ or citric acid. Preferred buffers contain 100-200 or 125-175 or approximately 150 (e.g. 153) mM physiological salts such as sodium chloride.
In preparing compositions of the invention, the antibodies and/ or fragments thereof can be dissolved in a vehicle, and sterilised, for example by filtration through a sterile filter using aseptic techniques, before filling into suitable sterile vials or ampoules and sealing. Advantageously additives such as buffering, solubilising, stabilising, preservative or bactericidal or suspending and/or local anaesthetic agents may be dissolved in the vehicle.
Dry powders, which are dissolved or suspended in a suitable vehicle prior to use, may be prepared by filling pre-sterilised ingredients into a sterile container using aseptic technique in a sterile area. Alternatively the ingredients may be dissolved into suitable containers using aseptic technique in a sterile area. The product is then freeze dried and the containers are sealed aseptically.
The dosage ranges for administration of the antibodies of the present invention are those to produce the desired therapeutic effect. It will be appreciated that the dosage range required depends on the precise nature of the antibody or composition, the nature of the formulation, the age of the patient, the nature, extent or severity of the patient's condition, contraindications, if any, and the judgement of the attending physician. Variations in these dosage levels can be adjusted using standard empirical routines for optimisation.
In one embodiment, typical daily dosages are in the range of 5-20mg (e.g. 8-15mg or approximately 10mg) per kg of body weight. The unit dosage can vary from less than 100mg, but typically will be in the region of 250-500 mg per dose, which may be administered daily (e.g. 1x, 2x, 3x or 4x per day) or less frequently (e.g. on alternative days, or say once per week).
An "antibody" is used in the broadest sense and specifically covers polyclonal antibodies and antibody fragments so long as they exhibit the desired biological activity. In particular, an antibody is a protein including at least one or two, heavy (H) chain variable regions (abbreviated herein as VHC), and at least one or two light (L) chain variable regions (abbreviated herein as VLC). The VHC and VLC regions can be further subdivided into regions of hypervariability, termed "complementarity determining regions" ("CDR"), interspersed with regions that are more conserved, termed "framework regions" (FR). The extent of the framework region and CDRs has been precisely defined (see, Kabat, E.A., et al. Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, 1991 , and Chothia, C. et al, J. Mol. Biol. 196:901-917, 1987, which are incorporated herein by reference). Preferably, each VHC and VLC is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy- terminus in the following order: FRI, CDRI, FR2, CDR2, FR3, CDR3, FR4.
The VHC or VLC chain of the antibody can further include all or part of a heavy or light chain constant region. In one embodiment, the antibody is a tetramer of two heavy immunoglobulin chains and two light immunoglobulin chains, wherein the heavy and light immunoglobulin chains are inter-connected by, e.g., disulfide bonds. The heavy chain constant region includes three domains, CHI, CH2 and CH3. The light chain constant region is comprised of one domain, CL. The variable region of the heavy and light chains contains a binding domain that interacts with an antigen. The constant regions of the antibodies typically mediate the binding of the antibody to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The term "antibody" includes intact immunoglobulins of types IgA, IgG, IgE, IgD, IgM (as well as subtypes thereof), wherein the light chains of the immunoglobulin may be of types kappa or lambda.
The term antibody, as used herein, also refers to a portion of an antibody that binds to a C. burnetii protein antigen of the invention, e.g., a molecule in which one or more immunoglobulin chains is not full length, but which binds to a C. burnetii protein antigen of the invention. Examples of binding portions encompassed within the term antibody include (i) a Fab fragment, a monovalent fragment consisting of the VLC, VHC, CL and CHI domains; (ii) a F(ab') 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fc fragment consisting of the VHC and CHI domains; (iv) a Fv fragment consisting of the VLC and VHC domains of a single arm of an antibody, (v) a dAb fragment (Ward et al, Nature 341 :544-546, 1989), which consists of a VHC domain; and (vi) an isolated complementarity determining region (CDR) having sufficient framework to bind, e.g. an antigen binding portion of a variable region. An antigen binding portion of a light chain variable region and an antigen binding portion of a heavy chain variable region, e.g., the two domains of the Fv fragment, VLC and VHC, can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VLC and VHC regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science ΙΑΙ-ΑΤΊ-ΑΙβ; and Huston et al. (1988) Proc. Natl. Acad. ScL USA 85:5879-5883). Such single chain antibodies are also encompassed within the term antibody. These are obtained using conventional techniques known to those with skill in the art, and the portions are screened for utility in the same manner as are intact antibodies.
The invention provides one or more of the aforementioned protein antigens of the invention, for use in the generation of antibodies that bind to said one or more protein antigens in a host animal.
The protein antigens of the invention may be used as immunogens separately or in combination, either concurrently or sequentially, in order to produce antibodies specific for individual C. burnetii antigens or combinations. For example, two or more protein antigens of the invention may be mixed together and used as a single immunogen. Alternatively a C. burnetii protein antigen of the invention may be used separately as a first immunogen on a first host, and another C. burnetii protein antigen of the invention may be used separately on a second host. The antibodies produced by separate immunisation may be combined to yield an antibody composition directed against C. burnetii antigens. Non-limiting examples of suitable adjuvants for animal/veterinary use include Freund's (complete and incomplete forms), alum (aluminium phosphate or aluminium hydroxide), saponin and its purified component Quil A.
A related aspect of the invention provides one or more antibodies that bind to one or more aforementioned protein of the invention, for use in the prevention, treatment or suppression of C. burnetii infection in a patient. Thus, the invention provides compositions comprising one or more antibodies of the invention.
In one embodiment, said antibodies have been generated by immunisation of a host with one or more of the aforementioned protein antigens of the present invention. The host is typically a non-human animal such as goat, sheep or horse).
The present invention includes a method of producing antibodies against C. burnetii protein antigens of the invention, for use in compositions of the invention. Said method generally involves (i) administering a protein antigen of the invention to a host animal, (ii) allowing sufficient time for the generation of antibodies in the host animal, and (iii) obtaining the antibodies from the host animal. Preferred host animals for the production of antibodies include sheep, goat or horse. The invention also provides a method of producing an antibody, said method comprising the following steps:
(i) administering to a host animal protein antigen of the invention;
(ii) allowing sufficient time for the generation of antibodies in the host animal; and
(iii) obtaining the antibodies from the host animal.
Said host animal is typically a mammal, preferably a sheep, goat or horse.
The invention also provides a method for producing an antibody, said method comprising the following steps:
(a) contacting a B cell with an effective amount of at least one protein antigen of the invention;
(b) fusing the B cell of step (a) with a myeloma cell to obtain a hybridoma cell; and
(c) isolating the antibody produced by the cultivated hybridoma cell.
The invention also provides an in vitro method for isolating antibodies that bind to C. burnetii protein antigen of the invention, said method comprising a) immobilising on a surface (for example on a matrix within a column) one or more C. burnetii protein antigen(s) according to any of the invention;
b) contacting the immobilised protein(s) with a solution containing antibodies that bind to the C. burnetii protein antigen(s);
c) allowing said antibodies to bind to said C. burnetii protein antigen(s), thereby forming a bound complex of antibody and protein antigen(s);
d) washing away any unbound antibody or protein; and
e) eluting the bound antibodies from the surface, thereby providing affinity-purified antibodies.
The invention also provides an antibody obtainable by a method described herein.
The antibody may be obtained from the serum. Thus, the procedures generate antisera containing antibodies capable of binding C. burnetii antigens. In a further embodiment, the antibodies are isolated and/or purified. Thus, another aspect of the present invention involves purifying the antibodies from antiserum.
The method of producing antibodies allows all modes of immunisation (i.e. to generate the antibodies of the invention), including subcutaneous, intramuscular, intraperitoneal, and intravenous. The invention also contemplates a wide variety of immunisation schedules. In one embodiment, an animal is administered protein antigen on day zero and subsequently receives protein antigen at intervals thereafter. It will be appreciated that the interval range and dosage range required depends on the route of administration, the nature of the formulation, and the judgement of the attending person. Variations in these dosage levels can be adjusted using standard empirical routines for optimisation. Similarly, it is not intended that the present invention be limited to any particular schedule for collecting antibody. The preferred collection time is someday after day 56. Levels of the specific antibody i.e. that which binds to the immunogen, preferably represents at least 3 g per litre of serum.
The antibodies of the invention may be modified as necessary after collection from the host animal, so that, in certain instances, they are less immunogenic in the patient to whom they are administered. For example, if the patient is a human, the antibodies may be despeciated by methods well known in the art. One example as to how an antibody can be made less immunogenic is to prepare the F(ab) 2 fragment. The antibodies of the invention may be used to produce such antibody fragments for which various techniques have been developed. For example, the fragments may be derived by proteolytic digestion of intact antibodies. Other techniques for their production will be apparent to the skilled practitioner.
The invention provides an in vitro method for confirming the presence or absence of C. burnetii in a patient sample (i.e. diagnosis of C. burnetii infection). The presence of a C. burnetii is confirmed by detecting the binding of an antibody of the invention to a C. burnetii antigen present in said sample, and wherein failure to detect the binding of said to a C. burnetii antigen in said sample confirms the absence of C. burnetii. Suitable methods for detecting antibody binding are well-known e.g direct or indirect detection methods, involving detection of a fluorescent or chromogenic signal.
In one embodiment, upon confirmation of C. burnetii in the patient sample, said patient is administered agent for treatment of suppression of C. burnetii infection. Said agent is typically selected from: (a) composition of the invention; (b) antibody of the invention; (c) bacteriostatic agent, such as co-trimoxazole; and/or (d) bactericidal agent, such as doxycycline and/or hydroxychloroquine and/or ciprofloxacin.
In one embodiment, protein antigens of the invention are for use as ligands for use in affinity chromatography procedures. In such procedures, protein antigens of the invention may be covalently immobilised onto a matrix, such as Sepharose, e.g. using cyanogen bromide- activated Sepharose. Such affinity columns may then be used to purify antibody from antisera or patially purified solutions of immunoglobulins by passing them through the column and then eluting the bound IgG fraction (e.g. by low pH). Almost all of the antibody in the eluted fraction will be directed against the protein antigens of the invention, with nonspecific antibodies such as IgG and other proteins having been removed. These affinity purified IgG fractions have applications both as immunotherapeutics and as a reagents in diagnostics. For immunotherapeutics, affinity purified antibodies enable a lower dose to be administered making adverse side effects less likely. For diagnostics, affinity purified agents often give improved specificity and fewer false positive results. BRIEF DESCRIPTION OF DRAWINGS
There now follows a brief description of the Figures, which illustrate aspects and/ or embodiments of the present invention.
Figure 1 - One-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 μΙ, 5 μΙ, and 2.5 μΙ 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).
Figure 2 - Western 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.
Figure 3 - Linear 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 BSA; S = 0.013; R 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.
Figure 4 - Coomassie-stained 2D PAGE separation of C. burnetii proteins across the wide pi range 3.0-11.0 (non-linear). Isoelectric point ranges from pi = 3 at the left side of the image to pi = 1 1 at the right side of the image.
Figure 5 - Coomassie-stained 2D PAGE separation of C. burnetii proteins across the narrower (acidic) pi range 3.0-5.6 (non-linear). Isoelectric point ranges from pi = 3 at the left side of the image to pi = 5.6 at the right side of the image.
Figure 6 - Coomassie-stained 2D PAGE separation of C. burnetii proteins across the narrower (basic) pi range 7.0-1 1.0 (non-linear). Isoelectric point ranges from pi = 7 at the left side of the image to pi = 11 at the right side of the image.
Figure 7 - Western Blue® (BCIP/NBT)-stained antibody-probed Western blot of C. burnetii proteins detected in wide pi 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.
Figure 8 - Western Blue® (BCIP/NBT)-stained antibody-probed Western blot of C. burnetii proteins detected in lower, narrow pi 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.
Figure 9 - Western Blue® (BCIP/NBT)-stained antibody-probed Western blot of C. burnetii proteins detected in higher, narrow pi 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.
Figure 10 - Output 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.
Figure 11 - Left-hand pane - 1 D 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 pane - Antibody-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.
Figure 12 - The left-hand pane is the Coomassie-stained 1 D 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.
Figure 13 - Western-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
IN VIVO EXPERIMENTS
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.
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.
Aerosol infection of guinea pigs and two strains of mouse
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.
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 1.5 x 10 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 (7 x BALB/c and 7 x A/J), 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 licence 30/2423.
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. burnef/V isocitrate-dehydrogenase (icd) gene.
