Veterinary vaccines and methods for the treatment of Pasteurella multocida infections in food-producing animals - Patents.com

Abstract

Novel animal vaccine compositions are disclosed that include P. multocida PmSLP proteins, or immunogenically equivalent portions thereof. The vaccine compositions can be used to ameliorate, treat, or prevent pathogenic infections in food-producing animals, such as bovine and porcine animals, caused by Pasteurella multocida. Related methods and uses are also disclosed.

Description

[Technical field]

[0001] Related Applications This application claims the benefit of priority to U.S. Provisional Application No. 63 / 332,966, filed April 20, 2022, the entire contents of which are incorporated herein by reference.

[0002] Incorporating sequence tables A computer-readable form of the sequence listing, "21806-P64683PC01_SequenceListing.xml" (148,616 bytes), was created on April 19, 2023, was submitted herewith by electronic submission, and is incorporated herein by reference.

[0003] Technical Field of the Disclosure The methods, uses and compositions disclosed herein relate to the treatment of infectious diseases. In particular, the methods, uses and compositions disclosed herein relate to veterinary vaccines for the prevention, treatment or amelioration of infectious diseases in food-producing animals caused by the infectious gram-negative bacterium Pasteurella multocida. [Background technology]

[0004] The following paragraphs are provided as background to the present disclosure, however, they are not an admission that all that is discussed therein is prior art or part of the knowledge of those skilled in the art.

[0005] Pasteurella multocida is a gram-negative bacterium that can colonize and infect a variety of different animals, including mammals and birds. The resulting diseases vary depending on the animal species and include respiratory disease in ruminants, bovine respiratory disease (BRD) and hemorrhagic septicaemia (HS) in cattle and Bovinae species, swine pneumonia pasteurellosis and porcine atrophic rhinitis (PAR) in pigs, and fowl cholera in birds.

[0006] P. multocida is a rod-shaped, non-flagellated bacterium frequently found in the oral cavity, nasal cavity, and respiratory tract of animals. However, disease is often accompanied by further organic or systemic dissemination of the bacteria and can include symptoms such as pneumonia, atrophic rhinitis, skin necrosis, cellulitis, abscesses, meningitis, and hemorrhagic septicemia. Furthermore, P. multocida infections can be either chronic or acute (Harper M. et al., 2006, FEMS Microbiol. Letters, 265(1), 1-10. doi:10.1111 / j.1574-6968.2006.00442.x; Wilson, B. & Ho, M., 2013, Clinical Microbiol. Rev., 26(3), 631. doi:10.1128 / CMR.00024-13).

[0007] Taxonomically, P. multocida can be divided into three subspecies, namely P. multocida subsp. multocida, P. multocida subsp. gallicida, and P. multocida subsp. septica (Mutters R. et al., 1985, Intern. J. of Syst. Evol. Microbiol., 35(3), 309-322. doi.org / 10.1099 / 00207713-35-3-309). Furthermore, P. multocida can be classified according to serogroups that represent different types of extracellular capsular polysaccharides. In this respect, five serogroups (serogroups A, B, D, E, and F) are generally distinguished (Carter, G. 1955, Rev Sci Tech, 19(2), 626-637. doi:10.20506 / rst.19.2.1236). P. multocida strains can be further classified into 16 serotypes (serotypes 1-16) based on the antigenic membrane lipopolysaccharide (LPS) components that different P. multocida strains can exhibit (Heddleston, K., 1972, Avian Dis, 16(4), 925-936).

[0008] Cattle-related diseases caused by P. multocida include bovine respiratory disease syndrome (BRD) and hemorrhagic septicemia (HS), with BRD being common in feedlots in North America and Europe and HS being a frequent cause of illness among smallholder farmers in Asia and Africa. Overall, BRD, also known as "shipping fever" in feedlot cattle, is considered the leading cause of morbidity and mortality in cattle in feedlots, and is estimated to account for 45-55% of feedlot deaths (Johnson, K. & Pendell, D., 2017, Frontiers in Vet. Sci., 4(189). doi:10.3389 / fvets.2017.00189). Moreover, BRD is estimated to be one of the most costly diseases in commercial feedlots in North America (Griffin, D., 1997, Vet. Clin. North Am. Food Animal Practice, 13(3), 367-377. doi:10.1016 / s0749-0720(15)30302-9). In this regard, BRD is commonly associated with pulmonary infections, causing pneumonia in weaned and feedlot cattle, lactating beef calves, feedlot dairy calves, and lactating dairy cows. BRD is common in herds kept in small plots, such as feedlots, or in large herds kept on a few acres. BRD is also more prevalent in stressed animals and animals with existing infections, such as preweaned calves and calves shipped to a new location immediately after weaning (Wilson et al., BK2017 J. Animal Sci., 95(5), 2170-2182. doi: 10.2527 / jas.2016.1006; Dubrovsky, S. et al., J Dairy Sci., 2019, 102(8): 7320-7328. doi: 10.3168 / jds.2018-15463).

[0009] Hemorrhagic septicemia (HS) is a rapidly progressing and highly fatal septic disease of cattle and buffaloes. HS causes significant economic losses in low- and middle-income countries in the tropical regions of the world, especially in Africa and Asia. HS has a devastating impact on smallholder farmers, whose husbandry and preventive care are often inadequate. P. multocida, which causes HS, can colonize the tonsils of a small proportion of healthy buffaloes and cattle (carrier cattle) and can be shed during periods of stress, such as high temperatures and humidity (Shivachandra, S. et al., 2011, Animal Health Res. Rev., 12(1), 67-82. doi:10.1017 / S146625231100003X). Outbreaks of the disease are most common during rainy seasons. Infection occurs by contact with infected oral or nasal secretions from either healthy carrier or diseased animals, or in some cases by ingestion of contaminated feed or water (OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, 8th Edition, 2018, Chapter 3.4.10, 1125-1138). The pathogenesis of disease in infected cattle is not fully understood but usually combines localized lesions in the neck with systemic spread, eventually leading to dissemination to various organs, tissue damage, cytokine storm, and toxic shock. Clinical signs may appear 1-3 days after the bacterium is first detected, and death may occur rapidly within 8-24 hours after the onset of symptoms. Economic losses due to HS are estimated to be around US$800 million per year in India (Singh, B. et al., 2014, Agric. Econ. Res. Rev., 27(347-2016-17135), 271-279).

[0010] Currently, there are limited veterinary vaccines available for ruminant-associated P. multocida infections, primarily based on traditional bacterin formulations (killing the entire bacterium) or attenuated bacteria (streptomycin-dependent mutants). Both of these vaccines offer limited protection, due in part to the specificity of the bacterial polysaccharide capsule of these vaccines and the possibility of mismatched circulating strains, resulting in unreliable efficacy profiles, but even when capsule-matched, efficacy is incomplete and poorly documented in the literature (Dabo, S. et al., 2007, Anim. Health Res. Rev., 8(2), 129-150). Furthermore, due to the limited effectiveness of existing vaccines against P. multocida and other BRD-causing bacteria, the prophylactic and semi-prophylactic use of antimicrobials is widespread in livestock production and may be a significant source of the development of antimicrobial resistance in pathogens (Cameron, A. & McAllister T., 2016, J. Animal Sci. and Biotechn., 7(1), 68. doi:10.1186 / s40104-016-0127-3).

[0011] P. multocida infection in pigs can cause porcine pneumonia pasteurellosis and progressive atrophic rhinitis (PAR) infections, which are of great economic importance worldwide (Adlam C. & Rutter J, 1989, Pasteurella multocida: molecular biology, toxins and infection (Vol. 361): Springer Science & Business Media). PARs have been associated with certain toxigenic strains of P. multocida, mostly serogroup D and to a lesser extent serogroup A (Eamens G.et al.,1988,Aust.Vet.J,65(4),120-123.doi:10.1111 / j.1751-0813.1988.tb14430.x; Foged N.et al.,1989,Vet.Rec.,125,7-11; Fussing,V.et al.,Vet.Microbiol,65(1),61-74.doi:10.1016 / s0378-1135(98)00288-0; and Sakano T.et al. al., 1992, J. Vet. Med. Sci., 54(3), 403-407. doi:10.1292 / jvms.54.403), whereas pneumonic pasteurellosis is generally caused by nontoxigenic strains, including serogroups A and D (Djordjevic, S. et al., 1998, J. Med. Microbiol., 47(8), 679-688. doi.org / 10.1099 / 00222615-47-8-679; Pijoan, C. et al., 1983, J. Clin. microbiol., 17(6), 1074-1076; and Zhao, G. et al., 1992, Infect. Immun., 60(4), 1401-1405). P. multocida-associated swine infections occur worldwide, including in North America, Europe, and Asia (VanderWaal D. & Deen J., 2018, PNAS 115(45), 11495. doi:10.1073 / pnas.1806068115). As in cattle farming, P. multocida-associated infections pose a significant economic burden, and management of these infections leads to increased antibiotic use.Swine vaccines against P. multocida are generally either bacterin- or toxoid-based vaccines for toxigenic and nontoxigenic strains (OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, 8th Edition, 2018, Chapter 3.8.2, 1540-1550). Vaccines against pneumonic pasteurellosis in pigs do not appear to be widespread.

[0012] P. multocida is also the causative agent of fowl cholera and is associated with a variety of infections. Acute infections can progress rapidly and often the sudden death of birds in a flock is the only sign that an infection is occurring in an establishment, although other symptoms may include oral and nasal discharge, cyanosis, generalized depression, and diarrhea (Christensen, J. & Bisgaard, M. 2000, Rev. Sci. Tech., 19(2), 626-637. doi:10.20506 / rst.19.2.1236). Fowl cholera can affect a variety of birds, with chickens, turkeys, ducks, and quails being the most economically important (Glisson, J. 1998, Poultry Science, 77(8), 1139-1142. doi.org / 10.1093 / ps / 77.8.1139). Fowl cholera is caused mainly by serogroup A strains of P. multocida, although serogroups F and D have also been reported (Dziva, F. et al., 2008, Vet. Microbiol., 128(1-2), 1-22. doi:10.1016 / j.vetmic.2007.10.018). Available vaccines include inactivated bacterins and live attenuated vaccines. Inactivated vaccines induce serotype-specific responses and therefore provide limited effective protection. On the other hand, inactivated live vaccines appear to induce broader responses beyond serotypes. However, live attenuated vaccines can cause chronic fowl cholera in chickens and turkeys (Glisson, J. 1998, Poultry Science, 77(8), 1139-1142. doi.org / 10.1093 / ps / 77.8.1139).

[0013] In light of the above, there is a need in the art for improved methods and compositions for treating P. multocida infections. In particular, there is a need in the art for improved vaccines for preventing disease caused by Pasteurella multocida infections in food-producing animals, including, but not limited to, ruminants such as cattle, swine, and avian food-producing animals. Summary of the Invention

[0014] The following paragraphs are intended to introduce the reader to a more detailed description and do not define or limit the claimed subject matter of the present disclosure.

[0015] In one aspect, the present disclosure relates to a vaccine formulation.

[0016] In another aspect, the present disclosure relates to a vaccine formulation for preventing or ameliorating disease in food-producing animals caused by the bacterial pathogen Pasteurella multocida (P. multocida).

[0017] The inventors have discovered that the veterinary vaccine formulations of the present disclosure can cross-protectively protect food-producing animals against infection with multiple P. multocida strains using a single immunogenic active agent. The immunogenic agents found to be effective when administered to food-producing animals in the veterinary vaccine formulations are in particular proteins selected from the class of P. multocida proteins known as PmSLP proteins.

[0018] Thus, in accordance with the teachings of the present specification, in at least one embodiment, the present disclosure provides an animal vaccine formulation for the prevention or amelioration of P. multocida infection in a food-producing animal susceptible to P. multocida infection, the vaccine formulation comprising an effective amount of at least one PmSLP protein, or an immunogenically equivalent portion thereof.

[0019] In at least one embodiment, in one aspect, the vaccine formulation may comprise at least one P. multocida PmSLP protein, or an immunogenically equivalent portion thereof, selected from the same phylogenetic cluster as a PmSLP protein present in an infecting P. multocida strain.

[0020] In at least one embodiment, in one aspect, the vaccine formulation may comprise a P. multocida PmSLP protein, or an immunogenically equivalent portion thereof, selected from the group of phylogenetic clusters consisting of PmSLP-1, PmSLP-2, PmSLP-3, PmSLP-4.1, and PmSLP-4.2, wherein the selected P. multocida PmSLP protein, or an immunogenically equivalent portion thereof, belongs to the same phylogenetic cluster as the PmSLP protein present in the infecting P. multocida strain.

[0021] In at least one embodiment, in one aspect, the food-producing animal may be a ruminant susceptible to infection with a P. multocida strain that causes respiratory disease, and the vaccine formulation comprises a P. multocida PmSLP protein, or an immunogenically equivalent portion thereof, derived from a P. multocida strain that causes respiratory disease, wherein the PmSLP protein is selected from the group of phylogenetic clusters consisting of PmSLP-1, PmSLP-2, PmSLP-3, and PmSLP-4.2, and wherein the selected P. multocida PmSLP protein, or an immunogenically equivalent portion thereof, belongs to the same phylogenetic cluster as the PmSLP protein present in the infecting P. multocida strain.

[0022] In at least one embodiment, in one aspect, the food-producing animal may be a bovine animal susceptible to infection with a P. multocida strain that causes BRD, and the vaccine formulation comprises a P. multocida PmSLP protein, or an immunogenically equivalent portion thereof, derived from a P. multocida strain that causes BRD, wherein the PmSLP protein is selected from the group of phylogenetic clusters consisting of PmSLP-1, PmSLP-2, and PmSLP-4.2, and wherein the selected P. multocida PmSLP protein, or an immunogenically equivalent portion thereof, belongs to the same phylogenetic cluster as the PmSLP protein present in the infecting P. multocida strain.

[0023] In at least one embodiment, in one aspect, the food-producing animal can be a bovine animal susceptible to infection with a P. multocida strain that causes HS, and the vaccine formulation comprises a P. multocida PmSLP protein, or an immunogenically equivalent portion thereof, derived from a P. multocida strain that causes HS, wherein the PmSLP protein is selected from phylogenetic cluster PmSLP-3, and wherein the selected P. multocida PmSLP protein, or an immunogenically equivalent portion thereof, belongs to the same phylogenetic cluster as the PmSLP protein present in the infecting P. multocida strain.

[0024] In at least one embodiment, in one aspect, the food-producing animal may be a swine animal susceptible to infection with a P. multocida strain that causes porcine atrophic rhinitis (PAR), and the vaccine formulation comprises a P. multocida PmSLP protein, or an immunogenically equivalent portion thereof, from a P. multocida strain that causes PAR, wherein the PmSLP protein is selected from the group of phylogenetic clusters consisting of PmSLP-2, PmSLP-4.1, and PmSLP-4.2, and wherein the selected P. multocida PmSLP protein, or an immunogenically equivalent portion thereof, belongs to the same phylogenetic cluster as the PmSLP protein present in the infecting P. multocida strain.

[0025] In at least one embodiment, in one aspect, the food-producing animal may be a swine animal susceptible to infection with a P. multocida strain that causes pneumonic pasteurellosis, and the vaccine formulation comprises a P. multocida PmSLP protein, or an immunogenically equivalent portion thereof, from a P. multocida strain that causes pneumonic pasteurellosis, wherein the PmSLP protein is selected from the group of phylogenetic clusters consisting of PmSLP-2, PmSLP-4.1, and PmSLP-4.2, and wherein the selected P. multocida PmSLP protein, or an immunogenically equivalent portion thereof, belongs to the same phylogenetic cluster as the PmSLP protein present in the infecting P. multocida strain.

[0026] In at least one embodiment, in one aspect, the food-producing animal may be an avian animal susceptible to infection with a P. multocida strain that causes fowl cholera, and the vaccine formulation comprises a P. multocida PmSLP protein, or an immunogenically equivalent portion thereof, derived from a P. multocida strain that causes fowl cholera, wherein the PmSLP protein is selected from the group of phylogenetic clusters consisting of PmSLP-3 and PmSLP-4.2, and wherein the selected P. multocida PmSLP protein, or an immunogenically equivalent portion thereof, belongs to the same phylogenetic cluster as the PmSLP protein present in the infecting P. multocida strain.

[0027] In at least one embodiment, in one aspect, the vaccine formulation may comprise a P. multocida PmSLP protein, or an immunogenically equivalent portion thereof, selected from the group of phylogenetic clusters consisting of PmSLP-1, PmSLP-2, PmSLP-3, PmSLP-4.1, and PmSLP-4.2, wherein the selected P. multocida PmSLP protein, or an immunogenically equivalent portion thereof, belongs to the same phylogenetic cluster as the PmSLP protein present in the infecting P. multocida strain, and the selected P. multocida PmSLP protein, or an immunogenically equivalent portion thereof, is further from a P. multocida strain belonging to a serogroup selected from the group consisting of serogroups A, B, D, E, and F, wherein the serogroup is the same as the serogroup of the infecting P. multocida strain.

[0028] In at least one embodiment, in one aspect, at least one PmSLP protein, or an immunologically equivalent portion thereof, (a) SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, or SEQ ID NO:96, (b) a nucleic acid sequence having at least 70% identity to any one of the nucleic acid sequences of (a); (c) a nucleic acid sequence which is substantially identical to any one of the nucleic acid sequences of (a) above, apart from the degeneracy of the genetic code; (d) a nucleic acid sequence complementary to any one of the nucleic acid sequences of (a); (e) a chimeric nucleic acid obtained by fusion between at least two of the nucleic acid sequences (a), (b), (c), and (d), or a part thereof; (f) a nucleic acid sequence complementary to any one of the nucleic acid sequences of (a); (g) a nucleic acid sequence encoding a polypeptide having any one of the amino acid sequences set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, or SEQ ID NO:97, or an immunogenically equivalent portion thereof; (h) a nucleic acid sequence encoding a functional variant of any one of the amino acid sequences set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, or SEQ ID NO:97, or an immunogenically equivalent portion thereof; and (i) a nucleic acid sequence that hybridizes under stringent conditions to any one of the nucleic acid sequences set forth in (a), (b), (c), (d), (e), (f), (g), or (h); The protein may be expressed by a nucleic acid sequence selected from the group of nucleic acid sequences consisting of:

[0029] In at least one embodiment, in one aspect, at least one P. multocida PmSLP protein, or an immunogenically equivalent portion thereof, may comprise any one of the amino acid sequences set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, or SEQ ID NO:91, or an immunogenically equivalent portion thereof, or is a chimeric protein resulting from the fusion of any one of the above sequences or portions thereof.

[0030] In at least one embodiment, in one aspect, the food-producing animal can be a ruminant species and the P. multocida infection causes respiratory disease.

[0031] In at least one embodiment, in one aspect, the food-producing animal may be a bovine species, and the P. multocida infection causes bovine respiratory disease (BRD) or hemorrhagic septicemia (HS).

[0032] In at least one embodiment, in one aspect, the food-producing animal may be a porcine species, and the P. multocida infection causes porcine pneumonia pasteurellosis or porcine atrophic rhinitis (PAR).

[0033] In at least one embodiment, in one aspect, the food-producing animal can be an avian animal and the P. multocida infection causes fowl cholera.

[0034] In at least one embodiment, in one aspect, the food-producing animal may be a ruminant species, and the P. multocida infection causes respiratory disease, and the PmSLP protein comprises SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, or SEQ ID NO:91, or an immunogenically equivalent portion thereof, to prevent or ameliorate the P. multocida infection causing respiratory disease.

[0035] In at least one embodiment, in one aspect, the food-producing animal may be a bovine species, and the P. multocida infection causes BRD, and the PmSLP protein comprises SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:77, or SEQ ID NO:91, or an immunogenically equivalent portion thereof, to prevent or ameliorate the P. multocida infection causing BRD.

[0036] In at least one embodiment, in one aspect, the food-producing animal may be a bovine species, the P. multocida infection causes HS, and the PmSLP protein comprises SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:53, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, or SEQ ID NO:89, or an immunogenically equivalent portion thereof, to prevent or ameliorate the P. multocida infection causing HS.

[0037] In at least one embodiment, in one aspect, the food-producing animal may be a porcine species, the P. multocida infection causes pneumonic pasteurellosis, and the PmSLP protein comprises SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:40, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:75, SEQ ID NO:77, or SEQ ID NO:91, or an immunogenically equivalent portion thereof, to prevent or ameliorate P. multocida infection causing pneumonic pasteurellosis.

[0038] In at least one embodiment, in one aspect, the food-producing animal may be a porcine species, and the P. multocida infection causes PAR, and the PmSLP protein comprises SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:40, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:75, SEQ ID NO:77, or SEQ ID NO:91, or an immunogenically equivalent portion thereof, to prevent or ameliorate the P. multocida infection that causes PAR.

[0039] In at least one embodiment, in one aspect, the food-producing animal may be an avian species, and the P. multocida infection causes poultry cholera, and the PmSLP protein comprises SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:40, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:61, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, or SEQ ID NO:89, or an immunogenically equivalent portion thereof, to prevent or ameliorate the P. multocida infection that causes poultry cholera.

[0040] In at least one embodiment, in some aspects, the PmSLP protein, or an immunogenically equivalent portion thereof, may be produced recombinantly in a microbial host organism.

[0041] In at least one embodiment, in one aspect, the animal vaccine formulation can be a cross-protective vaccine formulation comprising a PmSLP protein, or an immunogenically equivalent portion thereof, obtained from a first P. multocida strain, and the vaccine formulation is for administration to a food-producing animal to prevent or ameliorate infection caused by another P. multocida strain.

[0042] In at least one embodiment, in some aspects, the vaccine formulation may be substantially free of other P. multocida components.

[0043] In at least one embodiment, in some aspects, the PmSLP protein, or an immunologically equivalent portion thereof, may be a recombinantly produced protein and the vaccine formulation may be substantially free of host cell components.

[0044] In at least one embodiment, in some aspects, the vaccine formulation may further comprise a veterinary pharma- ceutically acceptable adjuvant.

[0045] In at least one embodiment, in certain aspects, the vaccine formulation may further comprise a veterinary pharma- ceutically acceptable excipient, carrier, or diluent.

[0046] In at least one embodiment, in certain aspects, the vaccine formulation may contain from about 0.001% to about 20% by weight of PmSLP protein or an immunogenically equivalent portion thereof, and a veterinary pharma- ceutically acceptable adjuvant comprising from about 0.1% to about 60% by weight or volume of the vaccine formulation.

[0047] In at least one embodiment, in some aspects, the vaccine formulation may include a second P. multocida PmSLP protein, or an immunologically equivalent portion thereof.

[0048] In at least one embodiment, in one aspect, the vaccine formulation may comprise a fusion polypeptide comprising a first P. multocida PmSLP protein and a second P. multocida PmSLP protein, or an immunologically equivalent portion thereof.

[0049] In at least one embodiment, in some aspects, a second P. multocida PmSLP protein, or an immunologically equivalent portion thereof, may belong to the same or a different phylogenetic cluster as a first P. multocida PmSLP protein, or an immunologically equivalent portion thereof.

[0050] In at least one embodiment, in certain aspects, the fusion polypeptide may comprise a fusion polypeptide selected from the group consisting of: (i) a PmSLP protein belonging to phylogenetic cluster 1, or an immunologically equivalent portion thereof, and a PmSLP protein belonging to phylogenetic cluster 3, or an immunologically equivalent portion thereof; (ii) a PmSLP protein belonging to phylogenetic cluster 1, or an immunologically equivalent portion thereof, and a PmSLP protein belonging to phylogenetic cluster 2, or an immunologically equivalent portion thereof; (iii) a PmSLP protein belonging to phylogenetic cluster 1, or an immunologically equivalent portion thereof, and a PmSLP protein belonging to phylogenetic cluster 4.1, or an immunologically equivalent portion thereof; and (iv) a PmSLP protein belonging to phylogenetic cluster 1, or an immunologically equivalent portion thereof, and a PmSLP protein belonging to phylogenetic cluster 4.2, or an immunologically equivalent portion thereof.

[0051] In at least one embodiment, in one aspect, the second P. multocida PmSLP protein, or an immunologically equivalent portion thereof, can be obtained from a P. multocida strain belonging to the same or a different serogroup as the P. multocida strain of the first P. multocida PmSLP protein, or an immunologically equivalent portion thereof.

[0052] In another aspect, the disclosure provides, in accordance with the teachings herein, in at least one embodiment, the use of an animal vaccine formulation for the prevention, treatment, or amelioration of P. multocida infection in a food-producing animal susceptible to P. multocida infection, the vaccine formulation comprising an effective amount of at least one PmSLP protein, or an immunogenically equivalent portion thereof. In another embodiment, the disclosure provides an animal vaccine formulation for use in the prevention, treatment, or amelioration of P. multocida infection in a food-producing animal susceptible to P. multocida infection, the animal vaccine formulation comprising an effective amount of at least one PmSLP protein, or an immunogenically equivalent portion thereof.

[0053] In at least one embodiment, in some aspects, the vaccine formulation may cause an improvement toward normal in one or more clinical parameters selected from the group consisting of: (i) rectal temperature, (ii) animal behavior, (iii) nasal discharge pattern, (iv) coughing pattern, (v) respiratory pattern, and (vi) overall clinical health status, compared to an animal production animal that has not been administered the veterinary vaccine formulation.

