Recombinant vaccine proteins for the prevention of avian necrotic enteritis

Abstract

Avian necrotic enteritis (NE) is a complex and multifactorial enteric disease of commercial chickens and turkeys that has major economic consequences on the poultry industry worldwide. There is thus a need for improved and efficient vaccines for the prevention of diseases associated to pathogenic Clostridium perfringens in the poultry industry. The present application discloses immunogenic polypeptides unique to pathogenic strains of Clostridium perfringens which causes avian NE, as well as vaccine compositions comprising one or more of the immunogenic polypeptides, immunogenic fragments thereof, or nucleic acids encoding such immunogenic polypeptides and fragments. Methods and uses of the vaccine compositions for inducing or eliciting an immune response against Clostridium perfringens, for preventing or treating infection by Clostridium perfringens, and / or for preventing or treating avian NE in an animal, such as a poultry, are also disclosed.

Description

[0001] TITLE OF THE INVENTION

[0002] RECOMBINANT VACCINE PROTEINS FOR THE PREVENTION OF AVIAN NECROTIC ENTERITIS

[0003] SEQUENCE LISTING

[0004] A sequence listing is submitted herewith as an XML file named G10875-00206_Seq Listing.xml, created on March 27, 2024, and having a size of ~70385 bytes. The content of the aforementioned file is hereby incorporated by reference in its entirety.

[0005] FIELD OF THE DISCLOSURE

[0006] The disclosure relates generally to the field of animal infections, and more particularly to the prevention and treatment of avian necrotic enteritis (NE) caused by Clostridium perfringens.

[0007] BACKGROUND OF THE DISCLOSURE

[0008] Avian necrotic enteritis (NE) is a complex and multifactorial enteric disease of commercial chickens and turkeys that has major economic consequences on the poultry industry worldwide [1 ,2], The primary cause is some pathogenic strains of type G Clostridium perfringens, a Gram-positive, anaerobic, and spore-forming bacterium that is widely distributed in various environments, such as soils, feces, foods, and intestine of both humans and animals [3], In broiler chickens, the sole presence of pathogenic strains of C. perfringens has been shown to be generally insufficient to reproduce NE in chicken disease models, and it is recognized that different predisposing factors including Eimeria infection, high amounts of non-starch polysaccharides, immunosuppression, and high amounts of animal proteins (e.g., fishmeal) in the diet are required for the onset of NE [4],

[0009] To date, various C. perfringens-associated toxins and other virulence factors have been identified as contributors to NE development, and a large proportion of them have also been evaluated for their ability to stimulate a protective immunity in the avian bird [5,6], Although some of these tested antigens have proven to stimulate a protective immune response, none of them has proven to be fully protective against the disease in broiler chickens [7,8],

[0010] When breaking NE pathogenesis down, the first step in the establishment of the intestinal infection by C. perfringens is the displacement of commensal C. perfringens by bacteriocins, with perfrin having been identified to play an important role [9,10], The subsequent phase would involve the attachment of disease-causing C. perfringens to the intestinal mucosa through the expression of different adhesion factors, including pili (FimA and FimB), collagen adhesion proteins (e.g., CnaA), and fibronectin-binding proteins (e.g., FbpA and FbpB) for which the role has recently been studied [11 ,12], Over the past few years, colonization followed by degradation of the mucus layer covering the small intestine of the chicken host by C. perfringens extracellular enzymes including zinc metalloproteases, collagenolytic enzymes, glycoside hydrolases, sialidases, and other degradative enzymes became a major key step of interest for scientists [9], As part of better understanding NE pathogenesis, the identification of genetic determinants of C. perfringens that could play a role in both the ability of the pathogen to cause the disease and to elicit a protective immune response has been established as a priority [13,14], Indeed, when adopting a global approach aiming at reducing the impacts of NE in the absence of antibiotic growth promoters (AGPs), vaccination represents the most cost-effective alternative

[0015] ,

[0011] As of today, most efforts have focused on designing vaccines based on alpha and NetB toxins. The only commercially available vaccine (Netvax®) which was based on alpha-toxin (CPA) toxoid and administered to broiler breeder hens subcutaneously, is no longer authorized [16,17], On the experimental side, the efficacy of the recombinant form of NetB (rNetB) as a single subunit vaccine candidate, of the formalin-treated bacterin and of the toxoid to protect chickens from NE was also investigated. Findings indicated that immunization with all of these components was not able to protect birds against a severe challenge, nor the immunization scheme used was applicable to field conditions [7], Immunization with Salmonella-vectored vaccines delivering nontoxic fragments of the alpha toxin, of NetB, of a fructose 1 ,6- bisphosphate aldolase (FBA)

[0018] , of a pyruvate-ferredoxin oxidoreductase (PFOR), and of a hypothetical protein (HP) provided partial protection against a challenge with virulent C. perfringens and required the improvement of antigen expression systems in Salmonella for effective delivery of antigens

[0019] , Other subunit vaccines involving TpeL, an endo-beta-N- acetylglucosaminidase (Naglu), phosphoglyceromutase (Pgm)

[0020] , and a glyceraldehyde 3- phosphate dehydrogenase (GPD) proteins

[0021] , and three predicted pilin structural subunits (CnaA, FimA, FimB) of C. perfringens have also been evaluated, with suboptimal results regarding the ability of these antigens to stimulate an immune response providing complete protection against NE in broiler chickens

[0022] ,

[0012] There is a need for improved and efficient vaccines for the prevention of diseases associated to pathogenic Clostridium perfringens in the poultry industry.

[0013] SUMMARY OF THE DISCLOSURE

[0014] In various aspects and embodiments, the present disclosure provides the following items 1 to 41 :

[0015] 1 . A vaccine composition comprising one or more immunogenic polypeptides comprising one or more of the amino acid sequences set forth in SEQ ID NOs: 1-14 and 57-60, or an immunogenic fragment thereof comprising at least 10 amino acids, and at least one vaccine excipient.

[0016] 2. The vaccine composition of item 1 , wherein the one or more immunogenic fragments comprises at least 15 amino acids. 3. The vaccine composition of any one of items 1 to 3, wherein the one or more immunogenic polypeptides or immunogenic fragments thereof comprise one or more of the following epitope sequences: (a) residues 6-19, 22-35, 41-54, 56-69, 72-85, 10-23, 54-67, 69-82, 5-18, and 51-64 of SEQ ID NO:1 ; (b) residues 13-26, 37-50, 15-28, 32-45, and 30-43 of SEQ ID NO:2; (c) residues 29-42, 44-57, 69-82, 85-98 and 115-128, 1-13, 36-49, 52-65, 67-80, 82-95, 114-127, 24-37, 47- 60, and 76-89 of SEQ ID NO:3; (d) residues 1-13, 21-34, 44-57, 84-97, 125-138, 8-21 , 46-59, 110-123, 129-142, 6-19, 31-44, 49-62, 71-84, and 124-137 of SEQ ID NO:4; (e) residues 3-16, 32-45, 113-126, 139-152, 154-167, 15-28, 31-44, 46-59, 92-105, 138-151 , 153-166, 27-40, 47- 60, 76-89, 136-149, and 151-164 of SEQ ID NO:5; (f) residues 14-27, 81-94, 110-123, 156-169, 189-202, 223-236, 241-254, 349-352, 409-422, 457-470, 482-495, 515-528, 530-543, 549-562, 579-592, 609-622, 651-664, 697-710, 45-58, 108-121 , 157-170, 234-247, 458-471 , 487-500, 552- 565, 581-594, 610-623, 662-675, 694-707, 721-734, 45-58, 63-76, 172-185, 188-201 , 225-238, 243-256, 514-527, 562-575, and 695-708 of SEQ ID NO:6; (g) residues 46-59, 93-106, 125-138, 177-190, 229-242, 228-241 , and 96-109 of SEQ ID NO:7; (h) residues: 34-47, 56-69, 74-87, 54- 67, 70-83, 15-28, 34-47, and 49-62 of SEQ ID NO:8; (i) residues 14-27, 51-64, 66-79, 81-94, 97- 110, 115-128, 130-143, 149-162, 215-228, 230-243, 249-262, 12-25, 50-63, 73-86, 97-110, 117-

[0017] 130, 141-154, 161-174, 230-243, 249-262, 14-27, 82-95, 118-131 , 148-161 , 174-187 and 199- 212 of SEQ ID NO:9; (j) residues 62-75, 80-93, 96-109, 112-125, 133-146, 159-162, 193-206, 243-259, 259-262, 277-290, 315-328, 330-343, 369-382, 384-397, 429-442, 444-457, 480-493, 40-53, 63-76, 86-99, 115-128, 137-150, 165-178, 201-214, 255-268, 313-326, 337-350, 362-375, 401-414, 429-442, 480-493, 63-76, 78-91 , 112-125, 153-166, 199-212, 234-247, 318-331 , 334- 347, 370-383, 391-404, 432-445, and 460-473 of SEQ ID NQ:10; (k) residues 26-39, 46-59, 94- 107, 113-126, 19-32, 46-59, 107-120, 21-34 and 108-121 of SEQ ID NO:11 ; (I) residues 24-37, 48-61 , 67-80, 117-130, 140-153, 168-181 , 184-197, 199-212, 221-234, 250-263, 269-282, 284- 297, 304-317, 319-332, 344-357, 374-387, 390-403, 417-430, 433-446, 479-492, 501-514, 519- 532, 571-584, 611-624, and 656-669, 30-43, 66-79, 98-111 , 126-139, 214-227, 234-247, 311- 324, 345-358, 360-373, 386-399, 552-565, 570-583, 626-639, 645-658, 47-60, 65-78, 133-146, 163-176, 179-192, 224-237, 242-255, 295-308, 352-365, 380-393, 423-436, 441-454, 469-482, 521-534, 564-577, 625-638, 643-656, and 661-674 of SEQ ID NO: 12; (m) residues 17-30, 32-45, 50-63, 65-78, 83-96, 118-131 , 133-146, 159-172, 193-206, 222-235, 257-270, 278-291 , 306-319, 32-45, 53-66, 71-84, 107-120, 134-147, 222-235, 275-288, 293-306, 310-323, 36-49, 51-64, 78- 91 , 98-111 , 125-138, 223-236, and 251-264 of SEQ ID NO: 13; and / or (n) residues 88-101 , US-

[0018] 131 , 158-161 , 180-193, 245-258, 278-291 , 18-31 , 76-89, 119-132, 141-154, 247-260, 2-15, 53- 66, 85-98, 112-125, 138-151 , 174-187, 249-262, and 268-281 of SEQ ID NO: 14.

[0019] 4. The vaccine composition of any one of items 1 to 3, wherein the one or more immunogenic polypeptide or immunogenic fragment thereof comprises a sequence having at least 70% identity with one or more of the amino acid sequences set forth in SEQ ID NOs: 1-14 and 57-60. 5. The vaccine composition of item 4, wherein the one or more immunogenic polypeptide or immunogenic fragment thereof comprises a sequence having at least 90% identity with one or more of the amino acid sequences set forth in SEQ ID NOs: 1-14 and 57-60.

[0020] 6. The vaccine composition of item 5, wherein the one or more immunogenic polypeptide or immunogenic fragment thereof comprises one or more of the amino acid sequences set forth in SEQ ID NOs: 1-14 and 57-60.

[0021] 7. The vaccine composition of any one of items 1 to 6, wherein the one or more immunogenic polypeptides comprises one or more of the amino acid sequences set forth in SEQ ID NOs: 1-7 and 57-60, or an immunogenic fragment thereof.

[0022] 8. The vaccine composition of any one of items 1 to 7, wherein the one or more immunogenic polypeptides comprises one or more of the amino acid sequences set forth in SEQ ID NOs: 1-5 and 57-58, or an immunogenic fragment thereof.

[0023] 9. The vaccine composition of any one of items 1 to 8, wherein the one or more immunogenic polypeptides comprises the amino acid sequence set forth in SEQ ID NO: 1 , or an immunogenic fragment thereof.

[0024] 10. The vaccine composition of any one of items 1 to 9, wherein the one or more immunogenic polypeptides comprises the amino acid sequence set forth in SEQ ID NO: 2, or an immunogenic fragment thereof.

[0025] 11. The vaccine composition of any one of items 1 to 10, wherein the one or more immunogenic polypeptides comprises the amino acid sequence set forth in SEQ ID NO: 3 or 57, or an immunogenic fragment thereof.

[0026] 12. The vaccine composition of any one of items 1 to 11 , wherein the one or more immunogenic polypeptides comprises the amino acid sequence set forth in SEQ ID NO: 4 or 58, or an immunogenic fragment thereof.

[0027] 13. The vaccine composition of any one of items 1 to 12, wherein the one or more immunogenic polypeptides comprises the amino acid sequence set forth in SEQ ID NO: 5, or an immunogenic fragment thereof.

[0028] 14. The vaccine composition of any one of items 1 to 13, wherein the vaccine composition comprises one immunogenic polypeptide or immunogenic fragment thereof.

[0029] 15. The vaccine composition of any one of items 1 to 14, wherein the vaccine composition comprises at least two immunogenic polypeptides or immunogenic fragments thereof.

[0030] 16. A vaccine composition comprising one or more nucleic acids encoding the one or more immunogenic polypeptides or immunogenic fragments thereof defined in any one of items 1-15, and at least one vaccine excipient.

[0031] 17. The vaccine composition of any one of items 1 to 16, wherein the at least one vaccine excipient comprises at least one vaccine adjuvant. 18. The vaccine composition of item 17, wherein the at least one vaccine adjuvant comprises a saponin-based adjuvant.

[0032] 19. The vaccine composition of item 18, wherein the saponin-based adjuvant is Quil-A® or QS-21.

[0033] 20. The vaccine composition of any one of items 1 to 19, wherein the at least one vaccine excipient comprises a buffer, a solvent, a preservative, an antibiotic, or any combination thereof.