Table 1- qPCR titres of the bacterial culture material in the Collison nebuliser (generator 1 - Cnebuiiser) samples taken prior to challenge of each group as well as the impinger samples (Cimpinger) and the derived C ae ro concentrations in copies/ml (derived from C im pi n ger 20 ml (the sample volume)/l/min (sample flow) x 10 min(sampling time)/1000).
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 aeiO x Inspired volume. The inhaled dose is an estimate of the number of organisms to which each animal was exposed.
To estimate the dose of organism inhaled by each animal (Table 2) the C aer o 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).
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. In addition, the following organs were harvested for qPCR analysis; spleen, kidney, liver, heart, lung and testicle. For the qPCR, approximately 1/3 of the total organ mass was removed from each organ. DNA was extracted and assessed by qPCR (data not shown).
RESULTS - in vivo Experiments
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 measureable febrile response.
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.
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.
Terminal blood was harvested from all animals and the anti-sera harvested and stored. PROTEIN ISOLATION
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.
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.
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. Growth of C. burnetii
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.
Optimisation of inoculation concentration
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 2.9 x 10 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.
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 1.0 x 10 2 copies/ml to 1.0 x 10 6 copies/ml in 10 increments. The plates were sealed into a 2.5 I 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.
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.
The growth curves for different C. burnetii seeding concentrations show that for inoculation concentrations in the range 1 x 10 2 to 1 x 10 4 copies/ml, growth was exponential (results not shown). For the two highest titres (1 x 10 5 and 1 x 10 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 1 x 10 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. Larger scale growth
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 1 x 10 4 copies/ml. This consisted of 35 μΙ 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.
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 12,000 x g 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 1.1 x 10 9 copies/ml (using icd real-time qPCR).
Quality assessments
Electron microscopy
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.
Immunofluorescent microscopy
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).
EXTRACTION OF PROTEINS FROM ACCM-2 GROWN C. BURNETII
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 16,000 x g and the PBS discarded. The pellets were re-suspended in 1.0 ml each of Bug Buster master mix (containing Benzonase Nuclease and rLysozyme; Novagen #71456-3 ) containing 1x 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 16,000 x g for 20 min. The soluble protein fraction was then filtered through a 0.1 μηι PVDF syringe filter (Millipore #SLW033RS) to remove any residual infectious particles.
Soluble C. burnetii protein was stored at -80 °C in small aliquots until required.
One dimensional protein separation and detection One dimensional protein separation
To produce a denatured, reduced protein preparation suitable for one-dimensional (1 D) PAGE, 50 μΙ of soluble protein was mixed with 50 μΙ of 2x concentrated Laemmli sample buffer (Sigma S3401-10VL). This mixture was heated in a heating block at 95 °C for 10 min.
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.
Coomassie staining of gels
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.
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). Silver staining of gels
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.
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.
Western blotting
To produce membranes with bound C. burnetii proteins, a Western Blot was performed. This was achieved using an iBIot® semi-dry blotting system (Life Technologies IB1001 UK) using a PVDF mini transfer stack (Life Technologies iBIot® IB4010-02). The pre-run PAGE gel was removed from the cassette, trimmed and floated off into deionised water. The iBIot anode stack was inserted into the iBIot device. The gel was carefully laid onto the PVDF membrane without introducing air-bubbles. An iBIot 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 iBIot (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.
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.
Antibody probing of transferred proteins
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 M7049-1 BTL) diluted in PBS containing 0.05 % (v/v) Tween-20 buffer (PBS-T; Thermo Scientific™ Pierce™ 20X PBS Tween™ 20 PI-28352) (5 % Milk/PBS-T) at room temperature for 1 - 2 h, or overnight at 8 °C. After blocking, the membranes were incubated with 5ml 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).
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.
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).
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).
Confirmation of antibody activity in convalescent sera
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 1 D 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.
RESULTS - One dimensional protein separation
Basic assessment of protein extraction
The initial assessment of the C. burnetii protein extraction process was performed by running three lanes of the reduced, denatured protein; 10 μΙ, 5 μΙ, 2.5 μΙ. The gel image (Figure 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. Confirmation of antibody activity in convalescent sera
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 (Figure 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.
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.
Two-dimensional protein separation and immune-reactive protein isolation
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.
Protein quantitation
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 μg. Two microcentrifuge tubes were also set up with 10 μΙ each of the C. burnetii protein. To all tubes, 500 μΙ of precipitant (containing trichloroacetic acid) was added, the tubes vortex-mixed and incubated at room temperature for 3 min. To all tubes, 500 μΙ of co-precipitant (containing deoxycholic acid) was added, the tubes vortex-mixed and the proteins pelleted by centrifugation at 10,000 x g for 5 min.
The supernatants were decanted to waste and the tubes re-centrifuged at 10,000 x g for a brief pulse. Any residual supernatant was removed with a micropipette. To all tubes, 100 μΙ of copper solution and 400 μΙ 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.
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.
Protein purification
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 pi = 3-1 1 NL IEF separations 17 μΙ (41 μg) and pi = 3-5.6 NL and pi = 7-1 1 NL separations 25 μΙ (60 μg) per strip were used.
The required number of μg of lysed C. burnetii material was pipetted into 1.5 ml microcentrifuge tubes. To each tube, 300 μΙ precipitant was added and the tubes vortex- mixed, the samples were then incubated on ice (4 °C) for 30 min. To each tube, 300 μΙ of co- precipitant was added and the contents vortex-mixed. The tubes were centrifuged at 12,000 x g 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 μΙ 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 12,000 x g at 4 °C for 10 min and the supernatant discarded using a micropipette.
Onto each pellet was pipetted 25 μΙ 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 μΙ 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 16,000 x g 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 μΙ 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.
Isoelectric focusing of C. burnetii proteins
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 poly- acrylamide 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 pi 3 - 1 1 NL 17-6004-40, pi 7 - 11 NL 17-6004-39, pi 3 - 5.6NL 17-6002-02) with the appropriate pi 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 μΙ of the protein preparation was pipetted. The protective backing was removed from the IPGstrip (GE Healthcare pi 3 - 1 1 NL 17-6003-73, pi 7 - 1 1 NL 17-6003-68, pi 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.
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 pi range (pi 3-3.5NL) or the anode with deionised water and the cathode with rehydration solution for basic pi ranges (pi 3-11 NL and pi 7-1 1 NL). 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.
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 μΑ/strip.
Second dimension protein separation
For second dimension separation of the pl-separated proteins according to their size, polyacrylamide gel electrophoresis (PAGE) was performed. Lithium dodecyl sulphate buffer (4X NuPAGE® LDS sample buffer; Life Technologies NP0008) was diluted to 1x with deionised water. The proteins in the IPG strips were reduced in immunoassay reagent troughs in 1X LDS sample buffer containing 1 :10 sample reducing agent (NuPAGE® sample reducing agent 10X; 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 1X LDS 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.
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 μΙ 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.
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.
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.
Isolation of immune-reactive protein spots
Parallel 2D PAGE gels were prepared of the C. burnetii proteins for three pi 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.
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. The spots and regions that were cut were collected into labelled 500 μΙ microcentrifuge tubes and stored at -80 °C prior to further processing.
RESULTS - Two-dimensional protein separation and immune-reactive protein isolation
Protein quantitation
The standard curve generated during the protein assay (Figure 3) yielded the line equation:
OD 480 nm = 0.856 - 0.003 X ig BSA
To calculate the quantity of protein present in the C. burnetii protein preparation this was rearranged to the form:
OD 480 nm - 0.856
The two samples of the C. burnetii extract gave protein quantities of 21 and 26 μg in the 10 μΙ tested. For the purposes of the 2D PAGE the mean value of 2.4 g/μΙ was used.
Two-dimensional protein separation
Protein separations in two-dimensions over the three pi ranges chosen showed a satisfactory range of spots in terms of both molecular weight and pi (Figures 4-7). There appears to be a slight bias towards the number of spots in the low pi (3-5.6) range compared to the higher (7-1 1) range.
Immune-probing of 2D PAGE Western Blots and protein spot excision
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 (Figures 7-9).
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 (pi). 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.
Immunoprecipitation of immune-reactive proteins
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.
Antibody affinity purification
Immunoprecipitation (IP) requires an affinity purified class G immunoglobulin (IgG) antibody to use as the capture antibody. This was prepared from 500 μΙ of guinea pig sera as follows.
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.
The guinea pig antisera (4427 1-4) was made up to 5 ml with binding buffer and filtered through a 0.45 μηι syringe filter. This was then injected into a 5ml 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.
Finally, the bound IgG was eluted by washing 5 ml of elution buffer through the column. Fractions (7 x 1 ml) were collected into tubes each containing 200 μΙ of 1 M Tris-HCI, pH 9.0 to rapidly neutralise the low pH of the elution buffer and minimise damage to the purified antibodies.
Dialysis and concentration of affinity purified antibody
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.
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 μΙ PBS for 10 min at 15,000 x g. The purified antibody was loaded onto the column 500 μΙ at a time and concentrated for 10 min at 15,000 x g until all of the antibody had been loaded and concentrated, and the final volume was approximately 50 μΙ. The concentrated antibody was quantitated by measuring the absorbance at 280 nm (NanoDrop ND-2000 spectrophotometer) using PBS as a blank. Purity and activity check of affinity purified antibody
Non-reducing and reducing 1 D 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).
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.
Preparation of proteins for immunoprecipitation
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; VS021 1) at 20 °C and 4,000 x g.
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.
Immunoprecipitation of C. burnetii immune-reactive proteins
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 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 μΙ of modified coupling buffer (MCB - consisting of 100 μΙ of 20X coupling buffer and 100 μΙ 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 μΙ of MCB containing 20 μg 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 temperature - vortexing every 10 min during incubation. The beads were collected with a magnetic stand and the supernatant discarded. The beads were washed with 100 μΙ of MCB, vortexed, magnetically collected and the supernatant discarded. This was repeated with a 300 μΙ volume of MCB twice more to remove any unbound antibody.
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 μΙ coupling buffer containing 20 μΜ 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 μΙ 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 μΙ washes in 8 °C IP lysis/wash buffer.
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 μΙ of IP lysis/wash buffer and once with 500 μΙ of deionised water. Bound proteins were eluted by adding 100 μΙ_ 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 μΙ of neutralisation buffer. The elution step was repeated twice and the supernatants pooled.
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 1x Laemmli buffer, heating to 95 °C for 10 min and subjecting to 1 D PAGE as described above. Western blots were also performed and the blots probed with guinea pig antisera.
The eluted protein lanes from the Coomassie-stained gel were cut into 7 x sections (each approximately 1 cm x 0.5 cm) and stored at -20 °C for mass spectrometric analysis.
RESULTS - Immunoprecipitation of immune-reactive proteins Antibody affinity purification
The output of the FPLC instrument is shown below (Figure 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/μΙ (A 2 so nm = 3.945).
The purity and activity assessment (Figure 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.
Immunoprecipitation
The protein results of the immunoprecipitation (IP) experiment (Figure 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.
Immunoprobing the Western-blot membrane with the IgG present in guinea pig sera (4427 A1-4) shows reactions with proteins present in all lanes (Figure 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.
PROTEIN IDENTIFICATION
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.
In gel tryptic digestion of proteins
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.
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/μΙ stock. This stock was divided into 10 μΙ 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. Each gel piece was incubated with 500 μΙ 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 μΙ 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.
The proteins were reduced by adding 50 μΙ 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 μΙ 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 μΙ digestion buffer. The gel pieces were then dehydrated by incubating twice with 500 μΙ 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.
Trypsin was thawed and diluted in 25 mM ammonium bicarbonate to a working concentration of 10 ng/μΙ. To each gel piece, 75 μΙ of working trypsin solution was added and the tubes incubated overnight at 37 °C at 300 rpm in a shaking incubator.
Peptides were extracted by centrifuging the gel pieces at 10,000 x g 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 μΙ of 0.1 % (v/v) aqueous trifluoroacetic acid (Fisher Scientific #1031 1725). The gel pieces were again centrifuged at 10,000 x g for 5 min, the trifluoroacetic acid aspirated off and combined and mixed with the retained trypsinised peptide solution.
The extracted peptides were frozen at -80 °C until required for mass spectrometric analysis. Mass spectrometry analysis
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 nl_C 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.).
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 μΙ) was loaded onto a reversed phase Nano Trap Column (Acclaim PepMap 100, 100 μηι i.d. x 2 cm long, Ci 8 , 5μm, 100 A) and further separated on an Ci 8 reversed-phase nanocolumn (Acclaim PepMap100, 75 μm i.d. χ 15 cm long, Ci 8 , 3 μm, 100 A; 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.
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.
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.
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 (NT) 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 NT 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.