[0054] In at least one embodiment, in one aspect, the vaccine formulation may be capable of inducing an immune response in a food-producing animal, wherein anti-PmSLP antibodies are detectable in the serum of the food-producing animal for a period of at least 7 days to 52 weeks from the date of use of the vaccine formulation.

[0055] In another aspect, the disclosure provides, in accordance with the teachings of the specification, in at least one embodiment, a method for preventing, treating, or ameliorating Pasteurella multocida (P. multocida) infection in a food-producing animal susceptible to P. multocida infection, the method comprising administering to the food-producing animal an animal vaccine formulation comprising a P. multocida PmSLP protein, or an immunogenically equivalent portion thereof, wherein the vaccine formulation is administered in an amount effective to prevent, treat, or ameliorate P. multocida infection.

[0056] In at least one embodiment, in some aspects, amelioration of P. multocida infection can include a reduction in clinical signs of any disease caused by P. multocida infection.

[0057] In at least one embodiment, in one aspect, the clinical signs may be selected from the group consisting of: (i) rectal temperature, (ii) animal behavior, (iii) nasal discharge pattern, (iv) coughing pattern, (v) respiratory pattern, and (vi) overall clinical health, compared to animal production animals not administered the animal vaccine formulation.

[0058] In at least one embodiment, in one aspect, the food-producing animal susceptible to Pasteurella multocida infection may be selected from the group consisting of ruminant species, porcine species, and avian species.

[0059] In at least one embodiment, in some aspects, the disease caused by Pasteurella multocida infection can be selected from the group consisting of respiratory disease, bovine respiratory disease (BRD), hemorrhagic septicemia (HS), porcine atrophic rhinitis (PAR), and fowl cholera.

[0060] In at least one embodiment, in one aspect, the food-producing animal may be a ruminant susceptible to P. multocida infection, and the animal vaccine formulation administered to the ruminant in an effective amount comprises at least one PmSLP protein, or an immunogenically equivalent portion thereof, derived from a P. multocida strain that causes respiratory disease.

[0061] In at least one embodiment, in one aspect, the food-producing animal susceptible to P. multocida infection may be a bovine species, and the veterinary vaccine formulation administered in an effective amount to the bovine species comprises at least one PmSLP protein, or an immunogenically equivalent portion thereof, derived from a P. multocida strain that causes BRD.

[0062] In at least one embodiment, in one aspect, the food-producing animal susceptible to P. multocida infection can be a ruminant, and the veterinary vaccine formulation administered to the ruminant in an effective amount comprises at least one PmSLP protein, or an immunogenically equivalent portion thereof, derived from a P. multocida strain that causes HS.

[0063] In at least one embodiment, in one aspect, the food-producing animal susceptible to infection with a P. multocida strain may be a swine animal, and the veterinary vaccine formulation administered in an effective amount to the swine animal comprises at least one PmSLP protein, or an immunogenically equivalent portion thereof, derived from P. multocida, which causes porcine atrophic rhinitis (PAR).

[0064] In at least one embodiment, in one aspect, the food-producing animal susceptible to infection with a P. multocida strain may be an avian animal, and the veterinary vaccine formulation administered to the avian animal in an effective amount comprises at least one PmSLP protein, or an immunogenically equivalent portion thereof, derived from a P. multocida strain that causes fowl cholera.

[0065] In at least one embodiment, in one aspect, the vaccine formulation administered in an effective amount to a food-producing animal may comprise at least one P. multocida PmSLP protein, or an immunogenically equivalent portion thereof, selected from the group of phylogenetic clusters consisting of PmSLP-1, PmSLP-2, PmSLP-3, PmSLP-4.1, and PmSLP-4.2.

[0066] In at least one embodiment, in one aspect, an animal vaccine formulation can comprise at least one P. multocida PmSLP protein, or an immunogenically equivalent portion thereof, which confers homologous protection to a food-producing animal susceptible to infection with P. multocida against P. multocida strains having a PmSLP protein from the same phylogenetic cluster.

[0067] In at least one embodiment, in one aspect, an animal vaccine formulation can comprise at least one P. multocida PmSLP protein, or an immunogenically equivalent portion thereof, which confers heterologous protection in a food animal susceptible to infection with P. multocida against P. multocida strains having PmSLP proteins from different phylogenetic clusters.

[0068] In at least one embodiment, in one aspect, the vaccine formulation comprises: (a) SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, or SEQ ID NO:96, (b) a nucleic acid sequence having at least 70% identity to any one of the nucleic acid sequences of (a); (c) a nucleic acid sequence which is substantially identical to any one of the nucleic acid sequences of (a) above, apart from the degeneracy of the genetic code; (d) a nucleic acid sequence complementary to any one of the nucleic acid sequences of (a); (e) a chimeric nucleic acid obtained by fusion between at least two of the nucleic acid sequences (a), (b), (c), and (d), or a part thereof; (f) a nucleic acid sequence complementary to any one of the nucleic acid sequences of (a); (g) a nucleic acid sequence encoding a polypeptide having any one of the amino acid sequences set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, or SEQ ID NO:97, or an immunogenically equivalent portion thereof; (h) a nucleic acid sequence encoding a functional variant of any one of the amino acid sequences set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, or SEQ ID NO:97, or an immunogenically equivalent portion thereof; and (i) a nucleic acid sequence that hybridizes under stringent conditions to any one of the nucleic acid sequences set forth in (a), (b), (c), (d), (e), (f), (g), or (h); The nucleic acid sequence may comprise at least one P. multocida PmSLP protein, or an immunogenically equivalent portion thereof, selected from the proteins encoded by a nucleic acid sequence selected from the group of nucleic acid sequences consisting of:

[0069] In at least one embodiment, in certain aspects, the vaccine formulation may comprise at least one P. multocida PmSLP protein, or an immunogenically equivalent portion thereof, having any one of the amino acid sequences set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, or SEQ ID NO:91, or an immunogenically equivalent portion thereof, or a chimeric protein obtained by fusion between any one of the above sequences or portions thereof.

[0070] In at least one embodiment, in some aspects, the PmSLP protein, or an immunogenically equivalent portion thereof, may be produced recombinantly in a microbial host organism.

[0071] In at least one embodiment, in some aspects, the PmSLP protein, or an immunologically equivalent portion thereof, may be substantially free of other P. multocida components.

[0072] In at least one embodiment, in some aspects, the PmSLP protein, or an immunologically equivalent portion thereof, can be a recombinantly produced protein, and the PmSLP protein, or an immunologically equivalent portion thereof, is substantially free of host cell components.

[0073] In at least one embodiment, in some aspects, the vaccine formulation may further comprise a veterinary pharma- ceutically acceptable adjuvant.

[0074] In at least one embodiment, in certain aspects, the vaccine formulation may include an excipient, carrier, or diluent that is pharma- ceutically acceptable for use in animals.

[0075] In another aspect, the disclosure provides, in at least one embodiment, a method for preparing an animal vaccine formulation for the prevention or amelioration of P. multocida infection in a food-producing animal susceptible to P. multocida infection in accordance with the teachings herein, the method comprising: (i) to diagnose P. multocida infections in food-producing animals; (ii) identifying a phylogenetic cluster to which the PmSLP proteins present in the infecting P. multocida belong, said phylogenetic cluster being selected from PmSLP-1, PmSLP-2, PmSLP-3, PmSLP-4.1, or PmSLP-4.2; and (iii) preparing a vaccine formulation comprising a P. multocida PmSLP protein belonging to the identified phylogenetic cluster, or an immunogenically equivalent portion thereof, together with a veterinary pharmaceutically acceptable adjuvant to form an veterinary vaccine formulation comprising an effective amount of a P. multocida PmSLP protein, or an immunogenically equivalent portion thereof, for treating a food-producing animal susceptible to P. multocida infection; The present invention provides a method comprising:

[0076] In at least one embodiment, in one aspect, the method may further include identifying the serogroup of the infecting P. multocida strain, wherein the serogroup is selected from the group consisting of serogroups A, B, D, E, and F, and the vaccine is prepared using a P. multocida PmSLP protein, or an immunogenically equivalent portion thereof, derived from the same or another P. multocida strain belonging to the selected serogroup.

[0077] In another aspect, the disclosure provides, in at least one embodiment, a method for preparing an animal vaccine formulation comprising a P. multocida PmSLP protein, or an immunogenically equivalent portion thereof, in accordance with the teachings herein, comprising: (a) as operably linked components: (i) a nucleic acid sequence encoding a P. multocida PmSLP protein, or an immunogenically equivalent portion thereof; and (ii) one or more nucleic acid sequences capable of controlling the expression of the nucleic acid sequence encoding the PmSLP protein, or an immunogenically equivalent portion thereof, in a host cell; providing a chimeric nucleic acid sequence comprising: (b) introducing the chimeric nucleic acid sequence into a host cell; (c) growing the host cell to produce the P. multocida PmSLP protein, or an immunogenically equivalent portion thereof; (d) recovering the P. multocida PmSLP protein, or an immunogenically equivalent portion thereof; and (e) formulating the P. multocida PmSLP protein, or an immunogenically equivalent portion thereof, with a veterinary pharma- tically acceptable adjuvant to form an animal vaccine formulation comprising an effective amount of the P. multocida PmSLP protein, or an immunogenically equivalent portion thereof, for treating a food-producing animal susceptible to P. multocida infection; The present invention provides a method comprising:

[0078] In at least one embodiment, in one aspect, the nucleic acid sequence comprises: (a) SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, or SEQ ID NO:96, (b) a nucleic acid sequence having at least 70% identity to any one of the nucleic acid sequences of (a); (c) a nucleic acid sequence which is substantially identical to any one of the nucleic acid sequences of (a) above, apart from the degeneracy of the genetic code; (d) a nucleic acid sequence complementary to any one of the nucleic acid sequences of (a); (e) a chimeric nucleic acid obtained by fusion between at least two of the nucleic acid sequences (a), (b), (c), and (d), or a part thereof; (f) a nucleic acid sequence complementary to any one of the nucleic acid sequences of (a); (g) a nucleic acid sequence encoding a polypeptide having any one of the amino acid sequences set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, or SEQ ID NO:97, or an immunogenically equivalent portion thereof; (h) a nucleic acid sequence encoding a functional variant of any one of the amino acid sequences set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, or SEQ ID NO:97, or an immunogenically equivalent portion thereof; and (i) a nucleic acid sequence that hybridizes under stringent conditions to any one of the nucleic acid sequences set forth in (a), (b), (c), (d), (e), (f), (g), or (h); The nucleic acid sequence may be selected from the group of nucleic acid sequences consisting of:

[0079] In another aspect, the present disclosure provides, in accordance with the teachings herein, in at least one embodiment, an expression vector comprising: (a) a nucleic acid sequence encoding a P. multocida PmSLP protein, or an immunogenically equivalent portion thereof; and (b) a nucleic acid sequence capable of controlling the expression in a host cell of a nucleic acid sequence encoding a P. multocida PmSLP protein, or an immunogenically equivalent portion thereof; An expression vector is provided, comprising:

[0080] In at least one embodiment, in one aspect, the nucleic acid sequence comprises: (a) SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, or SEQ ID NO:96, (b) a nucleic acid sequence having at least 70% identity to any one of the nucleic acid sequences of (a); (c) a nucleic acid sequence which is substantially identical to any one of the nucleic acid sequences of (a) above, apart from the degeneracy of the genetic code; (d) a nucleic acid sequence complementary to any one of the nucleic acid sequences of (a); (e) a chimeric nucleic acid obtained by fusing any of the nucleic acid sequences of (a), (b), (c), and (d), or a part thereof; (f) a nucleic acid sequence complementary to any one of the nucleic acid sequences of (a); (g) a nucleic acid sequence encoding a polypeptide having any one of the amino acid sequences set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, or SEQ ID NO:97, or an immunogenically equivalent portion thereof; (h) a nucleic acid sequence encoding a functional variant of any one of the amino acid sequences set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, or SEQ ID NO:97, or an immunogenically equivalent portion thereof; and (i) a nucleic acid sequence that hybridizes under stringent conditions to any one of the nucleic acid sequences set forth in (a), (b), (c), (d), (e), (f), (g), or (h); The nucleic acid sequence may be selected from the group of nucleic acid sequences consisting of:

[0081] In another aspect, the present disclosure provides, in accordance with the teachings herein, at least one embodiment, a method for producing a cellular membrane comprising: (a) a nucleic acid sequence encoding a P. multocida PmSLP protein, or an immunogenically equivalent portion thereof; and (b) a nucleic acid sequence capable of controlling the expression in a host cell of a nucleic acid sequence encoding a P. multocida PmSLP protein, or an immunogenically equivalent portion thereof; The present invention provides a host cell comprising the chimeric nucleic acid,

[0082] In at least one embodiment, in one aspect, the nucleic acid sequence comprises: (a) SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, or SEQ ID NO:96, (b) a nucleic acid sequence having at least 70% identity to any one of the nucleic acid sequences of (a); (c) a nucleic acid sequence which is substantially identical to any one of the nucleic acid sequences of (a) above, apart from the degeneracy of the genetic code; (d) a nucleic acid sequence complementary to any one of the nucleic acid sequences of (a); (e) a chimeric nucleic acid obtained by fusing any of the nucleic acid sequences of (a), (b), (c), and (d), or a part thereof; (f) a nucleic acid sequence complementary to any one of the nucleic acid sequences of (a); (g) a nucleic acid sequence encoding a polypeptide having any one of the amino acid sequences set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, or SEQ ID NO:97, or an immunogenically equivalent portion thereof; (h) a nucleic acid sequence encoding a functional variant of any one of the amino acid sequences set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, or SEQ ID NO:97, or an immunogenically equivalent portion thereof; and (i) a nucleic acid sequence that hybridizes under stringent conditions to any one of the nucleic acid sequences set forth in (a), (b), (c), (d), (e), (f), (g), or (h); The nucleic acid sequence may be selected from the group of nucleic acid sequences consisting of:

[0083] In another aspect, the disclosure provides, in at least one embodiment, the use of a P. multocida PmSLP protein, or an immunogenically equivalent portion thereof, in accordance with the teachings of the specification, to prepare an animal vaccine formulation comprising the P protein, or an immunogenically equivalent portion thereof, together with a pharma- ceutically acceptable adjuvant for animals.

[0084] Other features and advantages will become apparent from the following detailed description, which, however, illustrates preferred embodiments of the present disclosure, and is given by way of example only, and it should be understood that various changes and modifications that become apparent to those skilled in the art from this detailed description are within the spirit and scope of the present disclosure.

[0085] The present disclosure is described in the paragraphs provided below, by way of example, in connection with the accompanying figures. The figures provided herein are provided for a better understanding of the exemplary embodiments and to more clearly show how various embodiments may be implemented. These implementations are not intended to limit the present disclosure. [Brief description of the drawings]

[0086] [Figure 1] 1 shows certain aspects of an example of a recombinant PmSLP protein production and purification workflow, as well as exemplary analytical data and results obtained at different steps in carrying out an exemplary process of purification of PmSLP-3. A is a flow chart showing the schematic workflow for purification of recombinant PmSLP. B is a photographic image of a sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) gel showing sample material collected at different stages (1-6) of nickel nitrilotriacetic acid (NTA) purification of his-tagged PmSLP-3 (SEQ ID NO: 12) from E. coli lysate. C is an S75 gel filtration chromatogram of size exclusion chromatography after removal of the polyhistidine tag of PmSLP-3 (SEQ ID NO: 14). D is a photographic image of an SDS-PAGE gel of purified PmSLP-3 after carrying out a polishing step with MonoQ. Note that PmSLP-3 migrates in the gel at approximately 35 kDa after tag removal. [Diagram 2]Phylogenetic tree of PmSLP protein sequences collected from a specific local collection of PmSLP sequences, combining publicly available sequences from online databases with specific information related to the phylogenetic tree. Annotations regarding host species, confirmed disease status, geographical region, and serotype for each PmSLP variant are based on information available in the BioSample database of the National Center for Biotechnology Information (NCBI), literature search, and genome sequence analysis. Black circles on the lineage branches of the phylogenetic tree indicate sequences originating from bovine species. Small dots with light grey shaded areas represent other hosts. In addition, four ring structures are depicted around the periphery of the phylogenetic tree, representing (1) host (species), (2) disease, (3) (geographical) region, and (4) capsule. Each ring structure is composed of multiple rectangular segments, each corresponding to a single PmSLP variant in the phylogenetic tree. Thus, moving directly outward from a particular selected rectangular piece on the innermost ring structure representing a particular PmSLP variant sequence are (1) host species (a selected rectangular piece on the inner ring structure, e.g., rectangular piece (a)), (2) disease (a rectangular piece moving directly outward and immediately adjacent to the piece selected in (1), e.g., rectangular piece (b)), (3) geographic region (a rectangular piece moving further directly outward and immediately adjacent to the piece identified in (2), e.g., rectangular piece (c)), and (4) capsule (a rectangular piece moving further directly outward and immediately adjacent to the piece identified in (3), e.g., rectangular piece (d)). The shading of the rectangular pieces represents the host species on the innermost ring structure: black for Bovidae (including rectangular piece (a)), diagonal lines for Porcineae, medium grey shading for Avian, light grey shading for other species, and white for defects. For the ring structures immediately surrounding the most central disease ring, the rectangle gradation index is as follows; black for bovine hemorrhagic septicemia (HS), medium grey shades for bovine respiratory disease (BRD) (including rectangle (b)), and white for unconfirmed / unknown disease status.The ring structure immediately central to the most peripheral ring represents the geographic region where the sample was taken, with a gradation index of the rectangular pieces as follows: black for North America, medium gray shades for Asia, light gray shades for Europe (including rectangular piece (C)), and white for unknown regions. The most peripheral ring structure indicates the capsular serogroup of P. multocida (if known), with a gradation index of: black for serogroup A (including rectangular piece (d)), diagonal line pattern for serogroup B, medium gray shades for serogroup D, light gray shades for serogroup F, and white for unclassifiable. Thus, the exemplary selected PmSLP sequences corresponding to exemplary rectangular pieces (a), (b), (c), and (d) correspond to bovine PmSLPs isolated from a P. multocida strain causing BRD in Europe, which has serogroup A. The phylogenetic tree of PmSLP variants can be seen to be divided into five phylogenetic clusters (PmSLP-1, PmSLP-2, PmSLP-3, and PmSLP-4.1 and PmSLP-4.2). [Diagram 3] 1 is a graph showing results obtained in conducting an experiment, specifically an experiment evaluating antibody responses in mice administered PmSLP vaccine formulations. In this graph, the end point IgG titers are plotted as a function of time. Serum IgG titers of mice administered two doses of the vaccine were measured in serum samples taken over a period of 26 weeks. The data points on the graph represent the mean values, and the error bars represent the standard error. The dotted line at the end point IgG titer of 8,000 indicates the limit of detection, which is the lowest serum dilution measured. The IgG titers remained stable throughout the entire duration of the experiment, with no group showing any sign of significant decline. These experimental results demonstrate that long-lived antibody responses can be elicited using PmSLP-1 (SEQ ID NO: 14)-containing vaccine formulations. [Figure 4]1A-1C are further graphs showing the results obtained in carrying out certain experiments, in particular those evaluating immune responses in mice administered PmSLP vaccine formulations. In this example experiment, the efficacy of the PmSLP-1 (SEQ ID NO: 14) vaccine formulation was evaluated against a bovine respiratory disease (BRD) isolate of P. multocida strain H246, which carries a PmSLP gene from the same phylogenetic cluster. The graph shown in A shows the survival rate as a function of time, in particular the survival rate of mice after infection with P. multocida BRD strain H246 (the confirmed sequence of PmSLP of this strain is defined in SEQ ID NO: 22). Mice vaccinated with PmSLP-1 were 100% protected. The graph shown in B shows the clinical score as a function of time, in particular the clinical score of mice immunized with PmSLP-1 vaccine, per individual animal. The graph shown in C shows the clinical score as a function of time, in particular the clinical score of mice immunized with adjuvant alone, per individual animal. The graph in D shows the clinical score as a function of time, specifically the mean clinical score for both groups of mice. Error bars indicate standard error. In B, C, and D, a clinical score cutoff of 10 is considered the humane end point, at which point the animals are euthanized. The graph in E shows bacterial recovery as a function of time, specifically the recovery of bacteria from tail vein bleeding during infection for individual animals. The experimental results show that the PmSLP vaccine is effective against a relevant bovine P. multocida disease isolate in an acute mouse infection model. [Diagram 5]1A-B are further graphs showing results obtained in carrying out certain experiments, particularly those evaluating the immune response of mice administered PmSLP vaccine formulations. Serum samples were taken from mice immunized with two doses of either PmSLP-1 (SEQ ID NO: 14) vaccine or adjuvant prior to challenge. The graph shown in A shows α-PmSLP-1 IgG titers against purified antigens measured by protein ELISA. The graph shown in B shows α-PmSLP-1 IgG titers against whole bacteria measured by heat-inactivated whole cell ELISA. P. multocida BRD strain H246 (containing PmSLP as defined in SEQ ID NO: 22 and carrying PmSLP genes from the same phylogenetic cluster as the vaccine antigen) was used. In both A and B, individual points represent individual animals. Bars indicate the mean value and error bars indicate the standard error. The experimental results show that α-PmSLP-1 antibodies elicited in vaccinated mice are able to bind not only to antigens on the bacterial surface but also to purified antigens. [Figure 6]3A-3C are further graphs showing results obtained in certain experiments, particularly in the performance of experiments evaluating the immune response of mice administered PmSLP vaccine formulations. In this example, the efficacy of the PmSLP-3 (SEQ ID NO: 20) vaccine against porcine disease isolates carrying the PmSLP gene from the same phylogenetic cluster was evaluated. The graph shown in A shows the survival rate as a function of time, particularly the survival rate of mice after infection with P. multocida porcine strain H229 (containing PmSLP with the sequence defined in SEQ ID NO: 24). Mice vaccinated with PmSLP-3 were 100% protected. The graph shown in B shows the clinical score as a function of time, particularly the clinical score of mice immunized with the PmSLP-3 vaccine, for each individual animal. The graph shown in C shows the clinical score as a function of time, particularly the clinical score of mice immunized with adjuvant only, for each individual animal. The graph shown in D shows the clinical score as a function of time, particularly the average clinical score of mice in both groups. Error bars indicate standard error. In B, C, and D, a clinical score cutoff of 10 is considered the humane end point, at which point the animals are euthanized. The graph in E shows bacterial recovery as a function of time, specifically from tail vein bleeds during infection, for individual animals. Experimental results show that a vaccine containing PmSLPs is effective against a related porcine P. multocida disease isolate in an acute mouse infection model. [Figure 7]2A-2C are further graphs showing results obtained in carrying out certain experiments, particularly those evaluating the immune response of mice administered PmSLP vaccine formulations. Serum samples were taken from mice immunized with two doses of either PmSLP-3 (SEQ ID NO: 20) vaccine or adjuvant prior to challenge. The graph shown in A shows α-PmSLP-3 IgG titers against purified antigens measured by protein ELISA. The graph shown in B shows α-PmSLP-3 IgG titers against whole bacteria measured by heat-inactivated whole cell ELISA. A P. multocida swine isolate of P. multocida strain H229 was used, carrying PmSLP genes from the same phylogenetic cluster as the vaccine antigen (the confirmed sequence of PmSLP for this strain is defined in SEQ ID NO: 24). In both A and B, individual points represent individual animals. Bars indicate the mean value and error bars indicate the standard error. The experimental results show that α-PmSLP-3 antibodies elicited in vaccinated mice are able to bind not only to antigens on the bacterial surface but also to purified antigens. [Figure 8] 8A-8B are further graphs showing results obtained in carrying out certain experiments, particularly those evaluating the stability of PmSLP polypeptides. Thermal profiles were generated by measuring the intrinsic fluorescence (ratio 350 nm / 330 nm) emanating from tryptophan and tyrosine residues (graph shown in FIG. 8A) and calculating the thermal inflection temperature (Ti) (graph shown in FIG. 8B) of PmSLP-1 protein samples stored under the specified storage conditions. The results demonstrate that purified PmSLP-3 protein (SEQ ID NO:20) can be stable for one year under various storage conditions after lyophilization. [Figure 9]Graphs showing results obtained in certain experiments, particularly those evaluating protection from lethal challenge in mice administered PmSLP vaccine formulations. The graph shown in A shows survival as a function of time, particularly survival of mice after infection with antigen-matched P. multocida porcine strain H229 (the confirmed sequence of PmSLP for this strain is defined in SEQ ID NO: 24), with all mice administered PmSLP-3 vaccine formulations being fully protected from lethal challenge. Graphs shown in B, C, D, and E show clinical scores of individual mice as a function of time, particularly clinical scores of mice immunized with adjuvant, vaccine 1 (freshly formulated vaccine with proteins stored at -80°C until formulation), vaccine 2 (freshly formulated vaccine with lyophilized proteins stored at 4°C until formulation), and vaccine 3 (formulated vaccine prepared prior to dose 1 and stored at 4°C until dose 2), respectively. Each line shows the clinical scores of individual mice over the experimental challenge period, with clinical monitoring performed at multiple time points over a period of 36 hours post-infection. The dotted line at a clinical score of 10 indicates the clinical score cutoff that is considered a humane endpoint at which point animals are euthanized. Experimental results demonstrate that PmSLP-3 (SEQ ID NO: 20) vaccine preparations stored under various conditions can be effective in mouse models. [Figure 10] Figure 1 is a further graph showing results obtained in the conduct of certain experiments, particularly those evaluating immune responses in ruminant (bovine) species administered PmSLP vaccine formulations. In this example, zebu cattle were immunized subcutaneously with either PmSLP-1 (SEQ ID NO: 14) vaccine or adjuvant. Serum samples were taken at baseline or 2-3 weeks after the indicated doses and analyzed using a protein-based ELISA. Data points represent individual animals, bars represent mean α-PmSLP-1 IgG titers, and error bars represent standard error. These experimental results demonstrate that PmSLP vaccines have the potential to exert immunogenicity in animal host species relevant to the food production industry and affected by P. multocida infections. [Figure 11]Figure 1 is a further graph showing results obtained in the conduct of certain experiments, in particular experiments evaluating immune responses in ruminants (bovine species) administered PmSLP vaccine formulations. In this example, beef cattle were immunized via the intramuscular route with either the PmSLP-1 (SEQ ID NO: 14) vaccine or adjuvant. Serum samples were taken at baseline or 2-3 weeks after the indicated doses and analyzed using a protein-based ELISA. Data points represent individual animals, bars represent mean α-PmSLP-1 IgG titers, and error bars represent standard error. These experimental results demonstrate that PmSLP vaccines have the potential to exert immunogenicity in animal host species relevant to the food production industry and affected by P. multocida infections. [Figure 12] 1A-1C are further graphs showing results obtained in the conduct of certain experiments, particularly those evaluating immune responses in ruminant (bovine) species administered PmSLP vaccine formulations. In this example, zebu cattle were immunized subcutaneously with PmSLP-3 (SEQ ID NO: 20) formulated with two different adjuvants in a prime-boost schedule. The graph shown in A shows serum samples taken at baseline, pre-booster and pre-challenge and analyzed using a protein-based ELISA. Data points represent individual animals, bars represent mean α-PmSLP-3 IgG titers and error bars represent standard error. The graph shown in B shows the survival of animals as a function of time, particularly after lethal challenge. The graph shown in C records the local reactogenicity assessed after either vaccine administration. The exemplified experimental results demonstrate that PmSLP vaccines can be immunogenic and safe in animal host species relevant to the food production industry and susceptible to P. multocida infection, and that these vaccine formulations can be protective against experimental challenge with a lethal dose of P. multocida. [Figure 13A]FIG. 1 shows a specific experimental procedure, in particular experimental groups formed to determine the lethal dose of three clinical strains of Pasteurella multocida serogroup A (AFK 300 / 23, 143 / 23 and 283 / 23) isolated from pigs with severe lung disease and carrying the pfhA gene, a virulence marker for P. multocida. [Figure 13B] FIG. 1 is a graph depicting certain results obtained in certain experimental evaluation runs, including results for survival analysis of mice challenged with three different strains of Pasteurella multocida serogroup A. Mice (n=5) in each group were challenged intraperitoneally with 0.2 mL of PBS containing 10, 10, or 10. The number of dead / surviving mice was monitored and is expressed as percentiles of survival. [Figure 14A] Graphs depicting certain results obtained in particular experimental evaluation runs, in particular those relating to titration of mouse anti-PmSLP-2 IgG, in which air samples collected from two experimental groups were titrated using indirect ELISA based on PmSLP2 recombinant protein, statistical analysis was performed using indirect ELISA based on PmSLP2 recombinant protein, ANOVA test, and statistical differences (p<0.05), if any, are indicated. [Figure 14B] Graphs depicting certain results obtained in particular experimental evaluation runs, in particular a survival analysis of mice immunized with a PmSLP2-based vaccine and challenged with Pasteurella multocida A strain AFK 143 / 23, where the AFK 143 / 23 strain expresses PmSLP cluster 2 and therefore the challenge is considered homologous. [Figure 15]Graphs depicting certain results obtained in certain experimental evaluation runs, specifically those relating to survival analysis of pigs challenged with two different strains of Pasteurella multocida serogroup A. Each group of pigs (n=3) was challenged intranasally with 3 mL of PBS containing 10 or 10 bacteria, and the number of dead / survived pigs was monitored, as shown in the graph as percentile survival. [Figure 16A] FIG. 1 shows the specific experimental procedure, in particular the immunization protocol used to assess homologous protection, in which four groups of mice were immunized with different PmSLP-based vaccines or inoculated with sterile PBS on experimental days 0 and 14, and all mice were challenged with P. multocida (102 bacteria / mouse) on experimental day 28. [Figure 16B] Graphs depicting certain results obtained in certain experimental evaluation runs, in particular those relating to titration of mouse anti-PmSLP-1 and PmSLP-4.2 IgG. Serum samples collected from the four experimental groups were titrated in an indirect ELISA based on PmSLP-1 or PmSLP-4.2 recombinant protein, and statistical analysis was performed using a two-way ANOVA test. Statistical differences (p<0.05), if any, are indicated. [Figure 16C]Graphs showing certain results obtained in a particular experimental evaluation run, two graphs showing results related to survival analysis of mice immunized and challenged with two different strains of Pasteurella multocida serogroup A. The graph in panel (I) shows a first graph showing the results of an experiment in which mice (n=8) from each group (n=2) were challenged intraperitoneally with 0.2 mL of PBS containing 102 P. multocida A, strain AFK300 / 23. The graph in panel (II) shows a second graph showing the results of an experiment in which mice (n=8) from each group (n=2) were challenged intraperitoneally with 0.2 mL of PBS containing 102 P. multocida A, strain AFK 283 / 23. Here, the number of dead / surviving mice was monitored in each case and is graphed as percentile of survival. The different groups and strains and challenges used are described in panels (i) and (ii). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0087] The drawings, taken in conjunction with the following detailed description, will make apparent to those skilled in the art how the present disclosure may be practically practiced.