[0034] 21. The vaccine composition of any one of items 1 to 20 for use in inducing or eliciting an immune response against Clostridium perfringens in an animal.

[0035] 22. The vaccine composition of any one of items 1 to 20 for use in preventing or treating infection by Clostridium perfringens in an animal.

[0036] 23. The vaccine composition of any one of items 1 to 20 for use in preventing or treating avian necrotic enteritis (NE) in an animal.

[0037] 24. The vaccine composition for use according to any one of items 21-23, wherein the animal is a poultry.

[0038] 25. The vaccine composition for use according to item 24, wherein the poultry is a chicken or a turkey.

[0039] 26. The vaccine composition for use according to any one of items 21-25, wherein the vaccine composition is for oral administration, nasal administration, injection, or in ovo administration.

[0040] 27. A method for inducing or eliciting an immune response against Clostridium perfringens in an animal comprising administering to the animal an effective amount of the vaccine composition of any one of items 1 to 20.

[0041] 28. A method for preventing or treating infection by Clostridium perfringens in an animal comprising administering to the animal an effective amount of the vaccine composition of any one of items 1 to 20.

[0042] 29. A method for preventing or treating avian necrotic enteritis (NE) in an animal comprising administering to the animal an effective amount of the vaccine composition of any one of items 1 to 20.

[0043] 30. The method of any one of items 27-29, wherein the animal is a poultry.

[0044] 31 . The method of item 30, wherein the poultry is a chicken or a turkey.

[0045] 32. The method of any one of items 27-31 , wherein the administration is oral administration, nasal administration, injection, or in ovo administration.

[0046] 33. Use of the vaccine composition of any one of items 1 to 20 for inducing or eliciting an immune response against Clostridium perfringens in an animal.

[0047] 34. Use of the vaccine composition of any one of items 1 to 20 for the manufacture of a medicament for inducing or eliciting an immune response against Clostridium perfringens in an animal. 35. Use of the vaccine composition of any one of items 1 to 20 for preventing or treating infection by Clostridium perfringens in an animal.

[0048] 36. Use of the vaccine composition of any one of items 1 to 20 for the manufacture of a medicament for preventing or treating infection by Clostridium perfringens in an animal.

[0049] 37. Use of the vaccine composition of any one of items 1 to 20 for preventing or treating avian necrotic enteritis (NE) in an animal.

[0050] 38. Use of the vaccine composition of any one of items 1 to 20 for the manufacture of a medicament for preventing or treating avian necrotic enteritis (NE) in an animal.

[0051] 39. The use of any one of items 33-38, wherein the animal is a poultry.

[0052] 40. The use of item 39, wherein the poultry is a chicken or a turkey.

[0053] 41. The use of any one of items 33-40, wherein the vaccine composition is for oral administration, nasal administration, injection, or in ovo administration.

[0054] Other objects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings.

[0055] BRIEF DESCRIPTION OF THE DRAWINGS

[0056] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0057] In the appended drawings:

[0058] FIGs. 1A-E: SDS-PAGE gel showing purification of His-tagged recombinant proteins by imidazole step gradient. 15 pL of total cell lysate and fractions of P153 (FIG. 1A), P264 (FIG. 1B), P509 (FIG. 1C), P537 (FIG. 1D), and P509 (FIG. 1E) were observed by SDS-PAGE and Coomassie Blue staining. The size of the recombinant protein is 4 KDa higher than the expected size due to the inclusion of the His-tag and V5 tag at N-terminal. Negative control samples in FIG. 1A consist of total cell protein samples from BL21 cells that were transformed with pET empty vector.

[0059] FIGs. 2A-C: Western blot analysis of V5-tagged purified recombinant proteins: FIG. 2A: Negative control, P264, and P153 FIG. 2B: P537 and P264; and FIG. 2C: P561 and P509. Negative control sample in FIG. 2A consists of total cell protein samples from BL21 cells that were transformed with pET empty vector.

[0060] FIGs. 3A-C: Western blot analysis of chicken antibodies raised against recombinant forms of P537 and P509 (FIG. 3A), P153 and P264 (FIG. 3B), and P561 (FIG. 3C).

[0061] FIGs. 4A-F show the evaluation of the serum IgY on days 7, 14, 21 , and 35 by ELISA following immunization of chicken with P153 (FIG. 4A), P264 (FIG. 4B), P509 (FIG. 4C), P537 (FIG. 4D), P561 (FIG. 4E), and adjuvant (Quil-A) alone (FIG. 4F). Chickens were immunized intramuscularly with purified recombinant protein mixed with the adjuvant Quil-A. Birds from the negative control group were immunized with adjuvant alone. The error bars represent standard error on means of two repeats. *P < 0.05; **P < 0.01 ; ***P < 0.001 , determined by the likelihood ratio test (LRT), followed by a post-hoc Tukey test with a Benjamini-Hochberg correction.

[0062] FIGs. 5A-F: ELISA were performed with whole-cell lysate from C. perfringens MLG_7820 and P561 (FIG. 5A), P509 (FIG. 5B), P153 (FIG. 5C), P264 (FIG. 5D), and P537 (FIG. 5E) antibodies raised in immunized birds. Quil-A response is also included (FIG. 5F). Immuno plate MaxiSorp wells were coated with 0.5 pg of whole-cell lysate from C. perfringens MLG_7820. The error bars representing standard error. The asterisks refer to the level of significance: *P < 0.05; **P < 0.01 ; ***P < 0.001 , determined by the Mann-Whitney test.

[0063] FIG. 6: Distribution of candidate protein-encoding genes among analyzed commensal strains of C. perfringens.

[0064] FIG. 7A depicts the amino acid sequence of candidate protein P264-2 (SEQ ID NO:1) with the putative linear B-cell epitopes as determined by the Bepipred Linear Epitope Prediction 2.0 tool (Larsen JE, Lund O, Nielsen M. 2006. Improved method for predicting linear B-cell epitopes. Immunome Res 2:2) underlined, and FIG. 7B depicts the nucleotide sequence encoding the candidate protein P264-2 (SEQ ID NO: 15).

[0065] FIG. 8A depicts the amino acid sequence of candidate protein P153 (SEQ ID NO:2) with the putative linear B-cell epitopes as determined by the Bepipred Linear Epitope Prediction 2.0 tool underlined, and FIG. 8B depicts the nucleotide sequence encoding candidate protein P153 (SEQ ID NO:16).

[0066] FIG. 9A depicts the amino acid sequence of candidate protein P509 (SEQ ID NO:3) with the sequences putative linear B-cell epitopes as determined by the Bepipred Linear Epitope Prediction 2.0 tool underlined, and FIG. 9B depicts the nucleotide sequence encoding candidate protein P509 (SEQ ID NO:17).

[0067] FIG. 10A depicts the amino acid sequence of candidate protein P561 (SEQ ID NO:4) with the sequences of putative linear B-cell epitopes as determined by the Bepipred Linear Epitope Prediction 2.0 tool underlined, and FIG. 10B depicts the nucleotide sequence encoding the candidate protein P561 (SEQ ID NO: 18).

[0068] FIG. 11 A depicts the amino acid sequence of candidate protein P537 (SEQ ID NO:5) with the putative linear B-cell epitopes as determined by the Bepipred Linear Epitope Prediction 2.0 tool underlined, and FIG. 11B depicts the nucleotide sequence encoding the candidate protein P537 (SEQ ID NO:19).

[0069] FIG. 12A depicts the amino acid sequence of candidate protein P2091 (SEQ ID NO:6) with the putative linear B-cell epitopes as determined by the Bepipred Linear Epitope Prediction 2.0 tool underlined, and FIG. 12B depicts the nucleotide sequence encoding the candidate protein P2091 (SEQ ID NQ:20). FIG. 13A depicts the amino acid sequence of candidate protein P1074 (SEQ ID NO:7) with the putative linear B-cell epitopes as determined by the Bepipred Linear Epitope Prediction 2.0 tool underlined, and FIG. 13B depicts the nucleotide sequence encoding the candidate protein P1074 (SEQ ID NO:21).

[0070] FIG. 14A depicts the amino acid sequence of candidate protein P2232 (SEQ ID NO:8) with the putative linear B-cell epitopes as determined by the Bepipred Linear Epitope Prediction 2.0 tool underlined, and FIG. 14B depicts the nucleotide sequence encoding the candidate protein P2232 (SEQ ID NO:22).

[0071] FIG. 15A depicts the amino acid sequence of candidate protein P759 (SEQ ID NO:9) with the putative linear B-cell epitopes as determined by the Bepipred Linear Epitope Prediction 2.0 tool underlined, and FIG. 15B depicts the nucleotide sequence encoding the candidate protein P759 (SEQ ID NO:23).

[0072] FIG. 16A depicts the amino acid sequence of candidate protein P264-1 (SEQ ID NQ:10) with the putative linear B-cell epitopes as determined by the Bepipred Linear Epitope Prediction 2.0 tool underlined, and FIG. 16B depicts the nucleotide sequence encoding the candidate protein P264-1 (SEQ ID NO:24).

[0073] FIG. 17A depicts the amino acid sequence of candidate protein P804 (SEQ ID NO:11) with the putative linear B-cell epitopes as determined by the Bepipred Linear Epitope Prediction 2.0 tool underlined, and FIG. 17B depicts the nucleotide sequence encoding the candidate protein P804 (SEQ ID NO:25).

[0074] FIG. 18A depicts the amino acid sequence of candidate protein P1569 (SEQ ID NO: 12) with the putative linear B-cell epitopes as determined by the Bepipred Linear Epitope Prediction 2.0 tool underlined, and FIG. 18B depicts the nucleotide sequence encoding the candidate protein P1569 (SEQ ID NO:26).

[0075] FIG. 19A depicts the amino acid sequence of candidate protein P384 (SEQ ID NO: 13) with the putative linear B-cell epitopes as determined by the Bepipred Linear Epitope Prediction 2.0 tool underlined, and FIG. 19B depicts the nucleotide sequence encoding the candidate protein P384 (SEQ ID NO:27).

[0076] FIG. 20A depicts the amino acid sequence of candidate protein P891 (SEQ ID NO: 14) with the putative linear B-cell epitopes as determined by the Bepipred Linear Epitope Prediction 2.0 tool underlined, and FIG. 20B depicts the nucleotide sequence encoding the candidate protein P891 (SEQ ID NO:28).

[0077] DETAILED DESCRIPTION

[0078] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0079] The terms "comprising", "having", "including", and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to") unless otherwise noted.

[0080] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All subsets of values within the ranges are also incorporated into the specification as if they were individually recited herein.

[0081] The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed.

[0082] No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0083] Herein, the term "about" has its ordinary meaning. The term “about” is used to indicate that a value includes an inherent variation of error for the device or the method being employed to determine the value, or encompass values close to the recited values, for example within 10% or 5% of the recited values (or range of values).

[0084] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0085] The results presented herein show that candidate proteins from pathogenic C. perfringens have the ability to elicit an early immune response, make them attractive as vaccine targets that could confer immunity to broiler chickens before they become susceptible to NE in commercial conditions.

[0086] In an aspect, the present disclosure provides a vaccine composition comprising an immunogenic polypeptide comprising of the amino acid sequences set forth in SEQ ID NOs: 1- 14, preferably SEQ ID NOs: 1-5, or an immunogenic fragment thereof, and at least one vaccine excipient.

[0087] In another aspect, the present disclosure provides a vaccine composition comprising an immunogenic polypeptide comprising at least 10 amino acids (e.g., contiguous) from one or more of the amino acid sequences set forth in SEQ ID NOs: 1-14, preferably SEQ ID NOs: 1-7, more preferably SEQ ID NOs: 1-5, and at least one vaccine excipient.

[0088] The term “immunogenic polypeptide” or “immunogenic fragment” as used herein refers to a polypeptide (or a fragment thereof) that is capable of stimulating a host's immune system to make a humoral antibody response and / or a cellular antigen-specific immune response when the antigen is presented / administered to an animal, such as a bird, in a suitable amount. In an embodiment, the immunogenic polypeptide comprising at least 15 amino acids (e.g., contiguous) from one or more of the amino acid sequences set forth in SEQ ID NOs: 1-14, preferably SEQ ID NOs: 1-7, more preferably SEQ ID NOs: 1-5. In an embodiment, the immunogenic polypeptide comprising at least 20 amino acids (e.g., contiguous) from one or more of the amino acid sequences set forth in SEQ ID NOs: 1-14, preferably SEQ ID NOs: 1-7, more preferably SEQ ID NOs: 1-5. In an embodiment, the immunogenic polypeptide comprising at least 25 amino acids (e.g., contiguous) from one or more of the amino acid sequences set forth in SEQ ID NOs: 1-14, preferably SEQ ID NOs: 1-7, more preferably SEQ ID NOs: 1-5. In an embodiment, the immunogenic polypeptide comprising at least 30 amino acids (e.g., contiguous) from one or more of the amino acid sequences set forth in SEQ ID NOs: 1-14, preferably SEQ ID NOs: 1-7, more preferably SEQ ID NOs: 1-5. In an embodiment, the immunogenic polypeptide comprising at least 35 amino acids (e.g., contiguous) from one or more of the amino acid sequences set forth in SEQ ID NOs: 1-14, preferably SEQ ID NOs: 1-7, more preferably SEQ ID NOs: 1-5. In an embodiment, the immunogenic polypeptide comprises at least 40 amino acids from one or more of the amino acid sequences set forth in SEQ ID NOs: 1-14, preferably SEQ ID NOs: 1-7, more preferably SEQ ID NOs: 1-5. In an embodiment, the immunogenic polypeptide comprises at least 50 amino acids from one or more of the amino acid sequences set forth in SEQ ID NOs: 1-14, preferably SEQ ID NOs: 1-7, more preferably SEQ ID NOs: 1-5. In an embodiment, the immunogenic polypeptide comprises at least 60 amino acids from one or more of the amino acid sequences set forth in SEQ ID NOs: 1-14, preferably SEQ ID NOs: 1-7, more preferably SEQ ID NOs: 1-5. In an embodiment, the immunogenic polypeptide comprises at least 70 amino acids from one or more of the amino acid sequences set forth in SEQ ID NOs: 1-14, preferably SEQ ID NOs: 1-7, more preferably SEQ ID NOs: 1-5. In an embodiment, the immunogenic polypeptide comprises at least 80 amino acids from one or more of the amino acid sequences set forth in SEQ ID NOs: 1-14, preferably SEQ ID NOs: 1-7, more preferably SEQ ID NOs: 1-5. In an embodiment, the immunogenic polypeptide comprises at least 90 amino acids from one or more of the amino acid sequences set forth in SEQ ID NOs: 1-14, preferably SEQ ID NOs: 1-7, more preferably SEQ ID NOs: 1-5. In an embodiment, the immunogenic polypeptide comprises at least 100 amino acids from one or more of the amino acid sequences set forth in SEQ ID NOs: 1-14, preferably SEQ ID NOs: 1-7, more preferably SEQ ID NOs: 1-5. In an embodiment, the immunogenic polypeptide comprises at least 150 amino acids from one or more of the amino acid sequences set forth in SEQ ID NOs: 1-14, preferably SEQ ID NOs: 1-7, more preferably SEQ ID NOs: 1-5. In an embodiment, the immunogenic polypeptide comprises at least 200 amino acids from one or more of the amino acid sequences set forth in SEQ ID NOs: 1-14, preferably SEQ ID NOs: 1-7, more preferably SEQ ID NOs: 1-5.