In silico analyses and protein identification
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.org - containing 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 tolerance ± 10 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. Organisation of identified proteins
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 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.
Protein functional characterisation
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.
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 Gr amN, 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.
Table 5 - Tools used to predict subcellular localisation and functionality of identified C. burnetii proteins. RESULTS - protein identification
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.
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).
Energy metabolism - electron transport
CBU_0270 Short-chain alcohol L10, L1 1 , 44,875 5.8 dehydrogenase L13, L19, H7,
C5
CBU_0629 Proline dehydrogenase/Delta-1- H2 1 16,423 6.3 pyrroline-5-carboxylate
dehydrogenase
CBU_0974 Acetyl-CoA acetyltransferase H6, H7 42,243 7.7
Bifunctional NAD(P)H-hydrate
CBIM088 repair enzyme Nnr L16, L17, H7 51 ,699 5.7
CBIM 1 16 Alanine dehydrogenase H6 39,472 6.1
H5, C2, C3,
CBIM 193 Thioredoxin reductase C4 34,620 5.9
L15, H5, C1 ,
CBIM296 ATP-NAD kinase C2, C4 32,892 5.3
L10, L1 1 ,
L12, L13,
Succinyl-CoA synthetase beta L19, H3, H4,
CBIM397 chain H6, H7 42,333 5.5
Succinate dehydrogenase iron-
CBU_1400 sulfur protein C6 27,792 8.2
Succinate dehydrogenase H2, L21 , L22,
CBU_1401 flavoprotein subunit H3, H4 65,438 6.7
CBIM720 Aconitate hydratase H2, L22, H4 101 ,389 5.8
Intermediary metabolism and other metabolic pathways
CBU_0638 Dihydrolipoamide L2, L10, L1 1 , 40,846 5.2 acetyltransferase component of L12, L13, pyruvate dehydrogenase L19, H3, H4, complex H6, H7, C5
CBU_0640 Pyruvate dehydrogenase E1 L10, L1 1 , 41 , 138 5.3 component alpha subunit L12, L13,
L19, H3, H4,
H6, H7, C1
CBU_0962 Short chain dehydrogenase H1 , H5, H6, 25,567 6.9
H7
Posttranslational modification, degradation, protein turnover, chaperones
CBU_0073 Xaa-Pro aminopeptidase H2, L21 68, 185 5.6
CBU_0094 ClpB protein H2, L22 96,769 5.5
Membrane alanine
CBU_0338 aminopeptidase H2, L22, H4 103,023 6.1 Cell division, chromosome partitioning
27 CBIM352 Cell division protein ftsH L15 71 ,610 6.2
Protein and peptide secretion and trafficking
28 CBIM648 DotA protein H4, C8 86,867 5.4
29 CBIM652 IcmX protein H3, C3 41 ,352 6.0
Adaptation to atypical conditions - response to starvation
30 CBIM275 Starvation sensing protein rspA L10, L19, H2, 45,431 5.7
H3, H4, H6,
H7, C5
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.
Translation - protein biosynthesis
CBU_0234 SSU ribosomal protein S7P 21 ,291 10.3
CBU_0445 SSU ribosomal protein S16P 20,726 9.9
CBU_0808 Valyl-tRNA synthetase 106,648 8.6
CBU_0851 SSU ribosomal protein S15P 10,316 10.4
CBIM325 Bacterial Protein Translation Initiation Factor 19,456 9.9
3 (IF-3)
CBIM383 Ribosome Recycling Factor (RRF) 20,945 6.4
CBIM473 Aspartyl/glutamyl-tRNA(Asn/Gln) 11 , 102 4.7 amidotransferase subunit C
CBIM594 GatB/Yqey domain protein 16,744 6.0
CBIM841 Peptidyl-tRNA hydrolase 20,771 9.0
Amino acid biosynthesis and metabolism
CBIM970 Diaminopimelate epimerase 30,071 6.1
Energy metabolism - electron transport
CBU_0075 2-polyprenyl-6-methoxyphenol hydroxylase 45,231 9.7
CBU_2087 Thioredoxin 12,613 4.9
Intermediary metabolism and other metabolic pathways
CBU_0502 DNase, TatD family 28,627 5.9
CBU_0288 Phosphopantetheine adenylyltransferase 17,967 6.2
CBU_0928 Pyridoxamine 5'-phosphate oxidase 23,636 6.3
Posttranslational modification, degradation, protein turnover, chaperones
CBU_0738 ATP-dependent endopeptidase dp 21 ,602 6.1 proteolytic subunit clpP
CBU_2012 ATP-dependent endopeptidase hsl ATP- 52, 123 5.5 binding subunit hsIU
Lipopolysaccharide biosynthesis and metabolism
CBU_2092 Phosphoenolpyruvate carboxykinase [ATP] 56,809 5.8
Protein and peptide secretion and trafficking
CBU_0091 Peptidoglycan-associated lipoprotein OmpA- 21 ,357 9.5 like
CBU_0155 Type 4 pili biogenesis protein pilB (nuleotide- 57,831 8.6 binding protein)
Pathogenicity and pathogenesis
CBIM 136 Enhanced entry protein enhC, 117,740 9.3 tetratricopeptide repeat family
Detoxication and Resistance
CBU_0943 Rhodanese-related sulfurtransferases 16,593 8.7
CBU_1708 Superoxide dismutase 22,274 6.2
Adaptation to atypical condition - response to starvation
CBIM916 Universal stress protein A 15,779 6.6 Poorly characterised
68 CBU_01 14 Protein yajQ 18, 184 7.9
69 CBU_0510 Hypothetical protein 1 1 ,275 5.5
70 CBU_0656 Hypothetical transcriptional regulatory 12, 103 4.9
protein
71 CBU_2009 Hypothetical protein 50, 169 9.1
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.
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.
Further characterisation of unpublished proteins
Of the 71 novel immune reactive proteins identified, 19 were identified as having particularly advantageous features and/or a predicted non-cytoplasmic location.
36 CBU_075 Lipoprotein, ComL family + OM C 8
23 CBU_096 Short chain E C
2 dehydrogenase
64 CBIM 13 Enhanced entry protein + + 1 E ukn
6 enhC, tetratricopeptide
repeat family
15 CBIM 19 Thioredoxin reductase C P
3
27 CBIM 35 Cell division protein ftsH + + IM IM
2
18 CBIM40 Succinate IM ukn
0 dehydrogenase iron- sulfur protein
19 CBIM40 Succinate IM ukn
1 dehydrogenase
flavoprotein subunit
7 CBU_157 Trp repressor binding + ukn C
9 protein
28 CBIM64 DotA protein + + + 1 IM IM
8
29 CBU_165 IcmX protein + + + ukn P
2
66 CBIM 70 Superoxide dismutase + P C
8
71 CBU_200 Hypothetical protein + + ukn IM
9
Key: C = Cytoplasmic; IM = Inner/Cytoplasmic Membrane; P = Periplasmic; OM = Outer Membrane; E = Extracellular; ukn = unknown
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.
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. CBU 1648 (DotA, SEQ ID NO: 28) and CBU 1652 (IcmX, 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.
Further characterisation of mechanisms of immunity