[0088] Various compositions, methods, or processes are described below to provide examples of embodiments of each claimed subject matter. The embodiments described below do not limit the claimed subject matter, which may be directed to processes, compositions, or methods different from those described below. The claimed subject matter is not limited to compositions, processes, or methods having all of the features of any one composition, method, or process described below, or features common to more than one or all of the compositions, methods, or processes described below. A composition, method, or process described below may not be an embodiment of any claimed subject matter. Subject matter disclosed in the compositions, methods, or processes described below that is not claimed herein may be the subject of another defense, such as a pending patent application, and the applicant(s), inventor(s), or owner(s) do not intend to abandon, disclaim, or disclose such subject matter by disclosure herein.

[0089] As used in this specification and claims, singular forms such as "a," "an," and "the" include plural references and vice versa unless the context clearly dictates otherwise. Throughout this specification, unless otherwise indicated, "comprise," "comprises," and "comprising" are used inclusively rather than exclusively, whereby a recited integer or group of integers may include one or more other unrecited integers or groups of integers. The term "or" is inclusive unless modified, for example, by "either."

[0090] When ranges are used herein for physical properties such as molecular weights, or chemical properties such as chemical formulas, all combinations and subcombinations of ranges and specific embodiments therein are intended to be included. Except in the operating examples or where otherwise indicated, all numbers expressing amounts of ingredients or reaction conditions used herein should be understood to be modified in all cases by the term "about". The term "about" when referring to a numerical value or numerical range means that the numerical value or numerical range referred to is an approximation within experimental variability (or within statistical experimental error), and thus the numerical value or numerical range may vary by 1% to 15% of the specified numerical value or numerical range, as can be readily discerned from the context. Furthermore, any numerical range of values ​​described herein is intended to specifically include the limits of that range, and any intermediate values ​​or subranges within the given range, and all such intermediate values ​​and subranges are individually and specifically disclosed (e.g., the range of 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). Similarly, other terms of degree used herein, such as "substantially" and "approximately," imply a reasonable amount of deviation from the modified term such that the end result is not materially altered. These terms of degree should be construed to include deviations from the modified term if such deviations do not negate the meaning of the modified term.

[0091] Unless otherwise defined, scientific and technical terms used in connection with the formulations described herein shall have the meanings commonly understood by those skilled in the art. The terms used herein are intended only to describe certain embodiments and are not intended to limit the scope of the invention, which is defined only by the claims.

[0092] All publications, patents, and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

[0093] Terms and Definitions As used herein, the term "nucleic acid" or "nucleic acid sequence" refers to a sequence of nucleoside or nucleotide monomers consisting of naturally occurring bases, sugars, and intersugar (backbone) linkages. The term also includes modified or substituted sequences that include non-naturally occurring monomers or portions thereof. The nucleic acids of the present disclosure may be deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and may include naturally occurring bases including adenine, guanine, cytosine, thymidine, and uracil. Nucleic acids may also contain modified bases. Examples of such modified bases include aza and deaza adenine, guanine, cytosine, thymidine, and uracil, as well as xanthine and hypoxanthine. A sequence of nucleotide or nucleoside monomers may be referred to as a polynucleotide sequence, a nucleic acid sequence, a nucleotide sequence, or a nucleoside sequence.

[0094] The terms "polypeptide" and "protein", which may be used interchangeably herein, in conjunction with a reference SEQ ID NO: refer to any polypeptide and protein that (i) comprises a sequence of amino acid residues that is substantially identical to the amino acid sequence that constitutes a polypeptide having such reference SEQ ID NO:, or (ii) comprises a sequence of amino acid residues that is encoded by a nucleic acid sequence that is capable of hybridizing, under at least moderately stringent conditions, except for synonymous codon usage, to any nucleic acid sequence that encodes a polypeptide having such reference SEQ ID NO:. A sequence of amino acid residues may be referred to as an amino acid sequence, or a polypeptide sequence.

[0095] The terms "nucleic acid sequence encoding a polypeptide" and "nucleic acid sequence encoding a protein" as used herein in conjunction with a reference SEQ ID NO: refer to any nucleic acid sequence encoding a polypeptide or protein having such a reference SEQ ID NO. A nucleic acid sequence encoding a polypeptide in conjunction with a reference SEQ ID NO further includes (i) any nucleic acid sequence encoding a polypeptide substantially identical to a polypeptide having such a reference SEQ ID NO, or (ii) any nucleic acid sequence that hybridizes to any nucleic acid sequence encoding a polypeptide having such a reference SEQ ID NO under at least moderately stringent hybridization conditions or that would hybridize to them under at least moderately stringent conditions in the absence of synonymous codon usage.

[0096] The terms "nucleic acid sequence encoding PmSLP" and "nucleic acid sequence encoding a "PmSLP polypeptide", "nucleic acid sequence encoding a PmSLP protein", which may be used interchangeably herein, refer to any nucleic acid sequence encoding a PmSLP polypeptide, including, for example, SEQ ID NO: 1. Nucleic acid sequences encoding PmSLP polypeptides further include (i) any nucleic acid sequence encoding a polypeptide substantially identical to a PmSLP polypeptide sequence described herein, or (ii) all nucleic acid sequences that hybridize to any PmSLP nucleic acid sequence described herein under at least moderately stringent hybridization conditions, or, if synonymous codons are not used, hybridize thereto under at least moderately stringent conditions.

[0097] The term "PmSLP protein" or "PmSLP polypeptide" as used herein refers interchangeably to any protein that (i) comprises a sequence of amino acid residues that is substantially identical to the amino acid sequence constituting a PmSLP polypeptide described herein, e.g., comprising SEQ ID NO:2, or (ii) comprises a sequence of amino acid residues encoded by a nucleic acid sequence that is capable of hybridizing under at least moderately stringent conditions to a nucleic acid sequence encoding a PmSLP protein described herein, excluding the use of synonymous codons. PmSLP proteins may also be numbered, e.g., PmSLP-1, PmSLP-2, PmSLP-3, etc., to facilitate distinction between the different PmSLP proteins referred to herein.

[0098] The term "substantially identical" means that the two amino acid sequences are preferably at least 70% identical, more preferably at least 85% or 90% identical, and most preferably at least 95% identical, for example, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical. To determine the percentage of identity between two amino acid sequences, the amino acid sequences of the two sequences are aligned, for example, using the alignment method of Needleman and Wunsch (Adv. Appl. Math., 1981, 2:482), as revised by Smith and Waterman (J. Mol. Biol., 1970, 48:443), to obtain the highest degree of correspondence between the two sequences and to determine the number of amino acids that are identical between the two sequences. Methods for calculating the percentage of identity between two amino acid sequences are generally recognized in the art, such as those described by Carillo and Lipton (SIAM J. Applied Math., 1988, 48:1073) and those described in Computational Molecular Biology, Lesk, ed. Oxford University Press, New York, 1988, Biocomputing: Informatics and Genomics Projects. Generally, computer programs are used for such calculations. Computer programs that may be used in this regard include, but are not limited to, GCG (Devereux et al., Nucleic Acids Res., 1984, 12:387), BLASTP, BLASTN and FASTA (Altschul et al., J. Mol. Biol., 1990:215:403).A particularly preferred method for determining the percent identity between two polypeptides involves the Clustal W algorithm (Thompson, JD, Higgines, DG and Gibson, TJ, 1994, Nucleic Acid Res 22(22):4673-4680), in conjunction with a BLOSUM 62 scoring matrix (Henikoff S & Henikoff, JG, 1992, Proc. Natl. Acad. Sci. USA 89:10915-10919), using a gap opening penalty of 10 and a gap extension penalty of 0.1, to obtain the highest order match obtained between the two sequences where at least 50% of the total length of the two sequences is included in the alignment.

[0099] By "at least moderately stringent hybridization conditions" is meant that conditions are selected that promote selective hybridization between two complementary nucleic acid molecules in solution. Hybridization may occur to all or part of the nucleic acid sequence molecule. The hybridization portion is usually at least 15 (e.g., 20, 25, 30, 40 or 50) nucleotides in length. Those skilled in the art will recognize that the stability of a nucleic acid duplex or hybrid is determined by its Tm, and in a sodium-containing buffer, Tm is a function of sodium ion concentration and temperature (Tm=81.5°C-16.6(Log10[Na+])+0.41(%(G+C)-600 / l), or a similar formula). Thus, the parameters in the wash conditions that determine hybrid stability are sodium ion concentration and temperature. To identify molecules that are similar but not identical to a known nucleic acid molecule, it can be assumed that a 1% mismatch results in a decrease in Tm of about 1°C. For example, if a nucleic acid molecule with more than 95% identity is required, the final washing temperature is reduced by about 5°C. Based on these considerations, those skilled in the art can easily select appropriate hybridization conditions. In a preferred embodiment, stringent hybridization conditions are selected. As an example, the following conditions can be adopted to achieve stringent hybridization: hybridization with 5x sodium chloride / sodium citrate (SSC) / 5x Denhardt's solution / 1.0% SDS at Tm (based on the above formula)-5°C, followed by a 0.2x SSC / 0.1% SDS wash at 60°C. Moderately stringent hybridization conditions include a wash step with 3x SSC at 42°C. However, it is understood that equivalent stringency can be achieved by using alternative buffers, salts, and temperatures.Further guidance regarding hybridization conditions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, NY, 1989, 6.3.1.-6.3.6, and Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989, Vol. 3.

[0100] As used herein, the term "functional variant" in reference to a polynucleotide or polypeptide refers to a polynucleotide or polypeptide that can perform the same function as the described reference polynucleotide or polypeptide. Thus, for example, a functional variant of a polypeptide set forth in SEQ ID NO:2 refers to a polypeptide that can perform the same function as the polypeptide set forth in SEQ ID NO:2. Functional variants include modified polypeptides relative to the described reference polypeptide, where the modifications include substitution, deletion, or addition of one or more amino acids. In some embodiments, the substitution is to replace one amino acid with an amino acid having similar properties. Such substitutions include, but are not limited to, (i) glutamic acid and aspartic acid, (ii) alanine, serine and threonine, (iii) isoleucine, leucine and valine, (iv) asparagine and glutamine, and (v) tryptophan, tyrosine and phenylalanine.

[0101] The term "chimera" as used herein in the context of nucleic acids refers to at least two linked nucleic acids that are not naturally linked. Chimeric nucleic acids include linked nucleic acids of different natural origin. For example, a nucleic acid that constitutes a microbial promoter linked to a nucleic acid that encodes a plant polypeptide is considered a chimera. Chimeric nucleic acids may also be composed of nucleic acids of the same natural origin, so long as they are not naturally linked. For example, a nucleic acid that constitutes a promoter obtained from a particular cell type may be linked to a nucleic acid that encodes a polypeptide obtained from the same cell type, but is not normally linked to the nucleic acid that constitutes the promoter. Chimeric nucleic acids also include nucleic acids that include any naturally occurring nucleic acid linked to any non-naturally occurring nucleic acid.

[0102] The term "phylogenetic cluster" refers to a group of evolutionarily related polypeptide sequences. To determine whether two polypeptides belong to the same phylogenetic cluster, an evolutionary tree with multiple branches (e.g., at least 5, 7, 10, 15, or 20 branches) can be constructed using multiple more or less similar polypeptide sequences (e.g., preferably at least 25, at least 50, at least 100, or at least 1,000 polypeptide sequences). By examining the phylogenetic tree, the evolutionary relationship of the polypeptide sequences can be evaluated. Polypeptides that belong to the same phylogenetic cluster are polypeptides that are located on a specific branch on the phylogenetic tree that is derived from a common ancestor. Those skilled in the art will be familiar with software programs that support the automatic generation of phylogenetic trees based on polypeptide sequence input. Suitable phylogenetic tree construction software includes, for example, sequence alignment software such as MAFFT (v7.450) (Katoh, K. et al., 2002, Nucleic Acids Research, 30(14), 3059-3066) that can use the G-INS-I algorithm, evolutionary modeling software such as ProtTest (v3.4.2) (Darriba D, Taboada GL, Doallo R, Posada D. ProtTest 3: Fast selection of best-fit models of protein evolution. Bioinformatics, 27: 1164-1165, 2011) that identifies suitable evolutionary models, as well as PhyML (v3.3.20190909) (Guindon S., et al., 2010, Systematic Biology, 59(3): 307-21, 2010) and RAxML (Stamakis, A. Bioinformatics, Volume 30, Issue 1, 2010). 9, May 2014, Pages 1312-1313, doi.org / 10.1093 / bioinformatics / btu033) and other phylogenetic tree assembly software.Exemplary phylogenetic trees, such as an example of a phylogenetic tree of PmSLP polypeptide sequences showing phylogenetic clusters of PmSLP-1, PmSLP-2, PmSLP-3, PmSLP-4.1, and PmSLP-4.2, include the phylogenetic tree shown in FIG. 2. Exemplary phylogenetic cluster PmSLP-1, for example, includes at least a PmSLP polypeptide sequence having SEQ ID NO:2. Exemplary phylogenetic cluster PmSLP-2, for example, includes at least a PmSLP polypeptide sequence having SEQ ID NO:4. Exemplary phylogenetic cluster PmSLP-3, for example, includes at least a PmSLP polypeptide sequence having SEQ ID NO:6. Exemplary phylogenetic cluster PmSLP-4.1, for example, includes at least a PmSLP polypeptide sequence having SEQ ID NO:8. Exemplary phylogenetic cluster PmSLP-4.2, for example, includes at least a PmSLP polypeptide sequence having SEQ ID NO:10.

[0103] As used herein, the term "animal" refers to all species within the kingdom Animalia, excluding humans.

[0104] As used herein, the term "avian" refers to any animal species belonging to the class Aves, including poultry such as chickens, turkeys, geese, ducks, and quail.

[0105] The term "bovine" as used herein refers to any animal species belonging to the subfamily Bovidae, including, for example, cows, bulls, bison, and buffalo.

[0106] As used herein, the term "pig" refers to any animal species belonging to the family Suidae, including, for example, pigs, boars, and wild boars.

[0107] As used herein, the term "ruminant" refers to a herbivorous mammalian animal that has a digestive system that typically includes a multicompartment stomach system including the rumen, reticulum, omasum, and abomasum, and is capable of digesting plant material through a process of microbial fermentation. Ruminant animals include cows, oxen, bulls, goats, sheep, bison, and buffalo.

[0108] As used herein, the term "food-producing animals" refers to animals that are kept and farmed by humans for the production of food for humans. Food-producing animals include, but are not limited to, ruminant species, bovine species, porcine species, and avian species.

[0109] The term "Pesteurella multocida" or "P. multocida" as used herein refers to bacteria belonging to the taxonomic species of bacteria classified as such, including subspecies thereof, including P. multocida subsp. multocida, P. multocida subsp. gallicida, and P. multocida subsp. septica, and further includes strains, variants, serogroups (including serogroups A, B, D, E, and F), serotypes (including serotypes 1-16), or genotypes of P. multocida. It should be noted that P. multocida strains are sometimes referred to by serogroup and serotype. Thus, for example, a strain referred to as P. multocida A:3 refers to a P. multocida strain of serogroup A and serotype 3.

[0110] The term "effective amount" as used herein refers to an amount of an active agent or veterinary pharmaceutical preparation, including an veterinary vaccine preparation, sufficient to induce a desired biological or therapeutic effect, including a prophylactic effect. Such effects may include effects on the signs, symptoms or causes of a disorder or disease, or other desired changes in a biological system. The effective amount may vary depending on, for example, the health status of the animal being treated, the stage of injury, disorder or disease, the timing of administration, the method of administration, the age of the animal, the size of the animal, etc., all of which can be determined by one skilled in the art.

[0111] The term "immunologically equivalent" as used herein refers to a molecule that, when administered, can induce a humoral immune response in a subject animal in the form of the production of natural polyclonal antibodies, where the binding specificity for the natural polyclonal antibodies is equivalent to that of the natural polyclonal antibodies produced when the reference molecule is administered to the subject animal. For example, an immunologically equivalent portion of a reference full-length PmSLP polypeptide includes an immunogenic portion of a PmSLP polypeptide that, when administered to a subject animal, induces a humoral immune response in the form of the production of natural antibodies having a specificity for the PmSLP polypeptide equivalent to the binding specificity for the PmSLP polypeptide of the natural antibodies obtained when the reference full-length PmSLP is administered to the animal. An immunologically equivalent portion of a full-length PmSLP polypeptide can vary in length, and can include, for example, a polypeptide that includes or consists of at least 10, at least or up to 15, at least or up to 20, at least or up to 30, at least or up to 50, or at least or up to 60 consecutive amino acid residues that are identical to a portion of a PmSLP polypeptide. Furthermore, immunologically equivalent portions of a full-length PmSLP polypeptide include polypeptides that are at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% identical to the full-length PmSLP polypeptide. Radioimmunoassays (RIA) may be used to compare the binding specificity between a reference molecule and an immunologically equivalent molecule, and the extent of binding may be measured. The dissociation constant of an immunologically equivalent molecule is preferably at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the dissociation constant of the reference molecule.

[0112] The term "cross-protection" as used herein refers to a vaccine formulation that can provide protection against infection with multiple strains (e.g., 2, 3, 4, 5, or more strains) of a pathogenic microorganism, such as strains belonging to multiple serogroups or serotypes. A cross-protective vaccine formulation can contain multiple antigenic substances, or a single antigenic substance, such as multiple immunogenic polypeptides or a single immunogenic polypeptide. To assess cross-protection, a subject animal can be immunized with a vaccine formulation that contains an immunogenic substance, such as a PmSLP protein, from a selected microbial strain. The subject animal can then be challenged with another pathogenic strain of a pathogenic microorganism, and the animal's immunological response and development of disease symptoms can be evaluated. A vaccine formulation is said to be cross-protective if the infection results in an improved immune response or if the infected animal shows milder or no disease symptoms than unvaccinated animals exposed to the same infection.

[0113] As used herein, the term "veterinary pharma- ceutically acceptable" refers to a substance, including a carrier, diluent, or adjuvant, that is within the bounds of reasonable medical judgment compatible with other substances in an animal pharmaceutical preparation, including an animal vaccine preparation, and suitable for use in contact with animals without undue toxicity, allergic response, irritation, or other adverse reaction commensurate with a reasonable risk / benefit ratio.

[0114] As used herein, terms such as "treat" and "treatment" are intended to mean obtaining a desired physiological, pharmacological, or biological effect. This effect may result in the prevention (i.e., prophylactic treatment), suppression, amelioration, reduction, or reversal of the signs, symptoms, or causes of the disorder or disease resulting from the disorder or disease. Clinical evidence of treatment may vary depending on the disorder or disease, the animal, and the treatment selected. In the context of treating an indication, physiological effects may include, for example, improved respiratory ability or pulmonary function, reduced bleeding, reduced mucus nasal or oral secretions, improved rectal temperature, improved animal behavior, reduced coughing, or reduced pulmonary lesions.

[0115] The term "respiratory disease" as used herein refers to the veterinary accepted definition of respiratory disease and includes any disease involving the invasion and colonization of the respiratory tract, including the upper and lower respiratory tract, of an animal by P. multocida species, including, for example, BRD and HS.