[0089] In an embodiment, the immunogenic polypeptide or immunogenic fragment thereof comprises one or more putative B cell epitopes as identified using a suitable B cell epitope prediction tool / program. Examples of such tools / programs include the Bepipred Linear Epitope Prediction 2.0 tool (Larsen JE, Lund O, Nielsen M. 2006. Improved method for predicting linear B-cell epitopes. Immunome Res 2:2), BCPREDS (EL-Manzalawy Y, Dobbs D, Honavar V (2008) Predicting linear B-cell epitopes using string kernels. J Mol Recognit 21 : 243-255), FBCPREDS (EL-Manzalawy Y, Dobbs D, Honavar (2008) Predicting flexible length linear B-cell epitopes. 7thInternational Conference on Computational Systems Bioinformatics, Stanford, CA. pp. 121- 131), amino acid pair (AAP) antigenicity scale (Chen J, Liu H, Yang J, Chou K (2007) Prediction of linear B-cell epitopes using amino acid pair antigenicity scale. Amino Acids 33: 423-428). Table 1 below depicts predicted 14-amino acid epitopes from the polypeptides of SEQ ID NO:1-14 described herein as determined using the FBCPREDS, BCPREDS and AAP tools / programs.

[0090] Table 1

[0091] Thus, in an embodiment, the immunogenic polypeptide or immunogenic fragment thereof comprises one or more of the epitope sequences depicted in Table 1 above. In a further embodiment, the immunogenic polypeptide or immunogenic fragment thereof comprises an epitope sequence that is predicted by at least 2 of the B cell epitope prediction tools / programs as shown in Table 1.

[0092] In an embodiment, the immunogenic polypeptide or immunogenic fragment thereof comprises one or more of the following epitope sequences: residues 6-19, 22-35, 41-54, 56-69, 72-85, 10-23, 54-67, 69-82, 5-18, and 51-64 of SEQ ID NO:1. In a further embodiment, the immunogenic polypeptide or immunogenic fragment thereof comprises one or more of the following epitope sequences: residues 6-18, 56-67, and 69-82 of SEQ ID NO:1.

[0093] In an embodiment, the immunogenic polypeptide or immunogenic fragment thereof comprises one or more of the following epitope sequences: residues 13-26, 37-50, 15-28, 32-45, and 30-43 of SEQ ID NO:2. In a further embodiment, the immunogenic polypeptide or immunogenic fragment thereof comprises the following epitope sequence: residues 15-28 and 32-43 of SEQ ID NO:2.

[0094] In an embodiment, the immunogenic polypeptide or immunogenic fragment thereof comprises one or more of the following epitope sequences: residues 29-42, 44-57, 69-82, 85-98 and 115-128, 1-13, 36-49, 52-65, 67-80, 82-95, 114-127, 24-37, 47-60, and 76-89 of SEQ ID NO:57. In a further embodiment, the immunogenic polypeptide or immunogenic fragment thereof comprises one or more of the following epitope sequences: residues 1-13, 69-80, 85-95, and 114- 127 of SEQ ID NO:57.

[0095] In an embodiment, the immunogenic polypeptide or immunogenic fragment thereof comprises one or more of the following epitope sequences: residues 1-13, 21-34, 44-57, 84-97, 125-138, 8-21 , 46-59, 110-123, 129-142, 6-19, 31-44, 49-62, 71-84, and 124-137 of SEQ ID NO:58. In a further embodiment, the immunogenic polypeptide or immunogenic fragment thereof comprises one or more of the following epitope sequences: residues 46-57, 84-97, and 125-137 of SEQ ID NO:58.

[0096] In an embodiment, the immunogenic polypeptide or immunogenic fragment thereof comprises one or more of the following epitope sequences: residues 3-16, 32-45, 113-126, 139- 152, 154-167, 15-28, 31-44, 46-59, 92-105, 138-151 , 153-166, 27-40, 47-60, 76-89, 136-149, and 151-164 of SEQ ID NO:5. In a further embodiment, the immunogenic polypeptide or immunogenic fragment thereof comprises one or more of the following epitope sequences: residues 32-44, 47- 59, and 138-149 of SEQ ID NO:5.

[0097] In an embodiment, the immunogenic polypeptide or immunogenic fragment thereof comprises one or more of the following epitope sequences: residues 14-27, 81-94, 110-123, 156- 169, 189-202, 223-236, 241-254, 349-352, 409-422, 457-470, 482-495, 515-528, 530-543, 549- 562, 579-592, 609-622, 651-664, 697-710, 45-58, 108-121 , 157-170, 234-247, 458-471 , 487-500, 552-565, 581-594, 610-623, 662-675, 694-707, 721-734, 45-58, 63-76, 172-185, 188-201 , 225- 238, 243-256, 514-527, 562-575, and 695-708 of SEQ ID NO:6. In a further embodiment, the immunogenic polypeptide or immunogenic fragment thereof comprises one or more of the following epitope sequences: residues 110-121 , 157-169, 189-201 , 225-236, 243-254, 458-470, 515-527, 581-592, 610-622, and 697-708 of SEQ ID NO:6.

[0098] In an embodiment, the immunogenic polypeptide or immunogenic fragment thereof comprises one or more of the following epitope sequences: residues 46-59, 93-106, 125-138, 177-190, 229-242, 228-241 , and 96-109 of SEQ ID NO:7. In a further embodiment, the immunogenic polypeptide or immunogenic fragment thereof comprises one or more of the following epitope sequences: residues 96-106 and 229-241 of SEQ ID NO:7.

[0099] In an embodiment, the immunogenic polypeptide or immunogenic fragment thereof comprises one or more of the following epitope sequences: residues: 34-47, 56-69, 74-87, 54-67, 70-83, 15-28, 34-47, and 49-62 of SEQ ID NO:8. In a further embodiment, the immunogenic polypeptide or immunogenic fragment thereof comprises one or more of the following epitope sequences: residues 34-47 and 56-67 of SEQ ID NO:8.

[0100] In an embodiment, the immunogenic polypeptide or immunogenic fragment thereof comprises one or more of the following epitope sequences: residues 14-27, 51-64, 66-79, 81-94, 97-110, 115-128, 130-143, 149-162, 215-228, 230-243, 249-262, 12-25, 50-63, 73-86, 97-110, 117-130, 141-154, 161-174, 230-243, 249-262, 14-27, 82-95, 118-131 , 148-161 , 174-187 and 199-212 of SEQ ID NO:9. In a further embodiment, the immunogenic polypeptide or immunogenic fragment thereof comprises one or more of the following epitope sequences: residues 14-25, 51- 63, 97-110, 118-128, 149-161 , 230-243, and 249-262 of SEQ ID NO:9.

[0101] In an embodiment, the immunogenic polypeptide or immunogenic fragment thereof comprises one or more of the following epitope sequences: residues 62-75, 80-93, 96-109, 112-

[0102] 125, 133-146, 159-162, 193-206, 243-259, 259-262, 277-290, 315-328, 330-343, 369-382, 384- 397, 429-442, 444-457, 480-493, 40-53, 63-76, 86-99, 115-128, 137-150, 165-178, 201-214, 255- 268, 313-326, 337-350, 362-375, 401-414, 429-442, 480-493, 63-76, 78-91 , 112-125, 153-166, 199-212, 234-247, 318-331 , 334-347, 370-383, 391-404, 432-445, and 460-473 of SEQ ID NQ:10. In a further embodiment, the immunogenic polypeptide or immunogenic fragment thereof comprises one or more of the following epitope sequences: residues 63-75, 80-91 , 112-125, 201- 212, 315-326, 370-382, and 429-442 of SEQ ID NO: 10.

[0103] In an embodiment, the immunogenic polypeptide or immunogenic fragment thereof comprises one or more of the following epitope sequences: residues 26-39, 46-59, 94-107, US-

[0104] 126, 19-32, 46-59, 107-120, 21-34 and 108-121 of SEQ ID NO:11. In a further embodiment, the immunogenic polypeptide or immunogenic fragment thereof comprises one or more of the following epitope sequences: residues 21-32, and 46-59 of SEQ ID NO:11 .

[0105] In an embodiment, the immunogenic polypeptide or immunogenic fragment thereof comprises one or more of the following epitope sequences: residues 24-37, 48-61 , 67-80, 117- 130, 140-153, 168-181 , 184-197, 199-212, 221-234, 250-263, 269-282, 284-297, 304-317, 319- 332, 344-357, 374-387, 390-403, 417-430, 433-446, 479-492, 501-514, 519-532, 571-584, 611- 624, and 656-669, 30-43, 66-79, 98-111 , 126-139, 214-227, 234-247, 311-324, 345-358, 360- 373, 386-399, 552-565, 570-583, 626-639, 645-658, 47-60, 65-78, 133-146, 163-176, 179-192, 224-237, 242-255, 295-308, 352-365, 380-393, 423-436, 441-454, 469-482, 521-534, 564-577, 625-638, 643-656, and 661-674 of SEQ ID NO:12. In a further embodiment, the immunogenic polypeptide or immunogenic fragment thereof comprises one or more of the following epitope sequences: residues 48-61 , 67-78, 98-111 , 117-130, 168-181 , 184-197, 269-282, 284-297, 345- 357, 521-532, 571-583, 626-638 and 645-656 of SEQ ID NO:12.

[0106] In an embodiment, the immunogenic polypeptide or immunogenic fragment thereof comprises one or more of the following epitope sequences: residues 17-30, 32-45, 50-63, 65-78, 83-96, 118-131 , 133-146, 159-172, 193-206, 222-235, 257-270, 278-291 , 306-319, 32-45, 53-66, 71-84, 107-120, 134-147, 222-235, 275-288, 293-306, 310-323, 36-49, 51-64, 78-91 , 98-111 , 125-138, 223-236, and 251-264 of SEQ ID NO:13. In a further embodiment, the immunogenic polypeptide or immunogenic fragment thereof comprises one or more of the following epitope sequences: residues 32-45, 51-63, 134-146, and 222-235 of SEQ ID NO:13. In an embodiment, the immunogenic polypeptide or immunogenic fragment thereof comprises one or more of the following epitope sequences: residues 88-101 , 118-131 , 158-161 , 180-193, 245-258, 278-291 , 18-31 , 76-89, 119-132, 141-154, 247-260, 2-15, 53-66, 85-98, 112- 125, 138-151 , 174-187, 249-262, and 268-281 of SEQ ID NO:14. In a further embodiment, the immunogenic polypeptide or immunogenic fragment thereof comprises one or more of the following epitope sequences: residues 88-98, 119-131 , 141-151 , and 247-258 of SEQ ID NO:14.

[0107] In an embodiment, the immunogenic polypeptide or immunogenic fragment thereof comprises one or more of the regions underlined in FIGs. 7A-20A. The regions underlined correspond to putative B cell epitopes as determined by the Bepipred Linear Epitope Prediction 2.0 tool (Larsen JE, Lund O, Nielsen M. 2006. Improved method for predicting linear B-cell epitopes. Immunome Res 2:2).

[0108] As described in the Examples below, bioinformatics analyses have revealed some of the polypeptides described herein comprise putative transmembrane helix. For example, P509 has a predicted transmembrane helix between residues 12-34, P561 has a predicted transmembrane helix between residues 13-35, P2091 has two predicted transmembrane helices at positions 7- 29 and 668-690, and P1074 has a predicted transmembrane helix at position 331-350. In an embodiment, the immunogenic polypeptide or immunogenic fragment thereof is free of transmembrane domain. In an embodiment, the immunogenic polypeptide or immunogenic fragment thereof does not comprise residues 12-34 of SEQ ID NO:3. In an embodiment, the immunogenic polypeptide or immunogenic fragment thereof does not comprise residues 1-36 of SEQ ID NO:4. In an embodiment, the immunogenic polypeptide or immunogenic fragment thereof does not comprise residues 7-29 and 668-690 of SEQ ID NO: 12. In an embodiment, the immunogenic polypeptide or immunogenic fragment thereof does not comprise residues 331-350 of SEQ ID NO:13. In an embodiment, the immunogenic polypeptide comprises residues 41-168 of SEQ ID NO:3 (SEQ ID NO:57). In an embodiment, the immunogenic polypeptide comprises residues 37-186 of SEQ ID NO:4 (SEQ ID NO:58). In an embodiment, the immunogenic polypeptide comprises residues 30-667 of SEQ ID NO:6 (SEQ ID NO:59). In an embodiment, the immunogenic polypeptide comprises residues 1-330 of SEQ ID NO:7 (SEQ ID NQ:60).