Overlapping peptide pools representing the entire open reading frame (ORF) of four vaccine candidate proteins (CBU_0510, CBU_0091 , CBU_2009 and CBIM648) 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 ^g/peptide) were used to stimulate splenocytes harvested from acutely infected and recovered (convalescent) mice in an interferon-γ 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:
Table 9 - Further characterisation of mechanisms of immunity
The inventors found that peptide pools corresponding to CBU_0091 and CBIM648 gave highly statistically significant interferon-γ 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.
Summary of Results
Despite previous efforts in the literature to identify immune reactive proteins of C. burnetii, the inventors have surprisingly identified 71 new immune reactive proteins.
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.
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 + 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.
SEQUENCES:
SEQID NO: 1
MTISFVHLKIHSEYSIVDSVVRIDQLLQRAVDLKMPAVALTDEVNLFALVKFYRQAI NKGIKPIIGSELLLAEGDDVFRF
TALCQNQIGFRHLIQLLSRAYVEGRQRDHVLIQWEWLVQANEGLIILSGARRGNVGQ ALLQRRSPLAEERLTRWIN
HFPGRFYLELQRTRRDQEEEYIHSVIELALKHRVPVVATNEVCFLSQGDFEAHEARV CIHQGYLLQDVNRPREYSDQ
QYFKSAEEMTALFSDIPEALENTVEIAKRCSVPLSLDEVFLPKFPVPANLKVEDYFR AQAKQGLTRRLVGLEMKNNLT
HKDYEERLETEITVITKMGFASYFLIVADFIAWAKQHHIPVGPGRGSGAGSLVAYSL GITELDPLEHDLLFERFLNLER
VSMPDFDIDFCMEGRDRVIDYVAERYGQEAVAQIITYGTMAARAVLRDVGRVLGLPY GYVDKIAKLVPFELGVTLE
KALEQEEILAKRYAEDEEVKNLIDLAMKLEGLTRNAGKHAGGVVIAPTKLTDFVPLY SEPGSDHVVTQFDKDDVEAV
GLVKFDFLGLRTLTIINWAVQNINAKRKIQNETELDIGTIPLDDPKTYALLKSCATT AVFQLESRGMKELIRRLQPDNF
ADIMALVALFRPGPLQSGMVETFIACKHGEQSVHFLHPALEPILRTTYGVILYQEQV MQIAQVLAGYSLGAADVLR
HAMGKKKPEEMAKQRAVFLEGTKARGLKEALANQIFDLMEKFSGYGFNKSHSAAYAL IAYQTAWLKAHYPAEFM
AAVLSSDMDNTDKVVGFINECRDMNLELLPPNINWSHYPFTVNTKGQIVYGLGAIKG VGEAAAMNIVAYREAEGE
FKGLFNFCSRVDLRKVNRRAVEPLIRSGAMDTFGVSRASLFESLTKAFQAAEQRNRD MILGQHDLFGEEVKGIDED
YTEVPEWNDSDRLRGEKETLGLYVSGHPLQACIKEMKAVGAVPINHLSLSEKNSVVV AGMMMGMRTITTRSGKR
MAILSLEDQTGKIDVTLFNDLYQQVAADLTDHAILVIRGTVGRDDYTGGQKMVADML LTLDKVREQMVKRLLIRV
AGQDGVDQLLTELPPLIKPYVGGRCPVAIAYQSETAIAELLLGETWRVKLDDKLLSE LSKLYGKDQVELEY
SEQID N0:2
MRDLVKQLKSEKHTAEFDALRIKLASPEEVRSWSYGEVKKPETINYRTFKPEREGLF CAKIFGPIKDYECLCGKYKRLK
HRGVICEKCGVEVTLAKVRRERMGHIELASPVAHIWYLKSLPSRIGLLLDVTLRDIE RILYFEAYVVVDPGMTDLEPR
QLLSEEAYLDALEEYGDDFTALMGAEAIQRLLRDIDVEAEVEALRTELQTTTSETKT KKLTKRLKVLSAFLESGNKPEW
MILTVLPVLPPDLRPLVPLDGGRFATSDLNDLYRRVINRNNRLKRLLDLNAPDIIVR NEKRMLQEAVDALLDNGRRG
RAILGSNRRQLKSLADMIKGKSGRFRQNLLGKRVDYSGRSVIVVGPTLKLHQAGLPK KMALELFKPFIFSKLQLRGLA
TTVKAAKKLVENEGPEVWDILEEVIREHPILLNRAPTLHRLGIQAFEPVLVEGKAIQ LHPLVCTAYNADFDGDQMAV
HVPLTLEAQLEARSLMMSTNNVLHPANGEPIIVPSQDVVLGLYYITRDRVNAKGEGM RFADAQEVVRAYENDQV
DLHARITVRIKEGILNEAGEIEESDRLVNTAAGRILLWQIVPKGLPFALVDQPMTKK AVTKLLDFCYRNLGLKTTVIFA
DKLMYMGFHYATHSGVSIGINDLVVPDQKEAIISRAEDEVREIEKQYASGLVTHGER RNKVIDIWSRTNDQVAKA
MMEKIAVEKVKDAEGKEVAQSSFNSIYMMSDSGARGSAAQTRQLAGMRGLMARPDGT IIETPITANFREGLNVL
QYFISTHGARKGLADTALKTANSGYLTRRLVDVAQDLVVTEHDCGTEASIEMMPHIE GGDVVEPLRERVLGRILAEP
VMDPKSRKELLAKDTFLDERRVDILEEHSIDRVRVRSAITCEARYGICSMCYGRDLA RGHVVNVGEAIGVVAAQSIG
EPGTQLTMRTFHIGGAASRATAANNIGVKSTGKIKLRNLKTVEQAQGNLVAVSRSGE LVVQDLQGSEREHYKVPY
GATISVRDGDSVKAGQIVAQWDPHTHPIITEVAGTLRFVDLVDGVTMNRQTDELTGL SSIVITSTKQRSASGKELRP
MVKLVDKNDDDLFLPGGKVPAHYFLPEGTFLTKEDGTTVNIGDVLARIPQETSKTRD ITGGLPRVADLFEARRPKDA
AILAEISGVVSFGKDTKDKGRLIITAPDGTTHEELIPKWRHVSVFEGETVEKGEVIA DGPRDPHDILRLLGVNALANYI
VNEVQEVYRLQGVKINDKHIEVIVRQMLRKVKITQPGDTDLLQNEQVERTRVREENE KIIKKDGTVAKVEPILLGITK
ASLATESFISAASFQETTRVLTAASVAGKRDDLRGLKENVIVGRLIPAGTGFSYHQQ RRAVAGKSVEEKEIEEKRVTA
SEAEQALSEALKSSAPQEAKAAQKDE
SEQID N0:3
MNKIRKTFQYGKHEVTFETGEMARQATGAVVVRMGDTVLLVSVVAKKEAEEGRDFFP LTVNYQEKTYAAGKIPG GYFKREGRPTEKETLTSRLIDRPLRPLFPKGFTNEVQVIATVLSVDSKVPTDIPAILGAS AAIGLSGIPFNGSLGAARVG YRGGEYLLNPSLDELKDSALDLVVAGTRDAVLMVESEAQELPESVMLGAVLHGHQAMQVA IQAIAEFIQEAGGAK WEWEPPTVNTALEKWVVEKSEAPLKKAYQIQEKTARQAQIQAIRDQLLADRAAEREGEEN AVNEHELAVIFHELE
RRIVREQILTGQPRIDGRDTKTVRPITVKVGVLPRSHGSALFTRGETQALVVTTLGT ERDAQSIDDLDGDRQEEFIFH
YNFPPFCVGEVGFMSGPKRREIGHGRLAKRAVVPVVPTLDKFPYVIRVVSEILESNG SSSMASVCGSSLALMDAGV
PTKAPVAGIAMGLIKENDKYAVLSDILGDEDHLGDMDFKVAGTSNGVTALQMDIKIE GITKEIMEQALDQAKEGRL
HILSIMNKVLDKPRSQVSDLAPQYVTMKINPEKIRDVIGKGGVVIREITEATNCAID ISDDGTIKIAAHTTEEGEAAKR
RIEELTAEVELGKVYEGTVVKITDFGAFVQILPNTQGLVHISQIAQERVENVRDYLE EGQVIRVKVIEIDRQGRVRLS
MKQID
SEQIDN0:4
MLGVAEKCYDLTIMNILIIGNGGREHALAWKVAQSPRVEKIWVAPGNAGTARELKTQ NVPIGVTDIKSLIAFAKKN
QINLTLVGPEIPLAAGIVDHFQQENLIVFGPTQAAAQLETSKSFCKTFMRRHGIPTA RFEAFRNTSDAFSYLEQQSFPI
VIKASGLAAGKGVVIAQSLQEAKETVIAMMEEKQFGNAGAEIVIEEFLAGEELSFIA MVDGEHILPLAGSQDHKRRD
DGDRGPNTGGMGAYSPVPQLSDALQEKIMTTIMQPTVTALKSEGILYRGFLYAGIMI TLNNEPKVLEFNVRLGDPE
TQPLMMRLRSDLIELILSALSGRLNQTQSAWDSRAALTVVLAAGGYPAHYQKGDIIQ GLDQLSLPDVKVFHAGTQE
INHQVVTDGGRVLGVTALGKDLREAQQKAYQAAQLITWPNCYYRHDIGHRAIS
SEQID NO: 5
MCGIVGIIANGIVNQALYDALTILQHRGQDAAGIMTSDGERVFLRKSNGLVRDAIRE PHMLHLVGNMGIGHVRYP
TAGSESPAESQPFYVNSPYGLSLVHNGNLVNVKELTNDLIRSDLRHLNTTSDSEILL NVVAHELQHYGGVQLSPKQL
FKAMTKVYERVEGAFAAVMIITGYGVIGFRDPHAIRPLVYGRRDNGNGPEYMLASES IALDALGFELIDDVGPGEVI
YFDREGSVHRERCAKQVSHSPCIFEYIYLARPDSIIDGVPVYQARSGMGESLAQKIL RERPDHGIDVVIPIPDTSRNAA
QALARALDVPYSEGFVKNRYIGRTFIMPGQAKRRSSVRLKLNAIKAEFANKTVLLVD DSIVRGTTSKEIIQMARDVG
AKKVYFASAAPEVRYPNVYGIDMPTADELIAHNKSTEEVMHSIGADWLVYQNLEDVY QAINDAMGSRKPKIERFE
DSVFTGDYIAGNITKEYLAELAESRNDAAKMKKRALNEQEEANGLL
SEQID N0:6
MTNGPQPLYRRVLLKMSGEALMGKGLHAIDPNVLDRMAKDVTQVYQLGVQIAIVIGG GNFFRGAALQAAGINRI TGDYMGMLATLMNALALRDAFERSNLPVRILSAIPMTGVADAFHRRKAIHHLQQGRVVIF AAGTGNPLVTTDSAA SLRGIEINADVVLKATNVDGVYSDDPAKNPQAKLYKHLSYQEALKKELAVMDLAAFCQCR DYNMPLRVFNINKPG ALLSVI MNQE EGTLVDQGQ
SEQID N0:7
MPFILVLYYSRYGATAEMAEQVARGVERVNKIEARIRTVPSVSPKTEATEPDVPKDG PPYVTHDDLKNCVGLALGSP TRFGNMAAPLKYFLDTTSALWQSGSLIGKPAGFFTSTASLHGGQETTLLSMMMPLIHHGA IIVGVPYSETELFTTTA GGTPYGPSHMAGADSNWPLTQTEKNLCQALGKRLAEISLKLKA
SEQID NO: 8
MEWEPVIGLEVHVQLRTQSKIFSGAATAYGAEPNTQACAIDLGLPGVLPVLNKEAVKLAV CFGLSVNASIPPYSIFA
RKNYFYPDLPKGYQISQYNFPIVQNGHLDIENEDGTTKRIGITRAHLEEDAGKSFHE GMQGYSGIDFNRAGTPLLEIV
SEPDIRSAQEAVAYLKALHSLVRYIGVSDANMQEGAFRCDVNISLRPKSEEKFGTRA EIKNVNSFRFVERAILFEINRQ
KEILENGGTIVQETRLYDAVQDETRSMRTKEEAHDYRYFPDPDLLPVEIGPEFIEAV KNQLPELPWEKRKRFAASYQL
SNYDVKLLTTQIEIANYFETVLKIDKTIPPKLAANWINGDLAAALNKNNLSITQSPI NAEQLAGLLHRIADNTLSGSMG KQVFETMWGGEGDADTIIERHGLKQITDTEALEKIIDEVIENNPTQVEQYRSGKDKLIAF FVGQVMKATKGKANPQ QVNELFKKKL