[0116] As used herein, the term "bovine respiratory disease" or "BRD" refers to the veterinary accepted definition of bovine respiratory disease and generally includes bovine respiratory disease conditions caused by pathogenic P. multocida infection.

[0117] As used herein, the term "hemorrhagic septicemia" or "HS" refers to the veterinary accepted definition of hemorrhagic septicemia and generally includes the bovine respiratory disease condition caused by pathogenic P. multocida infection.

[0118] As used herein, the term "porcine atrophic rhinitis" or "PAR" refers to the veterinary accepted definition of porcine atrophic rhinitis and generally includes respiratory disease conditions in pigs caused by pathogenic P. multocida infection.

[0119] As used herein, the term "pneumonic pasteurellosis" refers to the veterinary accepted definition of pneumonic pasteurellosis and generally includes the respiratory disease condition in pigs caused by infection with pathogenic P. multocida.

[0120] The term "fowl cholera" as used herein refers to the veterinary accepted definition of fowl cholera and generally includes the avian respiratory disease condition caused by infection with pathogenic P. multocida.

[0121] As used herein, the terms "vaccine," "vaccine formulation," "veterinary vaccine," and "veterinary vaccine formulation" refer to a veterinary pharmacologic acceptable preparation that can be administered to an animal to elicit a humoral immune response (including the induction of a soluble antibody response) and / or a cellular immune response (including the induction of a cytotoxic T lymphocyte (CTL) response).

[0122] As used herein, the term "homologous protection" refers to protection conferred by a vaccine against infection with a strain of a microbial species, which infecting strain has an antigen, e.g., a PmSLP protein antigen, that belongs to the same phylogenetic cluster as an antigen, e.g., a PmSLP protein antigen, included in the vaccine formulation, or an immunologically equivalent portion thereof.

[0123] As used herein, the term "heterologous protection" refers to the protection conferred by a vaccine against infection with a strain of a microbial species, where the infecting strain has an antigen, e.g., a PmSLP protein antigen, that belongs to a different phylogenetic cluster than the antigen, e.g., a PmSLP protein antigen, contained in the vaccine formulation, or an immunologically equivalent portion thereof.

[0124] The terms "substantially pure" and "isolated", which may be used interchangeably herein, refer to a compound (e.g., a polypeptide) that is separated from components that naturally accompany it. Typically, a compound is substantially pure when at least 60%, more preferably at least 75%, even more preferably at least 90%, 95%, 96%, 97%, or 98%, and most preferably at least 99% of the total material in a sample (by volume, wet or dry weight, or by mole percent or mole fraction) is the compound of interest. Purity can be measured by any suitable method, such as, for example, in the case of a polypeptide, by chromatography, gel electrophoresis, or HPLC analysis.

[0125] General embodiment As previously described herein, the present disclosure relates to an animal vaccine formulation. In general, the methods and compositions provided herein can be used to prevent or ameliorate Pasteurella multocida infections in food-producing animals, including ruminant, bovine, porcine, and avian species. In this regard, the methods and compositions of the present disclosure can be used to treat, among other things, respiratory disease or hemorrhagic septicemia in cattle, pneumonic pasteurellosis or progressive atrophic rhinitis in swine, or avian cholera in poultry. The animal vaccine formulation of the present disclosure can provide long-term and effective protection against P. multocida infections.

[0126] The inventors have discovered that the veterinary vaccine formulations of the present disclosure are cross-protective and can protect food-producing animals from infection with multiple P. multocida strains. Surprisingly, the veterinary vaccine formulations of the present disclosure can be cross-protective using a single immunogenic active agent.

[0127] Furthermore, the vaccine formulations of the present disclosure can provide long-term protection, for example, where antibodies to the immunizing agent are detectable in the blood serum of the subject food-producing animal at least 26 weeks after administration of the vaccine formulation.

[0128] Furthermore, because the vaccine formulations of the present disclosure involve the use of polypeptide-based immunogenic active agents, the compositions and methods of the present disclosure do not require the use of live attenuated microbial species, thus avoiding the infection risks associated with the use of live vaccines.

[0129] Additionally, the vaccine formulations of the present disclosure may limit the administration of antibiotics to food-producing animals, thereby limiting the emergence of antibiotic-resistant microbial strains, and some consumers prefer foods derived from animals that have not been administered antibiotics.

[0130] Furthermore, the polypeptide-based immunogenic active agents included in the vaccine formulations of the present disclosure can be prepared using convenient recombinant production systems, and the immunogenic active agents can be stably stored.

[0131] The present inventors have found that a protein selected from a class of proteins known as PmSLP proteins can be used as an immunogenic active agent in the formulation of an animal vaccine to prevent or ameliorate P. multocida infection in food-producing animals. The PmSLP protein can be selected and obtained from a P. multocida strain. The P. multocida strain from which the PmSLP protein is selected is preferably a P. multocida strain that belongs to the same phylogenetic cluster as the P. multocida strain that causes infection in food-producing animals. Thus, the PmSLP protein does not have to be selected from the same P. multocida strain as the infectious P. multocida infection strain, but this other strain belongs to the same phylogenetic cluster.

[0132] Exemplary embodiments of the compositions and methods of the present disclosure are described below.

[0133] Thus, in at least one aspect, and in at least one embodiment, the present disclosure provides an animal vaccine formulation for the prevention or amelioration of P. multocida infection in a food-producing animal susceptible to P. multocida infection, the vaccine formulation comprising an effective amount of PmSLP protein, or an immunogenically equivalent portion thereof.

[0134] In another aspect, in at least one other embodiment, the present disclosure further provides a method for treating a food-producing animal susceptible to P. multocida infection, the method comprising administering to the food-producing animal an animal vaccine formulation comprising a PmSLP protein, or an immunogenically equivalent portion thereof, wherein the vaccine formulation is administered in an amount effective to prevent or ameliorate P. multocida infection.

[0135] Generally, according to one embodiment, animal vaccine formulations are prepared or obtained that contain a selected PmSLP protein or an immunogenically equivalent portion thereof. These formulations are administered in effective amounts to food-producing animals in need thereof. Accordingly, the following describes suitable preparations that contain a selected PmSLP protein or an immunogenically equivalent portion thereof, as well as methods for producing the selected PmSLP protein or an immunogenically equivalent portion thereof. This is followed by a description of animal vaccine formulations that contain a selected PmSLP protein or an immunogenically equivalent portion thereof, and methods for preparing and administering the animal vaccine formulations to food-producing animals in need thereof.

[0136] Thus, considering initially a PmSLP protein preparation, in one embodiment, a preparation containing a selected PmSLP protein, or an immunogenically equivalent portion thereof, may be prepared biosynthetically using a host cell system, In this regard, an isolated nucleic acid encoding an amino acid sequence corresponding to a PmSLP protein, or an immunogenically equivalent portion thereof, may be introduced into the host cell and expressed therein.

[0137] In general, any PmSLP protein may be used in accordance with the present specification. PmSLP protein may be obtained from P. multocida bacteria, including strains that constitute any PmSLP, including P. multocida strains belonging to any serogroup or serotype. In this regard, it is noted that P. multocida strains may be classified as belonging to different serogroups and / or serotypes, as is well known to those skilled in the art. In this regard, serogroups include serogroups A, B, D, E, and F, and serotypes include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and 16. When referring to different P. multocida strains, the serogroups and serotypes may be referenced accordingly. For example, P. multocida A:3 strain may be classified as belonging to serogroup A and serotype 3, P. multocida E:2 strain may be classified as belonging to serogroup E and serotype 2, and so on. Thus, according to this, the PmSLP protein may be a PmSLP protein obtained from a P. multocida strain selected from serogroups A, B, D, E, and F. Furthermore, the PmSLP protein may be a PmSLP protein obtained from a P. multocida strain selected from serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and 16. Thus, according to this specification, it is understood that the PmSLP protein may be selected from PmSLP proteins obtainable or obtained from a P. multocida strain belonging to any serogroup or any serotype, or any combination thereof.

[0138] According to certain aspects, in exemplary embodiments, nucleic acid sequences encoding PmSLP proteins may be selected, such nucleic acids comprising SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, or SEQ ID NO:96, as described herein.Examples of selected PmSLP proteins include SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, or SEQ ID NO:97; These are polypeptides encoded by SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, or SEQ ID NO:96, respectively.

[0139] In some embodiments, the PmSLP protein is (a) SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, or SEQ ID NO:96, (b) a nucleic acid sequence substantially identical to any one of the nucleic acid sequences of (a); (c) a nucleic acid sequence that is substantially identical to any one of the nucleic acid sequences in (a), apart from the degeneracy of the genetic code; (d) a nucleic acid sequence complementary to any one of the nucleic acid sequences of (a); (e) a nucleic acid sequence encoding a polypeptide having any one of the amino acid sequences set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, or SEQ ID NO:97, or an immunogenically equivalent portion thereof; (f) a nucleic acid sequence encoding a functional variant of any one of the amino acid sequences set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, or SEQ ID NO:97, or an immunogenically equivalent portion thereof; and (g) a nucleic acid sequence that hybridizes under stringent conditions to any one of the nucleic acid sequences set forth in (a), (b), (c), (d), (e), or (f); The protein may be expressed by a nucleic acid sequence selected from the group of nucleic acid sequences consisting of:

[0140] PmSLP proteins that may be used in accordance with the present invention include not only naturally occurring PmSLP proteins, but also PmSLP proteins that may be modified. Modifications in this regard include modifications to the amino acid sequence of a PmSLP polypeptide, including modifications of one or more specific individual amino acids, sometimes referred to as "site-directed mutations," such as, for example, the PmSLP proteins having SEQ ID NO:65 and SEQ ID NO:67.

[0141] Further modifications that can be made to the PmSLP protein include modifications that reduce the binding of the PmSLP protein to native host proteins (i.e., proteins in food-producing animals susceptible to P. multocida infection). Such reduced binding can generally be assessed by evaluating the affinity of the PmSLP protein for the native host protein. The affinity can be quantitatively evaluated by experimentally determining the dissociation constant (Kd) between the PmSLP protein and the native host protein. Thus, for example, in this respect, the Kd between the native PmSLP protein and the native host protein, e.g., the Kd between PmSLP-1 and bovine complement factor I, can be compared to the Kd between the modified PmSLP protein and the same native host protein. In general, the higher the Kd value, the weaker the affinity of the PmSLP protein for the native host protein. In some embodiments, the Kd between modified PmSLP and the native host protein may exceed the Kd between native, unmodified PmSLP and the native host protein by at least 2-fold, at least 5-fold, at least 10-fold, at least 25-fold, at least 50-fold, at least 100-fold, at least 250-fold, or at least 500-fold. Techniques for determining the Kd between two proteins are well known to those of skill in the art and include, for example, isothermal calorimetry, surface plasmon resonance, and biolayer interferometry (see also, e.g., Rich R. et al. 2007, Anal. Biochem 361:1-6; Abdiche, Y. et al., 2008, Anal. Biochem. 377:209-217; and Velazquez-Campoy, A. et al., 2004, Methods Mol. Biol. 261:35-54).

[0142] Examples of PmSLP polypeptides that may be used in this regard include PmSLP polypeptides having SEQ ID NO:65 and SEQ ID NO:67 (encoded by nucleic acid sequences having SEQ ID NO:64 and SEQ ID NO:66, respectively). In this regard, PmSLP polypeptides having SEQ ID NO:65 and SEQ ID NO:67 encode a PmSLP-1 polypeptide (SEQ ID NO:2) that includes a V214D mutation (replacing valine amino acid residue 214 with an aspartic acid amino acid residue) and an E240A mutation (replacing glutamic acid amino acid residue 240 with an alanic acid amino acid residue). The Kd between each of the modified PmSLPs having SEQ ID NO:65 and SEQ ID NO:67 and the native host protein was determined to be less than 2,000 nM, and the Kd between the wild-type PmSLP-1 protein (SEQ ID NO:2) and the native host protein was 30 nM±10 nM. Thus, the Kd between the modified PmSLP proteins of these two examples and the native host protein is at least 500-fold greater than the Kd between native, unmodified PmSLP and the native host protein.

[0143] According to certain aspects, suitable nucleic acid sequences include nucleic acid sequences encoding immunogenically equivalent portions of PmSLP polypeptides, and in particular include nucleic acid sequences encoding portions of PmSLP polypeptides that are at least immunologically equivalent to full-length PmSLP polypeptides including the polypeptides set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, or SEQ ID NO:97. Accordingly, further exemplary nucleic acid sequences that can be used in selected embodiments include nucleic acid sequences that encode an amino acid sequence that is at least immunologically equivalent to a PmSLP polypeptide, which corresponds to at least 10 contiguous amino acids and up to 150 amino acids, including SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, The PmSLP polypeptides may include 10, 20, 30, 40, 50, 55, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 amino acids of a full-length PmSLP polypeptide, including the PmSLP polypeptide set forth in SEQ ID NO:40, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, or SEQ ID NO:97.

[0144] In further embodiments, the PmSLP protein or an immunogenically equivalent portion thereof may be linked to another polypeptide to form a hybrid polypeptide, including, for example, a carrier protein or an extension to facilitate detection or purification, such as a polyhistidine extension (HIS tag) or a FLAG tag peptide extension, or another immunogenic polypeptide further comprising a second PmSLP or an immunogenically equivalent portion thereof.

[0145] Further examples of hybrid polypeptides include hybrid polypeptides comprising a first and a second PmSLP polypeptide, e.g., PmSLP-1 and PmSLP-3, and PmSLP-1 and PmSLP-2, as set forth in SEQ ID NO:51 (PmSLP-1 and PmSLP-2 hybrid), SEQ ID NO:53 (PmSLP-1, PmSLP-2, PmSLP-3 hybrid), and SEQ ID NO:55 (PmSLP-1, PmSLP-2, PmSLP-4.2 hybrid), which may be encoded by the nucleic acid sequences set forth in SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:53, and SEQ ID NO:54, respectively.

[0146] Further examples of hybrid polypeptides include hybrid polypeptides comprising first, second, and third PmSLP polypeptides, e.g., PmSLP-1, PmSLP-2, and PmSLP-3, and PmSLP-1, PmSLP-2, and PmSLP-4, as set forth in SEQ ID NO:53 (PmSLP-1, PmSLP-2, and PmSLP-3 hybrid polypeptides) and SEQ ID NO:55 (PmSLP-1, PmSLP-2, and PmSLP-3 hybrid polypeptides), which may be encoded by the nucleic acid sequences set forth in SEQ ID NO:52 and SEQ ID NO:54, respectively.

[0147] As known to those skilled in the art, biosynthesis of a protein by expression of a nucleic acid in a host cell can be achieved by providing one or more nucleic acids capable of controlling expression in the host cell and operably linking the one or more nucleic acids capable of controlling expression in the host cell to the nucleic acid to be expressed. Such operably linking of the nucleic acid controlling expression generally involves linking, in the 5' to 3' direction of expression, the nucleic acid capable of controlling expression in the host cell to the nucleic acid to be expressed, i.e., in the context of the present disclosure, the PmSLP protein. Nucleic acid sequences capable of controlling expression in a host cell that can be used herein include any transcription promoter capable of controlling expression of a polypeptide in a host cell. Generally, when a bacterial host cell is selected, a promoter derived from a bacterial cell is used, when a yeast host cell is selected, a yeast promoter is used, when a plant cell is selected, a plant promoter is used, and so on. The resulting nucleic acid comprising a promoter and a nucleic acid expressing a PmSLP protein is generally a chimeric nucleic acid. Additional nucleic acid elements that can be elements controlling expression in a host cell include transcription terminators, enhancers, etc., all of which can be included in the chimeric nucleic acid sequences of the present disclosure.

[0148] According to the present disclosure, the chimeric nucleic acid sequence can be incorporated into a recombinant expression vector that ensures good expression in a host cell, and the recombinant expression vector is suitable for expression in the host cell. The term "suitable for expression in a host cell" means that the recombinant expression vector comprises a chimeric nucleic acid linked to the necessary genetic elements to achieve expression in the cell. As mentioned above, such genetic elements may include transcription promoters, terminators, enhancers, etc. Additional genetic elements that may be included in the expression vector are one or more nucleic acid sequences encoding marker genes, and one or more origins of replication. In some embodiments, the expression vector can replicate freely in the host cell. In other embodiments, the chimeric nucleic acid may be integrated into the genomic DNA of the host cell. In some embodiments, the expression vector may further comprise the necessary genetic elements to integrate the vector or a part thereof into the genome of the host cell, for example, when a plant host cell is used, the vector may include T-DNA left and right border sequences that facilitate integration into the nuclear genome of the plant.

[0149] Marker genes that can be used according to the present disclosure include all genes that allow transformant cells to be distinguished from non-transformant cells, including all selectable and screenable marker genes.Marker genes can be, for example, resistance markers, such as antibiotic resistance markers for kanamycin, chloramphenicol, methotrexate, or ampicillin.In another example, marker genes can be genes that allow cells to produce essential nutrients, such as amino acids.

[0150] Thus, in one aspect, the disclosure provides, in an exemplary embodiment, an expression vector comprising: (a) a nucleic acid sequence encoding a P. multocida PmSLP protein, or an immunogenically equivalent portion thereof; and (b) a nucleic acid sequence capable of controlling the expression in a host cell of a nucleic acid sequence encoding a P. multocida PmSLP protein, or an immunogenically equivalent portion thereof; The present invention provides a host cell comprising a chimeric nucleic acid comprising:

[0151] In an exemplary embodiment, an expression vector may comprise a chimeric nucleic acid comprising a nucleic acid sequence encoding a promoter linked to a nucleic acid sequence encoding a PmSLP protein, or an immunogenically equivalent portion thereof.

[0152] Turning now to host cells, it should first be noted that any host cell that expresses a chimeric nucleic acid upon culture can be selected and used in accordance with the present disclosure. Suitable host cells in this regard include, for example, bacterial cells, microbial cells such as yeast cells, and algae or plant cells. A variety of techniques and methodologies exist for introducing nucleic acid sequences into cells and engineering host cells to achieve expression, and are well known to those skilled in the art. These methods include, for example, cation-based methods, such as lithium ion or calcium ion-based methods, electroporation, biolistics, and glass bead-based methods. As is well known to those skilled in the art, depending on the host cell selected, the method of introducing nucleic acid material into the host cell may vary, and the method may also be optimized for uptake of the nucleic acid material by the host cell, for example, by comparing the uptake of the nucleic acid material using different conditions. Detailed guidance can be found, for example, in Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press, 2012, Fourth Ed. It should be noted that a chimeric nucleic acid is a chimeric nucleic acid sequence that does not occur in nature, and can be said to be heterologous to the host cell.

[0153] One example of a host cell that can be conveniently used is Escherichia coli. Preparation of E. coli vectors can be accomplished using commonly known techniques such as restriction digestion, ligation, gel electrophoresis, DNA sequencing, polymerase chain reaction (PCR), and other methodologies. A wide variety of cloning vectors are available to carry out the necessary steps required to prepare recombinant expression vectors. Among the vectors with a replication system that functions in E. coli are pBR322, pUC series vectors, M13 mp series vectors, pBluescript, and other vectors. Suitable promoter sequences for use in E. coli include, for example, T7 promoter, T5 promoter, tryptophan (trp) promoter, lactose (lac) promoter, tryptophan / lactose (tac) promoter, lipoprotein (lpp) promoter, and lambda phage PL promoter. Cloning vectors usually contain a marker, for example, an antibiotic resistance marker, such as an ampicillin or kanamycin resistance marker, which allows for the selection of transformed cells. These vectors carry nucleic acid sequences that can be introduced into E. coli by preparing competent cells, electroporation, or other methods known to those skilled in the art. E. coli can be grown and harvested in an appropriate medium, such as Luria Broth medium. The recombinant expression vector can be easily recovered from the cells upon harvesting and lysis of the cells.

[0154] Another exemplary host cell that can be advantageously used is a yeast cell.Exemplary yeast host cells that can be used are yeast cells belonging to the genera Candida, Kluyveromyces, Saccharomyces, Schizosaccharomyces, Pichia, Hansenula, and Yarrowia.In certain exemplary embodiments, the yeast cell can be a Saccharomyces cerevisiae cell, a Yarrowia lipolytica cell, or a Pichia pastoris cell.

[0155] There are several vectors for expressing recombinant proteins in yeast host cells. Examples of vectors that can be used in yeast host cells include, for example, Yip-type vectors, YEp-type vectors, YRp-type vectors, YCp-type vectors, pGPD-2, pAO815, pGAPZ, pGAPZα, pHIL-D2, pHIL-S1, pPIC3.5K, pPIC9K, pPICZ, pPICZα, pPIC3K, pHWO10, pPUZZLE, and 2μm plasmid. Such vectors are known in the art and described, for example, in Cregg et al., Mol Biotechnol. (2000) 16(1): 23-52. Suitable promoter sequences for use in yeast host cells are also known and are described, for example, in Mattanovich et al., Methods Mol. Biol., 2012, 824:329-58, and Romanos et al., 1992, Yeast 8:423-488. Examples of promoters suitable for use in yeast host cells include triosephosphate isomerase (TPI), phosphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH or GAP) and its variants, lactase (LAC) and galactosidase (GAL), P. pastoris glucose-6-phosphate isomerase promoter (PPGI), 3-phosphoglycerate kinase promoter (PPGK), glycerolaldehyde phosphate dehydrogenase promoter (PGAP), translation elongation factor promoter (PTEF ), S. cerevisiae enolase (ENO-1), S. cerevisiae galactokinase (GAL1), S. cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2 / GAP), S. cerevisiae triosephosphate isomerase (TPI), S. cerevisiae metallothionein (CUP1), and S. cerevisiae 3-phosphoglycerate kinase (PGK), and glycolytic enzyme promoters such as the maltase gene promoter (MAL). Marker genes suitable for use in yeast host cells are also known in the art.Thus, antibiotic resistance markers such as ampicillin resistance markers can be used in yeast, as well as marker genes providing genetic functions for essential nutrients such as leucine (LEU2), tryptophan (TRP1 and TRP2), uracil (URA3, URA5, URA6), histidine (HIS3), etc. Methods for introducing vectors into yeast host cells are described, for example, in S. Kawai et al., 2010, Bioeng. Bugs 1(6): 395-403.

[0156] Yet another exemplary host cell that can be used according to the present specification is a plant cell. Methods for introducing nucleic acid into plant cells are known to those skilled in the art. Agrobacterium-mediated plant cell transformation methods are described, for example, in Gelvin S., Microbiol.Mol.Biol.Rev., 2003, 67(1):16-37, and methods based on physical transformation of plant cells are described in Rivera ALet al., 2012, Phys.Life Rev.9(3):308-345. Plant selection marker genes are known to those skilled in the art and include antibiotic resistance genes, such as kanamycin resistance genes, and herbicide resistance genes, such as bar genes and pat genes (Wohlleben et al., 1988, Gene 70:25-37). Screenable markers that can be used to identify plant transformants by visual inspection include β-glucuronidase (GUS) (U.S. Pat. Nos. 5,268,463 and 5,599,670) and green fluorescent protein (GFP) (Niedz et al., 1995, Plant Cell Rep., 14:403). Plant promoters are also known to those skilled in the art, and include, for example, constitutive promoters such as the 35S cauliflower mosaic virus (CaMV) promoter (Rothstein et al., 1987, Gene 53:153-161), the rice actin promoter (McElroy et al., 1990, Plant Cell 2:163-171; U.S. Pat. No. 6,429,357), ubiquitin promoters such as the maize ubiquitin promoter (U.S. Pat. Nos. 5,879,903 and 5,273,894), the parsley ubiquitin promoter (Kawalleck, P. et al., 1993, Plant Mol. Biol. 21:673-684), and, for example, the phaseolin promoter (Sengupta-Gopalan et al., 1985, Proc. Natl. Acad. Sci. USA 82:3320-3324), or seed specific promoters such as the oleosin promoter (U.S. Patent No. 5,792,922).

[0157] Further guidance on preparing expression vectors and introducing them into host cells, including E. coli cells, yeast cells, and other host cells, can be found, for example, in Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press, 2012, Fourth Ed.

[0158] Thus, in another aspect, the present disclosure provides, as one exemplary embodiment, (a) a nucleic acid sequence encoding a P. multocida PmSLP protein, or an immunogenically equivalent portion thereof; and (b) a nucleic acid sequence capable of controlling the expression in a host cell of a nucleic acid sequence encoding a P. multocida PmSLP protein, or an immunogenically equivalent portion thereof; The present invention provides a host cell comprising a chimeric nucleic acid comprising:

[0159] In an exemplary embodiment, an expression vector may comprise a chimeric nucleic acid comprising a nucleic acid sequence encoding a promoter linked to a nucleic acid sequence encoding a PmSLP protein, or an immunogenically equivalent portion thereof.

[0160] Thus, in brief summary, a host cell can be prepared according to the present disclosure that contains a chimeric nucleic acid comprising (i) a nucleic acid sequence encoding a PmSLP protein or an immunogenically equivalent portion thereof, and (ii) a nucleic acid sequence capable of controlling the expression of the nucleic acid sequence encoding the PmSLP protein or an immunogenically equivalent portion thereof in the host cell.