[0109] In an embodiment, the immunogenic polypeptide comprises an amino acid sequence having at least 70% sequence identity with one or more of the amino acid sequences set forth in SEQ ID NOs:1-14 and 57-60, preferably SEQ ID NOs:1-7 and 57-60, more preferably SEQ ID NOs:1-5 and 57-58. In an embodiment, the immunogenic polypeptide comprises an amino acid sequence having at least 75% sequence identity with one or more of the amino acid sequences set forth in SEQ ID NOs:1-14 and 57-60, preferably SEQ ID NOs:1-7 and 57-60, more preferably SEQ ID NOs:1-5 and 57-58. In an embodiment, the immunogenic polypeptide comprises an amino acid sequence having at least 80% sequence identity with one or more of the amino acid sequences set forth in SEQ ID NOs:1-14 and 57-60, preferably SEQ ID NOs:1-7 and 57-60, more preferably SEQ ID N0s:1-5 and 57-58. In an embodiment, the immunogenic polypeptide comprises an amino acid sequence having at least 85% sequence identity with one or more of the amino acid sequences set forth in SEQ ID NOs:1-14 and 57-60, preferably SEQ ID NOs:1-7 and 57-60, more preferably SEQ ID NOs:1-5 and 57-58. In an embodiment, the immunogenic polypeptide comprises an amino acid sequence having at least 90% sequence identity with one or more of the amino acid sequences set forth in SEQ ID NOs:1-14 and 57-60, preferably SEQ ID NOs:1-7 and 57-60, more preferably SEQ ID NOs:1-5 and 57-58. In an embodiment, the immunogenic polypeptide comprises an amino acid sequence having at least 95% sequence identity with one or more of the amino acid sequences set forth in SEQ ID NOs:1-14 and 57-60, preferably SEQ ID NOs:1-7 and 57-60, more preferably SEQ ID NOs:1-5 and 57-58. In an embodiment, the immunogenic polypeptide comprises an amino acid sequence having at least 96% sequence identity with one or more of the amino acid sequences set forth in SEQ ID NOs:1- 14 and 57-60, preferably SEQ ID NOs:1-7 and 57-60, more preferably SEQ ID NOs:1-5 and 57- 58. In an embodiment, the immunogenic polypeptide comprises an amino acid sequence having at least 97% sequence identity with one or more of the amino acid sequences set forth in SEQ ID NOs:1-14 and 57-60, preferably SEQ ID NOs:1-7 and 57-60, more preferably SEQ ID NOs:1-5 and 57-58. In an embodiment, the immunogenic polypeptide comprises an amino acid sequence having at least 98% sequence identity with one or more of the amino acid sequences set forth in SEQ ID NOs:1-14 and 57-60, preferably SEQ ID NOs:1-7 and 57-60, more preferably SEQ ID NOs:1-5 and 57-58. In an embodiment, the immunogenic polypeptide comprises an amino acid sequence having at least 99% sequence identity with one or more of the amino acid sequences set forth in SEQ ID NOs:1-14 and 57-60, preferably SEQ ID NOs:1-7 and 57-60, more preferably SEQ ID NOs:1-5 and 57-58. In an embodiment, the immunogenic polypeptide comprises one or more of the amino acid sequences set forth in SEQ ID NOs:1-14 and 57-60, preferably SEQ ID NOs:1-7 and 57-60, more preferably SEQ ID NOs:1-5 and 57-58.

[0110] “Identity” refers to sequence similarity / identity between two polypeptide molecules. The identity can be determined by comparing each position in the aligned sequences. A degree of identity between amino acid sequences is a function of the number of identical amino acids at positions shared by the sequences. As used herein, a given percentage of identity between sequences denotes the degree of sequence identity in optimally aligned sequences.

[0111] Optimal alignment of sequences for comparisons of identity may be conducted using a variety of algorithms, such as the local homology algorithm of Smith and Waterman, 1981 , Adv. Appl. Math 2: 482, the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443, the search for similarity method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85: 2444, and the computerized implementations of these algorithms (such as GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, Wl, U.S.A.). Sequence similarity or identity may also be determined using the BLAST algorithm, described in Altschul et al., 1990, J. Mol. Biol. 215: 403-10 (using the published default settings). Software for performing BLAST analysis may be available through the National Center for Biotechnology Information web site. The BLAST algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold. Initial neighborhood word hits act as seeds for initiating searches to find longer HSPs. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension of the word hits in each direction is halted when the following parameters are met: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST program may use as defaults a word length (W) of 11 , the BLOSUM62 scoring matrix (Henikoff and Henikoff, 1992, Proc. Natl. Acad. Sci. USA 89: 10915- 10919) alignments (B) of 50, expectation (E) of 10 (or 1 or 0.1 or 0.01 or 0.001 or 0.0001), M=5, N=4, and a comparison of both strands. One measure of the statistical similarity between two sequences using the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.

[0112] “Variant” as used herein refers to an immunogenic polypeptide in which one or more of the amino acids of the native sequence (SEQ ID NOs:1-14 and 57-60 or fragments thereof having at least 10 amino acids) has / have been modified, but which retains immunogenic properties. The modification may be, for example, a deletion of one or more consecutive or non-consecutive amino acids, a substitution of amino acids, one or more substitution(s) of a naturally occurring amino acid (L-amino acid) by a corresponding D-amino acid, an extension of the sequence by e.g., one, two, three or more amino acids. In an embodiment, the above-mentioned substitution(s) are conserved amino acid substitutions. As used herein, the term "conserved amino acid substitutions" (or sometimes “conservative amino acid substitutions”) refers to the substitution of one amino acid for another at a given location in the immunogenic polypeptide, where the substitution can be made without substantial loss of the relevant structure / function (e.g., ability to induce an immune response). In making such changes, substitutions of like amino acid residues can be made on the basis of relative similarity of side-chain substituents, for example, their size, charge, hydrophobicity, hydrophilicity, and the like, and such substitutions may be assayed for their effect on the structure / function of the immunogenic polypeptide by routine testing.

[0113] In some embodiments, conserved amino acid substitutions may be made where an amino acid residue is substituted for another having a similar hydrophilicity value (e.g., within a value of plus or minus 2.0), where the following may be an amino acid having a hydropathic index of about -1.6 such as Tyr (-1 .3) or Pro (-1 .6) are assigned to amino acid residues (as detailed in U.S. Patent. No. 4,554,101): Arg (+3.0); Lys (+3.0); Asp (+3.0); Glu (+3.0); Ser (+0.3); Asn (+0.2); Gin (+0.2); Gly (0); Pro (-0.5); Thr (-0.4); Ala (-0.5); His (-0.5); Cys (-1.0); Met (-1.3); Vai (-1.5); Leu (-1.8); He (-1.8); Tyr (-2.3); Phe (-2.5); and Trp (-3.4).

[0114] In other embodiments, conserved amino acid substitutions may be made where an amino acid residue is substituted for another having a similar hydropathic index (e.g., within a value of plus or minus 2.0). In such embodiments, each amino acid residue may be assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics, as follows: He (+4.5); Vai (+4.2); Leu (+3.8); Phe (+2.8); Cys (+2.5); Met (+1.9); Ala (+1.8); Gly (-0.4); Thr (-0.7); Ser (-0.8); Trp (-0.9); Tyr (-1 .3); Pro (-1 .6); His (-3.2); Glu (-3.5); Gin (-3.5); Asp (-3.5); Asn (-3.5); Lys (-3.9); and Arg (-4.5).

[0115] In other embodiments, conserved amino acid substitutions may be made where an amino acid residue is substituted for another in the same class, where the amino acids are divided into non-polar, acidic, basic and neutral classes, as follows: non-polar: Ala, Vai, Leu, lie, Phe, Trp, Pro, Met; acidic: Asp, Glu; basic: Lys, Arg, His; neutral: Gly, Ser, Thr, Cys, Asn, Gin, Tyr.

[0116] Conservative amino acid changes can include the substitution of an L-amino acid by the corresponding D-amino acid, by a conservative D-amino acid, or by a naturally-occurring, non- genetically encoded form of amino acid, as well as a conservative substitution of an L-amino acid. Naturally-occurring non-genetically encoded amino acids include beta-alanine, 3-amino-propionic acid, 2,3-diamino propionic acid, alpha-aminoisobutyric acid, 4-amino-butyric acid, / V- methylglycine (sarcosine), hydroxyproline, ornithine, citrulline, t-butylalanine, t-butylglycine, N- methylisoleucine, phenylglycine, cyclohexylalanine, norleucine, norvaline, 2-napthylalanine, pyridylalanine, 3-benzothienyl alanine, 4-chlorophenylalanine, 2-fluorophenylalanine, 3- fluorophenylalanine, 4-fluorophenylalanine, penicillamine, 1 ,2,3,4-tetrahydro-isoquinoline-3- carboxylix acid, beta-2-thienylalanine, methionine sulfoxide, homoarginine, N-acetyl lysine, 2- amino butyric acid, 2-amino butyric acid, 2, 4, -diamino butyric acid, p-aminophenylalanine, / V- methylvaline, homocysteine, homoserine, cysteic acid, epsilon-amino hexanoic acid, delta-amino valeric acid, or 2,3-diaminobutyric acid.

[0117] In other embodiments, conservative amino acid changes include changes based on considerations of hydrophilicity or hydrophobicity, size or volume, or charge. Amino acids can be generally characterized as hydrophobic or hydrophilic, depending primarily on the properties of the amino acid side chain. A hydrophobic amino acid exhibits a hydrophobicity of greater than zero, and a hydrophilic amino acid exhibits a hydrophilicity of less than zero, based on the normalized consensus hydrophobicity scale of Eisenberg et al. J. Mol. Biol. 179: 125-142, 1984). Genetically encoded hydrophobic amino acids include Gly, Ala, Phe, Vai, Leu, lie, Pro, Met and Trp, and genetically, encoded hydrophilic amino acids include Thr, His, Glu, Gin, Asp, Arg, Ser, and Lys.

[0118] Hydrophobic or hydrophilic amino acids can be further subdivided based on the characteristics of their side chains. For example, an aromatic amino acid is a hydrophobic amino acid with a side chain containing at least one aromatic or heteroaromatic ring, which may contain one or more substituents.

[0119] An apolar amino acid is a hydrophobic amino acid with a side chain that is uncharged at physiological pH and which has bonds in which a pair of electrons shared in common by two atoms is generally held, equally by each of the two atoms ( / .e., the side chain is not polar). Genetically encoded apolar amino acids include Gly, Leu, Vai, He, Ala, and Met. Apolar amino acids can be further subdivided to include aliphatic amino acids, which is a hydrophobic amino acid having an aliphatic hydrocarbon side chain. Genetically encoded aliphatic amino acids include Ala, Leu, Vai, and He.

[0120] A polar amino acid is a hydrophilic amino acid with a side chain that is uncharged at physiological pH, but which has one bond in which the pair of electrons shared in common by two atoms is held more closely by one of the atoms. Genetically encoded polar amino acids include Ser, Thr, Asn, and Gin.

[0121] An acidic amino acid is a hydrophilic amino acid with a side chain pKa value of less than 7. Acidic amino acids typically have negatively charged side chains at physiological pH due to loss of a hydrogen ion. Genetically encoded acidic amino acids include Asp and Glu. A basic amino acid is a hydrophilic amino acid with a side chain pKa value of greater than 7. Basic amino acids typically have positively charged side chains at physiological pH due to association with hydronium ion. Genetically encoded basic amino acids include Arg, Lys, and His.

[0122] The above classifications are not absolute, and an amino acid may be classified in more than one category. In addition, amino acids can be classified based on known behavior and or characteristic chemical, physical, or biological properties based on specified assays or as compared with previously identified amino acids. Amino acids can also include bifunctional moieties having amino acid-like side chains.

[0123] The present disclosure includes polypeptides having an amino acid sequence with one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more amino acid changes from an amino acid sequence described herein (SEQ ID NOs:1-14 and 57-60, preferably SEQ ID NOs:1-7 and 57-60, more preferably SEQ ID NOs:1-5 and 57-58), or fragments thereof. Such amino acid changes include, but are not limited to, conservative amino acid changes.

[0124] The immunogenic polypeptide may be fused to another domain, peptide or polypeptide, for example to a domain or peptide favoring the formation of aggregates, nanofilaments or nanofibrils, which typically increases the immunogenicity of polypeptide. Examples of domains or peptides favoring the formation of nanofilaments or nanofibrils include p-sheet-rich quaternary motifs, amyloid-derived peptides (e.g., amyloid-p peptide), coiled-coil motifs, Curli-specific gene A protein and multimerization domains (see, e.g., Zottig et al., Nanomaterials 2020, 10, 1008; doi:10.3390 / nano10051008).

[0125] In another aspect, the present disclosure provides a vaccine composition a vaccine composition comprising one or more nucleic acids encoding one or more of the immunogenic polypeptides or fragments thereof described herein, and at least one vaccine excipient.

[0126] In an embodiment, the one or more nucleic acids comprise one or more of the nucleotides sequences of SEQ ID NOs:15-28, preferably SEQ ID NOs:15-21 , more preferably SEQ ID NOs:15-19, or a fragment thereof.

[0127] In an embodiment, the one or more nucleic acids is / are DNA molecule(s). In another embodiment, the one or more nucleic acids is / are mRNA molecule(s).

