Veterinary vaccines and methods for treating pasteurella multocida infection in food producing animals

Abstract

The present invention discloses novel veterinary vaccine compositions comprising pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof. The vaccine compositions can be used to ameliorate, treat or prevent pathogen infections in food producing animals such as bovine and porcine animals caused by Pasteurella multocida. Related methods and uses are also disclosed.

Description

[0001] Related Applications

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 332,966, filed on April 20, 2022; the entire contents of U.S. Provisional Application No. 63 / 332,966 are hereby incorporated by reference.

[0003] Incorporation of Sequence Listing

[0004] A computer-readable form of the sequence listing "21806-P64683PC00_SequenceListing.xml" (149,526 bytes) was created on April 19, 2023, and is submitted electronically with this case and incorporated herein by reference. Technical Field

[0005] The methods, uses and compositions disclosed herein relate to the treatment of infectious diseases. Specifically, the methods, uses and compositions disclosed herein relate to veterinary vaccines for preventing, treating or ameliorating infections caused by the infectious Gram-negative bacteria Pasteurella multocida in food-producing animals. Background Art

[0006] The following paragraphs are provided by way of background to the present disclosure. The following paragraphs do not admit that anything discussed therein is prior art or part of the knowledge of a person skilled in the art.

[0007] Pasteurella multocida is a gram-negative bacterium that can colonize and infect a wide variety of different animals, including mammals and birds. The diseases caused vary by animal species and include respiratory disease in ruminants; bovine respiratory disease (BRD) and hemorrhagic septicemia (HS) in animals of the family Bovinae and Bovidae; porcine pneumonic pasteurellosis and porcine atrophic rhinitis (PAR) in suids; and fowl cholera in avian species.

[0008] Pasteurella multocida is a rod-shaped, non-flagellated bacterium that is commonly found in the oral cavity, nasal cavity, and respiratory tract of animals. However, disease is usually associated with further organic or systemic spread of the bacteria and may include symptoms such as pneumonia, atrophic rhinitis, skin necrosis, cellulitis, abscesses, meningitis, and hemorrhagic sepsis. In addition, Pasteurella multocida infection can be 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. and Ho, M., 2013, Clinical Microbiol. Rev., 26 (3), 631. doi: 10.1128 / CMR.00024-13).

[0009] According to taxonomy, Pasteurella multocida can be divided into three subspecies: P. multocida subspecies multocida, P. multocida subspecies gallicida and P. multocida subspecies septica (Mutters R. et al., 1985, Intern. J. of Syst. Evol. Microbiol., 35 (3), 309-322. doi.org / 10.1099 / 00207713-35-3-309). In addition, Pasteurella multocida can be classified according to serogroups, representing different types of extracellular capsular polysaccharides. In this regard, five serogroups are generally distinguished (serogroups A, B, D, E and F) (Carter, G. 1955, Rev Sci Tech, 19 (2), 626-637.docoi: 10.20506 / rst.19.2.1236). Based on the antigenic membrane lipopolysaccharide (LPS) components that may be exhibited by different strains of Pasteurella multocida, strains of Pasteurella multocida can be further divided into 16 serotypes (serotypes 1-16) (Heddleston, K., 1972, Avian Dis, 16 (4), 925-936).

[0010] Diseases associated with bovines caused by Pasteurella multocida include bovine respiratory disease complex (BRD), which is common in feedlots in North America and Europe, and hemorrhagic septicemia (HS), which is a common cause of disease among smallholder farmers in Asia and Africa. BRD, also known as "shipping fever" in feedlot cattle, is generally considered to be the leading cause of morbidity and mortality in feedlot cattle, and it is estimated that 45-55% of all deaths in feedlots are caused by BRD (Johnson, K. and Pendell, D., 2017, Frontiers in Vet. Sci., 4(189). doi: 10.3389 / fvets.2017.00189). Additionally, 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 often associated with lung infection, resulting in pneumonia in newly weaned and feedlot cattle, lactating beef calves, confined dairy calves, and lactating dairy cows. BRD is more common in herds of cattle raised in confined spaces such as feedlots or in large herds of cattle raised in several acres of space. BRD is also more common in stressed animals and animals with existing infections, such as calves before weaning and calves transported to a new location shortly after weaning (Wilson et al., BK 2017 J. Animal Sci., 95(5), 2170-2182. doi: 10.1016 / s0749-0720(15)30302-9. i:10.2527 / jas.2016.1006; Dubrovsky, S. et al., J Dairy Sci., 2019, 102(8):7320-7328. doi:10.3168 / jds.2018-15463).