SEQIDN0:9
MTFQKPCFPHCLPVYFPLLYHSNHKELRKMNDVLSVRAQQLEPSVTLAVSDLARELLNKG HDVISLSAGEPDFDTP
DFIKQSAIKAIQEGFTKYTNVDGTPALKAAIVHKLKRDNHLNYEPSEILVSGGAKQS IYNVLMGTLNAGDEAIIPAPY
WVSYPPMVQLAEAKPIIISATIDQNFKLTPGQLSQAITPQSRLLILNSPNNPSGVAY TESELKALADVLMEHPQILILS
DEIYEYILWGQNRFVNILNVCPELRDRTIIINGASKAYAMTGWRIGYAAGPKSIIQA MKKIQSQSTSSPNSIAQVAAT
TALGAQRGDFAYMYEAYKTRHDLVLKALNQMKGVHCIPADGAFYLFPDVSAAIQQLG LEDDIKLGTYLLDKTKVAV
VPGSAFGSPGHVRLSCATSTEKLQEALERLASVLDY
SEQID NO:10
MIVQPKVRGFICTTAHPEGCARHVGEWINYAKQEPSLTGGPQKVLIIGASTGFGLAS RIVAAFGAGAKTIGVFFERP
ASGKRTASPGWYNTAAFEKTALAAGLYAKSINGDAFSDEIKQQTIDLIQKDWQGGVD LVIYSIASPRRVHPRTGEIF
NSVLKPIGQTYHNKTVDVMTGEVSPVSIEPATEKEIRDTEAVMGGDDWALWINALFK YNCLAEGVKTVAFTYIGPE
LTHAVYRNGTIGRAKLHLEKTARELDTQLESALSGQALISVNKALVTQASAAIPVVP LYISLLYKIMKEKNIHEGCIEQ
MWRLFKERLYSNQNIPTDSEGRIRIDDWEMREDVQAEIKRLWESINTGNVETVSDIA GYREDFYKLFGFGLNGIDY
ERGVEIEKAIPSITVTPENPE
SEQID NO: 11
MTDTHLLFFEKAIAQNAIRPSLNKTYRMDETTCVNHLLKTIAFTPRLEAAVSRLAKELVT AVREQESEKGGIEGFMM
QYDLSTEEGILLMCLAEALLRVPDKETENLLIRDKLTSAEWNKYVGASESSFVNFAT WGLALSGKILKKEKDGQFKNV
WRNLVRRSGEPVIRKAVREAMKLMSEHFVLGRTIEEAVKRSQSAIKEGFRHSYDMLG EVARTQEDADRYYDSYHR
AISVLGKSHPTKSVHEAPGISVKLSALYPRYDFKKRELAVPFLIERVKELALHAKEQ KIGMTIDAEEADRLDISLDIFEAL
FTDEAFENWQGLGLAVQAYQKRAFYLIDWLIDLAQRQKRRIPVRLVKGAYWDTEIKL AQMEGLSGYPVFTRKVNT
DISYIACAQKMLNAQDAIYPQFATHNAYSVAAILNLMDHHYDNYEFEFQQLQGMGKA LHHYIVTKLKLPCRVYAP
VGYHEDLLPYLVRRLLENGANSSFVNRIADKTVPVDQLIESPVKKIEAFGDIPNPKI PLPKGIFKTRTNSSGIDLSNFAE
LMPLNEEIHHALEKEWEAAPFLQEIKNGKPVFDPTDNRRQIGVIELANESDVEKAIQ AGHSAFPNWDQKGISARAT
ILRKMADLLEKHKAELMAVVVREGGRTLQNALSEVREATDFCRYYAEQAEQHLSDKA LPGYTGESNTLRMNGRGII
LCISPWNFPIAIFTGQIAAALVTGNAVIAKPSGQTPLTGALVTRLFHEAGVPKEILQ LMPGSGKTVGQALIEDTKISGV
IFTGSDATARHIQKTLAARPGPIVPFVAETSGINAMIADSTALPEQLVNDVIVSAFD SAGQRCSALRILYIQEDIADDV
IKMLKGAMAEIKMGDPLLLSTDVGPVIDANAQKTLQKHQALMQKEAKLIYKVDLPRE TDFGTFVAPQAYELPNLGL
ITEEVFGPILHVIRYKRENLNKVIEEINGLGYGLTFGIQSRIDETVDYIQQRINAGN IYVNRNTVGAVVGVQPFGGSW
LSGTGPKAGGPHYLPRFCIESTLTINTTAAGGNASLMAMED
SEQID NO: 12
MENPIVIVSAARTPMGHYGGYFKEMPAPELGAAVIKAVVERAGLQPAEIDEVIMGCV LPAGQGQAPARQAALKA
GLPVSTPCTTINKMCGSGMKAIMLAHDEILADSYPHIIAGGMENMSRAPYLMMKARF GYRLGHDRIYDHMMLD
GLEDAYDKGKAMGVFAEKCVDKYQFTREALDKFAIESLLRAKKANENGSFAPEIVPI TITHQRETLTVDHDENAMK
ANPEKIPQLKPVFKADGAVTAANSSSISDGAAAVTLMRLSEAKRLNIQPLAKIIGHF TYAEDPSWFTTAPIGAIRGLLK
KISWKKEAVDLFEINEAFAAVTMAAMKEIGLAHNKVNIHGGACALGHPIGASGARIL VTLLYALQKNNLQRGIASLC
IGGGEATAIAIERGF SEQID NO: 13
MTVLYQNRQIRELERLAVESGISEYELMCRAGEAAFKALLARWPEAQEITVCCGKGN NGGDGLVLARLAYENGLKV
TVYLAGQRHQLKGAAAQAANACEASNLPILPFPEPLLFKGEVIVDALLGSGLSGEVK APYDHLIAAINQAGQYVLAL
DVPSGINVDSGEVQGTAVKANLTVTFIAPKRGLYTDKAPAYCGELIVDRLGLSESFF RAVFTDTRLLEWKGVFPLLPK
RARDAHKGSYGHVLVIGGDYGMGGAVRMAAEAAARVGAGLVTVATRPEHVPIVSGPR PELMCHQVAAADDLK
PLLTAATVVVIGPGLGKSDWAKSLLNKVLETDLPKVLDADSLNLLAESPSQREDWIL TPHPGEASRLLGISCNEVQRD
RFQAINDLQEKYQGVLVLKGVGTLIKDESQAYYVCPAGNPGMATGGMGDILSGIIGG LVAQRLSLASAAQAGVFIH
SMAADRAAEEGGERGLLATDLFPHLRVLVNP
SEQID NO: 14
MLIGVPKEVKIEEYRVGLTPYSVRELVLHGHQVIMERDAGNAINFTDEAYLAAGAKI VDTPVEVYQAEMIVKVKEP
QSSEYALIREGQILFTYLHLAPDPQQAQALIKSGCIAIAYETVTDNEGGLPLLSPMS QVAGRLAIQAGAHCLEKPEGG
SGILLGGVPGVYAGKVTVIGGGVVGSNAVRMAMGKKAQVTVLDKSLRRLQELDFQFG GRLNTAYSTESSIEHYVID
ADLVVGAVLVPGHSAPKLVGQDVLKKMRPGSVMVDVAIDQGGCFETSKPTTHKKPTY VIDGIVHYCVANMPGAV
PRTSTLALNNATLPYVIALADKGYRQAFLDDPHFLNGLNVYCGQITHKGVAQGLQQE FNPPLALL
SEQID NO: 15
MNKPQHHSLIILGSGPAGYTAAIYAARANLKPIMITGMEQGGQLMTTTDVDNWPGEA PGLQGPQLMERMQKH AERLDTQFIFDHINEADLNQRPFLLKGDNATYSCDALIIATGASARYLGLPSEKAYMGKG VSACATCDGFFYRGKKV AVVGGGNTAVEEALYLSHIASHVTLIHRRDKLRAEKMLSAQLIKKVEEGKVAIVWSHVIE EVLGDDQGVTGVHLKH VKEEKTQDLTIDGLFIAIGHDPNTKIFKEQLEMDEAGYLRAKSGLQGNATATNIPGVFAA GDVTDHVYRQAITAAG MGCMAALDAERYLDSLNQA
SEQID NO: 16
MLKIVSKPSFNRIALMGREGVEGVPETLAALKDYLVSLNREVILEENAAHMIDGSRL LTVPANDLKKKADLLIVVGG DGSLLNAAHIAVPQQLPVLGINRGRLGFLTDIPPNELTQISDILDGHYREEVRFLLEGTV EEGDEIVAQGIALNDIVLLP GNAPKMIEFDIFINDEFVCNQRADGLIITTPTGSTAYALSGGGPILHPQLNAMALVPMFP HTLSSRPIVVDAESQIKI TISPENDVSPYVSNDGQERVSIKPGGNVYTRKYHYPLHLIHPTDYNYYDTLRRKLDWEKR AAKV
SEQID NO: 17
MNLHEYQSKHLLKKYNIPVPASEVVFNPDAAVDAAAKIGGDRWVVKAQVHAGGRGKA GGVRLVKNKEELKSAVK
ALLGTRLVTYQTDERGQPVNQILVEQTSDIARELYLGAVIDRASQRIVFMASTEGGV EIEKVAEKSPEKILKVTIDPAI
GLQPFQCRQLFFGLGLQDLKQMRSFTDIVMGLYRLFTERDLSLLEINPLVITGSGEL ICLDAKINIDDSALYRQSELRE
MRDTTQEDEHETMAQQWELNYIKLDGNIGCMVNGAGLAMATMDLIKLSGGDPANFLD VGGSATKERVTEAFKI
IVSDKNVKGILVNIFGGIVRCDLIADGIISAVKEVGIDVPVVVRLEGNNAQLGAKKL ADSGMNIIAAKGFADAAEQIV
KQVGVIA
SEQID NO: 18
MNSKKSRIMTFSIMRFNPETDKKPYMQDFELDVSAIQGKMLLNALEALREKHPDIGLRRS CAEGVCGSDGMNING KNALACVTQLKDLPDRVVVRPLPGFPIIRDLIVDMEQFYAQYKKVKPYLLNDQEAPQKER LQSPEERAKLDGLYECIL CACCSSSCPSYWWNPDKFIGPAGLLWSYRFIADSRDSKEKERLDAMKDPYSVFRCRTIMD CATVCPKNLNPAKAIR KIRTEMLQETESGE SEQID NO: 19
MSSIRVKQYDALIVGAGGAGLRAALEMAQSRQYKVAVVSKVFPTRSHTVSAQGGIAA ALGNVVPDKPIWHMFDT
VKGSDYLGDQDAIQYMCEQAPPSVYELEHYGLPFSRLDDGRIYQRAFGGHTRDFGKE MARRTCACADRTGHAML
HTLYQKNVEAGTHFYYEWYGIDLVRGAQGGIAGMIAMNMETSELVFFKSRATIFATG GAGRIYETTSNAYTNTGD
GIGMVLRAGLPVQDMEFWQFHPTGIYGVGCLITEGARGEGGYLINKDGERFMERYSP HLKDLDCRDVVARSILQE
VMAGGGVGPKKDHVLLKLDHLGEKVLRERLPGIIELSEKFANVDITKEPIPILPTCH YMMGGIPTNIHGQALTVDEN
GKDQIIEGLFAAGECACVSVHGANRLGTNSLLDLVVFGRAIGLHLEEALKTELKHRS ENPDDIDAAIARLKRWEKPN
NVENPALLRQEMRKAMSEDFGVFREEQKMKQGLERLQKLNERLQRAKLTDTSRTFNN ARIEALELDNLMEVSYAT
AVSAQQRTESRGAHSRYDYKERDDANWLKHTVYFRDGHIAYRPVNMKPKGMDPFPPK SRD
SEQID NO: 20
MAGCGLTDFCRTFECVKLKRKIGCEVTMADSLKTRRELTAGGKTYHYHSLKAAEDAGLSN IHRLPYSLKILLENQLRH
EDGETVTQTHIEAFAHWLKDKHSDREIAYRPARVLMQDFTGVPAVVDLAAMRDAMAR MKGDPTKINPHCPVDL
IIDHSVQVDEFGNEEAFRDNVRIEMERNHERYTFLKWGQQAFRHFQLVPPGTGICHQ VNLEYLGRGVWSSQQDG
EWLAYPDTLVGTDSHTTMINGLGVLGWGVGGIEAEAAMLGQPISMLIPEVIGFYLSG QLCEGITATDLVLTVTQML
RQKGVVGKFVEFYGPGLAELPLADRATIGNMAPEYGATCGLFPIDAETIKYLELTGR DAEAIELVKAYSKAQGTWHD
ENTPEPIFSDTLSLDLSTVEPSLAGPKRPQDRVPLAKLKKTIEGVIATAERDQELDH SFQSTGDFDLHHGDVVIAAITS
CTNTSNPSVMLAAGLLAKNAVEKGLQRKPWVKSSLAPGSKVVTDYLHKTGLIDYLEK IGFYLVGYGCTTCIGNSGPL
PETVAKTVTENDLIVSSVLSGNRNFEGRIHPLVKTNWLASPPLVVAFALAGTTRIDL TKDPLGHNDRGEPIFLNDIWP
SNAEIAKTVMQVRNDMFRKEYADVFEGDEEWQRIHVSAGDTFSWQTNSTYVKNPPFF ENMSAKPEPLKNIIDARI
LAILGDSVTTDHISPAGAIKADSPAGKYLIEHGIDIKDFNSYGSRRGNHEVLMRGTF ANIRIRNEMLSKVEGGFTKHF
PDGEQLPIYDAAMKYHSENIPLVVIAGKEYGTGSSRDWAAKGPRLLGVKAVVAESFE RIHRSNLVGMGVLPLEFKN
DDNRHSLKLEGNEVIDITGLENDLQPGGDVIMTVKRKDGTIEKIPLHCRIDTQNELA YYQHGGILQFVLRQMLRSS