[0128] A nucleic acid of the disclosure may be used for recombinant expression of the immunogenic polypeptides or fragments thereof described herein, and may be included in a vector or plasmid, such as a cloning vector or an expression vector, which may be transfected into a host cell. In an embodiment, the disclosure provides a cloning, expression or viral vector or plasmid comprising one or more nucleic acids encoding one or more of the immunogenic polypeptides or fragments thereof described herein. The vector or plasmid contains the necessary elements for the transcription and translation of the inserted coding sequence, and may contain other components such as resistance genes, cloning sites, etc. Methods that are well known to those skilled in the art may be used to construct expression vectors containing sequences encoding peptides or polypeptides and appropriate transcriptional and translational control / regulatory elements operably linked thereto. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described in Sambrook. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y. "Operably linked" refers to a juxtaposition of components, particularly nucleotide sequences, such that the normal function of the components can be performed. Thus, a coding sequence that is operably linked to regulatory sequences refers to a configuration of nucleotide sequences wherein the coding sequences can be expressed under the regulatory control, that is, transcriptional and / or translational control, of the regulatory sequences. "Regulatory / control region" or "regulatory / control sequence", as used herein, refers to the non-coding nucleotide sequences that are involved in the regulation of the expression of a coding nucleic acid. Thus, the term regulatory region includes promoter sequences, regulatory protein binding sites, upstream activator sequences, and the like. The vector (e.g., expression vector) may have the necessary 5' upstream and 3' downstream regulatory elements such as promoter sequences such as CMV, PGK and EF-1 a promoters, ribosome recognition and binding TATA box, and 3' UTR AAUAAA transcription termination sequence for the efficient gene transcription and translation in its respective host cell. Other suitable promoters include the constitutive promoter of simian vims 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), HIV LTR promoter, MoMuLV promoter, avian leukemia virus promoter, EBV immediate early promoter, and Rous sarcoma vims promoter. Human gene promoters may also be used, including, but not limited to the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. In certain embodiments inducible promoters are also contemplated as part of the vectors expressing the TAP. This provides a molecular switch capable of turning on expression of the polynucleotide sequence of interest or turning off expression. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, or a tetracycline promoter. Examples of vectors are plasmid, autonomously replicating sequences, and transposable elements. Additional exemplary vectors include, without limitation, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or Pl-derived artificial chromosome (PAC), bacteriophages such as lambda phage or M13 phage, and animal viruses. Examples of categories of animal viruses useful as vectors include, without limitation, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpesvirus (e.g., herpes simplex virus), poxvirus, baculovirus, papillomavirus, and papovavirus (e.g., SV40). Examples of expression vectors are Lenti-X™ Bicistronic Expression System (Neo) vectors (Contech), pCIneo vectors (Promega) for expression in mammalian cells; pl_enti4 / V5-DEST™, pl_enti6 / V5-DEST™, and pLenti6.2N5-GW / lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells. The coding sequences of the immunogenic polypeptides or fragments thereof described herein can be ligated into such expression vectors for the expression of the immunogenic polypeptides or fragments thereof in mammalian cells.

[0129] The immunogenic polypeptides or fragments thereof may be produced by expression in a host cell comprising a nucleic acid encoding the immunogenic polypeptides or fragments thereof (recombinant expression) or by purification from natural sources, e.g., from a pathogenic C. perfringens strain.

[0130] In an embodiment, the immunogenic polypeptides, fragments thereof, or nucleic acids described herein may be encapsulated, for example in nanoparticles. In some embodiments, nanoparticles may be prepared from mucosal adhesive polymers, including, for example, chitosan, methylglycol chitosan, glycol chitosan, high molecular weight chitosan, collagen, albumin, gelatin, alginates, cyclodextrines, dextran, agarose, hyaluronic acid, starch, cellulose, polylactic acid, polyglycolic acid, polyhydroxyl butyrate, polycaprolactone, poly doxanones, polyadipic acid, polyterphthalicacid, polysebacic acid, poly iminocarbonates, poly amino acids, polyphosphates, polyphosphonates, polyphosphazenes, poly cyanocrylates, poly urethanes, poly ortho esters, polyacetals, or combinations thereof. In some embodiments, other related cross linkers for chitosan nanoparticle formation can also be used, for example: curdlan sulfate, sodium citrate, sulfosuccinic acid, oxalic acid, glutaraldehyde, epichlorohydrin, rimethylpropane triglycidyl ether, ethylene glycol diglycidyl ether, or combinations thereof. In some embodiments, the nanoparticle is a liposome or a lipid nanoparticle. The term “lipid nanoparticle” refers to liposomelike structure that may include one or more lipid bilayer rings surrounding an internal aqueous medium similar to liposomes, or micellar-like structures that encapsulates molecules (e.g. , nucleic acids) in a non-aqueous core. Lipid nanoparticles typically contain cationic lipids, such as ionizable cationic lipids. Examples of cationic lipids that may be used for LNPs include DOTMA, DOSPA, DOTAP, ePC, DLin-MC3-DMA, C12-200, ALC-0315, CKK-E12, Lipid H (SM-102), OF- Deg-Lin, A2-lso5-2DC18, 306Oi10, BAME-O16B, TT3, 9A1 P9, FTT5, COATSOME® SS-E, COATSOME® SS-EC, COATSOME® SS-OC and COATSOME® SS-OP (see, e.g., Hou et al., Nature Reviews Materials, volume 6, pages 1078-1094 (2021); Tenchov et al., ACS Nano, 15, 16982-17015 (2021). Liposomes and lipid nanoparticles typically include other lipid components such as lipids, lipid-like materials, and polymers that can improve liposome or nanoparticle properties, such as stability, delivery efficacy, tolerability and biodistribution. These include phospholipids (e.g., phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, and phosphatidylglycerol) such as 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and DOPE, sterols (such as cholesterol and cholesterol derivatives), PEGylated lipids (PEG-lipids) such as 1 ,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG2000-DMG) and 1 ,2- distearoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG2000-DSG).

[0131] The nanoparticles according to the present disclosure may be any of a variety of sizes. In some aspects, nanoparticles are of a size that is effectively taken up by macrophages and other immune presenting cells through phagocytosis. See, for example, Pratten and Lloyd, 1986, Biochim Biophys Acta, 881 (3):307-13. For example, in some aspects, nanoparticles of the compositions described herein may be about 100 nanometers (nm), about 200 nm, about 300 nm, about 400 nm, about 500 nm, about 600 nm, about 700 nm, about 800 nm, about 900 nm, about 100 nm, about 1100 nm, or any range thereof in size. For example, in some aspects, nanoparticles may be from about 100 nm to about 1 ,100 nm in size, about 200 nm to about 800 nm in size, about 300 nm to about 1100 nm in size, about 100 nm to about 500 nm in size, or about 150 nm to about 200 nm in size. In some aspects, nanoparticles may less than about 500 nm in size.

[0132] In an embodiment, the at least one vaccine excipient comprises a vaccine adjuvant. The term “vaccine adjuvant (or simply "adjuvant") refers to a substance which, when added to an immunogenic agent such as the immunogenic polypeptide or fragment thereof as described herein, nonspecifically improves, enhances or potentiates an immune response to the agent in the host upon exposure to the mixture. Adjuvants can be used to improve, enhance or potentiate the immune response to vaccine antigens for several different purposes, including: (1) increasing the immunogenicity of weak antigens; (2) enhancing the speed and duration of the immune response; (3) modulating antibody avidity, specificity, isotype or subclass distribution; (4) stimulating cell mediated immunity; (5) promoting the induction of mucosal immunity; (6) enhancing immune responses in immunologically immature or senescent individuals; (7) decreasing the dose of antigen in the vaccine to reduce costs.

[0133] Examples of adjuvants commonly used in the field of vaccines include (1) mineral salts (aluminum salts such as aluminum phosphate and aluminum hydroxide, calcium phosphate gels), squalene, (2) oil-based adjuvants such as oil emulsions and surfactant based formulations, e.g., MF59 (microfluidised detergent stabilised oil-in-water emulsion), QS-21 (purified saponin), Quil- A® (saponin), AS02 [SBAS2] (oil-in-water emulsion + MPL + QS-21), (3) particulate adjuvants, e.g., virosomes (unilamellar liposomal vehicles incorporating influenza haemagglutinin), AS04 ([SBAS4] aluminum salt with MPL), ISCOMS (structured complex of saponins and lipids), polylactide co-glycolide (PLG), (4) microbial derivatives (natural and synthetic), e.g., monophosphoryl lipid A (MPL), Detox (MPL + M. Phlei cell wall skeleton), AGP [RC-529] (synthetic acylated monosaccharide), DC_Chol (lipoidal immunostimulators able to self-organize into liposomes), OM-174 (lipid A derivative), CpG motifs (synthetic oligonucleotides containing immunostimulatory CpG motifs), modified Cholera toxin (CT) and Escherichia coli enterotoxin (LT) (genetically modified bacterial toxins to provide non-toxic adjuvant effects), (5) endogenous human immunomodulators, e.g., hGM-CSF or hlL-12 (cytokines that can be administered either as protein or plasmid encoded), Immudaptin (C3d tandem array) and / or (6) inert vehicles, such as gold particles, and the like.

[0134] Several adjuvants for veterinary applications are commercially available such as the oil- in-water (O / W) emulsion adjuvants Montanide ISA, Emulsigen® (MVP Technologies, USA), CAvant® WO-60 (CAVAC, Daejeon, Korea)) and MetaStim® (Fort Dodge Laboratories, USA), saponin-based adjuvants such as Quil-A® and QS-21 , polymer-based adjuvants such as Carbopol® (polyacrylic acid polymers (carbomers)), Polyoxidonium® (copolymer of 1 ,4- ethylenepiperazine, 1 ,4-ethylenepiperazine-N-oxide, and (N-carboxymethylene)-1 ,4- ethylenepiperazinium bromide), as well as other adjuvants such as TiterMax® Gold Adjuvant from Sigma-Aldrich (a mixture of a block copolymer, CRL-8300, squalene (a metabolizable oil) and a sorbitan monooleate).

[0135] The vaccine may comprise a combination of 2 or more adjuvants.

[0136] In an embodiment, the adjuvant comprises a saponin-based adjuvant. In a further embodiment, the saponin-based adjuvant is Quil-A® (a saponin mixture derived from an aqueous extract from the bark of Quillaja Saponaria) or QS21 (a fraction from Quil-A®).

[0137] Saponin-based adjuvants are sometimes combined with cholesterol and phospholipids in immune-stimulating complexes (ISCOMs). The vaccine according to the present disclosure may comprise other vaccine excipients, such as a suitable solvent, for example an aqueous buffer or a phosphate buffer. The vaccines of the present disclosure may further be formulated with one or more further additives to maintain isotonicity, physiological pH and stability, for example, a buffer such as physiological saline (0.85%), phosphate-buffered saline (PBS), citrate buffers, Tris (hydroxymethyl aminomethane (TRIS)), Tris-buffered saline and the like, or an antibiotic, for example, neomycin or streptomycin.

[0138] In a particular embodiment, the vaccine comprises the following excipients: LABRAFAC™ (Caprylic / Capric Triglyceride), cholesterol, and Quil-A®. In another embodiment, the vaccine comprises the following excipients: LABRAFAC™, CARBOPOL™ (acrylic acid polymer), saline, cholesterol, ethanol, Quil-A® and sodium hydroxide. In a further embodiment, the vaccine comprises the following excipients: LABRAFAC™, CARBOPOL™ 974P, saline, vegetable-derived cholesterol, ethanol, Quil-A®, and sodium hydroxide. In a particular embodiment, the vaccine comprises the following excipients: LABRAFAC® Lipophile WL1349 and CARBOPOL® 974P NF Polymer.

[0139] The compositions and vaccines described herein may be administered as the active component to immunize a subject to elicit an immune response.

[0140] Thus, in another aspect, the present disclosure provides a method for inducing or eliciting an immune response against Clostridium perfringens in an animal comprising administering to the animal an effective amount of the compositions and vaccines described herein. The present disclosure also provides the use of the compositions and vaccines described herein for inducing or eliciting an immune response against Clostridium perfringens in an animal. The present disclosure also provides the use of the compositions and vaccines described herein for the manufacture of a medicament for inducing or eliciting an immune response against Clostridium perfringens in an animal. The present disclosure also provides the compositions and vaccines described herein for use for inducing or eliciting an immune response against Clostridium perfringens in an animal.

[0141] Inducing or eliciting an immune response may include the induction of a higher level of protection in a population after vaccination compared to an unvaccinated group. The immune response may, or may not, confer protective immunity. An immune response may, for example, include one or more of a cell mediated immune response, which involves the production of lymphocytes in response to exposure to the antigen and / or a humoral immune response, which involves production of plasma lymphocytes (B cells) in response to antigen exposure with subsequent antibody production. Immunization may result in the reduction, inhibition, or prevention of one or more of the symptoms of Clostridium perfringens infection and necrotic enteritis. Such symptoms may include one or more of death, body weight suppression, decrease in egg production, reductions in growth and feed conversion rates, mortality, damage to the intestinal epithelium, necrotic lesions in the intestinal epithelium, severe depression, decreased appetite, diarrhea, closed eyes, and ruffled feathers.

[0142] Thus, in another aspect, the present disclosure provides a method for preventing or treating infection by Clostridium perfringens in an animal comprising administering to the animal an effective amount of the compositions and vaccines described herein. The present disclosure also provides the use of the compositions and vaccines described herein for preventing or treating infection by Clostridium perfringens in an animal. The present disclosure also provides the use of the compositions and vaccines described herein for the manufacture of a medicament for preventing or treating infection by Clostridium perfringens in an animal. The present disclosure also provides the compositions and vaccines described herein for preventing or treating infection by Clostridium perfringens in an animal. in another aspect, the present disclosure provides a method for preventing or treating necrotic enteritis (NE) in an animal comprising administering to the animal an effective amount of the compositions and vaccines described herein. The present disclosure also provides the use of the compositions and vaccines described herein for preventing ortreating NE in an animal. The present disclosure also provides the use of the compositions and vaccines described herein for the manufacture of a medicament for preventing or treating NE in an animal. The present disclosure also provides the compositions and vaccines described herein for preventing or treating NE in an animal.