[0011] Hemorrhagic septicemia (HS) is a rapidly progressive, highly lethal septicemia of cattle and buffaloes. HS has caused significant economic losses in tropical regions of the world, particularly in low- and middle-income countries in Africa and Asia. HS is particularly devastating for smallholder farmers, whose animal husbandry and preventive measures are often inadequate. The Pasteurella multocida bacteria that cause HS can colonize the tonsils of a small percentage of healthy buffaloes and cattle (carriers) and can be shed during stressful periods such as high temperatures and high humidity (Shivachandra, S. et al., 2011, Animal Health Res. Rev., 12(1), 67-82. doi: 10.1017 / S146625231100003X). Disease outbreaks are most frequent during the rainy season. Infection occurs through contact with infected oral or nasal secretions from healthy carriers or sick animals, or may occur through 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 disease progression of colonized cattle is not fully understood, but it is usually manifested as local involvement of the neck and systemic spread, ultimately leading to the spread of various organs, tissue damage, cytokine storms, and toxic shock. Clinical signs may appear within 1-3 days after the first detection of the organism, and may die rapidly within 8-24 hours after the onset of symptoms. It is estimated that the economic losses caused by HS in India are close to $800 million each year (Singh, B. et al., 2014, Agric. Econ. Res. Rev., 27 (347-2016-17135), 271-279).

[0012] Current veterinary vaccines for the treatment of ruminant-associated Pasteurella multocida infections are limited and focus primarily on traditional bacterial vaccine formulations (killed whole bacteria) or live attenuated bacteria (streptomycin-dependent mutant strains). Both vaccines are unreliable in efficacy due to limited protection, in part due to the bacterial polysaccharide capsule specificity of these vaccines, and the potential for mismatched circulating strains, but even with capsule matching, efficacy is incomplete and not well documented in the literature (Dabo, S. et al., 2007, Anim. Health Res. Rev., 8(2), 129-150). In addition, due to the limited effectiveness of existing vaccines against Pasteurella multocida and other bacteria that cause BRD, the preventive and metaphylactic use of antimicrobial drugs is common in the cattle industry and is a potential important source of antimicrobial resistance in pathogens (Cameron, A. and McAllister T., 2016, J. Animal Sci. and Biotechn., 7(1), 68. doi:10.1186 / s40104-016-0127-3).

[0013] Infection of pigs with Pasteurella multocida causes porcine pneumonic pasteurellosis and progressive atrophic rhinitis (PAR) infection, both of which are of considerable economic significance worldwide (Adlam C. and Rutter J, 1989, Pasteurella multocida: molecular biology, toxins and infection (Vol. 361): Springer Science & Business Medi a). PAR is associated with certain toxigenic strains of Pasteurella multocida, which are usually 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 Saka no. T. et al., 1992, J. Vet. Med. Sci., 54(3), 403-407. doi:10.1292 / jvms.54.403), while pneumococcal disease is generally caused by non-toxigenic strains, but also includes 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, Inf ect. Immun., 60(4), 1401-1405). Pasteurella multocida-associated swine infections occur worldwide, including in North America, Europe, and Asia (VanderWaal D. and Deen J., 2018, PNAS 115(45), 11495. doi: 10.1073 / pnas.1806068115). As in the cattle industry, Pasteurella multocida-associated infections represent a significant economic burden, and the management of these infections results in increased antibiotic use.Vaccines for pigs against Pasteurella multocida are generally bacterial vaccines (bacterin) of toxigenic and non-toxigenic strains, or toxoid-based vaccines (OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, 8th Edition, 2018, Chapter 3.8.2, 1540-1550). Vaccines against porcine pneumonic Pasteurella infection do not seem to be widely available yet.