SEQID NO: 21
MKVFKLPDLGEGLPDATIREWYIAVGDEVKIDQPLVAMETAKALVDVPSPLAGKIEK LFGEVGDVIETGSPLIGFEGE
AETEEPKDTGTVVGAIETSDTVLEESGAGIPVKKAAEKKNFKATPAVRMLAKQLGVD LTKITPKSSLISAEEVKQAAQ
ITKTGKTQKIEGELTPLSPVRRAMAQSMSQSHREVVPVSLMDDGDLSAWKGEQDITL RIIRAIEAACQAVPIMNAH
FDGETLGYKLNETINIGIAVDTPQGLYVPVLKDVSHQDDTALRNQINRFKELAQSRS FPPEDLRDATIMLSNFGAFA
GRYANPILLPPMVTIIGVGRTRDEIVPVDGKPAVHRILPLSVTSDHRVITGGEIARF LKQLIDSLEKAS
SEQID NO: 22
MTPKTTTVANFTIRYLQFLDANSNPTQPFPDFADPDMLLYLYRRMALIRQLDNKAIN LQRTGKMGTYPSSRGQEA
VGIGMGSAMQKEDIFCPYYRDQGALFEHGIKLSEILAYWGGDERGSRYANPDVKDDF PNCVPIAGQLLHAAGVAY
AVKYRKQARAVLTICGDGGTSKGDFYEAINLAGCWQLPLVFIINNNQWAISVARGEQ THCQTLAQKAIAGGFEGW
QVDGNDVIAVRYAVSKALEKARDGGGPTLIEALSYRLCDHTTADDATRYIPQEEWKV AWQKEPIARLGYYLESQGL
WSREKEAVLQKELAQEVDQVVEEFLTMPPPKATDMFDYLYAELPVSLEKQREELADN KPSHPSGREG
SEQID NO: 23
MKRILITGANRGIGLELVKQYLAAGWHVDGCYRDKKASNSLFELAAEKKQSLTLHEL DVLDEKAIQALGEHLKNQPI DILFNNAGVSAKNLREFGSIHDTENACEVFKINTIAPLLMVQALLESVEKSEKKLIINMS SEMGSIAQNVNGNYYVYR ASKSALNAITKSLAIDLKRRGITVISMNPGWVRTDMGGEQAPLDVISSVRGMREVIERVD IKSTGGFLGYDGGEMP W
SEQID NO: 24
MRTLQLREGNMTNLIADRLAALRRLMHEIGVDYYYVPSSDPHKNEYVPSCWQRRAWI SGFTGSAGDVVVGIDKA
FLWTDPRYFLQAEQQLDDSLYHLMKMGQGETPAIDQWLTQQRNGIVFAVDPRLINLQ QSEKIQRALEKQNGKLL
ALDENLIDRVWKDQPPLPQSAIQLQPLQYAGLSAEDKLAALRQTLQKESADAIVLNT LDAIAWLFNIRGNDVAYNP
LVISYAVITQNEASLFVDPHKITEGDRSYFKKIPVHIEPYEGIGKLLESLSGSVWLD PGATNLWLRDQLKNTASLILKPS
PITLAKALKNPVEQKGAREAHIIDAIAMIQFLHWLENHWQSGVSEISAAEKLEFFRR GDSRCLDLSFPSISGFGPHGA
IVHYSATTDTDATINDSAPYLIDSGGQYHYGTTDITRTIHLGTPTEEEKRLYTLVLK GHLAIRQAVFPKGTCGEHLNAL
AHQFLWREALDYGHGTGHGVGSYLCVHEGPQAITSRYTGIPLQPGMIVSNEPGVYLT HKYGIRIENLCLVTEKFTVD
DSLTGDGPFYSFEDLTLVPYCRKLINPNLLTSEEIQQINDYHQRVDQTLRDLLPANE LNDWLHEATAPL
SEQID NO: 25
MRIDKFTTAFQTALADAQSLAVGRDHQFIEPAHVMKVLLEQTQGTVAPLLEQSKVNL SRLIDGVNKAIDSYPQVEG
TGGEVHVSRELSKILTLMDKFAQQNKDQYISSEWFIPAALEAKGQLRDVLIEAGADK KAIEKNIMNLRKGERVTEQS
AEDQRQALAKYTIDLTEKAETGKLDPVIGRDEEIRRTVQVLQRRTKNNPVLIGEPGV GKTAIVEGLAQRIVNGEVPE
GLKQKRLLALDMGALIAGAKFRGEFEERLKAVLKDIAKEEGRVILFIDELHTMVGAG KAEGAMDAGNMLKPALAR
GELHCVGATTLDEYRKYIEKDAALERRFQKVLVEEPSTEDAIAILRGLKERYEVHHG VEITDPAIIAAATLSQRYITDRN
LPDKAIDLIDEAASQIRMEMDSKPVELDRLERRLIQLKIEREALKKETDEASKKRLS DLETEIKNVEKEYSDLEEVWKSE
KASLHGTQQIKEELEQARIELEAAGRAGDLARMSELQYGIIPELDKKLKAASQKEEQ FHDHKLLRSRVTEEEVAEVVS
KWTHIPVSKMLEGEREKLLHMETELHKRVIGQDEAVNAVANAIRRSRAGLSDPNRPV GSFLFLGPTGVGKTELCKA
LAVFLFDTEDAMVRIDMSEFMEKHSVARLIGAPPGYVGYEEGGYLTEAIRRRPYSVI LLDEIEKAHNDVFNVLLQVLD
DGRLTDGQGRTVDFRNTVIVMTSNLGSDLIREFSGENYDKMKDAVMEVVAQHFRPEF INRIDEAVVFHSLKKEQIR
NIAIIQIDRIKKRLKEKDYQLTISDDALDYLSELGYDPVYGARPLKRVLQQQLENPL SQKILEGKFVPGSLINIEKKGEQL
EFKEA
SEQID NO: 26
MGLEAFCLSSLQCQISFETAEPKMSNQKPRTVYLKDYRPSDFLVDTVHLYFDLHEEE THVKTILNLQRNPEGNATAP
LALTGEAMTLKKVALDGQTLASSDYTLDASSLTIANVPNEFTLETEVVIKPQENTQL MGLYKSRGNFCTQCESHGFR
RITYFLDRPDVMARYTTTITADKNKYPFLLSNGNLIETKILSDNRHWAHWEDPSKKP CYLFALVAGDFDLLEDTFVT
QSGREIALRLYLEKGFKDQGPFSLAALKKAMRWDEKRFGREYDLDIYMIVAVSDFNM GAMENKGLNIFNTKYILAN
PQSATDDNYVAIESVIGHEYFHNWSGNRVTCRDWFQITLKEGLTVFREQLFTEDTTS KGVARIGTVNILRNSQFPED
AGPMAHPIRPRSYIEVNNFYTTTVYNKGSEVIRMVQTLLGEALFRKAMDLYFSRYDG QAVTTENFIQAMEDASGK
NLEQFKRWYDQAGTPVLDLNSEYNANDKTLTLTVKQSCPPTPGQSEKLPFHLPLTLG FVGPECQDMPTQLAGEKK
AIPGTRVLEIKDAETEFKFVNVNHKPTLSLLRGFSAPVRLNYPYSDEELVWLFQCDS DPFARYEAGQIFAQRLIFKLID
DSYQGKPLKIDERFIDAHRKIIAGPHRDHWYEAALLQLPSINYLMQLMKKMDVEALH TIRQFVKKALSNALVDDLKI
QYEHHQLPLYEYTPADIGKRKLKNICLAYLTESDDTQFRQVAYQQFKKSDNMTDTVG ALSALLNHDCKERHQALDE
FYQQWKDQPLVVNKWLMLHASSTLPSTLEAVRKLTKHPAFDVKNPNNVYSLLGTFGA NAVCFHEGSGEGYRLIAD
YVLAIDPANPQVAARVLQPLTRWQMMDKKRQELMKAELNRIAKAERLSSDVYEIVTK SLL
SEQID NO: 27
MNSMIKNLLLWLVIAVVLITVFSNFGSRQSDVQPYSYSQFVQAVNNDKVSSVVIQGH EIKGVTKDNKHFTTYLPME DQALLNQLMAKGVSVKGEPPKQQSMFLHILISWLPFLILIFVWILFMRQMQGGGRGGGPM SFGRSKARLLSQDQ VKVTFDDVAGVDEAKEEVKELVEFLRDPGKFQRLGGKMPCGVLLVGPPGTGKTLLAKAVA GEAKVPFFTISGSDFV
EMFVGVGASRVRDMFDQAKKQAPCIIFIDEIDAVGRHRGAGLGGGHDEREQTLNQLL VEMDGFEGKEGIIVMAA
TNRPDVLDPALLRPGRFDRQVVVPLPDIKGREYILKVHMNKLPLAKDVKASVIARGT PGFSGADLANIVNEAALFAA
RENKKDVSMSEFERAKDKIMMGAERRSMVMSDDEKKLTAYHEAGHAIVGLHMLEHDP VYKVTIIPRGRALGVTM
FLPEHDRYSMTKRRLECQLAGLFGGRIAEEIIFGPDLVTTGASNDIEKATEIARNMV TKWGLSQKLGPLTYREEEGEV
FLGRSVTQRKDISDATNKEIDSEVRRIVDTAYTTAKQTLEEHIEQLHLMAKALIKYE TIGEAQIKEILAGKEPSPPPDW
KEENGSASAHKENSEKELSEEKGEEKTVNPSRPRPAEDG
SEQID NO: 28
MKKLVSSLLASISLFLISAAAWADNLPTDFTDNTAMNTHHDLSVTYLSQVFGTVGNV LHGMSGQMLGHLFYRLNE
GIIVVAGMWLVYTVFTIVLRAAQDGSFMGPNKNVALVFLKIAFGFSLLVPNPATGYS LLQDVVMKVVVEGVGLAD
QTWEYGLTYINNGGSLWRRPETNGAGKDIISQSTVNSVLGGNSQNKEGPGQKIFASA VCMYSSDDNQSPLKSNN
NNIGPAVNGGPTVKYTYDVITDDSAHQFEFPGSGDTPPFKPGDDSCGAVTWDINNAC TGAGSNSTKCTMAKEAV
SELVTSLLPAAKKYYCSQHSSSDLCLGVTHNDAFAENETSFFGALLNYVNTIVPLVQ FNSGKSADEAKRFIDEAQNEG
WLSAGRYYWDLSQIQSHYDNVSNVDSYYPRTVDPTVNGNPEDDYQAALKQSLGYIYG VIDTANPHPIPVKGSVLY
QLAQYAQSQHSGDTGGGEENWGHGGLDAGIALIGGIFSETIYDIYKLIHTFTTGSDG AMGPDPILFLHKIGIRAISVA
ADIWFGFLGIMAIALFATGVCTATYNAQTPVQALLGWIKPLLMVVAVGLWGTGFVLA YYVPLYPYMLYTFGVIGW
IIVVIEAMVAAPLIAFGLTHPEGHDFLGEAKQGGMLLLGVFLRPVLMVVGLIAGMIL SYVALRIVVYTFSGLAVDLFA
NTPSSGPASGSILHAATALMSNSMATAGSVTGAIVSLMVFPLVLIIFTILVYVVTTQ SFSLIFALPDNVMRWIGIPGQ
RSEYDRMATQLESKVGGFASSTGRSGGLQASERIGKGAANANLGKQLHLGPSKK
SEQID NO: 29
MKNFRVLGIASFLALGVASTSALADIDPMSGVIKAIKEVGLEVQALAIASKKSVSNM KYQLDKNLDLALQADVEKNN
ALQTVKNNAGTNTQNQISGTLLQFPEQVINASQLNDAQMAATIKNRKNLIPNLTTAI PASDTLYLTDAEDPLANTY
GVAKPDSLYDNYFNFDSLFAPSAYNSDQQQAATTYLQYLTKPYQSLTDNIHFSELKD NLNKLSAEKRADKLKSFLNN
PAYQKFQLAVRSLIATKSLAIDNFNTLLNERVPVKGLGAKVGMPDDPHLPKGYASPL QVENYIANQRINSPDWFKQ
MKTASPAVVAREQVLILAEIESQLERNHLDNERLLATLSLMALQGTKNSEMELQTNT AADLNKLIDQIGK
SEQID NO: 30
MKITDAKVFVCSPGRNFVTVKIYTDEGIYGLGDGTLNGRELAVASYLEDHLLPCLIGKDP SQIEDIWQYFYKGAYWR
RGPVTMSAIGAIDMALWDIKGKALKTPVYNLLGGRSRKGVMVYGHANGKDVEETVDE VGKYIEKGYLAIRAQTGV
PGLPSTYGVSPDKLFYEPAEKGLPPENVWSTEKYLNHVPKLFKKLRDVYGDDPHLLH DCHHRLTPIEAGRLGKELEP
YHLFWLEDTVPAELQEGFRIIRNHTTTPLAVGEVFNVIYDCTTLITEQLIDYIRMSI VHGGGLTPMMKIASFADIYHVR
TGCHGPTDVSPVTMAAALHFETAINNFGIQEFMRHTPETDEVFPHHYYFENGYLNVK DEPGLGVDFDEKLAAKYP
YERAYLPINRKLDGTMYNW
SEQID NO: 31
MRIITLNLNGIRAAARRGFFDWLKRQKADIVCLQETKACLEITNGDQFHPKGYHCYY HDAEKSGYSGVGIYCREKPD RVTTRLGWEHADKEGRYIQADFGSLSVASLYMPSGTTGEHRQKIKFDFMDRYMKRLKNIV HSKRSFIICGDWNIVH KEIDIKNFKSNQKYSGCLPEERAWLDEVFTKVGLVDAFRVVNQKPDQYTWWSSRGRAWEK NVGWRIDYQVITSD LKNSVKSERIYKDKRFSDHAPLIIDYEREISD SEQID NO: 32