[0143] The term “preventing” as used herein refers to the administration of the compositions and vaccines described herein to an animal that is not yet infected by Clostridium perfringens (prophylactic administration) to prevent or delay infection, to prevent or delay the development of a Clostridium perfringens related disease (e.g., avian NE), to prevent or reduce the severity of one or more symptoms of Clostridium perfringens infection and / or avian NE, and / or to prevent or reduce the risk of death caused by Clostridium perfringens infection and / or avian NE.

[0144] The term “treating” as used herein refers to the administration of the compositions and vaccines described herein to an animal that is already infected by Clostridium perfringens (therapeutic administration) to prevent or delay the development of a Clostridium perfringens related disease (e.g., avian NE), to prevent or reduce the severity of one or more symptoms of Clostridium perfringens infection and / or avian NE, and / or to prevent or reduce the risk of death caused by Clostridium perfringens infection and / or avian NE.

[0145] The route of administration can be any route including oral (e.g., gel drop, in feed, in water), ocular (e.g., by eyedrop), oculo-nasal administration using aerosol (e.g., spray), intranasal, cloacal, in ovo, or by injection (e.g., intravenous, subcutaneous, intramuscular, intradermal, and / or intraperitoneal) vaccination. The skilled person will easily adapt the formulation of the vaccine composition for each type of route of administration. Compositions and vaccines of the present disclosure may be administered to birds of any of a variety of avian species that are susceptible to infection with Clostridium perfringens.

[0146] The term “avian” is intended to encompass all kinds of avians, such as birds of the class of Aves, i.e., vertebrate animals which are feathered, winged, bipedal, endothermic and egglaying. In the context of the invention, avians or avian species refer more particularly to birds with economical and / or agronomical interests, such as poultry (such as chickens, turkeys, hens, guinea fowl, quail, partridge and pigeon), waterfowl poultry (such as ducks and geese) and ornamental birds (such as swans, parrot and psittacines). Chickens include, but are not limited to, hens, roosters, broilers, roasters, layers, breeders, the offspring of breeder hens, and layers.

[0147] As used herein, the term “susceptible to” means the possibility or actuality of a detrimental response to the referenced microorganism, when compared to a non-susceptible individuals or groups, and / or one or more pathological state(s) indicative of Clostridium perfringens infection and / or necrotic enteritis.

[0148] The vaccines and compositions of the present disclosure may be administered to poultry before or after hatching. Poultry may receive a vaccine at a variety of ages. For example, broilers may be vaccinated in ovo, at one-day-old, or at 2-3 weeks of age. Laying stock or reproduction stock may be vaccinated, for example, at about 6-12 weeks of age and boosted at about 16-20 weeks of age. Such laying stock or reproduction stock may be vaccinated at about 6, at about 7, at about 8, at about 9, at about 10, at about 11 , or at about 12 weeks of age. Such laying stock or reproduction stock may be boosted at about 16, at about 17, at about 18, at about 19, or at about 20 weeks of age. The offspring of such laying stock or reproduction stock may demonstrate an antibody titer to an immunogenic polypeptide as described herein, which may prevent or mitigate the symptoms of a Clostridium perfringens infection and / or necrotic enteritis in the offspring. In ovo vaccination may take place, for example, at about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, or at any range thereof.

[0149] Chickens may be vaccinated at any suitable age and are usually about one to three days old before first vaccination. The chickens may be vaccinated only once. Or, if two doses of vaccine are used, the first is given, for example, when the chickens are 3 days to a week old and subsequently after a further 1-10 weeks. Multiple doses of the vaccine or composition can be administered throughout the life of the chicken. As maternal immunity is a primary source of providing protection to broiler progeny, breeder chickens may be vaccinated.

[0150] EXAMPLES

[0151] Example 1 : Materials and Methods Bioinformatics analyses. The immunogenicity of the vaccine candidate proteins was predicted earlier

[0031] using the Vaxijen v2.0 tool (Irini A Doytchinova and Darren R Flower. BMC Bioinformatics. 2007; 8: 4. Published online 2007 Jan 5. doi: 10.1186 / 1471-2105-8-4) with a threshold of 0.5. For each candidate protein, the GRAVY score which represents the hydrophobic nature of the protein was calculated using the Sequence Manipulation Suite (https: / / www.bioinformatics.org / sms2 / protein_gravy.html; Stothard P (2000) The Sequence Manipulation Suite: JavaScript programs for analyzing and formatting protein and DNA sequences. Biotechniques 28:1102-1104) and the prediction of both signal peptides and transmembrane regions was also made using the SignalP 5.0 (https: / / services. healthtech. dtu.dk / service. php?SignalP-5.0; Jose Juan Almagro Armenteros et al., Nature Biotechnology, 37, 420-423, doi: 10.1038 / S41587-019-0036-z (2019)) and TMHMM 2.0 (https: / / services. healthtech. dtu.dk / services / TMHMM-2.0 / ; E. L.L. Sonnhammer et al., A hidden Markov model for predicting transmembrane helices in protein sequences. In J. Glasgow, T. Littlejohn, F. Major, R. Lathrop, D. Sankoff, and C. Sensen, editors, Proceedings of the Sixth International Conference on Intelligent Systems for Molecular Biology, pages 175-182, Menlo Park, CA, 1998. AAAI Press) tools from DTU Health Tech. A BLAST search was done to compare the resultant sequences with in silico identified proteinencoding genes. One candidate with a low predicted antigenic score was included as a negative control to validate in vivo the reverse vaccinology pipeline used in this paper.

[0152] Validation of in silico studies, amplification, and cloning of candidate protein-encoding genes. To validate in silico studies, forward and reverse primers were designed for each candidate protein-encoding gene. The list and sequence of primers used are presented in Table 2 below.

[0153] Table 2. Selected candidate proteins with their respective primer sequences and amplicon size after PCR amplification.

[0154] Genomic DNA was extracted from a virulent C. perfringens strain from our collection, MLG_7820, by the Chelex® DNA extraction method. Briefly, C. perfringens MLG_7820 was grown overnight on 5% sheep blood agar plates (Fisher, Ottawa, ON) at 37°C under anaerobic condition (Oxoid AnaeroGen gas packs (Thermo Fisher Scientific, MA, USA)). Seven to ten colonies were suspended in 1 mL of ddH2O, followed by centrifugation at 10000 x g for 2 minutes (Eppendorf 5415C centrifuge). A 200 pL volume of a 10% Chelex® 100 solution (BioRad, CA, catalog no. 143-2832) was added to the pellet and samples were vortexed before being heated at 56°C for 30 minutes (Dry Bath Incubator, Fisher Scientific) with a subsequent boiling step in water for 10 minutes. The DNA was collected after centrifugation at 13,000 x g for 2 minutes and used for the amplification of the candidate protein-encoding genes by PCR. A unique PCR program was used for all genes and cycling conditions were as follows: a hot start step of 2 minutes at 95°C, followed by 30 cycles of 20 seconds at 95°C, 20 seconds at 41.3°C, and 2 minutes at 72°C, with a final elongation step of 10 minutes at 72°C. A volume of 5 pL of each PCR product was analyzed by migration on a 1% (w / v) agarose gel containing 0.01% SYBR Safe DNA gel stain (Fisher, Ottawa, ON), followed by visualization under UV light and using a Tracklt™ 1 Kb Plus DNA ladder (Invitrogen, Carlsbad, CA). Sequencing of PCR products was conducted at McGill University (Quebec, Canada) and Genome Quebec Innovation Centre (Montreal, QC, Canada), before cloning into pET151 / D-TOPO vector using E. coli TOP10 cells (Champion™ pET151 Directional TOPO Expression Kit, Invitrogen, CA, USA) according to the manufacturer’s instructions. Ten colonies were picked up randomly and analyzed by colony PCR to screen for transformants that contained the recombinant plasmid. Briefly, single colonies were transferred to new LB agar plates supplemented with 100 pg / mL ampicillin (Fisher Scientific New Jersey, USA), followed by overnight incubation at 37°C. One loop of bacterial cells was collected and suspended in 1 mL of ddH2O, followed by vortex and centrifugation at 10000 x g for 3 minutes. A 200 pL volume of 10% Chelex® 100 was added to the pellet, with a subsequent vortex and boiling in water for 10 minutes. Samples were then centrifuged at 10000 x g for 2 minutes and 3 pL of supernatant was used as a template for PCR amplification using the same primers and PCR program described above. Positive transformants were sent for direct colony sequencing to the Centre Hospitalier Universitaire de Quebec-Universite Laval Research Center (Quebec, Canada). Recombinant plasmids were extracted using the QIAprep™ Spin Miniprep Kit (Qiagen, Hilden, Germany, catalog no. 27106) according to the manufacturer’s instructions. For the candidate protein P509, synthesis and cloning into pET- 24a(+) vector was done by GenScript (Piscataway, NJ, USA). To do that, the protein-encoding gene was first synthesized by Oligo Synthesizer (Biolytic Lab Performance, Inc. Fremont, USA) and then cloned into the vector using BamHI and Nde\ enzymes.

[0155] Expression and purification of candidate proteins. Ten ng of plasmid DNA was transformed into BL21 Star™(DE3) One Shot® cells via a heat-shock method according to the manufacturer’s instructions (Invitrogen, CA, USA, catalog no. K151 -01). One single transformed colony was grown in 30 mL of Luria Bertani broth (BD Difco, MD, USA, catalog no. DF0446- 07-5) supplemented with 100 pg / mL ampicillin (Fisher Scientific New Jersey, USA) for 17 hours, at 37°C with shaking (180 rpm). The overnight culture was then added to 600 mL Luria Bertani broth (supplemented with 100 pg / mL ampicillin) and incubated for 2 hours at 37°C with shaking (180 rpm) until an OD600 of 0.6 was reached. The expression of recombinant proteins was induced by the addition of isopropyl p-D-1 -thiogalactopyranoside (IPTG) (Invitrogen, CA, USA, catalog no. 15529019) to a final concentration of 1 mM. After five hours of incubation, all cultures were harvested by centrifugation at 3000 x g, 4°C for 20 minutes (Thermo Scientific Sorvall Legend XTR with F14-6 x 250y Fixed-Angle Rotor). The pellet was preserved at -20°C until purification. Purification of all proteins, except for P561 , was conducted under native conditions since they were all soluble proteins. To do so, the stored pellet was resuspended in 20 mL of lysis buffer (50 mM NaH2PO4, 300 mM NaCI, 10 mM imidazole, pH 8) supplemented with 1X protease inhibitor cocktail (Roche, Mannheim, Germany), and lysozyme ((1 mg / mL) Thermo Scientific, MA, USA, catalog no. J60701), followed by sonication on ice using a sonicator with a microtip probe (Ultrasonic processor, model: GE130). A protocol involving six 10 seconds bursts at 250 W with 10 seconds cooling intervals between each burst was used. The lysate was then centrifuged (15000 x g, 4°C, 20 minutes (Thermo Scientific Sorvall Legend XTR with Fiberlite™ F15-8 x 50cy Fixed Angle Rotor)) and the supernatant was loaded onto a 5 mL HisTrap™ HP column (GE Healthcare, Montreal, CA, catalog no. 17-5248-02) preequilibrated with the same lysis buffer mentioned above. His-tagged proteins were purified using a FPLC AKTA-purifier system (GE-Healthcare, Uppsala, Sweden) and a 50-500 mM imidazole gradient (Sigma-Aldrich, USA, catalog no. 792527). One mL fractions were collected, and since proteins showed strong absorption in the ultraviolet (UV) region at UV 280 nm, fractions showing a 280 nm peak were subjected to SDS-PAGE. Samples containing the protein of interest were desalted using PD-10 desalting columns packed with Sephadex™ G-25 resin (GE Healthcare, USA, catalog no. 17-0851-01). For the purification of P561 under denaturing conditions, the pellet was resuspended in 20 mL of lysis buffer (100 mM NaH2PO4, 10 mM Tris Ci, 8 M urea, pH 8) and stirred (Barnstead Thermolyne, Roto Mix-Type 50800) for 60 minutes at room temperature before being sonicated as described above. The lysate was centrifuged at 10000 x g for 30 minutes at room temperature (Thermo Scientific Sorvall Legend XTR with Fiberlite™ F15-8 x 50cy Fixed Angle Rotor) and the supernatant was loaded onto a 5 mL HisT rap HP column in the AKTA-purifier system. The protein of interest was eluted with an elution buffer (100 mM NaH2PO4, 10 mM Tris Ci, 8 M urea, pH 4.5), after washing the column with wash buffer (100 mM NaH2PO4, 10 mM Tris Ci, 8 M urea, pH 6.3). Individual fractions were collected, analyzed by SDS-PAGE, and desalted using PD-10 desalting columns. Quantitation of the purified candidate proteins was performed using the Qubit Protein Assay kit (Invitrogen, CA, USA, catalog no. Q33211) and Denovix QFX Fluorometer (Froggabio, Toronto, ON), according to the manufacturer’s instructions. Aliquots of proteins were stored at -80°C until used in the in vivo assays.

[0156] Preparation of whole-cell lysate from C. perfringens MLG_7820. C. perfringens cells were grown at 37°C in 30 mL of a medium made of 25% nutrient broth (EMD Chemicals Inc., Gibbstown, NJ), 50% tryptic soy broth (BD Biosciences, Franklin, NJ), and 25% peptone water (Biokar Diagnostics, France) under anaerobic conditions. The resulting culture was harvested after 24 hours and cells were pelleted by centrifugation (5000 x g, 4°C, 20 minutes (Thermo Scientific Sorvall Legend XTR with Fiberlite™ F15-8 x 50cy Fixed Angle Rotor)). The recovered pellet was then resuspended in 5 mL of phosphate-buffered saline (PBS), followed by eight repeated freeze-thaw cycles in liquid nitrogen

[0055] , The protein concentration was measured (Qubit™ Protein Assay kit and Denovix QFX Fluorometer), and aliquots of proteins were stored at -80°C until the vaccination trial.