[0014] Pasteurella multocida is also the causative agent of fowl cholera and is associated with a variety of infections. Acute infections may progress rapidly, and often sudden death of birds in a flock is the only sign of infection within a facility, although other symptoms may include oral and nasal discharge, cyanosis, general depression, and diarrhea (Christensen, J. and Bisgaard, M. 2000, Rev. Sci. Tech., 19(2), 626-637. doi: 10.20506 / rst.19.2.1236). Fowl cholera may affect a variety of poultry species, with chickens, turkeys, ducks, and quail being the most economically significant (Glisson, J. 1998, Poultry Science, 77(8), 1139-1142. doi.org / 10.1093 / ps / 77.8.1139). Fowl cholera is mainly caused by serogroup A strains of Pasteurella multocida, but there are also reports that it is caused by serogroups F and D (Dziva, F. et al., 2008, Vet. Microbiol., 128 (1-2), 1-22. doi: 10.1016 / j.vetmic.2007.10.018). Available vaccines include inactivated bacterial vaccines and live attenuated vaccines. Inactivated vaccines cause serotype-specific reactions and therefore provide limited protection. On the other hand, inactivated vaccines do cause extensive serotype cross-reactions. However, live attenuated vaccines have the potential to induce chronic fowl cholera in chickens and turkeys (Glisson, J. 1998, Poultry Science, 77 (8), 1139-1142. doi.org / 10.1093 / ps / 77.8.1139).

[0015] In view of the above, there is a need in the art for improved methods and compositions for treating Pasteurella multocida infection. Specifically, there is a need in the art for improved vaccines for preventing diseases caused by Pasteurella multocida infection in food-producing animals, including but not limited to ruminants, such as bovine, porcine, and avian food-producing animals. SUMMARY OF THE INVENTION

[0016] The following paragraphs are intended to introduce the reader to a more detailed description without defining or limiting the claimed subject matter of the present disclosure.

[0017] In one aspect, the present disclosure relates to vaccine formulations.

[0018] In another aspect, the present disclosure relates to vaccine formulations for preventing or ameliorating disease in food producing animals caused by the bacterial pathogen Pasteurella multocida.

[0019] The inventors of the present invention have discovered that the veterinary vaccine formulations of the present invention can provide protection against infection by multiple strains of Pasteurella multocida to food-producing animals using a single immunogenic active agent, including providing protection in a cross-protective manner. Specifically, the immunogenic agent that was found to be effective when administered to food-producing animals in the form of a veterinary vaccine formulation is a protein selected from a class of Pasteurella multocida proteins known as PmSLP proteins.

[0020] Therefore, according to the teachings herein, in at least one embodiment, the present disclosure provides a veterinary vaccine formulation for preventing or ameliorating Pasteurella multocida infection in food-producing animals susceptible to Pasteurella multocida infection, the vaccine formulation comprising an effective amount of at least one PmSLP protein, or an immunogenic equivalent portion thereof.

[0021] In at least one embodiment, in one aspect, the vaccine formulation may comprise at least one Pasteurella multocida PmSLP protein, or an immunogenic equivalent portion thereof, selected from the same phylogenetic cluster as the PmSLP proteins present in an infecting Pasteurella multocida strain.

[0022] In at least one embodiment, in one aspect, the vaccine formulation can comprise a Pasteurella multocida PmSLP protein, or an immunogenic 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 Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof belongs to the same phylogenetic cluster as the PmSLP protein present in the infecting Pasteurella multocida strain.

[0023] In at least one embodiment, in one aspect, the food producing animal can be a ruminant susceptible to infection with a Pasteurella multocida strain that causes respiratory disease, and the vaccine formulation comprises a Pasteurella multocida PmSLP protein from a Pasteurella multocida strain that causes respiratory disease, or an immunogenic equivalent portion thereof, wherein the PmSLP protein is selected from the group of phylogenetic clusters consisting of PmSLP-1, PmSLP-2, PmSLP-3 and PmSLP-4.2, wherein the selected Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof belongs to the same phylogenetic cluster as the PmSLP protein present in the infecting Pasteurella multocida strain.

[0024] In at least one embodiment, in one aspect, the food-producing animal can be a bovine susceptible to infection with a strain of Pasteurella multocida that causes BRD, and the vaccine formulation comprises a Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof from the strain of Pasteurella multocida 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, wherein the selected Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof belongs to the same phylogenetic cluster as the PmSLP protein present in the infecting Pasteurella multocida strain.