[0161] According to the present disclosure, the host cell is grown to express the chimeric nucleic acid. Expression of the chimeric nucleic acid results in the biosynthetic production of an immunogenically equivalent portion of the PmSLP protein in the host cell. Growth media and conditions may vary depending on the host cell selected, as will be readily understood by those skilled in the art. Growth media typically include a carbon source, one or more nitrogen sources, essential salts such as potassium, sodium, magnesium, phosphate, sulfate, trace metals, water-soluble vitamins, and processing aids including, but not limited to, antifoaming agents, protease inhibitors, stabilizers, ligands, and inducers. Exemplary carbon sources are, for example, monosaccharides or disaccharides. Exemplary nitrogen sources are, for example, ammonia, urea, amino acids, yeast extract, corn steep liquor, and fully or partially hydrolyzed proteins. Exemplary trace metals are, for example, Fe, Zn, Mn, Cu, Mo, and H3BO3. Exemplary water-soluble vitamins are, for example, biotin, pantothenic acid, niacin, thiamine, p-aminobenzoic acid, choline, pyridoxine, folic acid, riboflavin, and ascorbic acid. Further examples of specific media include liquid media for the growth of yeast cells and bacterial cells, including Luria-Bertani (LB) broth for bacterial cell culture, and yeast extract peptone dextrose (YEPD or YPD) for yeast cell culture. Further media and growth conditions can be found in Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press, 2012, Fourth Ed.

[0162] Once the PmSLP protein or its immunogenically equivalent portion is produced by the host cell, the PmSLP protein or its immunogenically equivalent portion can be harvested from the host cell and separated from other components, such as, for example, cell debris and medium components. Separation techniques are known to those skilled in the art and include a variety of different protein purification techniques, including, for example, ion exchange chromatography, size exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, reverse phase chromatography, gel filtration, and the like. Further general guidance on protein purification can be found, for example, in Cutler, P. Protein Purification Protocols, Humana Press, 2004, Second Ed. Thus, substantially pure preparations of the PmSLP protein or its immunogenically equivalent portion can be obtained. The recovered PmSLP protein may be obtained in a more or less pure form, for example, a preparation of PmSLP protein or an immunogenically equivalent portion thereof having a purity of at least about 60% (w / v), about 70% (w / v), about 80% (w / v), about 90% (w / v), about 95% (w / v), or about 99% (w / v) may be obtained.

[0163] Furthermore, it should be noted that recombinant production of the PmSLP protein, or an immunogenically equivalent portion thereof, in a host cell system allows the PmSLP protein, or an immunogenically equivalent portion thereof, to be produced in a manner that is substantially free of other P. multocida constituents, such as other P. multocida proteins, membrane material, lipopolysaccharides, etc., that are naturally associated with the PmSLP protein.

[0164] It should be noted that in some embodiments, the cells may secrete a portion of the produced PmSLP protein, or an immunogenically equivalent portion thereof, into the cell growth medium, and thus a portion of the produced PmSLP protein, or an immunogenically equivalent portion thereof, may be recovered from the cells and a further portion of the PmSLP protein, or an immunogenically equivalent portion thereof, may be recovered from the growth medium.

[0165] It is further noted that the animal vaccine formulations of the present disclosure may contain the PmSLP protein or an immunogenically equivalent portion thereof in a more or less pure form. Thus, in accordance with the present disclosure, substantially pure PmSLP protein or an immunogenically equivalent portion thereof may be obtained and used to prepare an animal vaccine formulation. Thus, for example, in some embodiments, the PmSLP protein may be substantially free of other host cell constituents, such as host cell proteins, membrane material, lipopolysaccharides, etc. In other embodiments, cruder preparations containing the PmSLP protein or an immunogenically equivalent portion thereof may be obtained and used to prepare the vaccine formulation. Thus, for example, in such embodiments, host cells, host cell lysates, or host cell fractions containing the PmSLP protein or an immunogenically equivalent portion thereof may be used to prepare the vaccine formulation.

[0166] It is noted that any PmSLP or immunogenically equivalent portion thereof may be used to formulate an animal vaccine formulation of the present disclosure, including a PmSLP protein having any one of the amino acid sequences set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, or SEQ ID NO:97, or an immunogenically equivalent portion thereof.

[0167] Additionally, in one aspect of the present disclosure, when preparing a vaccine formulation, it is preferable to take into consideration P. multocida strains that cause infections in food-producing animals, preferably when PmSLP proteins are selected.

[0168] In particular, when preparing a vaccine formulation for treating animals infected with P. multocida strains having PmSLP proteins belonging to a particular phylogenetic cluster, the vaccine is preferably prepared by selecting a PmSLP polypeptide belonging to the same phylogenetic cluster, including a PmSLP polypeptide belonging to the same phylogenetic cluster, another PmSLP polypeptide belonging to the same phylogenetic cluster, and including the PmSLP polypeptide thus selected in the vaccine formulation. Thus, for example, referring to FIG. 2, preferably, when preparing a vaccine formulation for treating animals infected with P. multocida strains having PmSLP polypeptides belonging to phylogenetic clusters PmSLP-1, PmSLP-2, PmSLP-3, PmSLP-4.1, or PmSLP-4.2, a PmSLP polypeptide from the same phylogenetic cluster is selected to prepare the vaccine. Thus, for example, if an animal is infected with a P. multocida strain having a PmSLP polypeptide belonging to phylogenetic cluster PmSLP-1, for example, SEQ ID NO: 2, the vaccine formulation is preferably prepared by selecting a PmSLP polypeptide belonging to phylogenetic cluster PmSLP-1, for example, SEQ ID NO: 2, or an immunogenically equivalent part thereof, or another PmSLP polypeptide belonging to phylogenetic cluster PmSLP-1, for example, and included in the vaccine formulation. As a further example, if an animal is infected with a P. multocida strain having a PmSLP protein belonging to phylogenetic cluster PmSLP-3, for example, SEQ ID NO: 6, the vaccine formulation is preferably prepared by selecting a PmSLP polypeptide belonging to phylogenetic cluster PmSLP-3, for example, SEQ ID NO: 6, or an immunogenically equivalent part thereof, or another PmSLP polypeptide belonging to phylogenetic cluster PmSLP-3, for example, SEQ ID NO: 6, and included in the vaccine formulation. The strain from which the PmSLP protein is selected may be the same P. multocida strain, or another strain from the P. multocida infection strain, provided that this other strain belongs to the same phylogenetic cluster. Thus, the PmSLP protein does not necessarily have to be selected from the same P. multocida strain as the infecting P. multocida strain it infects, provided that this other strain belongs to the same phylogenetic cluster.

[0169] Moreover, the host and clinical indication are preferably taken into consideration when selecting the PmSLP polypeptide. In particular, when preparing a vaccine formulation for treating an animal host infected with a P. multocida strain that causes a particular symptom, the vaccine is preferably prepared by selecting a PmSLP polypeptide from a P. multocida strain capable of causing symptoms in the host, comprising the same PmSLP polypeptide or another PmSLP polypeptide isolated from a P. multocida strain capable of causing symptoms in the host, and including the PmSLP polypeptide thus selected in the vaccine formulation. Thus, for example, referring again to FIG. 2, preferably, when preparing a vaccine formulation for treating an animal infected with a P. multocida strain having a PmSLP polypeptide belonging to the phylogenetic clusters PmSLP-1, PmSLP-2, PmSLP-3, PmSLP-4.1, or PmSLP-4.2, a PmSLP polypeptide from the same phylogenetic cluster is selected to prepare the vaccine, and further, the PmSLP is obtained from a P. multocida strain known to cause the indication being treated in the host species being treated. Thus, as an example, if a bovine animal is diagnosed with BRD caused by infection with a P. multocida strain having a PmSLP polypeptide belonging to phylogenetic cluster PmSLP-2 (e.g., SEQ ID NO: 4), the vaccine formulation is preferably prepared by selecting a PmSLP polypeptide belonging to phylogenetic cluster PmSLP-2 (e.g., SEQ ID NO: 4) or an immunogenically equivalent portion thereof, or another PmSLP polypeptide belonging to phylogenetic cluster PmSLP-2, and more preferably, the PmSLP-2 polypeptide is obtained from a P. multocida strain known to cause BRD in bovine animals, and not from, for example, a porcine animal that may be infected by a P. multocida strain containing PmSLP-2, as can be seen in Figure 2.

[0170] In an exemplary embodiment, the food-producing animal may be a ruminant susceptible to infection with a P. multocida strain that causes respiratory disease, and the vaccine formulation is prepared to contain at least one P. multocida PmSLP protein or immunogenically equivalent portion thereof selected from the group of phylogenetic clusters consisting of PmSLP-1, PmSLP-2, PmSLP-3, and PmSLP-4.2, wherein the selected P. multocida PmSLP protein or immunogenically equivalent portion thereof belongs to the same phylogenetic cluster as the PmSLP protein present in the infecting P. multocida strain.

[0171] In an exemplary embodiment, the food-producing animal may be a bovine animal susceptible to infection with a P. multocida strain that causes BRD, and the vaccine formulation is prepared to contain at least one P. multocida PmSLP protein or an immunogenically equivalent portion thereof selected from the group of phylogenetic clusters consisting of PmSLP-1, PmSLP-2, and PmSLP-4.2, wherein the selected P. multocida PmSLP protein or an immunogenically equivalent portion thereof belongs to the same phylogenetic cluster as the PmSLP protein present in the infecting P. multocida strain.

[0172] In an exemplary embodiment, the food-producing animal may be a bovine animal susceptible to infection with a P. multocida strain that causes HS, and the vaccine formulation is prepared to contain at least one P. multocida PmSLP protein or an immunogenically equivalent portion thereof selected from the group of phylogenetic clusters consisting of PmSLP-3, wherein the selected P. multocida PmSLP protein or an immunogenically equivalent portion thereof belongs to the same phylogenetic cluster as the PmSLP protein present in the infecting P. multocida strain.

[0173] In an exemplary embodiment, the food-producing animal may be a swine animal susceptible to infection with a P. multocida strain causing porcine atrophic rhinitis (PAR), and the vaccine formulation is prepared to contain at least one P. multocida PmSLP protein or immunogenically equivalent portion thereof selected from the group of phylogenetic clusters consisting of PmSLP-2, PmSLP-4.1, and PmSLP-4.2, wherein the selected P. multocida PmSLP protein or immunogenically equivalent portion thereof belongs to the same phylogenetic cluster as the PmSLP protein present in the infecting P. multocida strain.

[0174] In an exemplary embodiment, the food-producing animal may be a swine animal susceptible to infection with a P. multocida strain causing pneumonic pasteurellosis, and the vaccine formulation is prepared to contain at least one P. multocida PmSLP protein or immunogenically equivalent portion thereof selected from the group of phylogenetic clusters consisting of PmSLP-2, PmSLP-4.1, and PmSLP-4.2, wherein the selected P. multocida PmSLP protein or immunogenically equivalent portion thereof belongs to the same phylogenetic cluster as the PmSLP protein present in the infecting P. multocida strain.

[0175] In an exemplary embodiment, the food-producing animal may be an avian animal susceptible to infection with a P. multocida strain that causes fowl cholera, and the vaccine formulation is prepared to contain a P. multocida PmSLP protein, or an immunogenically equivalent portion thereof, selected from the group of phylogenetic clusters consisting of PmSLP-3 and PmSLP-4.2, wherein the selected P. multocida PmSLP protein, or an immunogenically equivalent portion thereof, belongs to the same phylogenetic cluster as the PmSLP protein present in the infecting P. multocida strain.

[0176] It should be noted that to identify P. multocida strains, they may be isolated and cultured from infected food-producing animals, and the sequence of the PmSLP polypeptide present therein may be determined using techniques known to those skilled in the art.See, for example, Pasteurellaceae: Biology, Genomics and Molecular Aspects, published by Caster Academic Press, edited by Peter Kuhnert (Institute of Veterinary Bacteriology, Vetsuisse Faculty University of Bern, Langgass-Str. 122, 3001 Bern, Switzerland), and Henrik Cristensen (Department of Veterinary Pathobiology, Faculty of Life Science, Copenhagen University, Dyrlaegevej 88, 1870 Frederiksberg, Denmark).

[0177] Furthermore, in an exemplary embodiment, when preparing a vaccine formulation, in order to treat an animal infected with a P. multocida strain belonging to a particular serogroup, the vaccine is preferably prepared using a PmSLP polypeptide obtained from a P. multocida strain belonging to the same serogroup. Thus, for example, when preparing a vaccine formulation, a vaccine formulation comprising a PmSLP polypeptide obtained from a P. multocida strain of the same serogroup is preferably selected for inclusion in the vaccine to treat an animal infected with a P. multocida strain belonging to serogroups A, B, C, D, E, or F. Thus, for example, if an animal is infected with a P. multocida strain of serogroup A, the vaccine formulation is preferably prepared by selecting a PmSLP polypeptide obtained from a P. multocida strain of serogroup A or an immunogenically equivalent portion thereof for inclusion in the formulation. As a further example, if an animal is infected with a P. multocida strain belonging to serogroup F, the vaccine formulation is preferably prepared by selecting a PmSLP polypeptide obtained from a P. multocida strain belonging to serogroup F or an immunogenically equivalent portion thereof for inclusion in the formulation. The strain may be the same or a different strain, so long as it belongs to the same serogroup.

[0178] In a further preferred exemplary embodiment, both phylogenetic clusters and serogroups are taken into consideration. Thus, preferably, when preparing a vaccine formulation, in order to treat an animal infected with a P. multocida strain that contains a PmSLP polypeptide belonging to a particular phylogenetic group and a particular serogroup, a PmSLP polypeptide belonging to a strain belonging to the same phylogenetic group and the same serogroup as the infecting strain is selected and used to prepare the vaccine formulation. Thus, referring again to FIG. 2, for example, in preparing a vaccine formulation, in order to treat an animal infected with a P. multocida strain that contains a PmSLP protein belonging to phylogenetic clusters PmSLP-1, PmSLP-2, PmSLP-3, PmSLP-4.1, or PmSLP-4.2, and a P. multocida strain that belongs to serogroups A, B, D, E, or F, a vaccine formulation containing a PmSLP polypeptide from the same phylogenetic cluster and obtained from a strain of the same serogroup is selected for inclusion in the vaccine. The PmSLP polypeptide may be identical to the PmSLP contained in the infecting strain or may be different, provided that the PmSLP polypeptide belongs to the same phylogenetic cluster and is obtained from a strain of the same serogroup as the infecting strain. Thus, by way of example, if an animal is infected with a P. multocida strain containing a PmSLP polypeptide belonging to phylogenetic group PmSLP-2 and the infecting strain belongs to serogroup A, the vaccine formulation is preferably prepared to contain a PmSLP polypeptide or an immunogenically equivalent portion thereof obtained from a P. multocida strain containing a PmSLP polypeptide belonging to phylogenetic group PmSLP-2 and a strain belonging to serogroup A. If an animal is infected with a P. multocida strain containing a PmSLP polypeptide belonging to phylogenetic cluster PmSLP-3 and the strain belongs to serogroup F, the vaccine formulation is preferably prepared to contain a P. multocida strain containing a PmSLP polypeptide belonging to phylogenetic cluster PmSLP-3 and a PmSLP polypeptide or an immunogenically equivalent portion thereof obtained from a strain belonging to serogroup F. This strain may be the same strain or a different strain, so long as it belongs to the same phylogenetic group and the same serogroup.

[0179] Thus, one embodiment now becomes apparent, wherein the vaccine formulation may comprise a P. multocida PmSLP protein, or an immunogenically equivalent portion thereof, selected from the group of phylogenetic clusters consisting of PmSLP-1, PmSLP-2, PmSLP-3, PmSLP-4.1, and PmSLP-4.2, wherein the selected P. multocida PmSLP protein, or an immunogenically equivalent portion thereof, belongs to the same phylogenetic cluster as the PmSLP protein present in the infecting P. multocida strain, and wherein the selected P. multocida PmSLP protein, or an immunogenically equivalent portion thereof, is further from a P. multocida strain belonging to a serogroup selected from the group consisting of serogroups A, B, D, E, and F, wherein the serogroup is the same as the serogroup of the infecting P. multocida strain.

[0180] Furthermore, the PmSLP protein can be a PmSLP protein obtained from a P. multocida strain selected from serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and 16, such that the food-producing animal is susceptible to infection with a P. multocida strain of the selected serotype. Thus, in one embodiment, when preparing a vaccine formulation, to treat an animal infected with a P. multocida strain that contains a PmSLP protein belonging to a particular phylogenetic cluster, a particular serogroup, and a particular serotype, the vaccine is preferably prepared using a PmSLP polypeptide belonging to the same phylogenetic cluster, the same serogroup, and the same serotype. Thus, for example, if P. multocida infection causes hemorrhagic septicemia in bovine species and the strain is a B:2 strain, the PmSLP protein can be obtained from the B:2 strain, or, for example, if P. multocida infection causes progressive atrophic rhinitis in porcine species and the strain belongs to serogroup D, the PmSLP protein can be obtained from a P. multocida strain belonging to serogroup D, and so on. It should be noted that a particular P. multocida strain can be obtained (e.g., from a collection of microbial species such as the American Type Culture Collection (ATCC)) and isolated, for example, from an infected food-producing animal, and the serogroup and / or serotype of the strain can be determined using methods known to those skilled in the art (see, for example, Wilson, M. et al., 1992, J. Clin. Microbiol, 1518-1524; Arumugam, N., et al. 2011, Tropical Biomed. 28(1) 55-63).

[0181] In a further exemplary embodiment, the food-producing animal may be selected to be a ruminant species and the vaccine formulation may be prepared for administration to the ruminant species, where P. multocida infection causes respiratory disease, and the PmSLP protein comprises SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, or SEQ ID NO:91, or an immunogenically equivalent portion thereof, to prevent or ameliorate P. multocida infection causing respiratory disease.

[0182] In a further exemplary embodiment, the food-producing animal may be selected to be a bovine species and the vaccine formulation may be prepared for administration to the bovine species, where P. multocida infection causes BRD and the PmSLP protein comprises SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:77, or SEQ ID NO:91, or an immunogenically equivalent portion thereof, to prevent or ameliorate P. multocida infection causing BRD.

[0183] In a further exemplary embodiment, the food-producing animal may be selected to be a bovine species, and the vaccine formulation may be prepared for administration to the bovine species, wherein a P. multocida infection causes HS, and the PmSLP protein comprises SEQ ID NO:6, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:53, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, or SEQ ID NO:89, or an immunogenically equivalent portion thereof, to prevent or ameliorate the P. multocida infection causing HS.

[0184] In a further exemplary embodiment, the food-producing animal may be selected to be a porcine species and the vaccine formulation may be prepared for administration to the porcine species, where the P. multocida infection causes pneumonic pasteurellosis, and the PmSLP protein comprises SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:40, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:75, SEQ ID NO:77, or SEQ ID NO:91, or an immunogenically equivalent portion thereof, to prevent or ameliorate the P. multocida infection causing pneumonic pasteurellosis.

[0185] In a further exemplary embodiment, the food-producing animal may be a porcine species, and the vaccine formulation may be prepared for administration to the porcine species, wherein a P. multocida infection causes PAR, and the PmSLP protein comprises SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:40, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:75, SEQ ID NO:77, or SEQ ID NO:91, or an immunogenically equivalent portion thereof, to prevent or ameliorate the P. multocida infection causing PAR.

[0186] In a further exemplary embodiment, the food-producing animal may be a bird, and the vaccine formulation may be prepared for administration to the bird, where P. multocida infection causes poultry cholera, and the PmSLP protein comprises SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:40, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:61, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, or SEQ ID NO:89, or an immunogenically equivalent portion thereof, to prevent or ameliorate P. multocida infection that causes poultry cholera.

[0187] However, it should be noted that the vaccine formulations of the present disclosure may be used to ameliorate or prevent infection by P. multocida strains other than the strain for which the PmSLP protein is selected for inclusion in the veterinary vaccine formulation. Thus, surprisingly, the vaccine formulations of the present disclosure do not necessarily need to contain PmSLPs from multiple P. multocida strains for use in treating food-producing animals, even if the food-producing animals may be exposed to, have been exposed to, or may be infected with, multiple P. multocida strains. Thus, for example, a vaccine formulation containing a PmSLP protein obtained from a P. multocida strain of a first serotype may be used to treat a food-producing animal against infection caused by a P. multocida strain of another serotype. In this respect, an veterinary vaccine formulation may be said to be cross-protective and may confer homologous or heterologous protection, even if it contains a single PmSLP or an immunogenically equivalent portion thereof.

[0188] Notwithstanding the foregoing, in some embodiments, a vaccine formulation may include two or more PmSLP proteins. Thus, in an exemplary embodiment, a vaccine formulation may include PmSLP proteins from two or more P. multocida strains, each belonging to a single serogroup selected from serogroups A, B, D, E, or F, or a veterinary vaccine formulation may include PmSLP proteins from two P. multocida strains belonging to two or more serogroups selected from serogroups A, B, D, E, or F. In such embodiments, a vaccine may prevent or reduce infection by more than one P. multocida strain, and such a vaccine may be said to be cross-protective.

[0189] In further exemplary embodiments, a vaccine formulation may include two or more PmSLP proteins from two or more strains each belonging to a single phylogenetic cluster, PmSLP-1, PmSLP-2, PmSLP-3, PmSLP-4.1, and PmSLP-4.2, or a veterinary vaccine formulation may include PmSLP proteins from two or more P. multocida strains belonging to two or more phylogenetic clusters, PmSLP-1, PmSLP-2, PmSLP-3, PmSLP-4.1, and PmSLP-4.2. In such embodiments, the vaccine may prevent or reduce infection by two or more P. multocida strains, and such a vaccine may be said to be cross-protective.

[0190] In further exemplary embodiments, the vaccine formulation can include two or more PmSLP proteins obtained from two P. multocida strains, each of which belongs to a single serotype selected from serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, or the veterinary vaccine formulation can include PmSLP proteins obtained from two or more P. multocida strains, each of which belongs to two or more serotypes selected from serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In such embodiments, the vaccine can prevent or ameliorate infection by strains of two or more P. multocida strains, and such a vaccine can be said to be cross-protective.

[0191] In further exemplary embodiments, the vaccine formulation may comprise two or at least two, three or at least three, four or at least four, five or at least five PmSLP proteins having any one of the amino acid sequences set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, or SEQ ID NO:97, or an immunogenically equivalent portion thereof.

[0192] Thus, in brief overview, in accordance with the present disclosure, a P. multocida PmSLP polypeptide, or an immunogenically equivalent portion thereof, may be selected for inclusion in an animal vaccine formulation for treating a food-producing animal infected with P. multocida. Preferably, the selected P. multocida PmSLP polypeptide belongs to the same phylogenetic cluster as the PmSLP polypeptide contained by the infecting P. multocida strain.

[0193] Turning now to the preparation of vaccine formulations, in one embodiment of the invention, to prepare a vaccine formulation, a preparation comprising a PmSLP protein, or an immunogenically equivalent portion thereof, can be combined with at least one other veterinary pharma- ceutically acceptable ingredient, including, but not limited to, a diluent, excipient, carrier, adjuvant, or mixtures thereof, whereby the PmSLP protein, or an immunogenically equivalent portion thereof, and the at least one other ingredient are mixed, blended, homogenized, or otherwise prepared together until a vaccine formulation is formed.

[0194] The amount of PmSLP protein or its immunogenically equivalent part in the animal vaccine formulation may vary. Generally, the dose to be administered to the animal is taken into consideration. The dose of PmSLP protein or its immunogenically equivalent part can be formulated to contain PmSLP protein or its immunogenically equivalent part in an amount ranging from about 1 pg / kg animal body weight to about 0.25 mg / kg animal body weight, preferably from about 1 μg / kg animal body weight to about 100 μg / kg animal body weight. Furthermore, the vaccine formulation preferably comprises a vaccine comprising a vaccinia virus having a dosage of at least about 0.001% by weight or about 0.001% by volume, at least 0.025% by weight or about 0.025% by weight, at least 0.05% by weight or about 0.05% by weight, at least 0.1% by weight or about 0.1% by weight, at least 0.5% by weight or about 0.5% by weight, at least 1% by weight or about 1% by weight, at least 5% by weight or about 5% by weight, at least 10% by weight or about 10% by weight, at least 15% by weight or about 15% by weight, at least or at least about 20% by weight PmSLP protein, such that the weight or volume ratio of PmSLP to other vaccine components (e.g., adjuvants, diluents, carriers, excipients) in the vaccine formulation is at least 0.001:99.999, 0.025:99.975, 0.05:99,95, 0.01:99.99, 0.5:99.5, 1:99, 5:95, 15:85, 20:80, or 25:75, respectively, although the exact amount required will vary depending on the species, age, general condition, and severity of the condition being treated, the particular formulation administered, the site of administration, the subject species, and other factors. In this regard, animal vaccine formulations may vary in terms of the amount of PmSLP protein, or immunogenically equivalent portion thereof, contained in a dose, depending in particular on the species of food-producing animal to which the vaccine formulation is administered. A suitable effective amount can be easily determined by one skilled in the art. Thus, a therapeutically effective amount of PmSLP protein, or immunogenically equivalent portion thereof, contained in an animal vaccine formulation of the present disclosure is an amount sufficient to cause amelioration or prevention of symptoms of a disease or condition, and falls within a relatively broad range that can be determined through routine testing.