[0157] Birds housing and feeding. This project was accepted and carried out under the guidelines of the Animal Ethics Committee of the Faculte de Medecine Veterinaire of the Universite de Montreal (certificate #21 -Rech-2070) and under the ARRIVE guidelines

[0056] , In this study, 140 commercial day-old male Ross broiler chickens bought directly from a commercial hatchery were randomly divided into seven experimental groups (n = 20): 1) Adjuvant-only (Quil- A), 2) P561 , 3) P509, 4) P537, 5) P153, 6) P264, and 7) Hyperimmunized group (wholecell lysate from C. perfringens MLG_7820). Over a period of 35 days, birds were fed a commercial diet void of any antibiotics and anticoccidials (Meunerie Benjamin, St-Cesaire, Quebec, Canada) and corresponding to the starter, grower, and finisher stages of a regular commercial broiler diet. Birds were euthanized at 35 days of age using cervical dislocation with prior sedation with 0.8 mL / kg of a xylazine (100 mg / mL) I ketamine (100 mg / mL) blend.

[0158] Preparation of recombinant proteins and immunization of broiler chickens. All recombinant proteins were prepared early in the morning. Briefly, proteins were thawed on ice and the concentration of each candidate was measured using the Qubit Protein Assay kit (Invitrogen, CA, USA, catalog no. Q33211) and Denovix™ QFX Fluorometer (Froggabio, Toronto, ON), according to the manufacturer’s instructions. Each bird was immunized intramuscularly in the pectoral muscle with 200 pL of PBS containing each recombinant protein (50 pg) and Quil-A™ adjuvant ((50 pg) InvivoGen, CA, USA) at days 7, 14, and 21 days of age. Birds from the hyperimmunized group were vaccinated with 200 pL of PBS containing 50 pg of proteins from the whole cell lysate preparation and Quil-A™ adjuvant (50 pg). Birds from the adjuvant control group received 50 pg of Quil-A™ in a 200 pL volume of PBS. Blood samples were collected from all birds priorto each immunization and at day 35 (end of the trial) in blood collection tubes (Covidien, Monoject blood collection tube, MA, USA, catalog no. 8881301215). Blood tubes were maintained at room temperature for 3 to 4 hours and then centrifuged (3000 rpm, 19°C, 10 minutes, Beckman Coulter with SX4750A Rotor) before the serum was collected. Serum samples were stored at -20°C until analysis by ELISA.

[0159] Western blot analysis of recombinant proteins. Recombinant proteins were separated on a 15% gel using an SDS-PAGE approach under reducing conditions, followed by a transfer onto a PVDF membrane (BIO-RAD, CA, USA, catalog no. 1620177) at 100 for 1 hour in IXtransfer buffer (48 mM Tris, 39 mM glycine, 20% methanol, 0.1% SDS). The membrane was blocked with blocking buffer (5% skim milk, 1% TBS, 0.1% Tween™-20) with gentle agitation for 45 minutes at room temperature. The membrane was then incubated with StrepTactin-Horseradish Peroxidase (HRP) conjugate (Bio-Rad, USA; (previously diluted 1 / 5 in ddH2O and added at a ratio of 2:15000 in blocking buffer)) to detect the protein ladder (Precision Plus Protein WesternC standards; Bio-Rad, USA), and Anti-V5-HRP Antibody (Invitrogen, USA; (at the same proportion described above)) to identify the protein of interest, with shaking for 1 hour at room temperature.

[0160] To investigate whether the raised antibodies in broiler chickens can recognize the recombinant form of the candidate proteins, the generated antibodies in birds (diluted 1 :200 in blocking buffer) and goat anti-chicken IgY horseradish peroxidase (HRP)-conjugated polyclonal antibody (Bethyl Laboratories, Montgomery, TX, USA; (diluted 1 :5000 in blocking buffer)) were submitted to the same procedure explained above as the primary and secondary antibodies, respectively, with one hour of incubation at room temperature for each step. A chemiluminescent detection was performed with Clarity Western ECL Substrate (BIO-RAD, CA, USA, catalog no. 1705060) according to the manufacturer’s instructions (Fusion FX imaging system (Vilber Lourmat; SU, Germany)). Measurement of serum antibody levels by ELISA. Antibody titers against the recombinant proteins as well as the whole-cell lysate proteins of C. perfringens MLG_7820 were analyzed by ELISA. To do that, the recombinant purified proteins or whole-cell lysate proteins of C. perfringens MLG_7820 were diluted to 5 pg / mL in 50 mM carbonate / bicarbonate coating buffer (pH 9.6), and 100 pL of this dilution was added to each well of a 96 well flat-bottom immuno plate MaxiSorp™ (Thermo Scientific, NY, USA, catalog no. 475094). Plates were then incubated for 1 hour at 37°C, followed by an overnight incubation at 4°C. After being washed three times with washing buffer (phosphate-buffered saline [PBS] containing 0.05% Tween™ 20), plates were blocked (blocking buffer made of PBS containing 0.05% Tween™ 20 and 0.3% casein (Sigma- Aldrich, USA, catalog no. C7078)) and incubated for 2 hours at 37°C. The plates were washed again three times with the same washing buffer before all the wells were coated with 100 pL of serum sample, using a two-fold serial dilution series (ranging from 1 :50 to 1 : 3,276,800) in blocking buffer. After incubation for 2 hours at 37°C and three washes with washing buffer, 100 pL of goat anti-chicken IgY horseradish peroxidase (HRP)-conjugated polyclonal antibody ((Bethyl Laboratories, Montgomery, TX, USA, catalog no. A30-104P); diluted 1 :8000 in wash buffer) was added to each well, followed by an incubation step at 37°C for 1 hour and three washes (wash buffer). Subsequently, 100 pL of 3,3',5,5'-tetramethylbenzidine (TMB; Life Technologies, Inc., CA, USA) substrate was applied to each well and plates were incubated in the dark at room temperature. The absorbance was measured periodically at 650 nm until the OD of the positive control (serum collected from birds immunized with the whole cell lysate from C. perfringens MLG_7820) reached 0.45. At this point, 100 pL of 0.18 M H2SO4was added to each well and the final OD reading was taken at 450 nm by a spectrophotometer (EZ Read 400, Biochrom, UK), according to the manufacturer’s instructions.

[0161] Statistical analysis. In this study, data distributions were not normal (Shapiro's tests < 0.05) but were rather of the Gamma type. Consequently, we used generalized linear models (GLMs) with Gamma family for statistical analysis of ELISA results generated from serum samples collected at days 7, 14, 21 , 35 days of age. Results were then extracted via likelihood ratio tests (LRTs). Tukey’s post-hoc tests with Benjamini-Hochberg correction on the P-values was carried out due to multiple comparisons. A Mann-Whitney test was used for ELISA results obtained from the analysis of serum samples on days 7 and 35 against the whole cell lysate of C. perfringens MLG_7820. The non-parametric Wilcoxon test was used to compare the antibody titers of Quil-A™ control group on days 14 and 35 against the whole cell lysate of C. perfringens MLG_7820 as well as the comparison between the antibody titers of Quil-A™ and vaccinated groups on day 35 against the whole cell lysate of C. perfringens MLG_7820, followed by the Bonferroni correction due to multiple comparisons. All the statistical analyzes were carried out with the software R Version 4.0.3 (R Core Team, 2020)

[0057] and the significance a threshold was set at 0.05.

[0162] Example 2: Bioinformatics analysis.

[0163] Using a comparative and subtractive reverse vaccinology (CSRV) approach on sixteen C. perfringens strains isolated from either healthy or NE-affected broiler chickens, fourteen proteins unique to six NE-causing C. perfringens strains and absent from ten commensal C. perfringens strains from this same collection were previously identified

[0031] ,

[0164] Further bioinformatics analyses were performed to assess the immunogenic potential of the proteins depicted in Table 3.

[0165] Table 3: Candiate antigenic proteins identified from NE-causing C. perfringens

[0166] Bioinformatics analyses performed on P509 predicted a transmembrane helix between residues 12-34 and an N-terminal signal peptide which cleaved between positions 47 and 48. One transmembrane helix was also predicted between residues 13-35 in P561. The GRAVY score attributed to the full-length sequence of P509 and P561 was -0.026 and -0.33, respectively. When excluding transmembrane regions, these scores were modified to -0.286 and -0.732 for P509 and P561 , respectively, which means that the truncated forms for P509 and P561 were approximately ten and two times more soluble than the full-length protein, respectively. Two transmembrane helices at positions 7-29 and 668-690 were found in the sequence of P2091 , while the sequence of P1074 revealed one transmembrane helix at position 331-350. The GRAVY score of P1074 and P2091 were -0.46 and -0.44, respectively. On the other hand, no transmembrane helices and signal peptide were found in P153, P264, and P537. The GRAVY score attributed to P153, P264, and P537 were -1.212, -0.917, and -0.816, respectively. The VaxiJen score for all proteins, except for P537 which was selected on the basis of its low immunogenic score as a negative control to be included in the in vivo immunization assay, was above 0.5. These proteins were then considered as candidate antigens for immunization. Vaxijen scores are presented in Table 4 below.

[0167] Table 4. Name, size, cell localization, and VaxiJen score of candidate proteins that were produced.

[0168] Name Size Location VaxiJen

[0169] (KDa) Score

[0170] P153 5.6 Extracellular / Cytoplasmicmembrane 0.69

[0171] P264 9.2 Extracellular / Cytoplasmic 1.47

[0172] P509 17.3 Extracellular 0.86

[0173] P537 21 Cytoplasmic 0.21

[0174] P561 (truncated 17 Extracellular 0.64 form)

[0175] Example 3: Validation of in silico studies, amplification, and cloning of candidate protein-encoding genes.

[0176] PCR results confirmed the presence of all 14 candidate protein-encoding genes, identified in the previous study, in each of the six virulent strains of C. perfringens screened. A BLAST alignment revealed 100% sequence similarity between 10 protein-encoding gene amplicons derived from virulent C. perfringens strains and in silico identified protein-encoding genes. PCR results also confirmed the existence of half of the candidate protein-encoding genes in commensal strains of C. perfringens (see FIG. 6). The BLAST alignment showed 97%-99% sequence homology between these seven protein-encoding gene amplicons derived from commensal C. perfringens strains and in silico identified protein-encoding genes (see Table 4 below). These candidate proteins were then excluded from further analysis on the basis of the ubiquitous presence of C. perfringens in the gastrointestinal tract of broiler chickens and on the probability of this DNA similarity in protein sequences to lead to the expression of proteins showing identical functions (Table 5 below). When taking PCR and sequencing results together, seven candidate proteins were identified as promising vaccine candidates because they do not seem to be expressed in commensal strains of C. perfringens. P153, P264-2, P509, P537, P561 , P1074, and P2091 . Among them, five candidate protein-encoding genes were successfully cloned into pET151 / D-TOPO® vector.

[0177] Table 5. Name of candidate protein-encoding genes and the DNA and protein similarity percentage between in silica identified protein-encoding genes and amplicons derived from commensal C. perfringens strains.

[0178] Example 4: Expression and purification of candidate proteins

[0179] Different amounts of purified proteins varying between 2 and 3 pg / pL were obtained: 2.5 pg / pL for P537, and 3 pg / pL and 2 pg / pL for P153 and P264, respectively. Despite several attempts to optimize protein expression for P2091 , P509, and P561 , low amounts of these expressed proteins were obtained and the presence of other contaminants also eluting with the 10 mM imidazole concentration used prevented measurement of the concentrations specific to these recombinant proteins. Transmembrane regions-encoding sequences (nucleotides 1-108 encoding the first 36 amino acids) were excluded from P561 gene sequence that led to an increase in the yield of expression to 17 pg / pL. Even after the deletion of the transmembrane region in P561 , the purification of this candidate protein in sufficient amounts under native conditions was not successful. Thus, the purification of this candidate protein was conducted under denaturing conditions. The full-length form of the P509 protein was used to immunize birds, although the solution contained some contaminants (FIG. 1C). The protein concentration of the whole-cell lysate obtained from C. perfringens MLG_7820 was 14.6 pg / pL. The name, size, location, and VaxiJen score of the proteins that were produced and used for the immunization trial are presented in Table 3 above.

[0180] Results of Western blotting indicated that the eluted purified proteins were detected by Anti-V5-HRP Antibody (FIGs. 2A-C). Serum samples collected from vaccinated birds at day 35 revealed the presence of antibodies able to recognize their respective recombinant protein (FIGs. 3A-C).