MESLTPKRDAFTVLSYNIHKGFSARYRRFVLPDIREALRAIDADIVLLQEVQGKHHK SRLKKFAHADLPQTEFIAESK WPHYMYGKNAVYGSAHHGNALLSNFPFKMVENINVSLSQRASRSILHAIIDYEPTVELHV ICIHLGLFRAERDYQLIT LSKRIEAHVPSHAPLIIAGDFNDWRRGAFNYMEKELELKEVYKVLEGKHAKTYPASRPTL EVDRIYYRGLKLLSGEIFN ESYWKKLSDHLPLHAKFAIE
SEQID NO: 33
MPKHFYFYFLRKMTMSQNKIYVGSLSYDVTADELQSFFGQYGEIEEAKLIMDRETGR SKGFAFITYGTQDAAQEAV SKANGIDLQGRKIRVNIARENTGDRRRDGGSGGRGGRGGRF
SEQID NO: 34
MDFSDDNLIWLDLEMTGLDPERDRIIEIATIVTNSHLDILAEGPAFAIHQPDKLLTA MDNWNTSHHTASGLLERVK NSSVDEVEAETLTLAFLEKYVSAGKSPLCGNSVCQDRRFLSRYMPRLNQFFHYRHLDVTT LKILAQRWAPQIAAAHI KESQHLALQDIRDSIEELRYYRAHLLNL.SK
SEQID NO: 35
MMFELFKEIFMKKIIQLISAVLITSLVFSAQAKPASEVIKNKLHRHAAVSTQKTGPV DINTADATLLTTLKGIGVKKAK AIIAYRKKEGNFKSIEALSSVPGISQKTVARLIRNNPHRLVVNP
SEQID NO: 36
MFYNGRICLALNPEEGPMKKILFLATLLLILSGCVRKDVDPYQAYRGKTSAELFTSG ERALAKKDYSEAVKNFEALDAI YPFGPHAEQAQLDIIYAYYKNNDTSSAIAAADRYIRLYPRGRNVDYAYYMRGVISFDLGL SWLQKLARVSPVSRDVS TLQQSFTSFATLAEVFPHSRYTPDALTRMRYIRNLMAQREIMIAEFYMKRRAYVAAANRG SYVVQHFQGSPQVAK ALAIMVQAYRALGLPKMADASNHLLQTNYPHTLEARKLRKA
SEQID NO: 37
MNLTDLKQKSVPELMQIAQEMNLEYVSRTRKQDIIFAVLKAHAKKGEDIFGDGVLEI LQDGFGFLRSADSSYLAGPD
DIYVSPSQIRRFNLRTGDTVSGKIRPPKESERYFALLQVNEINLEKPEASKGKILFE NLTPLFPNEQIRMETGNGSTEDI
TARIIDLISPIGKGQRGLIVSPPKAGKTMMLQNIAHSITTNHPECVLIVLLIDERPE EVTEMDRSVKGEVVASTFDEPA
SRHVQVAEMVIEKAKRLVEHKKDVVILLDSITRLARAYNTVIPASGKVLTGGVDANA LQRPKRFFGAARNVEEGGSL
TIIATALVETGSKMDDVIYEEFKGTGNMEIHLDRRIAEKRTFPAININRSGTRREEL MMPQDVLQKVWILRKILHPM
DELAASEFLIDRLKLTKTNNDFFDSMKG
SEQID NO: 38
MLEIVLASQNSSKLAEMQELLRDLEIKFIPQTEFSVPDIEETGSTFVENAIIKARHAAKQ TGLPALADDSGLTIAALNSA PGVFSSRYAGKNATDAERIQKVLEALEAADDSDRSASFHCVIALMENENDPAPLICHGVW EGEIAREPRGKNGFGY DPIFYVPSHQRTAAELDPQEKNAISHRGQALEQLSTVLTEAFLV
SEQID NO: 39
MCNNDFMNQATEIAKLLLNIKAVTLNLHEPYRYTSGILSPIYCDNRLIISYPEKRKMIIE AFLQLIEKNHLSFDIVAGTAT
AGIPHAAWIADRLDLPMIYVRAKAKTHGKQNQIEGRIRKGQRALIVEDLISTGKSAL AAGLALREKGVTVTDCIAIFS
YQLPQAQQNFSDANINCHALSHFDTLIEMAVDEGYIDEIEKQKALAWNKDPEHWQP SEQIDNO:40
MEKPDPKVIVAIDAGTVEQARAQINPLTPELCHLKIGSILFTRYGPAFVEELMQKGYRIF LDLKFYDIPQTVAGACRA VAELGVWMMNIHISGGRTMMETVVNALQSITLKEKPLLIGVTILTSLDGSDLKTLGIQEK VPDIVCRMATLAKSAGL DGVVCSAQEAALLRKQFDRNFLLVTPGIRLETDEKGDQKRVMTPRAAIQAGSDYLVIGRP ITQSTDPLKALEAIDKDI KTR
SEQIDN0:41
MYSIISCIPLRSIRATPILLKHDDLGSRMLFLQGSHVYTPFRHQQILFRLKQKQNTV RSVEAIYGYFVDGEKLLSRAEQE
RLERLLPKAYFSDYPKSAENFSVWVTPRLGTISPWSSKATDIAHNCEIPINRIERGI YFIIDGIAKRDKKAIEKVASELYD
PLTESLLFDAEDLAQLFQHPAPKTFNDIPVLGKGEAALKEADQNLGLALSDPDIHYL LRAFHQLNRNPTDIELMMFA
QVNSEHCRHKIFNAQWTIDGKEKKESLFDMIRYTYKTHPEKILVAYKDNAAVIEGFN CESFLINPSNHSYEKQKGRL
HTVLKVETHNHPTAIAPFAGAATGSGGEIRDEAATGRGAQSLAGLAGFSVSHLRIPD FLQPWEKAPSKKSLHSDSKP
KTLASALDIMLQGPIGAASFNNEFGRPTICGYFRTLEHLSSKTLKWGYHKPIMIAGG IGHIRESQIEKQSFTEGALLVV
LGGPAMAIGLGGGSASSRTSGESTEALDFASVQRANPEMQRRAQEVINACLSLGDDN PILSLHDVGAGGLSNAFP
ELVHATECGGEFELRHIPNAEPGMSPLEIWCNEAQERFVLAIKPESLKVFSGIAERE RCPFAVVGRAKEEKKLILNDA
HFHNRPIDLPLSFLFEDMPPMKREDKRVFSGETAWNISKINWADAVKRVLQYPCVAD KSFLITIGDRTVGGMVAR
DQMVGPWQIPVADVAVTAHSFTGYEGQALAMGERSPIAIVHPAASARMAVGEAITNI AAAPIKAISDIVLSANW
MAAPDQPGEGAGLYEAVQTVAKELCPALGICIPVGKDSLSMQTSLEKEIVTAPLSLI ITATAPVSDVRHALTPQLQTD
VGETRLLLIDLGQGANFLGGSCLAQTYNLLGKQPPDVDDPLLLRRFFEAIQSLNQKN LLLAYHDRSDGGLLATLCEM
AFTAHVGITIKLDSLGDDALASVFNEELGAVIQVKEKNIDIVFEILKSHKLQAHSHV IGELNQLDEIIFNFRGQTLYQET
RTTLQRWWSETSYRLQSLRDNPECAKQQYDGLLDKKDTGLFTKITFDNNEDIALPYI NSGKRPRVAILREQGTNGH
REMAAAFHLAGFESVDVHMSDLLNERVNLMDFKGAVAGGGFSYGDVLGAGRGWAQVI LMHPKIRDKFSLFFES
KDRFALGVCNGCQLFSHLKSLIPGALHWPAFQRNVSEQFEARLSMVEIPQSPSLFFQ GMAGSQLPVAVAHGEGRV
VFEKNTQEFENEKLIALRYVNYAGQPTENYPANPNGSPKGITGLTTPDGRITILMPH PERVFRTVQFSWHPKQWSE
MSPWMRIFKNARKWVG
SEQIDNO:42
MTACVFCKIAKGEIGELIYEDKQVVAFNDAAPQAPIHILVIPHRHIETINDVTPGDE DLLGHMVVVATRLAHDKNM AADGYRLVMNCNRNGGQAVFHIHLHLLGGRQMHWPPG
SEQIDNO:43
MPNVDDIRIFHGSANPSLAENVAKELNTTIGNALISRFSDGEIRFEIEENVRGRDIY LIQSTGHPTNEHVMELILMGD AFRRASAASITAVVPYFGYARQDRRVRSSRVPISAKVVADMMQKVGFSRLITVDLHADQI QGFFYMPVDNIYASIT ALEEYRLLDKLETPMIVSPDVGGVVRARAIAKRLNDSDLAIIDKRRPAPNQAEVMNVIGN VQNRHCVIVDDIVDTA GTLCHAASALKEKGALTVSSYCTHPVLSGNAVKNIMDSDIDELIVTDTIPLHEEAAKCRK ITQISLSRLIAETISRINQKE SVSSMFLD
SEQIDNO:44
MARRKAAPKRETLPDPLFHSELLAKFINAVMRNGKKSVAEKIVYGALDVVAKRVQNK SGEQGDGDGESGGKAGGI KKRSLGDIRTDENARALALETFKGALDKVMPNVEVKSRRVGGSTYQVPVEIRMARRQALA RRWLVEYANKRNEKT MVLRLAHEILDAVEGRGGAIKKREDVHRMAKANQAFAHYKW SEQIDNO:45
MNDLNSDGLFLFHFQAHLRWTRLALACPHQFRIKYPTLTNTGTHMVVIRLARGGSKK NPFYHIVVADRRKPRDGR FIERVGYYNPMARGQDIRLQLEKERISHWLNQGAQTSLRVKHLIKKLEKSPEEAQKGGMR KGEFKRLQAEQAAKA QKKAVATEEPKAEEAKEAPPAESQAAEGKEE
SEQIDNO:46
MEKTYDPKAIEKKWADYWEKRQLSKPTAQGSPYCIMLPPPNVTGTLHMGHGFQQTLM DTLIRYHRMKGERTLW
QGGTDHAGIATQMVVEQQLAQEDLTREDLGRQAFIKRVWEWRERSGGKITHQMRRLG VSIDWSRERFSMDEG
LSRATTEAFIRLHHEGLIYRGKRLVNWDPKLNTAISDLEVVTEEVEGHLWHIRYPLA EGSGHLIIATTRPETLLGDVAI
AVHPQDERYQPFVGKKVRLPLTDRTIPVIADEAVDKEFGTGSLKITPGHDFNDYEIG QRHQLPLINILTSEGYLNENV
PEPYRGLERFEARKKIIADLQRENLLEKTEPYRVPVPRGERSGVIIEPLLTDQWFIK MEALAKPAMEAVESGELKFIPK
NWEKTYLQWLSNIQDWCISRQLWWGHRLPVWYDEEKNSYVGRSREEILKKYHLSPDV KLQQETDVLDTWFSASL
WPFATLGWPEKTESFKTFYPTQVLVTGFDIIFFWVARMVMMGLKLTHKIPFHSVYIH GLIRDSQGRKMSKSKGNVI
DPIDIIDGISLDALIEKRTHALLQPKMAKTIEKMTRKEFPNGIASFGTDALRFTFCA LASRGRDINFDMGRIDGYRNFC
NKIWNAARFVTMNTQEKDLNPEKPLSYSAADEWIRTRLQQTIKNAEEALSQYRFDLL AQTLYEFTWNEYCDWYVE
FAKCILYDKQAKPAQLRGTRVALLEVLEILLRLLHPVMPFITEEIWQTVAPLAGKEG KSIMVEHWPQFNIHEMNYDA
KVEIEWVKNVITAIRTLRAEIGISPAKRIPVIFGKGDEKDKKRIAKMKSYIKTLGKV SQLRFAKHDDCFSATATGIVERL
EIHIPLAGVIDKQTEIARLKKEISKLQKEEEKSLKKLDNPNYLQRAPQEVVEKERLS LEKTQNALKKLQSQYASIESL
SEQIDNO:47
MSLASAETAKIVKEYQLGKDDTGSPEVQVAILTAKIIKLTDHMKAHKHDHHSRRGLL RMVSQRRKLLNFLKRNDLQ RYLKLIERLGLRS
SEQIDNO:48
MRLIDEKGEQVGVVRTDRALTMAEEAGLDLVEISPTAKPPVCRIMNFGKYQFEQSKRKAA QKKKQRLVHLKEVKF RPGTDVGDYQVKLRKIATFLDRGDKVKVSLRFRGREMQHRELGLELLGRVKRDLGNIVVE QEPRLEGRQMTMVV MKAKGEGNKTKREDHAEIKD
SEQIDNO:49
MINDIINDSKSRMEKSLGSLKTELAKLRTCRAHPSLLEHIKVDYYNVETPLSQVASI AIENPRTLSITPWEKNMVGPIE KAIQKADLGLNPATVGMVIRVPLPPLTEERRKELARVVREEAEHARVAIRNIRREANNDL KELMKEKEISEDEERRA QTAIQKLTDAQIAEVDKMASQKEADLMAV
SEQID NO: 50
MALLKSRDIDKIANLSKLIIPKNENDALLEALNKTFDLVIKMDKVDTSAVDPLAHPY NETQPLREDHVTESNQRDLFQ KSAPQVEAGLYMVPVVIDNEG
SEQID NO: 51
MTESLKNRIQEDMKAAMRAQEKGRLGTIRLLLAAIKQREIDEQITLDDAGVMKVIEK MIKQRRDSITQYEAGNRPD LAEKEKQEIDVLQAYLPEALSDAEIDIAVKQAIEETGATSMKDMGQLMGVLKGKLQGRVD MSMVSKKVKEHLS SEQID NO: 52