[0195] The animal vaccine formulations containing the PmSLP protein or immunogenically equivalent portion thereof of the present disclosure are preferably further prepared by combining the PmSLP protein or immunogenically equivalent portion thereof with, for example, carriers, excipients, diluents, and auxiliary substances, such as wetting or emulsifying agents, pH buffer substances, and the like. These carriers, excipients, diluents, and auxiliary substances are veterinary medicament acceptable ingredients. Veterinary medicament acceptable excipients include, but are not limited to, liquids such as water, saline, polyethylene glycol, hyaluronic acid, glycerol, and ethanol. Veterinary medicament acceptable salts, for example, mineral acid salts such as hydrochloride, phosphate, sulfate, and organic acid salts such as acetate, propionate, benzoate, and the like, can also be included in the formulation. In addition, although not required, the vaccine formulation preferably includes a veterinary medicament acceptable carrier that functions as a stabilizer, particularly to stabilize the polypeptide of the present disclosure. Examples of suitable carriers that also act as stabilizers for the peptides include, but are not limited to, veterinary grade dextrose, sucrose, lactose, sorbitol, inositol, dextran, etc. Other suitable carriers include, but are not limited to, starch, cellulose, sodium or calcium phosphate, citric acid, glycine, polyethylene glycol (PEG), and combinations thereof. Carriers, excipients, and diluents may comprise, for example, about 10% to about 95% by weight or volume of the vaccine formulation.

[0196] Additionally, auxiliary agents such as lyophilization stabilizers, wetting or emulsifying agents, pH buffering agents, gelling or viscosity enhancing additives, and preservatives may also be included in the vaccine formulations of the present disclosure. The vaccine formulations generally contain less than about 5% by weight of such auxiliary agents.

[0197] To enhance the immune response in the animal, the veterinary vaccine formulations provided herein further preferably contain one or more adjuvants, such as pharmacological agents, cytokines, etc. Suitable adjuvants include any substance that enhances the immune response of a recipient animal to the immunogenic PmSLP protein of the present disclosure, or an immunogenically equivalent portion thereof. Non-limiting examples of adjuvants include cytokines (e.g., IL-1, IL-2, IL-12, IL-6), as well as inorganic salts (e.g., aluminum hydroxide, aluminum phosphate, and calcium phosphate), oil emulsions (e.g., mineral oil, MF59, QS-21, Montanide™ ISA51, Montanide™ ISA61, Montanide™ ISA 61 VG, Montanide™ Gel 02, Montanide™ ISA-720, or Emulsigen D®), Isocoms (e.g., ISCOMATRIX), microbial derivatives (e.g., monophosphoryl lipid A (MPLA), macrophage activating protein-2, virosomes, LT / CT, CpG), natural polymers (e.g., polysaccharides), and synthetic polymers (e.g., polyanhydrides and polyesters), or nucleic acid analogs (e.g., Poly I:C). Adjuvants can be administered, for example, before or after administration of a polypeptide antigen, contemporaneously (e.g., by inclusion in the vaccine formulation) as a protein or other macromolecule. When included in a vaccine formulation, the adjuvant can constitute, for example, 0.1% or about 0.1%-50% or about 50%, 0.1% or about 0.1%-20% or about 20%, or 1% or about 1%-10% or about 10% of the weight or volume of the vaccine formulation.

[0198] In light of the above, it will be appreciated that the present disclosure provides, in another aspect, a method for preparing a vaccine formulation. In this regard, the present disclosure provides, in one embodiment, a method for preparing an animal vaccine formulation for the prevention or amelioration of P. multocida infection in a food-producing animal susceptible to P. multocida infection, in accordance with the teachings herein, comprising: (i) to diagnose P. multocida infections in food-producing animals; (ii) identifying a phylogenetic cluster selected from PmSLP-1, PmSLP-2, PmSLP-3, PmSLP-4.1, or PmSLP-4.2 to which the PmSLP proteins contained in the infecting P. multocida belong; and (iii) preparing a vaccine formulation comprising a P. multocida PmSLP protein, or an immunogenically equivalent portion thereof, belonging to the identified phylogenetic cluster, together with a veterinary pharma- ceutically acceptable adjuvant to prepare an animal vaccine formulation comprising an amount of P. multocida PmSLP protein, or an immunogenically equivalent portion thereof, effective to treat a food-producing animal susceptible to P. multocida infection; The present invention provides a method comprising:

[0199] In one embodiment, in one aspect, the method may further include identifying the serogroup of the infecting P. multocida strain, wherein the serogroup is selected from the group consisting of serogroups A, B, D, E, and F, and the vaccine is prepared using a P. multocida PmSLP protein, or an immunogenically equivalent portion thereof, derived from the same or another P. multocida strain belonging to the selected serogroup.

[0200] In a further exemplary embodiment, the disclosure provides a method for preparing an animal vaccine formulation comprising a PmSLP protein, or an immunogenically equivalent portion thereof, comprising: (a) as operably linked components: (i) a nucleic acid sequence encoding a P. multocida PmSLP protein, or an immunogenically equivalent portion thereof; and (ii) one or more nucleic acid sequences capable of controlling expression in a host cell; To provide (b) introducing the chimeric nucleic acid sequence into a host cell; (c) growing the host cell to produce the PmSLP protein, or an immunogenically equivalent portion thereof; (d) recovering the PmSLP protein, or an immunogenically equivalent portion thereof; and (e) formulating the PmSLP protein, or an immunogenically equivalent portion thereof, with an adjuvant to form an animal vaccine formulation containing an effective amount of the PmSLP protein, or an immunogenically equivalent portion thereof, for treating a food-producing animal susceptible to P. multocida infection; The present invention provides a method comprising:

[0201] The vaccine formulations of the present disclosure can be used to prevent infection or disease caused by pathogenic infectious P. multocida in food-producing animals. The vaccine formulations can be used to immunize any food-producing animal, including any bovine, porcine, or avian species.

[0202] The animal vaccine formulation of the present disclosure may be administered to food-producing animals using any convenient administration means. Thus, for example, the animal vaccine formulation may be injected, for example, intramuscularly or subcutaneously, or the vaccine formulation may be administered orally to animals, for example, as a dietary supplement. In this regard, it will be understood that administration means and techniques, such as, for example, in the case of injection, the gauge of the needle, may vary from animal to animal. The dosage of the animal vaccine formulation will depend on the disease, the route of administration, the animal species, the body weight, and other standard factors. In this regard, the skilled person can easily titrate the appropriate dosage to the effective amount, and select the appropriate administration method.

[0203] Additionally, it should be noted that the animal vaccine formulations of the present disclosure may be administered prophylactically, i.e., to prevent P. multocida infection in food-producing animals, or to ameliorate symptoms associated with P. multocida infection after the occurrence of infection in a food-producing animal.

[0204] Administration of the animal vaccine formulation of the present disclosure generally induces an immune response in the target food-producing animal.Therefore, antibodies against the PmSLP protein contained in the vaccine formulation can be formed by the food-producing animal.In some embodiments, anti-PmSLP antibodies can be detected in the serum of the food-producing animal at least 7 days, at least 2 weeks, at least 5 weeks, at least 10 weeks, at least 13 weeks, at least 26 weeks, or at least 52 weeks after administration of the vaccine formulation.

[0205] In light of the above, it will be appreciated that in another aspect, the disclosure provides, in an exemplary embodiment, the use of a PmSLP protein, or an immunogenically equivalent portion thereof, for the preparation of an animal vaccine formulation comprising the PmSLP protein, or an immunogenically equivalent portion thereof, together with a pharma- ceutically acceptable adjuvant for animals.

[0206] In light of the above, it will be appreciated that in another aspect, the present disclosure provides, in an exemplary embodiment, the use of an animal vaccine formulation comprising a PmSLP protein, or an immunogenically equivalent portion thereof, together with a pharma- ceutically acceptable adjuvant for animals, for ameliorating or preventing P. multocida infection in food-producing animals susceptible to P. multocida infection.

[0207] As can be appreciated herein, veterinary vaccine formulations can be prepared that include a PmSLP protein, or an immunogenically equivalent portion thereof, that can be administered to food-producing animals to ameliorate or prevent infection of the animals with P. multocida.

[0208] Of course, the above exemplary embodiments of the present disclosure are intended to be illustrative and not limiting in any way. The present embodiments are subject to numerous modifications or configurations, details, and sequences of operation. The present invention and the present disclosure are intended to encompass all such modifications within their scope, as defined by the claims, and should be accorded the broadest interpretation consistent with the description as a whole.

[0209] Overview of Arrays SEQ ID NO:1 and SEQ ID NO:2 show the polynucleotide sequence and deduced amino acid sequence, respectively, of mature PmSLP-1 of P. multocida (belonging to phylogenetic cluster 1).

[0210] SEQ ID NO:3 and SEQ ID NO:4 show the polynucleotide sequence and deduced amino acid sequence, respectively, of mature PmSLP-2 of P. multocida (belonging to phylogenetic cluster 2).

[0211] SEQ ID NO:5 and SEQ ID NO:6 show the polynucleotide sequence and deduced amino acid sequence, respectively, of mature PmSLP-3 of P. multocida (belonging to phylogenetic cluster 3).

[0212] SEQ ID NO:7 and SEQ ID NO:8 show the polynucleotide sequence and deduced amino acid sequence, respectively, of mature PmSLP-4 of P. multocida (belonging to phylogenetic cluster 4.1).

[0213] SEQ ID NO:9 and SEQ ID NO:10 show the polynucleotide sequence and deduced amino acid sequence, respectively, of mature PmSLP-4 of P. multocida (belonging to phylogenetic cluster 4.2).

[0214] SEQ ID NO:11 and SEQ ID NO:12 show the polynucleotide sequence and deduced amino acid sequence, respectively, of P. multocida PmSLP-1 having the 14-residue anchor cleavage of PmSLP, a thrombin cleavage site, and a C-terminal polyhistidine tag (belonging to phylogenetic cluster 1).

[0215] SEQ ID NO:13 and SEQ ID NO:14 show the polynucleotide sequence and deduced amino acid sequence, respectively, of P. multocida PmSLP-1 having a 14-residue anchor truncation (belonging to phylogenetic cluster 1).

[0216] SEQ ID NO:15 and SEQ ID NO:16 show the polynucleotide sequence and deduced amino acid sequence, respectively, of P. multocida PmSLP-1 with a 126-residue anchor cleavage, a thrombin cleavage site, and a C-terminal polyhistidine tag (belonging to phylogenetic cluster 1).

[0217] SEQ ID NO:17 and SEQ ID NO:18 show the polynucleotide sequence and deduced amino acid sequence, respectively, of P. multocida PmSLP-1 with a 94-residue anchor truncation, an introduced SERp1 mutation, a thrombin cleavage site, and a C-terminal polyhistidine tag (belonging to phylogenetic cluster 1).

[0218] SEQ ID NO:19 and SEQ ID NO:20 show the polynucleotide sequence and deduced amino acid sequence, respectively, of P. multocida PmSLP-3 having a 15-residue anchor truncation and a valine to methionine mutation at the 16th residue (belonging to phylogenetic cluster 3).

[0219] SEQ ID NO:21 and SEQ ID NO:22 show the polynucleotide sequence and deduced amino acid sequence, respectively, of PmSLP-1 from P. multocida challenge strain H246 (belonging to phylogenetic cluster 1).

[0220] SEQ ID NO:23 and SEQ ID NO:24 show the polynucleotide sequence and deduced amino acid sequence, respectively, of PmSLP-3 from P. multocida challenge strain H229 (belonging to phylogenetic cluster 3).

[0221] SEQ ID NO: 25 and SEQ ID NO: 26 show the polynucleotide sequence and deduced amino acid sequence, respectively, of C-terminally FLAG-tagged PmSLP-1 from P. multocida (belonging to phylogenetic cluster 1).

[0222] SEQ ID NO:27 and SEQ ID NO:28 show the polynucleotide sequence and deduced amino acid sequence, respectively, of PmSLP-1 from P. multocida (belonging to phylogenetic cluster 1) with a 14-residue anchor cleavage, a thrombin cleavage site, and a C-terminal polyhistidine tag from P. multocida.

[0223] SEQ ID NO:29 and SEQ ID NO:30 show the polynucleotide sequence and deduced amino acid sequence, respectively, of PmSLP-3 from P. multocida (belonging to phylogenetic cluster 3) with a 15-residue anchor truncation, a valine to methionine mutation at the 16th residue, a thrombin cleavage site, and a C-terminal polyhistidine tag from P. multocida.

[0224] SEQ ID NO:31 and SEQ ID NO:32 show the polynucleotide sequence and deduced amino acid sequence of PmSLP-1 from P. multocida (belonging to phylogenetic cluster 1) with a 14-residue anchor truncation as a fusion protein with PmSLP-3 (belonging to phylogenetic cluster 3) with a 15-residue anchor truncation and a valine to methionine mutation at residue 16. This construct lacks a signal peptide and contains a thrombin cleavage site between the two PmSLP proteins, as well as a second thrombin cleavage site at the C-terminus followed by a polyhistidine tag.

[0225] SEQ ID NO:33 and SEQ ID NO:34 show the polynucleotide sequence and deduced amino acid sequence of PmSLP-1 (belonging to phylogenetic cluster 1) from P. multocida with a 14-residue anchor truncation as a fusion protein with PmSLP-3 (belonging to phylogenetic cluster 3) with a 15-residue anchor truncation and a valine to methionine mutation at residue 16. This construct contains no signal peptide, a serine-alanine linker peptide between the two PmSLP proteins, a C-terminal thrombin cleavage site followed by a polyhistidine tag.

[0226] SEQ ID NO:35 and SEQ ID NO:36 show the polynucleotide sequence and deduced amino acid sequence, respectively, of the full-length PmSLP-1 protein (belonging to phylogenetic cluster 1) from P. multocida belonging to phylogenetic cluster 1, including the endogenous signal peptide as a fusion protein with PmSLP-3 (belonging to phylogenetic cluster 3), with a 15-residue anchor truncation and a valine to methionine mutation at residue 16. The construct contains a serine-alanine linker peptide between the two PmSLP proteins, a C-terminal thrombin cleavage site, followed by a polyhistidine tag.

[0227] SEQ ID NO:37 and SEQ ID NO:38 show the polynucleotide sequence and the deduced amino acid sequence, respectively, of mature PmSLP of P. multocida from strain 1500E (belonging to phylogenetic cluster 1).

[0228] SEQ ID NO: 39 and SEQ ID NO: 40 show the polynucleotide sequence and the deduced amino acid sequence, respectively, of PmSLP-4 from P. multocida (belonging to phylogenetic cluster 4.2) from strain HS-Canada1.

[0229] SEQ ID NO:41 shows the thrombin cleavage site which is contained once in SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57 and twice in SEQ ID NO:32.

[0230] SEQ ID NO:42 shows the endogenous signal sequence contained within SEQ ID NO:36.

[0231] SEQ ID NO:43 shows the polypeptide portion contained in SEQ ID NO:34 and SEQ ID NO:36 that includes a serine-alanine (SA) linker.

[0232] SEQ ID NO: 44 shows the first nucleic acid primer sequence.

[0233] SEQ ID NO: 45 shows the second nucleic acid primer sequence.

[0234] SEQ ID NO: 46 shows the third nucleic acid primer sequence.

[0235] SEQ ID NO: 47 shows the fourth nucleic acid primer sequence.

[0236] SEQ ID NO: 48 shows the sequence of a fifth nucleic acid primer.

[0237] SEQ ID NO: 49 shows the sixth nucleic acid primer sequence.

[0238] SEQ ID NO:50 and SEQ ID NO:51 show the polynucleotide sequence and deduced amino acid sequence of PmSLP-1 from P. multocida (belonging to phylogenetic cluster 1) with a 14-residue anchor truncation as a fusion protein with PmSLP-2 (belonging to phylogenetic cluster 2) with a 14-residue anchor truncation and a lysine to methionine mutation at residue 15. This construct contains no signal peptide, a serine-alanine linker peptide between the two PmSLP proteins, a C-terminal thrombin cleavage site followed by a polyhistidine tag.

[0239] SEQ ID NO:52 and SEQ ID NO:53 show the polynucleotide sequence and deduced amino acid sequence of PmSLP-1 from P. multocida with a 14-residue anchor truncation (belonging to phylogenetic cluster 1) as a fusion protein with PmSLP-2 with a 14-residue anchor truncation and a lysine to methionine mutation at the 15th residue (belonging to phylogenetic cluster 2) and with PmSLP-3 with a 15-residue anchor truncation and a valine to methionine mutation at the 16th residue (belonging to phylogenetic cluster 3), respectively. The construct contains a serine-alanine linker peptide between each of the three PmSLP proteins, a C-terminal thrombin cleavage site, followed by a polyhistidine tag.

[0240] SEQ ID NO:54 and SEQ ID NO:55 show the polynucleotide sequence and deduced amino acid sequence of PmSLP-1 from P. multocida (belonging to phylogenetic cluster 1) with a 14-residue anchor truncation as a fusion protein with PmSLP-2 (belonging to phylogenetic cluster 2) with a 14-residue anchor truncation and a lysine to methionine mutation at residue 15, and with PmSLP-4 (belonging to phylogenetic cluster 4.2) with an N-terminal truncation of 41 residues and an alanine to methionine mutation at residue 42, respectively. The construct contains a serine-alanine linker peptide between each PmSLP protein, a C-terminal thrombin cleavage site, followed by a polyhistidine tag.

[0241] SEQ ID NO:56 and SEQ ID NO:57 show the polynucleotide sequence and deduced amino acid sequence, respectively, of P. multocida PmSLP-2 from P. multocida (belonging to phylogenetic cluster 2) with anchor truncation at 14 residues and a lysine to methionine mutation at residue 15. The construct contains a C-terminal thrombin cleavage site followed by a polyhistidine tag.

[0242] SEQ ID NO:58 and SEQ ID NO:59 show the polynucleotide sequence and deduced amino acid sequence, respectively, of PmSLP-2 from P. multocida (belonging to phylogenetic cluster 2) having a 14-residue anchor truncation and a 15th residue lysine to methionine mutation.

[0243] SEQ ID NO:60 and SEQ ID NO:61 show the polynucleotide sequence and deduced amino acid sequence, respectively, of PmSLP-4 from P. multocida (belonging to phylogenetic cluster 4.2) with a 15-residue anchor cleavage.

[0244] SEQ ID NO:62 and SEQ ID NO:63 show the polynucleotide sequence and deduced amino acid sequence, respectively, of PmSLP-4 from P. multocida (belonging to phylogenetic cluster 4.1) with a 15-residue anchor cleavage.

[0245] SEQ ID NO:64 and SEQ ID NO:65 show the polynucleotide sequence and deduced amino acid sequence, respectively, of PmSLP-1 from P. multocida (belonging to phylogenetic cluster 1) having a 14-residue anchor truncation, a valine to aspartic acid mutation at residue 214, a C-terminal thrombin cleavage site followed by a polyhistidine tag.

[0246] SEQ ID NO:66 and SEQ ID NO:67 show the polynucleotide sequence and deduced amino acid sequence, respectively, of PmSLP-1 from P. multocida (belonging to phylogenetic cluster 1) having a 14-residue anchor truncation, a glutamic acid to alanine mutation at residue 240, a C-terminal thrombin cleavage site followed by a polyhistidine tag.

[0247] SEQ ID NO:68 and SEQ ID NO:69 show the polynucleotide sequence and deduced amino acid sequence, respectively, of mature PmSLP-4 from P. multocida (belonging to the unclassified phylogenetic cluster).

[0248] SEQ ID NO: 70 and SEQ ID NO: 71 show the polynucleotide sequence and deduced amino acid sequence, respectively, of mature PmSLP-1 from P. multocida (belonging to phylogenetic cluster 1).

[0249] SEQ ID NO:72 and SEQ ID NO:73 show the polynucleotide sequence and deduced amino acid sequence, respectively, of mature PmSLP-1 from P. multocida (belonging to phylogenetic cluster 1).

[0250] SEQ ID NO:74 and SEQ ID NO:75 show the polynucleotide sequence and deduced amino acid sequence, respectively, of mature PmSLP-4 from P. multocida (belonging to phylogenetic cluster 4.1).

[0251] SEQ ID NO:76 and SEQ ID NO:77 show the polynucleotide sequence and deduced amino acid sequence, respectively, of mature PmSLP-4 from P. multocida (belonging to phylogenetic cluster 4.2).

[0252] SEQ ID NO:78 and SEQ ID NO:79 show the polynucleotide sequence and deduced amino acid sequence, respectively, of mature PmSLP-3 from P. multocida (belonging to phylogenetic cluster 3).

[0253] SEQ ID NO:80 and SEQ ID NO:81 show the polynucleotide sequence and deduced amino acid sequence, respectively, of mature PmSLP-3 from P. multocida (belonging to phylogenetic cluster 3).

[0254] SEQ ID NO:82 and SEQ ID NO:83 show the polynucleotide sequence and deduced amino acid sequence, respectively, of mature PmSLP-3 from P. multocida (belonging to phylogenetic cluster 3).

[0255] SEQ ID NO:84 and SEQ ID NO:85 show the polynucleotide sequence and deduced amino acid sequence, respectively, of mature PmSLP-3 from P. multocida (belonging to phylogenetic cluster 3).

[0256] SEQ ID NO:86 and SEQ ID NO:87 show the polynucleotide sequence and deduced amino acid sequence, respectively, of mature PmSLP-3 from P. multocida (belonging to phylogenetic cluster 3).

[0257] SEQ ID NO:88 and SEQ ID NO:89 show the polynucleotide sequence and deduced amino acid sequence, respectively, of mature PmSLP-3 from P. multocida (belonging to phylogenetic cluster 3).

[0258] SEQ ID NO:90 and SEQ ID NO:91 show the polynucleotide sequence and deduced amino acid sequence, respectively, of mature PmSLP-2 from P. multocida (belonging to phylogenetic cluster 2).

[0259] SEQ ID NO: 92 and SEQ ID NO: 93 show the polynucleotide sequence and deduced amino acid sequence, respectively, of PmSLP-1 from P. multocida (belonging to phylogenetic cluster 1).

[0260] SEQ ID NO:94 and SEQ ID NO:95 show the polynucleotide sequence and deduced amino acid sequence, respectively, of mature PmSLP-2 from P. multocida (belonging to phylogenetic cluster 2).

[0261] SEQ ID NO:96 and SEQ ID NO:97 show the polynucleotide sequence and deduced amino acid sequence, respectively, of mature PmSLP-4 from P. multocida (belonging to phylogenetic cluster 4.2).

[0262] Below are provided examples of specific embodiments for carrying out the methods of the present disclosure, and embodiments representing compositions of the present disclosure. These examples are illustrative only and are not intended to limit the scope of the present disclosure in any way. EXAMPLES

[0263] Example 1 - Gene cloning, protein production, and purification of PmSLP This example describes the general workflow involved in producing recombinant PmSLP protein in E. coli. Cloning and site-directed mutagenesis Amino-terminal truncations of PmSLP (SEQ ID NO: 12) for protein expression and purification were designed based on key structural residues predicted by XtalPred (Slabinski et al., 2007) and synthesized by unrestricted cloning (van den Ent & Lowe, 2006). All primers were synthesized from Sigma Aldrich, and Taq DNA polymerase (Thermo Fisher Scientific) was used in PCR reactions under conditions recommended by the manufacturer. Megaprimer was amplified from pET52b containing PmSLP with a carboxy-terminal thrombin cleavage site and a decahistidine tag using forward primers (15 amino acid truncation amplified with primer (SEQ ID NO: 44), 95 amino acid truncation amplified with primer (SEQ ID NO: 45), and 121 amino acid truncation amplified with primer (SEQ ID NO: 46) with complementarity to the truncated amino terminus) and reverse primer (reverse primer (SEQ ID NO: 47) for vector plasmid pET52b). The megaprimers were then purified by gel electrophoresis and excision according to the manufacturer's instructions (Geneaid). The purified gene fragments were used in linear amplification reactions with empty pET52B plasmid template. The reactions were incubated with 1U Dpn1 (Thermo Fisher Scientific) for 1 hour at 37°C to degrade the methylated template. 5 μL of the reaction was used to transform E. coli MM294 cells by heat shock and recovery was carried out in LB for 1 hour with shaking at 37°C. Transformants were selected on LB agar containing 100 μg / mL ampicillin and grown overnight at 37°C. A single colony was used to inoculate a 5 mL LB culture and grown overnight at 37°C with shaking. The plasmids were then purified using a MiniPrep kit according to the manufacturer's instructions (Geneaid). Charged residues to be mutated in PmSLP were predicted by the UCLA MBI SERp server (Goldschmidt, Cooper, Derewenda, & Eisenberg, 2007).The megaprimer containing the mutation was amplified using a forward primer (SEQ ID NO: 48) with 10 nt or more of complementarity flanking the mutation, and a reverse primer (SEQ ID NO: 49) to the vector. A secondary PCR was performed using the purified megaprimer and pET52b template containing the construct. The construct was transformed and grown in E. coli MM294 and extracted by MiniPrep kit as described above. Mutagenesis was confirmed by Sanger sequencing (TCAG) using T7 forward and reverse primers. PmSLP-1. 94 The SERp1 mutant form is presented in SEQ ID NO:17.