[0181] Example 5: Measurement of serum antibody levels by ELISA following immunization

[0182] Statistical analyzes showed a significant interaction between time and candidate proteins (GLM: P-value < 0.001), indicating different responses over time between different candidates (FIGs. 4A-E). ELISA results evaluating antibody levels showed significant differences in the serum antibody response (Post-hoc tests: P-values < 0.001) on days 14, 21 , and 35 in comparison to day 7 for the birds immunized with P537. A similar trend was also observed for birds from group P264 (Post-hoc tests: P-values < 0.01), whereas a third immunization was able to significantly increase antibody response in birds immunized with the other candidate proteins. Globally, the fold-change in IgY titers between day 7 and day 35 in immunized birds varied from 8.8, 16.8, 23.8, 53, and 9110 for the P153, P561 , P264, P509, and P537 groups, respectively. The highest mean IgY titers were documented in birds from groups P509 (8.28E+04 and 2.41 E+05) and P537 (5.08E+05 and 9.11 E+05) on days 21 and 35, respectively. Birds from these two groups however presented with distinct fold- changes in IgY titers between day 7 and day 35, this foldchange being more than 170 greaterforthe birds immunized with P537. The average IgY antibody titers on days 21 and 35 were the lowest in birds immunized with P153 and P561 , although a significant difference was noted between these days and days 14 and 7 in birds from groups P153 and P561 , respectively (Post-hoc tests: P-values < 0.01). It should be pointed out that P561 was the only protein that was purified under denaturing conditions in order to improve its solubility, and this denaturation might have acted as a contributing factor to the low levels of specific IgY antibodies generated in immunized birds [39, 40],

[0183] The assessment of the antibodies raised in immunized birds using ELISA confirmed their ability to recognize the recombinant form of their related candidate protein (FIGs. 4A-E). ELISA results revealed IgY antibody titers showing fold changes ranging from 8.8 to 9110 between the first immunization and the end of the trial at 35 days of age, with IgY antibody titers varying from 1 .0E+02 to 4.54E+03 at 7 days of age and from 1 .47E+03 to 9.11 E+05 at time of slaughter, an increase that was shown to be statistically significant for all the candidate proteins evaluated (P153, P509, P264, and P561 (Post-hoc tests: P-values < 0.01), P537 (Post-hoc tests: P-values « 0.001)). The IgY titers documented in birds from the Quil-A™ control group at day 35 were inconsequential (FIG. 4F). To document the capacity of the IgY antibodies raised in birds immunized with the recombinant form of the different candidate proteins to recognize the native forms of these proteins, the same ELISA approach was used, except that wells of the ELISA plate were coated with the whole cell lysate prepared from C. perfringens MLG_7820. IgY antibody titers varied from 8.5E+02 to 1 .05E+03 for the different groups at day 7 (which was considered as nonspecific background), whereas these same titers showed minimum and maximum values of 1.34E+04 and 3.5E+04 at day 35 (FIGs. 5A-E). According to the group, titers showed fold changes ranging from 12.8 to 41.2 between days 7 and 35. Regarding the Quil-A group, the average IgY antibody titers on days 14 and 35 were 7.64E+02 and 4.75E+03, and even if a slight increase was shown, it revealed not to be significant (Wilcoxon: P-value < 0.001). Average IgY antibody titers at day 35 measured from the birds immunized with the different candidate proteins were all found to be significantly higher when compared to the titers generated in birds receiving Quil-A™ (Wilcoxon: P-values varying from < 0.001 to 0.007 (Table 6 below).

[0184] Table 6. Comparison between the antibody titers of Quil-A™ control group and groups immunized with the candidate proteins, on day 35, against the whole cell lysate of C. perfringens MLG_7820, and their respective p-values.

[0185] Compared groups Adjusted P-value (Bonferroni method)

[0186] Quil-A™ vs. P153 0.007

[0187] Quil-A™ vs. P264 0.001

[0188] Quil-A™ vs. P537 < 0.001

[0189] Quil-A™ vs. P509 0.001

[0190] Quil-A™ vs. P561 0.007

[0191] As will be understood by a skilled person, other variations and combinations may be made to the various embodiments of the invention as described herein above.

[0192] The scope of the claims should not be limited by the preferred embodiments set forth herein above; but should be given the broadest interpretation consistent with the description as a whole.

[0193] The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.

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Claims

CLAIMS:1 . A vaccine composition comprising one or more immunogenic polypeptides comprising one or more of the amino acid sequences set forth in SEQ ID NOs: 1-14 and 57-60, or an immunogenic fragment thereof comprising at least 10 amino acids, and at least one vaccine excipient.

2. The vaccine composition of claim 1 , wherein the one or more immunogenic fragments comprises at least 15 amino acids.

3. The vaccine composition of any one of claims 1 to 3, wherein the one or more immunogenic polypeptides or immunogenic fragments thereof comprise one or more of the following epitope sequences: (a) residues 6-19, 22-35, 41-54, 56-69, 72-85, 10-23, 54-67, 69-82, 5-18, and 51-64 of SEQ ID NO:1 ; (b) residues 13-26, 37-50, 15-28, 32-45, and 30-43 of SEQ ID NO:2; (c) residues 29-42, 44-57, 69-82, 85-98 and 115-128, 1-13, 36-49, 52-65, 67-80, 82-95, 114-127, 24-37, 47-60, and 76-89 of SEQ ID NO:3; (d) residues 1-13, 21-34, 44-57, 84-97, 125- 138, 8-21 , 46-59, 110-123, 129-142, 6-19, 31-44, 49-62, 71-84, and 124-137 of SEQ ID NO:4; (e) residues 3-16, 32-45, 113-126, 139-152, 154-167, 15-28, 31-44, 46-59, 92-105, 138-151 , 153- 166, 27-40, 47-60, 76-89, 136-149, and 151-164 of SEQ ID NO:5; (f) residues 14-27, 81-94, 110- 123, 156-169, 189-202, 223-236, 241-254, 349-352, 409-422, 457-470, 482-495, 515-528, 530- 543, 549-562, 579-592, 609-622, 651-664, 697-710, 45-58, 108-121 , 157-170, 234-247, 458-471 , 487-500, 552-565, 581-594, 610-623, 662-675, 694-707, 721-734, 45-58, 63-76, 172-185, 188- 201 , 225-238, 243-256, 514-527, 562-575, and 695-708 of SEQ ID NO:6; (g) residues 46-59, 93- 106, 125-138, 177-190, 229-242, 228-241 , and 96-109 of SEQ ID NO:7; (h) residues: 34-47, 56- 69, 74-87, 54-67, 70-83, 15-28, 34-47, and 49-62 of SEQ ID NO:8; (i) residues 14-27, 51-64, 66- 79, 81-94, 97-110, 115-128, 130-143, 149-162, 215-228, 230-243, 249-262, 12-25, 50-63, 73-86, 97-110, 117-130, 141-154, 161-174, 230-243, 249-262, 14-27, 82-95, 118-131 , 148-161 , 174- 187 and 199-212 of SEQ ID NO:9; (j) residues 62-75, 80-93, 96-109, 112-125, 133-146, 159-162, 193-206, 243-259, 259-262, 277-290, 315-328, 330-343, 369-382, 384-397, 429-442, 444-457, 480-493, 40-53, 63-76, 86-99, 115-128, 137-150, 165-178, 201-214, 255-268, 313-326, 337-350, 362-375, 401-414, 429-442, 480-493, 63-76, 78-91 , 112-125, 153-166, 199-212, 234-247, 318- 331 , 334-347, 370-383, 391-404, 432-445, and 460-473 of SEQ ID NQ:10; (k) residues 26-39, 46-59, 94-107, 113-126, 19-32, 46-59, 107-120, 21-34 and 108-121 of SEQ ID NO:11 ; (I) residues 24-37, 48-61 , 67-80, 117-130, 140-153, 168-181 , 184-197, 199-212, 221-234, 250-263, 269-282, 284-297, 304-317, 319-332, 344-357, 374-387, 390-403, 417-430, 433-446, 479-492, 501-514, 519-532, 571-584, 611-624, and 656-669, 30-43, 66-79, 98-111 , 126-139, 214-227, 234-247, 311-324, 345-358, 360-373, 386-399, 552-565, 570-583, 626-639, 645-658, 47-60, 65-78, 133- 146, 163-176, 179-192, 224-237, 242-255, 295-308, 352-365, 380-393, 423-436, 441-454, 469- 482, 521-534, 564-577, 625-638, 643-656, and 661-674 of SEQ ID NO:12; (m) residues 17-30, 32-45, 50-63, 65-78, 83-96, 118-131 , 133-146, 159-172, 193-206, 222-235, 257-270, 278-291 ,306-319, 32-45, 53-66, 71-84, 107-120, 134-147, 222-235, 275-288, 293-306, 310-323, 36-49, 51-64, 78-91 , 98-111 , 125-138, 223-236, and 251-264 of SEQ ID NO: 13; and / or (n) residues 88- 101 , 118-131 , 158-161 , 180-193, 245-258, 278-291 , 18-31 , 76-89, 119-132, 141-154, 247-260, 2-15, 53-66, 85-98, 112-125, 138-151 , 174-187, 249-262, and 268-281 of SEQ ID NO:14.

4. The vaccine composition of any one of claims 1 to 3, wherein the one or more immunogenic polypeptide or immunogenic fragment thereof comprises a sequence having at least 70% identity with one or more of the amino acid sequences set forth in SEQ ID NOs: 1-14 and 57-60.

5. The vaccine composition of claim 4, wherein the one or more immunogenic polypeptide or immunogenic fragment thereof comprises a sequence having at least 90% identity with one or more of the amino acid sequences set forth in SEQ ID NOs: 1-14 and 57-60.

6. The vaccine composition of claim 5, wherein the one or more immunogenic polypeptide or immunogenic fragment thereof comprises one or more of the amino acid sequences set forth in SEQ ID NOs: 1-14 and 57-60.

7. The vaccine composition of any one of claims 1 to 6, wherein the one or more immunogenic polypeptides comprises one or more of the amino acid sequences set forth in SEQ ID NOs: 1-7 and 57-60, or an immunogenic fragment thereof.

8. The vaccine composition of any one of claims 1 to 7, wherein the one or more immunogenic polypeptides comprises one or more of the amino acid sequences set forth in SEQ ID NOs: 1-5 and 57-58, or an immunogenic fragment thereof.

9. The vaccine composition of any one of claims 1 to 8, wherein the one or more immunogenic polypeptides comprises the amino acid sequence set forth in SEQ ID NO: 1 , or an immunogenic fragment thereof.

10. The vaccine composition of any one of claims 1 to 9, wherein the one or more immunogenic polypeptides comprises the amino acid sequence set forth in SEQ ID NO: 2, or an immunogenic fragment thereof.

11. The vaccine composition of any one of claims 1 to 10, wherein the one or more immunogenic polypeptides comprises the amino acid sequence set forth in SEQ ID NO: 3 or 57, or an immunogenic fragment thereof.

12. The vaccine composition of any one of claims 1 to 11 , wherein the one or more immunogenic polypeptides comprises the amino acid sequence set forth in SEQ ID NO: 4 or 58, or an immunogenic fragment thereof.

13. The vaccine composition of any one of claims 1 to 12, wherein the one or more immunogenic polypeptides comprises the amino acid sequence set forth in SEQ ID NO: 5, or an immunogenic fragment thereof.

14. The vaccine composition of any one of claims 1 to 13, wherein the vaccine composition comprises one immunogenic polypeptide or immunogenic fragment thereof.

15. The vaccine composition of any one of claims 1 to 14, wherein the vaccine composition comprises at least two immunogenic polypeptides or immunogenic fragments thereof.

16. A vaccine composition comprising one or more nucleic acids encoding the one or more immunogenic polypeptides or immunogenic fragments thereof defined in any one of claims 1-15, and at least one vaccine excipient.

17. The vaccine composition of any one of claims 1 to 16, wherein the at least one vaccine excipient comprises at least one vaccine adjuvant.

18. The vaccine composition of claim 17, wherein the at least one vaccine adjuvant comprises a saponin-based adjuvant.

19. The vaccine composition of claim 18, wherein the saponin-based adjuvant is Quil-A® or QS-21.

20. The vaccine composition of any one of claims 1 to 19, wherein the at least one vaccine excipient comprises a buffer, a solvent, a preservative, an antibiotic, or any combination thereof.

21. The vaccine composition of any one of claims 1 to 20 for use in inducing or eliciting an immune response against Clostridium perfringens in an animal.

22. The vaccine composition of any one of claims 1 to 20 for use in preventing or treating infection by Clostridium perfringens in an animal.

23. The vaccine composition of any one of claims 1 to 20 for use in preventing or treating avian necrotic enteritis (NE) in an animal.

24. The vaccine composition for use according to any one of claims 21-23, wherein the animal is a poultry.

25. The vaccine composition for use according to claim 24, wherein the poultry is a chicken or a turkey.

26. The vaccine composition for use according to any one of claims 21-25, wherein the vaccine composition is for oral administration, nasal administration, injection, or in ovo administration.

27. A method for inducing or eliciting an immune response against Clostridium perfringens in an animal comprising administering to the animal an effective amount of the vaccine composition of any one of claims 1 to 20.

28. A method for preventing or treating infection by Clostridium perfringens in an animal comprising administering to the animal an effective amount of the vaccine composition of any one of claims 1 to 20.

29. A method for preventing or treating avian necrotic enteritis (NE) in an animal comprising administering to the animal an effective amount of the vaccine composition of any one of claims 1 to 20.

30. The method of any one of claims 27-29, wherein the animal is a poultry.31 . The method of claim 30, wherein the poultry is a chicken or a turkey.

32. The method of any one of claims 27-31 , wherein the administration is oral administration, nasal administration, injection, or in ovo administration.

33. Use of the vaccine composition of any one of claims 1 to 20 for inducing or eliciting an immune response against Clostridium perfringens in an animal.

34. Use of the vaccine composition of any one of claims 1 to 20 for the manufacture of a medicament for inducing or eliciting an immune response against Clostridium perfringens in an animal.

35. Use of the vaccine composition of any one of claims 1 to 20 for preventing or treating infection by Clostridium perfringens in an animal.

36. Use of the vaccine composition of any one of claims 1 to 20 for the manufacture of a medicament for preventing or treating infection by Clostridium perfringens in an animal.

37. Use of the vaccine composition of any one of claims 1 to 20 for preventing or treating avian necrotic enteritis (NE) in an animal.

38. Use of the vaccine composition of any one of claims 1 to 20 for the manufacture of a medicament for preventing or treating avian necrotic enteritis (NE) in an animal.

39. The use of any one of claims 33-38, wherein the animal is a poultry.

40. The use of claim 39, wherein the poultry is a chicken or a turkey.

41. The use of any one of claims 33-40, wherein the vaccine composition is for oral administration, nasal administration, injection, or in ovo administration.

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