MSGGVKLIAG LGNPGDQYARTRHNVGAWFLETLAQQRNQSLAKENKFHGFVAKCNDYWLLKPTTFMNESG QA VAALAHFYKIKPSEILIAHDELDFPAGDIRLKEGGGHGGHNGLRNIIQHLGSSDFYRLRI GINHPGHKDRVTPYVLSPP SENDRIAILAAIEKGLRLIPELVQGDFQKVMRELHS
SEQID NO: 53
MKVNFTKMQGSGNDFVVIDATKTPFQLTTSQIQKMANRRFGVGFDQLLVIEPPKNNS VDFHFRIFNADGSEVGQ CGNGARCIARFIRAHQLSDREELRVSTLNEVLELKIQPDGKVSVKMGVPRFEPTEIPFIA SGVANFYDIAVDNQIVKL GVVNIGNPHAIIPVERINAEEVGKLGARLSVHECFPEGANVGFMQVIDPQNIRLRVYERG TGETLACGSNACAAVA VGRRCG LLQERVVVSQPGGSLTI DWQG PLTPVTMTG PATTVFCG EWLD
SEQID NO: 54
MNQTDIIIIGAGLVGTSVAVALQGHGIKIKILEHHLPSAAVTSSNDVRPLTLSFGSY QILKNLGVEADLANEACPISTV
HVSDQGALGALRFRASEFNVPALGYVVSFAKLQQSLYQRAALQKNAEIVPISTIDDI QCNTNHAQVTFSTINGQQQ
LQADLLIAADGTHSTARRLLKIPVEEENRNEVALIALLRLKQPHNHIAYERFTSQGT LALLPLFQANQCRLVWTLPKT
KADEIEQLSDDEFRAVLHRVFKPYIGAIQSVERGKRFPLQMLIAQEQVRPSFVMLGN ASHTLYPIAAQGFNLGLRDA
AVLSEVLIDARRQLKPLGDIRFLQEYSRWRKTDQARITGLTRGLSQWFGVQLPLANQ ARGLGLLATGLLPPFKKRLA
KRLMGLSGRLPQLMRGLKLDDAI
SEQID NO: 55
MSEHVHTASDENFETEVLQADMPVLVDFWAEWCQPCKMISPVVEEIAKEYAGRVKVF KLNVDENAQTPTKYGV RGIPSLLIFREGEVVDRKVGALNKSQLAAFLDESLHFSS
SEQID NO: 56
MFVDSHCHLNMLDLSPYEGDLGALIDKAKSMGVEHILCVGVDLTHAQTVIEIAARFE NVSASVGLHPSEKVDHEPT VQELVEVANHPKVVAIGETGLDYYYNHSELGKMRDRFRCHVQAALKLKKPLIIHSRSAQT DTIQIMQEENAQSVGG VMHCFTESWEMAEQAMKLGFYISFSGIVTFKNAKNVAEVAKKVPLEKMLIETDAPYLAPV PYRGKKNEPQYIPYVA ERIAELKNIPLNEVARKTTENYYHLFG
SEQID NO: 57
MKPIAIYPGTFDPLTNGHVDIIERALPLFNKIIVACAPTSRKDPHLKLEERVNLIAD VLTDERVEVLPLTGLLVDFAKTH
QANFILRGLRAVSDFDYEFQLAHMNYQLSPEIETIFLPAREGYSYVSGTMVREIVTL GGDVSPFVPPLVARHLQKRRE
K
SEQID NO: 58
MFRLDLLSDPLEQFKLWYDEAIRHETLHPDAMVLATADSKGKPSARNVLYKGISKGGFLI FTNYHSRKAHELDENPQ
AAWVFYWPKTYKQVRGEGRVERLTQEESEAYFETRSYESQIAAWVSEQSQEIPDREY LITRYKKYREKFQDDVRCP
EFWGGFRLIPDRMEFWVGQEHRLHDRFCYLKENQEWKIIRLAP SEQID NO: 59
MSVLVPMVVEQTSRGERAYDIYSRLLKDRVIFLVGQVEDHMANLAIAQMLFLESENP NKDINLYINSPGGAVTSA
MAIYDTMQFVKPDVRTLCIGQAASAGALLLAGGAKGKRHCLPHSSVMIHQVLGGYQG QGTDIQIHAKQTQRVSD
QLNQILAKHTGKDIERVEKDTNRDYFLTPEEAVEYGLIDSIFKERP
SEQID NO: 60
MADLNHSYLTENAPLAAQMTMTPREIVAELDKFIIGQNDAKRAVAIALRNRWRRMQLGEE LRREIFPKNILMIGPT
GVGKTEIARRLSDLAGAPFLKIEATKFTEVGYVGRDVESIIRDLVDVAVKMTREKAI RQVKSLAEEAAEERVLDALIPP
ARGGFQGEPTAEEKPTEKKESATRQLFRKKLRNGELDDKEIEVEVSAHPSFEIMGPP GMEEMVSQLQGIMSSMSS
RRSKSRRLKVKDALRILGEEEAAKLVDEDQIKSTALASVEQNGIVFIDEIDKIVKRE GAVGADVSREGVQRDLLPLVEG
STVFTKYGMVKTDHILFIASGAFHIAKPSDLVPELQGRFPIRVELKALTADDFVRIL TEPKASLTEQYTELLKTENFGLSF
TKDGIKRLAEIAYQVNDRSENIGARRLHTIMERLLEEVSFEATDKQGESITIDADYV NKQLKKLAEDEDLSRYIL
SEQID NO: 61
MEQIAARVTYINLSPDELIQHAVKNGEGVLSSTGALAVTTGKRTGRSPKDRFIVKDE QTADQVAWGNINQPVEQR
TFDQLWERALRYLSERAVYISHLQVGADDNYFLPLKVVTEFAWHNLFACDLFIRPSG DHANGKPSWVILSAPGLKT
DPERDGVNSDGAVMINLSQRRVLLVGMPYAGEMKKAMFSVLNYLLPPHDVLPMHCAA NAGQSGDVALFFGLS
GTGKTTLSADPHRFLIGDDEHGWSATSVFNFEGGCYAKCIDLSQEREPMIWNAIRHG AIMENVVLDENGVPDYAD
ARLTQNSRAAYPREYIPLRVENNRGRPPDAVLFLTCDLDGVLPPVALLTKEQAAYYF LSGYTALVGSTEVGSVKGVTS
TFSTCFGAPFFPRPPTVYAELLMKRIEATGCQVYLVNTGWTGGAYGEGGERFSIPTT RAIVNAVLSGKLKEGPTEVLS
GFNLTIPKSALGVDDHLLNPRKTWEDVSAYDARAQRLIQKFRENFEKFKVLAAIREA GPSDVH
SEQID NO: 62
MSRKFTDKIKGIVMNNLVKNSGLAVIALATLNLSGCKHH PAGAN AATGLSDGTGAQAYALAEGKGYQGQLKKDSE GRIINPLVAPANQTYYFDFDSTQLRSLDLGAIRVQANYLATHSTAKVRLEGNTDNRGSRE YNIGLGWRRDQAVARIL EQEGVAPKQIDMVSYGKERPAVMGNNENAWRLNRRVNLIYEAY
SEQID NO: 63
MQTKVEGLAHILLQTNALTNSQIARAIEQAAGAQSPLLHYLVTEKIVSSEKIAEACA TYFGLEAINLQTQPLNPSLCHE
IPRKYLMRYAFIPLAVKSPTLAISDPLYFPLIEELQFQTNKQYKIVFAPYKSFAALI NNFVSRQIYETVSQGEASIVELVN
QVLTDAIYREASDVHFEPMQQHYRIRMCIDGILHTTTLLPNTQSPAMSSRLKVLAEL DISEKRLPQDGRFYFTTLTHL
KRDCRLSSCPTLFGEKIVIRLLNPVHHLLKFEELGLEEKPKQLIMKKIKQLQGLILV TGPTRSGKTVSLYAALNQINSTQ
KNISTVEDPIEIQLAGVTQVNIRPKAGLNFAAVLRVFLRQDPDVIMVGEIRDFETAS IAVRAAHTGHLVLSTLHTNSA
VECITRLIDMGIEPFNLASVLKLVVAQRLVRQLCAHCQATKISCPFCLNGYQGRTGI YEVLPITPSIIELILQKRSAQEIN
ACAIQEGMQTLWQAALNKAKTGITNLNEIYRVIQSENNYA
SEQID NO: 64
MKRIAVFILTLSFFSISYSDKNPVFQEYYEGNYRAAETGLKQLAEKNNGEATFYLAT MYMNGFGVRRDFEKGFDYM
TRAAELKYLPAQLYLGNYYFQQQKDLEKAVPWFKKAADAGDAGAQLFTGISYLNGYG VKKNIDIARKYFIRAAQNEI
PMGQYELAKIFLASRHAGDRRMGRIWLTKAADKYNYPDAQYLLGTMLYTGNEAEKDP VKGVEWLEKAAANGSK
EASKTLDKINRINTSDAKANSENRSEPTPWQIMVGLMQKAGVQLNNPITVTASINNF TKTPKSMALDKNSIIKLNLN
LVNSKDIPPEKILSYMTQLNYKEEKFDLTVPAYPFEMPPGANNYKEAFQSLSRVANY GYAQSLFRLGQMYENGLGV
QKDPETAFQLYMKAAEQNYLKAQYAIGTYYLQGKGVPQDYEKAISWFIRAALKGSLQ AQFVLGNIYERGIKASNNK ILFKNFDRAKAMYSLAVGGNLPIAAYRLAELYVSGFLNPDNNVSLETQNWKKAYALYQKA AKSGLEKADVALGYFY
LQQNQTTLAEKTFEIAQKAYQTNDPEAAMLLAILYDRGFGVNRNSRKSAEILEKLSK QNNAIAQFMLGNYYLKNKR
KENIAISLLEKSANQGNGYAKYNLAILAKQNKYTKPGENFLSLLIRAANHYDKIKEI LADYYLLDTPVPGSEKKAVAIYQ
ELANKQDPAAELKLGFMNEHGLLFPKDYHKAEEWYQKSAEQGNPIAQYLLGNMYYLG RGVDRDVNKAIDWLKKS
AAQNYVPAKVGLGFIYEMSKHNYPEAKKWYTLASKFHNPQALYNLGLMYEYGKGVKS DPQKAFRLYKDAAQNGL
DLAAVQVAGMYLKGTGIGFDPNTALKMYSQAAQKNNSFATYQLGLMSESGVAQKIDL NKARLYYEKAAKEGSVE
AQLALARFYEFGISVPADISKSINFYQAAAAEGNEFAKQQLTRLSNQGKSSSNAMPF QCVNQVALEKVKNSFWKK
VTDWIAPVPNIDYMNAIDYLNSGKVEQATTALQKIIKVRPNFQPARETVSHYFCQKA DRK
SEQID NO: 65
MLETEKCTKIFLSFSLNSRRIIMNLSLTQDPQKAKEFFEKKMAFTTGPVEVSGMLKK NAKIQVVDVRAAEDYKKGHV PGAINLPSNEWEKAAEKLDKEKTNIIYCYSQVCHLAAKAAVKFAEQGFPVMEMEGGFKTW TEHKLETEK
SEQID NO: 66
MAFELPDLPYKLNALEPHISQETLEYHHGKHHRAYVNKLNKLIEGTPFEKEPLEEII RKSDGGIFNNAAQHWNHTFY
WHCMSPDGGGDPSGELASAIDKTFGSLEKFKALFTDSANNHFGSGWAWLVKDNNGKL EVLSTVNARNPMTEGK
KPLMTCDVWEHAYYIDTRNDRPKYVNNFWQVVNWDFVMKNFKS
SEQID NO: 67
MDNYKKILVALALDPNSDRPLVEKAKELSANRDAQLYLIHAVEHLSSYGAAYGVAAG VDVEDMLLEEAKKRMNEIA SQLNISSDHQIVKVGPAKFLILEQAKNWGVDLIIVGSHGRHGIQLLLGSTSNAVLHGAKC DVLAVRIKGS
SEQID NO: 68
MPSFDIQSELNKHEVSNAVDQANREVATRFDFKGSGATYKYEGNSITLQAETDFQLKQMI DILQNKFAKRQIDVAH MKLEDPIIQHKSAQQTVMLLEGIDQTAAKKIIKLIKDQKLKVQAAIQGEKVRVTGKKRDD LQSVIGLLKEQEIGLPLQ FDNFRD
SEQID NO: 69
MSNSGKKFDFQGVLNNIKSMISPESNTPSPDPSDAIGMKIAELSVLAQQLTKSHEEQ AKELANVNRLLNDLFKDLEA FRNPPENKTEEKQKDKKEETKKD
SEQID NO: 70
MIGGKFNLGSLMKNAKKIQEMMQKAQDELAKIRVTGESGAGMVKLTMTAQHEVVEMNLDD ELLKESKEVIEDLI KAALNDANQKILKITQEKMMSAGSLFGGNESDNEET
SEQID NO: 71
MIRSGKMRKLINSIIGVALIVVIVLLVLPLGMSFWLKNNYPSILTRLSQAHNVSLKL INFDRGWFASKAVIQVIIPNSED
KTTQPIKFTINQHIFNGPFIFSKNNHKVKLHCAKALVYTTSNDPNFTFHSSTLLRFN NSSKSSLYASNVNVANGQEQI
VLKDTNLEILYNPLTQRLVLNAVIKSALISEQQKTILIMDNITWRNDLHHATPLWEG KRSLSLNKFTYYLTPEQLIEVK
NFILENQQNAANDTTTFTFSSHADSIKDTSLNLAPLDIKFSLTQMNTAALVNLINTA LNENHLKLNPQQLHQFHTPA
INLLAQGLEVSLAHLTFGTEEGQVSVQGQLHLPAQNQSPDLSQIMVNAKGNLQAKMP MAWLKKELSRIYEDKKV
ELDDQALTPEQIADQQIQYWINNKKLIPQNQDVELTINYDKGKLLVNNLPSHAPQQ
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