[0264] Expression and purification of recombinant PmSLP Plasmids containing the PmSLP constructs were transformed into E. coli T7 expression via heat shock for 45 seconds and recovery in LB at 37°C with shaking for 1 hour. Transformants were selected on LB agar containing 100 μg / mL ampicillin. Seed cultures in 20 mL LB containing 100 μg / mL ampicillin were inoculated with multiple colonies and grown at 37°C with shaking for 16 hours. These overnight cultures were centrifuged at 4,500 × g for 4 minutes and the pelleted cells were used to inoculate 2 L of LB and this larger culture was grown at 37°C with shaking for approximately 3 hours or until OD 600= 0.5. Isopropyl β-d-1-thiogalactopyranoside (IPTG) was added to a final concentration of 5 mM to induce protein expression, and cells were continued to grow overnight at 20°C with shaking. Cells were pelleted at 4500 × g and resuspended in 40 mL of lysis buffer (50 mM Tris-HCl [pH 8.0], 300 mM NaCl) containing 10 mM imidazole, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM benzamidine, 1 mg / mL lysozyme, and 0.03 mg / mL DNase I. Cells were lysed by sonication (Branson) for 2.5 min and centrifuged at 30,000 × g for 45 min to remove cellular debris. The supernatant was passed through a 0.45 μm syringe filter and incubated overnight at 4°C with shaking with 2 mL of HisPur Ni-NTA resin (ThermoFisher Scientific). Beads were pelleted at 700xg for 2 min, loaded onto a gravity flow column (Econo-Pac® Bio Rad) and washed with 100 mL of cold wash buffer (lysis buffer with 20 mM imidazole). Protein was eluted with 12 mL of cold elution buffer (lysis buffer with 400 mM imidazole) and incubated with 2 U of bovine thrombin (Sigma Aldrich, catalog no. T4648) and dialyzed against 500 mL of 25 mM Tris-HCl [pH 8.0] and 100 mM NaCl overnight at 4°C. The dialyzed sample was incubated with 100 μL of HisPur Ni-NTA resin (Thermo Fisher Scientific) and 100 μL of p-aminobenzamide-agarose (Sigma Aldrich) for 1 h at 22°C with shaking. The cleaved protein was filtered through a 0.22 μm syringe filter, concentrated to 20 mg / mL with a 10K MWCO concentrator (Thermo Fisher Scientific), and purified by size-exclusion chromatography (Superdex 75 10 / 300 GL, GE Healthcare). For antigen testing, PmSLP protein was further purified on a strong anion-exchange chromatography column (MonoQ 5 / 50 GL, GE Healthcare) to remove endotoxins (see Figure 1A for workflow).

[0265] Selenomethionine-incorporated PmSLP was expressed and purified as described above, and showed a marked difference in the growth medium. Starter cultures were inoculated into 50 mL of minimal medium (M9 containing 0.2% glucose, 1 mM MgSO4, 1 mM thiamine, 0.02 mg / mL essential L-amino acids at final concentrations) supplemented with 0.02 mg / mL selenomethionine and 100 μg / mL ampicillin, pelleted, and subcultured into 2 L of minimal medium (ACROS, Thermo Fisher Scientific) supplemented with 0.02 mg / mL selenomethionine.

[0266] FIG. 1 shows the workflow of PmSLP protein production and purification with an example gel of purified PmSLP-3. Specifically, FIG. 1A shows a flow chart illustrating the schematic workflow for purification of recombinant PmSLP. FIG. 1B shows an SDS PAGE gel showing samples collected at different stages of nickel-NTA purification of histidine-tagged PmSLP-3 (SEQ ID NO: 12) from E. coli lysate (lane 1: total cell lysate, lane 2: cell pellet, lane 3: soluble fraction, lane 4: flow-through fraction, lane 5: wash fraction, lane 6: elution fraction). FIG. 1C shows an S75 gel filtration chromatogram of size-exclusion chromatography after removal of the polyhistidine tag of PmSLP-3 (SEQ ID NO: 14). FIG. 1D shows an SDS-PAGE gel of different fractions of PmSLP-3 (B9, B10, B11, B12, C1, C2) purified after performing a polishing step on a MonoQ column. PmSLP-3 migrates in the gel at approximately 35 kDa after tag removal.

[0267] Example 2 - Phylogenetic analysis of PmSLP clusters and associated disease types This example illustrates the variability of PmSLP sequences in P. multocida isolates from bovine respiratory disease, bovine hemorrhagic septicemia, porcine Pasteurella infection, and fowl cholera infection, as well as other host species, by constructing a phylogenetic tree based on multiple PmSLP sequences. Evaluation of the phylogenetic tree allows the selection of PmSLP polypeptides or immunogenic fragments for inclusion in veterinary vaccine formulations.

[0268] PmSLP genes were obtained from 263 publicly available assembled P. multocida genomes retrieved from public repositories on June 24, 2020. These genomes originated from China (n=45), the United States (n=56), Iran (n=1), India (n=9), the United Kingdom (n=6), Thailand (n=3), Pakistan (n=9), Bangladesh (n=1), Canada (n=1), France (n=16), Sri Lanka (n=1), Kazakhstan (n=3), and Myanmar (n=1). When available, information was included on which host animal the P. multocida strains were obtained from, including cattle (n=87), bison / buffalo (n=17), alpaca (n=2), chicken (n=10), duck (n=11), turkey (n=10), dog (n=1), human (n=12), sheep (n=4), goat (n=2), rabbit (n=20), rodent (n=2), wolf (n=2), cat (n=2), horse (n=2), wild boar (n=2), pig (n=59), and goose / anatidae / bird (n=8). For sequences from cattle samples, sequences were stratified by disease (BRD vs. HS) when available, and by capsular serotype when annotated.

[0269] PmSLP protein sequences were aligned by MAFFT (v7.450) using the G-INS-I algorithm. ProtTest (v3.4.2) was used to identify the most suitable evolutionary model, which turned out to be WAG+I+G+F. PhyML (v3.3.20190909) was used to generate a phylogenetic tree, which is shown in Figure 2.

[0270] Referring to FIG. 2, it can be seen that the phylogenetic tree of PmSLP variants is divided into five distinct clusters (PmSLP-1, PmSLP-2, PmSLP-3, PmSLP-4.1, and PmSLP-4.2). On the phylogenetic tree, variants originating from bovine species are shown as larger dark circles. Furthermore, the four ring structure in FIG. 2 shows (1) host species, (2) disease, (3) geographic region, and (4) capsule type in the order of the inner to outer ring structure. As can be seen, BRD-PmSLP variants represent two of the four major clusters (PmSLP-1 and PmSLP-2), while HS-PmSLP variants represent a single cluster (PmSLP-3). BRD-PmSLP variants were mainly isolated from samples originating from North America or Europe, while HS-PmSLP variants were mainly isolated from samples originating from Asia and Africa. The PmSLP sequences of each cluster include PmSLP-1 from cluster PmSLP-1 (e.g., SEQ ID NO:2), PmSLP-2 from cluster PmSLP-2 (e.g., SEQ ID NO:4), PmSLP-3 from cluster PmSLP-3 (e.g., SEQ ID NO:6), PmSLP-4.1 from cluster PmSLP-4.1 (e.g., SEQ ID NO:8), and PmSLP-4.2 from cluster PmSLP-4.2 (e.g., SEQ ID NO:10). PmSLP-1 is at least 99.7% identical to the cluster PmSLP-1 sequence. PmSLP-2 is at least 99.7% identical to the cluster PmSLP-2 sequence, and on average is 100.0% identical to the cluster PmSLP-2 sequence. PmSLP-3 is at least 93.9% identical to the cluster PmSLP-3 sequence, and on average is 99.4% identical to the cluster PmSLP-3 sequence. PmSLP-4.1 is a minimum of 99.7% identical to cluster PmSLP-4.1 sequences and an average of 99.8% identical to cluster PmSLP-4.1 sequences. PmSLP-4.2 is a minimum of 96.8% identical to cluster PmSLP-4.2 sequences and an average of 97.2% identical to cluster PmSLP-4.2 sequences.

[0271] Example 3 - Evaluation of IgG responses and duration of immunogenicity using various PmSLP-1 containing vaccine formulations in mice This example demonstrates that PmSLP proteins are immunogenic in mammalian hosts and can be formulated with a variety of vaccine adjuvants to induce strong and long-lasting antibody responses.

[0272] Vaccine formulations consisting of purified PmSLP-1 protein (SEQ ID NO: 14) formulated with various adjuvants were administered to 4-6 week old C57BL / 6 mice (Charles River) to assess immunogenicity. Each formulation was 100 μL total per dose and consisted of 20 μg PmSLP-1 protein formulated with (i) 20% (v / v) Montanide™ Gel 02 (Seppic) + 3 μg Poly(I:C) (Invivogen), (ii) 20% (v / v) Montanide™ Gel 02 (Seppic), (iii) 100 μg aluminum hydroxide (Invivogen), (iv) 20% (v / v) Emulsigen D® (MVP adjuvant), (v) 50% (v / v) Montanide™ ISA61 (Seppic). Two doses of each vaccine were administered by subcutaneous injection, 21 days apart. Serum samples were collected periodically, 20 days after dose 1, 2 weeks after dose 2, and up to 26 weeks after dose 2.

[0273] Serum antibody titers against PmSLP-1 protein were measured using ELISA (enzyme-linked immunosorbent assay). Total anti-PmSLP-1 IgG as well as anti-PmSLP-1 IgG subclasses (IgG1, IgG2b, IgG2c) were measured in samples taken after one and two doses of vaccine (Figure 9). All five vaccine formulations were able to induce PmSLP-1 specific IgG in serum after only one dose, and IgG titers were further elevated after the second dose. PmSLP-1 specific IgG was not detected in unvaccinated naive control mice. This suggests that the PmSLP-1 antigen is immunogenic in vivo and can be used in combination with various adjuvants. Antibody subclass analysis showed that the predominant subclass induced in mice was IgG1, followed by IgG2B, while IgG2C antibodies were consistently induced only in groups administered vaccines containing Montanide™ Gel02+Poly (I:C) or Montanide™ ISA61 adjuvants, suggesting that the type of immune response induced by the PmSLP-1 vaccine may be further controlled through the choice of adjuvant, as effector functions differ for each IgG subclass.

[0274] The duration of immunogenicity after two doses of five different PmSLP-1 formulations was evaluated for up to 26 weeks (Figure 3). PmSLP-1-specific IgG titers in serum were stable over the evaluated period in all groups and showed no signs of significant decline, suggesting that long-lasting antibody responses can be elicited by PmSLP-1-based vaccines.

[0275] Example 4 - Evaluation of the efficacy of PmSLP-1 against antigen-matched bovine P. multocida isolates in a mouse model of invasive infection This example illustrates the efficacy of a vaccine formulation containing PmSLP-1. This vaccine formulation was used to immunize mice and then challenge them with a P. multocida isolate that has a PmSLP gene that is different from the PmSLP-1 protein contained in the vaccine but that belongs to the same phylogenetic cluster. In this example, a bovine respiratory disease (BRD) P. multocida isolate was used for the challenge. This example further illustrates that a vaccine formulation can be prepared that is cross-protective against P. multocida infections caused by different strains.

[0276] PmSLP-1 (SEQ ID NO: 14) was formulated as a vaccine using 20 μg of protein mixed with Montanide™ Gel 02 (Seppic) in combination with Poly(I:C) (Invivogen) to a final volume of 100 μL per dose. Four- to six-week-old male C57Bl / 6 mice (Charles River) were administered the vaccine by subcutaneous injection twice, 3 weeks apart. Pre-challenge bleeds were collected prior to infection.

[0277] Two weeks after the second dose, anesthetized animals were given approximately 10 4Mice were infected intraperitoneally with logarithmic growth phase CFUs of P. multocida strain H246 (an antigenically compatible BRD isolate; the verified sequence of PmSLP for this strain is defined in SEQ ID NO:22. Strain H246 is a serogroup A strain). Note that SEQ ID NO:14 and SEQ ID NO:22 show 100% sequence identity to the portion of the antigen contained in the immunizing protein. Mice were monitored every 6-12 hours post-infection for clinical symptoms including lethargy, respiration, movement, dehydration, diarrhea, posture, and weight loss. A total clinical score of 10 or greater was considered the clinical end point, at which point mice were humanely euthanized (Figures 4B, 4C, and 4D). Tail vein blood was collected at 3, 24, 48, and 72 hours post-infection, or at the clinical end point, and plated onto selective media for bacterial enumeration (Figure 4E). All PmSLP-1 vaccinated mice survived infection, whereas all animals vaccinated with adjuvant alone reached the end point between 24 and 32 h postinfection (Fig. 4A). Mice immunized with adjuvant alone had extensive bacteremia, whereas mice receiving PmSLP-1 either had no detectable bacteremia or had cleared bacteremia within 72 h postinfection.

[0278] Prechallenge serum samples were evaluated against purified PmSLP-1 (Fig. 5A) or whole heat-killed P. multocida strain H246 (Fig. 5B). All mice vaccinated with the PmSLP-1 vaccine had high prevaccination levels of anti-PmSLP-1 and anti- P. multocida serum IgG, whereas mice vaccinated with adjuvant alone had low serum IgG levels against either protein antigen or whole bacteria.

[0279] These results suggest that purified PmSLP-1 protein is a highly effective vaccine antigen against P. multocida strains harboring PmSLP genes from the same phylogenetic cluster.

[0280] Example 5 - Protection of PmSLP-3 in an acute mouse infection model against an antigen-matched porcine P. multocida isolate This example illustrates the efficacy of a vaccine formulation containing PmSLP-3. This vaccine formulation was used to immunize and then challenge mice with a P. multocida isolate that has a PmSLP gene that is different from the PmSLP-3 protein included in the vaccine but that belongs to the same phylogenetic cluster. In this example, a porcine P. multocida isolate was used for the challenge. This example further illustrates that a vaccine formulation can be prepared that is cross-protective against P. multocida infections with different strains. PmSLP-3 (SEQ ID NO:20) was formulated as a vaccine using Montanide™ Gel 02 (Seppic) in combination with Poly(I:C) (Invivogen) and 20 μg of protein mixed to a final volume of 100 μL per dose. Male C57Bl / 6 mice (Charles River), 4-6 weeks of age, were administered the vaccine by subcutaneous injection twice, 3 weeks apart. Pre-challenge bleeds were collected prior to infection.

[0281] Two weeks after the second dose, anesthetized animals were given approximately 10 4Mice were infected by intraperitoneal injection with logarithmic growth phase CFUs of P. multocida strain H229 (an antigenically compatible swine isolate; the verified sequence of PmSLP for this strain is defined in SEQ ID NO:24. Strain H229 is a serogroup A strain). Note that SEQ ID NO:20 and SEQ ID NO:24 show 99.4% sequence identity to the portion of the antigen contained in the immunization protein. Mice were monitored every 6-12 hours post-infection for clinical symptoms including lethargy, respiration, movement, dehydration, diarrhea, posture, and weight loss. A total clinical score of 10 or greater was considered the clinical end point, at which point mice were humanely euthanized (Figures 6B, 6C, and 6D). Blood was collected from the tail vein at 3, 24, 48, 78, 100, and 124 hours post-infection, or at the clinical end point, and plated onto selective media for bacterial enumeration (Figure 13E). All PmSLP-3 vaccinated mice survived infection, whereas all adjuvant-only vaccinated animals reached the end point between 24 and 32 h postinfection (Fig. 6A). Mice immunized with adjuvant alone were highly bacteremic, whereas PmSLP-3 vaccinated mice had no detectable bacteremia or only subclinical bacteremia.

[0282] Prechallenge serum samples were evaluated against purified PmSLP-3 (Fig. 7A) or whole heat-killed P. multocida strain H229 (Fig. 7B). All mice vaccinated with the PmSLP-3 vaccine had high prevaccination levels of anti-PmSLP-3 and anti- P. multocida serum IgG, whereas mice vaccinated with adjuvant alone had low serum IgG levels against either protein antigen or whole bacteria.

[0283] These results suggest that purified PmSLP-3 protein is a highly effective vaccine antigen against P. multocida strains harboring PmSLP genes from the same phylogenetic cluster.

[0284] Example 6 - Stability and efficacy of PmSLP antigen under different storage conditions This example takes into account the practical aspects of utilizing PmSLP as a vaccine antigen and examines the effect of different storage conditions and lyophilization of the protein. The stability and efficacy of the PmSLP antigen were evaluated using both biophysical methods and protection studies in mouse models.

[0285] In a biophysical approach, the stability of purified PmSLP-3 protein (SEQ ID NO: 20) stored at 4°C or room temperature for 1 year after lyophilization was assessed by thermal denaturation using Tycho (NanoTemper) and compared to a reference antigen. In this approach, a heat lamp is applied to the sample and the intrinsic fluorescence emitted from tryptophan and tyrosine residues (detected at both 350 nm and 330 nm) is measured as the protein begins to unfold. The change in the fluorescence signal (350 nm / 330 nm ratio) indicates a transition in the unfolded state of the protein, and the temperature at which the transition occurs is called the inflection temperature (T i Comparison of the thermal denaturation profiles of samples stored under different conditions with Ti allows for a rapid biophysical assessment of the structural integrity of proteins.

[0286] PmSLP-3 protein that was lyophilized and stored at 4°C or room temperature (RT) for 1 year was compared to a reference PmSLP-3 protein, a sample freshly prepared at a concentration of 100 μg / mL in buffer (phosphate buffered saline, pH 7.4). The lyophilized protein was reconstituted at 100 μg / mL in the same buffer prior to analysis. Thermal denaturation profiles and T i assessed the fluorescent signal and showed no significant changes even after storage at room temperature or 4 °C for 1 year (Figure 8A,B), indicating that the lyophilized protein maintained its structural integrity over this period.

[0287] The impact of lyophilization of the antigen and storage at 4°C was further evaluated in vivo in a mouse infection model. PmSLP-3 protein (SEQ ID NO:20) was formulated as a vaccine using Montanide™ Gel 02 (Seppic) in combination with Poly(I:C) (Invivogen) and 20 μg of protein mixed to a final volume of 100 μL per dose. Male C57Bl / 6 mice (Charles River) aged 4-6 weeks were vaccinated by subcutaneous injection twice, 3 weeks apart. Vaccine group 1 was administered a freshly prepared formulation prior to each dose using PmSLP-3 protein aliquots stored at -80°C. Vaccine group 2 was administered a freshly prepared formulation prior to each dose using PmSLP-3 protein aliquots lyophilized and stored at 4°C. Vaccine group 3 was administered a freshly prepared formulation prior to the first dose using PmSLP-3 protein stored at -80°C. The mixed formulation was stored at 4°C for 3 weeks and then administered again at the second dose.

[0288] Two weeks after the second dose, anesthetized animals were given approximately 10 4Mice were infected by intraperitoneal injection with logarithmic growth phase CFUs of P. multocida strain H229 (an antigenically compatible porcine isolate; the PmSLP-verified sequence for this strain is defined in SEQ ID NO: 24). Mice were monitored every 6-12 hours post-infection for clinical signs including lethargy, respiration, movement, dehydration, diarrhea, posture, and weight loss. A combined clinical score of 10 or greater was considered the clinical end point, at which point mice were humanely euthanized (Figure 9). All mice receiving either of the PmSLP-3 vaccines were completely protected from infection compared to all animals receiving adjuvant alone, which reached the end point at 21 hours post-infection (Figure 9A). Clinical scores for individual mice that received adjuvant only (Figure 9B), vaccine 1 (freshly prepared vaccine with proteins stored at -80°C until immediately before each dose; Figure 9C), vaccine 2 (freshly prepared vaccine with proteins that were lyophilized and stored at 4°C until immediately before each dose; Figure 9D), or vaccine 3 (vaccine prepared with proteins that had been stored at -80°C prior to dose 1, then the remaining vaccine was stored at 4°C for 3 weeks until the second dose; Figure 9E).

[0289] Example 7 - Immunogenicity of PmSLP-1 in cattle This example describes the immunogenicity of a PmSLP vaccine in food-producing animals affected by P. multocida infection. In this example, two different strains of cattle were used to evaluate the immunogenicity of a PmSLP-1 vaccine. In this example, a PmSLP-1 vaccine in a bovine host was selected because this variant is expressed by the majority of P. multocida isolates that cause bovine respiratory disease (BRD).

[0290] Healthy Zebu cattle (4-6 months old) that were blood-negative to all serotypes of P. multocida were randomized into two groups. 200 μg of lyophilized PmSLP-1 protein (SEQ ID NO: 14) was reconstituted in PBS immediately prior to vaccination and formulated with 30 μg Poly(I:C) (Invivogen) and 20% v / v Montanide™ Gel 02 (Seppic) in a final volume of 2 mL / dose. Animals were randomized into groups and administered either PmSLP-1 vaccine or adjuvant alone by subcutaneous injection three times at three-week intervals. Blood was collected prior to vaccination (baseline) and approximately 2-3 weeks after the first, second, and third doses. Serum samples were assessed for the presence of α-PmSLP-1 IgG using ELISA (enzyme-linked immunosorbent assay). Figure 10 shows the endpoint titers in cattle immunized with either PmSLP-1 vaccine or adjuvant. As shown, there is a detectable increase in PmSLP-1 specific antibodies after one dose, which increases significantly after two doses. The boosting effect is minimal with the third dose.

[0291] The second bovine immunization was performed using healthy beef cattle (approximately 10 months of age) that were seronegative to all serotypes of P. multocida. Animals were randomly assigned to two groups of nine animals each. Animals received either three doses of PmSLP-1 (SEQ ID NO: 14) vaccine or adjuvant. Vaccines were formulated prior to each immunization by mixing 200 μg of PmSLP-1 protein stored at −80° C. with 30 μg of Poly(I:C) (Invivogen) and 20% v / v Montanide™ Gel 02 (Seppic) to a final volume of 2 mL / dose. Animals were vaccinated intramuscularly at 3-week intervals and bled prior to the first immunization (baseline), and 2-3 weeks after the first, second, and third doses. Serum was assessed for the presence of α-PmSLP-1 IgG. Figure 11 shows endpoint titers at baseline and after 1, 2 and 3 doses of vaccine in cattle immunized with either PmSLP-1 vaccine or adjuvant. As shown, there was a detectable increase in specific antibodies after the first dose, which increased significantly after the second dose, with only a small boosting effect after the third dose.

[0292] Overall, the PmSLP-1 vaccine was immunogenic in both cattle strains, with vaccine titers peaking after two doses of this formulation given intramuscularly or subcutaneously.

[0293] Example 8 - Protection of PmSLP-3 against serogroup B strains of P. multocida in cattle This example describes the immunogenicity, safety, and protective efficacy of PmSLP vaccines in food-producing animals affected by P. multocida infection. In this example, zebu breeding cattle were used to evaluate the immunogenicity and safety of two PmSLP-3 vaccine formulations, as well as the protective efficacy of the vaccines against a lethal challenge with P. multocida of serogroup BP. For this example in bovine hosts, the PmSLP-3 vaccine was selected because this variant is expressed by all known P. multocida isolates that cause hemorrhagic septicemia (HS).

[0294] Healthy Zebu cattle (4-6 months old) that were seronegative to all serotypes of P. multocida were randomly divided into three groups. 200 μg PmSLP-3 (SEQ ID NO:20) was reconstituted in PBS immediately prior to vaccination and formulated with 1 mg aluminum hydroxide (Alhydrogel, Sigma-Aldrich) or 30 μg Poly(I:C) (Invivogen) and 20% v / v Montanide™ Gel 02 (Seppic) in a final volume of 2 mL / dose. A control group receiving adjuvant only was used as a negative control. Animals were vaccinated subcutaneously twice, 3 weeks apart, and bled before the first immunization (baseline), before the second immunization (post-first dose), and before challenge (post-second dose). Serum was assessed for the presence of α-PmSLP-1 IgG. After vaccination, local reactions at the injection site were monitored. 14 days after the booster vaccination, 4.4 × 10 4 CFU / mL of a serogroup B strain of P. multoda was injected subcutaneously into cattle and monitored for 8 days postinfection.

[0295] FIG. 12A shows the endpoint titers of cattle immunized with PmSLP-3 formulated with aluminum hydroxide, PmSLP-3 formulated with Montanide™ Gel 02+Poly(I:C), or adjuvant alone. As shown, there is detectable serum IgG against PmSLP-3 after two vaccinations with both PmSLP-3 vaccine formulations, but PmSLP-3 formulated with aluminum hydroxide elicited detectable serum IgG against PmSLP-3 after one vaccination. FIG. 12B shows survival of cattle after challenge with serogroup B P. multocida. As shown, zebu cattle fed only adjuvant were completely vulnerable to infection and died one day post-infection. In comparison, 87.5% (7 of 8) of cattle vaccinated with PmSLP-3 formulated with aluminum hydroxide survived the challenge, and 75% (6 of 8) of cattle vaccinated with PmSLP-3 formulated with Montanide Gel 02 + Poly (I:C) survived the challenge. Figure 12C shows a table showing reactogenicity after one or two doses of the vaccine. As shown, PmSLP-3 formulated with aluminum hydroxide did not cause any local reactions after either dose, whereas PmSLP-3 formulated with Montanide Gel 02 + Poly (I:C) or Montanide Gel 02 alone caused local swelling in 50% of the animals after the first dose.

[0296] Overall, PmSLP-3 vaccines formulated with either aluminum hydroxide or Montanide Gel 02+Poly(I:C) were safe, immunogenic, and protective in zebu cattle after two doses of vaccine when delivered subcutaneously.

[0297] Example 9 - Evaluation of the virulence potential of Brazilian field strains of Pasteurella multocida serogroup A in mice Mouse challenge test The aim of the study described in this Example 9 was to characterize the pathogenicity of two Pasteurella multocida isolates, AFK143 / 23 cluster 2 and AFK283 / 23 cluster 4.2, in mice.

[0298] Bacterial Growth and Infectious Dose Formulations Three AFK isolates of P. multocida serogroup A (AFK AFK 300 / 23 PmSLP-1, AFK 143 / 23 PmSLP-2, and AFK 283 / 23 PmSLP-4) were used in this study. The bacterial isolates were cultured overnight at 37°C on defibrinated blood agar plates. From the plates, the bacteria were inoculated into Erlenmeyer medium containing liquid PPLO (multiple pneumonia-like organisms, Oxoid, USA). The initial optical density (OD) was assessed by spectrophotometry (filter 660 nm) and adjusted to 0.1. The bacteria were then cultured at 37°C under constant agitation (180 rpm) until the OD reached 0.5. The bacteria were then harvested by centrifugation (4,000xg, 15 min at 4°C) and the pellet was resuspended in phosphate-buffered saline (PBS) pH 7.4. The absolute number of bacteria in the suspension was measured by flow cytometry (Baldasso, DZ, Guizzo, JA, Dazzi, CC, Paraboni Frandoloso, GC, Feronato, C., von Berg, S., Carvalho Guedes, RM, Wilson, HL, Kreutz, LC, Frandoloso, R., 2023. Development and validation of a flow cytometry antibody test for Lawsonia intracellularis. Front Immunol 14, 1145072) and bacterial viability was simultaneously assessed by staining with propidium iodide (PI). All assays were performed in triplicate. 10 2 , 10 3 , and 10 4 Three infection doses containing 1000 P. multocida were prepared. The bacteria were then diluted with PBS (pH 7.4) to a final volume of 0.2 mL.

[0299] Experimental infection of mice Forty-five 8-week-old C57BL / 6 mice were used in this study. The mice were randomly and equally assigned to 9 groups and housed in the Central Animal Facility of the University of Passo Fundo with a 12 h / 12 ​​h light / dark photoperiod, commercial ratios, and water ad libitum. As shown in Figure 13A, the 9 groups of mice were infected intraperitoneally with P. multocida serogroup A. The inoculum was suspended in 0.2 mL and administered intraperitoneally. The number of dead / surviving mice was then monitored.

[0300] result As shown in Figure 13B, all challenged mice died after inoculation with the evaluated strains. 3 Pieces and 10 4 Animals challenged with 10 bacteria 2 Animals challenged with 100% lethal dose (LD 100 ) was 10 for the three strains analyzed. 2 was defined as a bacterium.

[0301] Example 10 - Clinical efficacy study in mice. Animals immunized with PmSLP2-based vaccine and challenged with P. multocida strain 143 / 23 Animal models and groups Ten C57BL / 6 mice were evenly divided into two groups: 5 mice were immunized with a vaccine formulated with PmSLP2 recombinant protein (SEQ ID NO: 95) and the other 5 mice were inoculated with PBS plus adjuvant.

[0302] Vaccine Production and Immunization Protocols The immunizing antigen (PmSLP2) was diluted in PBS (pH 7.4) and mixed with Emulsigen D (MVP Adjuvants, Teaneck, USA) to a final concentration of 20% adjuvant and 0.1 mg / mL antigen. The antigen-adjuvant mixture was homogenized with a magnetic stirrer (600 rpm) for 2 h and then stored refrigerated (4°C) until use. The vaccine was packaged in flasks containing 2 mL (final volume) for 25 doses. The dose volumes defined in this step were used for the experiments performed in pigs.

[0303] Mice were immunized according to standard protocols. Briefly, five mice were inoculated intraperitoneally with 0.2 mL of vaccine (20 μg PmSLP2), corresponding to 10% of the immunization dose used in pigs. The other five mice received sterile PBS and adjuvant (control group). The immunization protocol was repeated in all mice at 14-day intervals (booster injections).

[0304] Blood collection Blood samples were collected from all mice before priming (day 0), at the time of revaccination (day 14), and on day 28. In addition, on day 28, all mice were challenged with Pasteurella multocida serogroup A as described below.

[0305] IgG titration in mouse blood Anti-P. multocida antibody titers were assessed by indirect ELISA. Briefly, plates were sensitized with PmSLP-2 (1 μg / well) overnight (4°C). Then, wells were washed and blocked with 5% nonfat milk (SK) in PBS containing 0.05% Tween-20 (PBS-T) for 1 h (37°C). After washing again, mouse sera were serially diluted (in PBS-T 1% SK), added to the plates, and incubated at 37°C for 1 h. After removing serum and washing the plates once more, peroxidase-conjugated anti-mouse IgG was added. After incubation for 1 h (37°C), plates were washed again and substrate (3,3′,5,5′-tetramethylbenzidine liquid substrate) was added. The reaction was stopped and plates were read in an ELISA microplate reader (450 nm filter). Serum samples were considered positive if the OD reading of the samples was at least two-fold higher than the mean OD of the negative control samples.

[0306] Bacterial inoculation and experimental challenge The bacterial isolate (AFK 143 / 23-Pasteurella multocida serogroup A, PmSLP cluster 2) was grown overnight at 37 °C on defibrinated blood agar plates. Bacteria from the plates were inoculated into Erlenmeyer medium with liquid PPLO (Prolific Pneumonia-Like Organisms, Oxoid, USA). The initial optical density (OD) was assessed by spectrophotometry (filter 660 nm) and adjusted to 0.1. The bacteria were then grown at 37 °C with constant stirring (180 rpm) until the OD reached 0.5. The bacteria were then harvested by centrifugation (4,000 x g, 15 min at 4 °C) and the pellet was resuspended in phosphate-buffered saline (PBS) pH 7.4. The absolute number of bacteria in the suspension was determined by flow cytometry (Baldasso et al., 2023) and the viability of the bacteria was simultaneously assessed by staining with propidium iodine (PI). All assays were performed in triplicate. For mouse challenge studies, bacteria were diluted in PBS (pH 7.4) to 10 per infectious dose. 2 The culture contained 10 bacteria.

[0307] All mice were challenged on day 28 (14 days after the boost). The challenge inoculum (lethal dose - LD 100% ) intraperitoneally in a final volume of 200 μL. 2 The mice were then monitored twice daily for up to 7 days after exposure.

[0308] result Serology As shown in FIG. 14A, the PmSLP2-based vaccine induced anti-PmSLP-2 IgG production in mice. A slight increase in anti-PmSLP-2 IgG titers was observed between the first and second doses of the vaccine, but 14 days after the second dose (D28), the IgG titers were significantly increased (p>0.0001) with respect to the two previous time points analyzed (D0 and D14). At D28, the IgG titers were 45-fold higher than those observed at D14. At the same time points, the levels of IgG were different compared to unvaccinated animals (p>0.0001). Thus, the results of this analysis demonstrate that the PmSLP-2 recombinant protein in combination with the oil-based adjuvant Imulsigen D is highly immunogenic in mice.

[0309] Clinical Efficacy As shown in Figure 14B, the PmSLP2-based vaccine induced clinical protection in 80% of mice challenged with a lethal dose of P. multocida A (strain AFK143.23). This level of protection was statistically superior to the negative control group (0% survival). These results indicate that the PmSLP antigen is protective against a lethal challenge with Pasteurella multocida serogroup A.

[0310] Example 11 - Evaluation of the pathogenicity of Brazilian field strains of Pasteurella multocida serogroup A in pigs The aim of the study described in this Example 11 was to determine the pathogenicity of two Pasteurella multocida isolates, AFK143 / 23 (PmSLP-2) and AFK283 / 23 (PmSLP-4), in pigs.

[0311] Experimental infection in pigs Twelve colostrum-deficient pigs were used in this study. Colostrum-deficient piglets were generated as previously described (Guizzo, JA, Chaudhuri, S., Prigol, SR, Yu, RH, Dazzi, CC, Balbinott, N., Frandoloso, GP, Kreutz, LC, Frandoloso, R., Schryvers, AB, 2018). Amino acids selected to generate mutant TbpB antigens defective in binding to transferrin may impair protective capacity in vivo (Scientific Reports 8, 7372). For the challenge study, all pigs (63 days old) were anesthetized with a cocktail of 0.3 mg / kg acepromazine (Syntec do Brasil, Brazil), 0.3 mg midazolam (Laboratorio Teuto Brasileiro, Brazil), and 15 mg / kg ketamine (Ceva Sante Animale, Brazil) administered via the intramuscular route. The anesthetized pigs were then placed upright and inoculated intranasally. Each P. multocida isolate was used to inoculate six pigs: three pigs with 10 8 10 for each pig 9 The animals were inoculated with 100 ml of bacteria in a final volume of 3 mL PBS, pH 7.4. After challenge, pigs were monitored for clinical signs up to 10 days after challenge.

[0312] result P. mulutocidaAFK283 / 23(10 9 Two of the three pigs inoculated with the 1000-mg dose of bacteria / pig died by 20 hours after challenge. The other pig in this group died 2 hours later with severe respiratory distress. 8Pigs challenged with 100% swine (bacteria / pig) developed severe respiratory distress within 12 hours after challenge, with one pig dying by 36 hours after challenge. The other two pigs only had severe respiratory distress until 5 days after challenge and then improved (see Figure 15).

[0313] Within the cohort of pigs challenged with the AFK143 / 23 strain, 10 9 Only one animal inoculated with the bacteria died by 20 hours after challenge. The remaining pigs in this group had respiratory distress (dyspnea, coughing, weakness) until 4 days after challenge. Clinical signs subsequently resolved (see Figure 15).

[0314] During necropsy, all animals that died after experimental infection or were euthanized at the end of the study exhibited severe bronchopneumonia with extensive areas of necrosis in the diaphragmatic lobes. Severe pleurisy was observed in all animals euthanized at the end of the study. Pasteurella multocida A was recovered as pure and abundant cultures from the lungs of all challenged animals.

[0315] Considering the infection profile of P. multocida A in the field, the appropriate infection dose for the AFK283 / 23 strain was 10 8 bacteria, and 10 for 143 / 23 strains. 9 It was concluded that these doses were responsible for the production of the bacterium. Using these doses it was possible to induce respiratory clinical signs and typical pulmonary macroscopic lesions in the pigs tested.

[0316] Thus, this Example 11 demonstrates that strains AFK143 / 23 (PmSLP-2) and AFK283 / 23 (PmSLP-4) are pathogenic for pigs and act as major agents of severe pulmonary disease in this animal species. Studies reported by other Brazilian researchers have described the circulation in Brazil of strains of P. multocida serogroup A that are highly pathogenic and capable of causing severe pneumonia in pigs (Chitarra, CS, Oliveira Filho, JX, Mores, N., Silva, M., Candido, SL, Cezarino, PG, Nakazato, L., Dutra, V., 2018. Identification of Pasteurella multocida transcribed genes in porcine lungs through RNAseq. Microbial pathogenesis 122, 180-183; Oliveira Filho, JX, Mores, MAZ, Rebellato, R., Kich, JD, Cantao, ME, Klein, CS, Guedes, RMC, Coldebella, A., Barcellos, D., Mores, N., 2018. Pathogenic variability among Pasteurella multocida type A isolates from Brazilian pig farms.BMC veterinary research 14,244).

[0317] Furthermore, sera from convalescent animals were assessed for the presence of antibodies against PmSLP-2 and PmSLP4 proteins. This analysis was performed to understand whether these antigens are naturally expressed by P. multocida A during the infection process and, consequently, whether these proteins stimulate antibody production in pigs.

[0318] Interestingly, all pigs that survived challenge with AFK143 / 23 (n=5) and AFK283 / 23 (n=2) had IgG titers against the PmSLP protein. The presence of these antibodies was not observed before challenge. The antibodies are therefore specific. This result indicates that i) the PmSLP protein is an antigen involved in the pathogenesis of P. multocida infection in pigs, ii) it is an antigen that is naturally accessible to B lymphocytes and therefore immunogenic and antigenic. These provide the utility of PmSLP-based antigens in the preparation of vaccines against P. multocida.

[0319] Example 12 - Efficacy testing of vaccines based on PmSLP proteins in mice This Example 12 describes the evaluation of the protective capacity of two other vaccines, one based on PmSLP-1 and the other on PmSLP-4.2, in which mice were challenged with a P. multocida strain expressing a PmSLP homologous to that used in the vaccine formulation, as shown in Figure 16A.

[0320] For this purpose, 32 male and female C57BL / 6 mice were used, evenly distributed in 4 groups (G1 to G4). Mice were housed in the Central Animal Facility of the University of Passo Fundo under a 12 h / 12 ​​h light / dark photoperiod regime. The antigens used and the immunization protocol are shown in Figure 16A. The vaccine was formulated as described in Example 10. A challenge with homologous PmSLP-expressing bacteria was performed on day 28 (14 days after the booster vaccination) and further immunization with 10 2 Intraperitoneal injections were performed using 1000 bacteria / mouse.

[0321] result As shown in Figure 16B, both vaccines administered to mice stimulated the production of systemic antibodies (serological tests were performed as described in Example 10). In this example, serum samples (before revaccination) were collected 12 days after revaccination, and IgG titers at the time of collection were significantly higher than those observed at the prevaccination time point in all animals. Thus, all vaccines were immunogenic and considered safe, with no animals showing systemic reactions in the postvaccination period.

[0322] As shown in FIG. 16C, P. multocida A strains AFK300 / 23 (PmSLP-1) and AFK283 / 23 (PmSLP-4.2) were lethal to mice inoculated with PBS (groups 3 and 4). In contrast, a vaccine based on the PmSLP-1 protein (SEQ ID NO: 93) conferred 100% protection against the AFK300 / 23 strain (group 1). A vaccine based on the PmSLP-4.2 protein (SEQ ID NO: 97) protected 87.5% of mice challenged with the AFK283 / 23 strain (group 2). These data, together with the data shown in FIG. 14B, indicate that PmSLP1, 2, and 4.2 proteins induce greater than 80% protection against challenge with a 100% lethal dose of virulent P. multocida A. The protection shown in this study is homologous, as the vaccine antigen belongs to the same PmSLP cluster as the challenge strain.

Claims

1. A veterinary vaccine formulation for the prevention, treatment, or improvement of P. multicida infection in food-producing animals susceptible to P. multicida infection, comprising an effective amount of at least one PmSLP protein or an immunogenically equivalent portion thereof, together with veterinary pharmaceutically acceptable excipients, carriers, or diluents, and possibly comprising a veterinary pharmaceutically acceptable adjuvant.

2. The animal vaccine formulation according to claim 1, wherein the vaccine formulation comprises a P. multicida PmSLP protein or an immunogenically equivalent portion selected from a group of systematic clusters consisting of PmSLP-1, PmSLP-2, PmSLP-3, PmSLP-4.1, and PmSLP-4.2, and the selected P. multicida PmSLP protein or an immunogenically equivalent portion belongs to the same systematic cluster as the PmSLP protein present in the infected P. multicida strain.

3. The animal vaccine formulation according to claim 1, wherein the food-producing animal is a porcine susceptible to infection by a P. multicida strain that causes porcine atrophic rhinitis (PAR) or pneumonic pasteurellosis, and the vaccine formulation comprises a P. multicida PmSLP protein or an immunogenic equivalent portion derived from a P. multicida strain that causes PAR or a P. multicida strain that causes pneumonic pasteurellosis, respectively, and the PmSLP protein is selected from a group of systematic clusters consisting of PmSLP-2, PmSLP-4.1, and PmSLP-4.2, and the selected P. multicida PmSLP protein or an immunogenic equivalent portion belongs to the same systematic cluster as the PmSLP protein present in the infected P. multicida strain.

4. The animal vaccine preparation according to claim 1, wherein the vaccine preparation is further a P. multicida strain belonging to a serogroup selected from the group consisting of serogroups A, B, D, E, and F, and the serogroup is the same as the serogroup of the infecting P. multicida strain.

5. The aforementioned at least one PmSLP protein or an immunologically equivalent portion thereof (a) SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, or SEQ ID NO: 96, (b) A nucleic acid sequence having at least 70% identity with any one of the nucleic acid sequences in (a), (c) A nucleic acid sequence that is substantially identical to any one of the nucleic acid sequences in (a) above, apart from the degeneracy of the genetic code. (d) A nucleic acid sequence complementary to any one of the nucleic acid sequences in (a) above, (e) A chimeric nucleic acid obtained by fusion between at least two nucleic acid sequences of (a), (b), (c), and (d), (f) A nucleic acid sequence complementary to any one of the nucleic acid sequences in (a) above, (g) A nucleic acid sequence encoding a polypeptide having any one of the amino acid sequences described in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, or SEQ ID NO: 97, or an immunogenically equivalent portion thereof. (h) A nucleic acid sequence encoding any one functional variant of the amino acid sequence described in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, or SEQ ID NO: 97, or an immunogenically equivalent portion thereof, and (i) A nucleic acid sequence that hybridizes under severe conditions to any one of the nucleic acid sequences described in (a), (b), (c), (d), (e), (f), (g), or (h) above, The animal vaccine formulation according to claim 1, which is a protein expressed by a nucleic acid sequence selected from the group of nucleic acid sequences consisting of the above.

6. The animal vaccine formulation according to claim 1, wherein the food-producing animal is a pig, the P. multicida infection causes pneumonic pasteurellosis or PAR, and the PmSLP protein includes SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 40, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 75, SEQ ID NO: 77, or SEQ ID NO: 91, or an immunogenically equivalent portion thereof, to prevent or improve the P. multicida infection that causes pneumonic pasteurellosis or PAR.

7. The animal vaccine formulation according to claim 1, wherein the animal vaccine formulation is a cross-protective vaccine formulation comprising a PmSLP protein obtained from a first P. multicida strain or an immunogenically equivalent portion thereof, and the vaccine formulation is a formulation for administration to a food-producing animal to prevent or improve an infection caused by another P. multicida strain.

8. The animal vaccine formulation according to claim 1, wherein the vaccine formulation substantially does not contain other P. multicida components.

9. The veterinary vaccine preparation according to claim 1, wherein the vaccine preparation comprises about 0.001% by weight to about 20% by weight of PmSLP protein or an immunogenically equivalent portion thereof, and a pharmaceutically acceptable adjuvant for veterinary use constituting about 0.1% by weight to about 60% by volume of the vaccine preparation.

10. The animal vaccine formulation according to claim 1, wherein the vaccine formulation comprises a second P. multicida PmSLP protein or an immunologically equivalent portion thereof.

11. The animal vaccine formulation according to claim 10, wherein the second P. multicida PmSLP protein or its immunologically equivalent portion belongs to the same or a different phylogenetic cluster as the first P. multicida PmSLP protein or its immunologically equivalent portion, and may belong to the same phylogenetic cluster as the first P. multicida PmSLP protein or its immunologically equivalent portion.

12. The animal vaccine formulation according to claim 11, wherein the fusion polypeptide comprises a fusion polypeptide selected from the group consisting of (i) a PmSLP protein belonging to systematic cluster 1 or an immunologically equivalent portion thereof, and a PmSLP protein belonging to systematic cluster 3 or an immunologically equivalent portion thereof, (ii) a PmSLP protein belonging to systematic cluster 1 or an immunologically equivalent portion thereof, and a PmSLP protein belonging to systematic cluster 2 or an immunologically equivalent portion thereof, (iii) a PmSLP protein belonging to systematic cluster 1 or an immunologically equivalent portion thereof, and a PmSLP protein belonging to systematic cluster 4.1 or an immunologically equivalent portion thereof, and (iv) a PmSLP protein belonging to systematic cluster 1 or an immunologically equivalent portion thereof, and a PmSLP protein belonging to systematic cluster 4.2 or an immunologically equivalent portion thereof.

13. The animal vaccine formulation according to claim 12, wherein the second P. multicidaPmSLP protein or its immunologically equivalent portion is obtained from a P. multicida strain belonging to the same or a different serogroup as the first P. multicidaPmSLP protein or its immunologically equivalent portion of the P. multicida strain.

14. A veterinary vaccine formulation according to any one of claims 1 to 13, for use in the prevention, treatment, or improvement of P. multicida infection in food-producing animals susceptible to P. multicida infection.

15. A method for preventing, treating, or improving a Pasteurella multicida (P. multicida) infection in a food-producing animal susceptible to the infection, the method comprising administering to the food-producing animal a veterinary vaccine formulation according to any one of claims 1 to 13, wherein the vaccine formulation is administered in an amount effective to prevent, treat, or improve the P. multicida infection.

16. A method for preparing animal vaccine formulations for the prevention, treatment, or improvement of P. multicida infection in food-producing animals susceptible to P. multicida infection, (i) Steps for diagnosing P. multicida infection in food-producing animals, (ii) A step of identifying a systematic cluster to which PmSLP proteins present in infected P. multicida belong, wherein the systematic cluster is selected from PmSLP-1, PmSLP-2, PmSLP-3, PmSLP-4.1, or PmSLP-4.2, and (iii) P - A step of preparing a vaccine formulation according to any one of claims 1 to 13, comprising P. multicida PmSLP protein or an immunogenically equivalent portion thereof belonging to the identified systematic cluster, together with a pharmaceutically acceptable adjuvant for veterinary use, in order to treat food-producing animals susceptible to multicida infection, the vaccine formulation comprising an effective amount of P. multicida PmSLP protein or an immunogenically equivalent portion thereof, Methods that include...

17. A method for preparing an animal vaccine formulation according to any one of claims 1 to 13, comprising the P. multicida PmSLP protein or an immunogenically equivalent portion thereof, (a) as operably connected components, (i) P. multicida PmSLP protein, or a nucleic acid sequence encoding an immunogenically equivalent portion thereof, and (ii) One or more nucleic acid sequences capable of controlling the expression of the nucleic acid sequence encoding the PmSLP protein, or an immunogenically equivalent portion thereof, in a host cell. To provide a chimeric nucleic acid sequence containing, (b) Introducing the chimeric nucleic acid sequence into a host cell, (c) Proliferating the host cells to produce the P. multicida PmSLP protein or an immunogenically equivalent portion thereof. (d) Recovering the P. multicida PmSLP protein or an immunogenically equivalent portion thereof, and (e) Formulating the P. multicida PmSLP protein, or the immunogenic equivalent thereof, together with a pharmaceutically acceptable adjuvant for veterinary use, to form a veterinary vaccine formulation containing an effective amount of the P. multicida PmSLP protein, or the immunogenic equivalent thereof, for the treatment of food-producing animals susceptible to P. multicida infection. Methods that include...

18. (i) a nucleic acid sequence encoding the P. multicida PmSLP protein or an immunogenically equivalent portion thereof, (ii) A nucleic acid sequence capable of controlling the expression of the P. multicida PmSLP protein or a nucleic acid sequence encoding an immunogenically equivalent portion thereof in host cells. An expression vector comprising, The nucleic acid sequence encoding the P. multicida PmSLP protein or its immunogenically equivalent portion is (a) SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, or SEQ ID NO: 96, (b) A nucleic acid sequence having at least 70% identity with any one of the nucleic acid sequences in (a), (c) A nucleic acid sequence that is substantially identical to any one of the nucleic acid sequences in (a) above, apart from the degeneracy of the genetic code. (d) A nucleic acid sequence complementary to any one of the nucleic acid sequences in (a) above, (e) A chimeric nucleic acid obtained by fusion between at least two nucleic acid sequences of (a), (b), (c), and (d), (f) A nucleic acid sequence complementary to any one of the nucleic acid sequences in (a) above, (g) A nucleic acid sequence encoding an immunogenically equivalent portion of a polypeptide having any one of the amino acid sequences described in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, or SEQ ID NO: 97 (h) A nucleic acid sequence encoding any one functional variant or immunogenically equivalent portion of any of the amino acid sequences described in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, or SEQ ID NO: 97, and (i) A nucleic acid sequence that hybridizes under severe conditions to any one of the nucleic acid sequences described in (a), (b), (c), (d), (e), (f), (g), or (h) above, A group of nucleic acid sequences consisting of the following may be selected: Expression vector.

19. A host cell comprising the chimeric nucleic acid described in Claim 18.

20. Use of P. multicida PmSLP protein or an immunogenically equivalent portion thereof for preparing an animal vaccine formulation according to any one of claims 1 to 13.

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