[0025] In at least one embodiment, in one aspect, the food-producing animal can be a bovine susceptible to infection with a strain of Pasteurella multocida that causes HS, and the vaccine formulation comprises a Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof from the strain of Pasteurella multocida that causes HS, wherein the PmSLP protein is selected from the phylogenetic cluster PmSLP-3, wherein the selected Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof belongs to the same phylogenetic cluster as the PmSLP protein present in the infecting Pasteurella multocida strain.

[0026] In at least one embodiment, in one aspect, the food-producing animal can be a porcine animal susceptible to infection with a Pasteurella multocida strain that causes porcine atrophic rhinitis (PAR), and the vaccine formulation comprises a Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof from the Pasteurella 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, wherein the selected Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof belongs to the same phylogenetic cluster as the PmSLP protein present in the infecting Pasteurella multocida strain.

[0027] In at least one embodiment, in one aspect, the food-producing animal can be a porcine animal susceptible to infection with a strain of Pasteurella multocida that causes pneumonic pasteurellosis, and the vaccine formulation comprises a Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof from a strain of Pasteurella multocida 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, wherein the selected Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof belongs to the same phylogenetic cluster as the PmSLP protein present in the infecting Pasteurella multocida strain.

[0028] In at least one embodiment, in one aspect, the food-producing animal can be an avian animal susceptible to infection with a Pasteurella multocida strain that causes avian cholera, and the vaccine formulation comprises a Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof from a Pasteurella multocida strain that causes avian cholera, wherein the PmSLP protein is selected from the group of phylogenetic clusters consisting of PmSLP-3 and PmSLP-4.2, wherein the selected Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof belongs to the same phylogenetic cluster as the PmSLP protein present in the infecting Pasteurella multocida strain.

[0029] In at least one embodiment, in one aspect, the vaccine formulation can comprise a Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof, wherein the PmSLP protein is 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 Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof belongs to the same phylogenetic cluster as the PmSLP protein present in the infecting Pasteurella multocida strain, and wherein the selected Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof is also a PmSLP protein of a Pasteurella 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 Pasteurella multocida strain.

[0030] In at least one embodiment, in one aspect, the at least one PmSLP protein or an immunologically equivalent portion thereof can be a protein expressed by a nucleic acid sequence selected from the group consisting of:

[0031] (a) SEQ.ID NO:1, SEQ.ID NO:3, SEQ.ID NO:5, SEQ.ID.NO:7, SEQ.ID NO:9, SEQ.IDNO: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.IDNO:54, SEQ.ID NO:56, SEQ.ID NO:58, SEQ.ID NO:60, SEQ.ID NO:62, SEQ.ID NO:64, SEQ.IDNO:66, SEQ.ID NO:68, SEQ.ID NO:70, SEQ.ID NO:72, SEQ.ID NO:74, SEQ.ID NO:76, SEQ.IDNO:78, SEQ.ID NO:80, SEQ.ID NO:82, SEQ.ID NO:84, SEQ.ID NO:86, SEQ.ID NO:88, SEQ.IDNO:90, SEQ.ID NO:92, SEQ.ID NO:94 or SEQ.ID NO:96;

[0032] (b) a nucleic acid sequence that is at least 70% identical to any of the nucleic acid sequences in (a);

[0033] (c) a nucleic acid sequence substantially identical to any of the nucleic acid sequences in (a) except for the degeneracy of the genetic code;

[0034] (d) a nucleic acid sequence complementary to any one of the nucleic acid sequences in (a);

[0035] (e) a chimeric nucleic acid obtained by fusion of at least two nucleic acid sequences of (a), (b), (c) and (d) or parts thereof;

[0036] (f) a nucleic acid sequence complementary to any one of the nucleic acid sequences in (a);

[0037] (g) a nucleic acid sequence encoding a polypeptide having any of the amino acid sequences shown in the following, or an immunogenic equivalent portion thereof: 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:64, SEQ.ID NO:65 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;

[0038] (h) a nucleic acid sequence encoding a functional variant of any one of the amino acid sequences shown below, or an immunogenic equivalent thereof: 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:64, SEQ.ID NO:65 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

[0039] (i) A nucleic acid sequence that hybridizes under stringent conditions to any one of the nucleic acid sequences shown in (a), (b), (c), (d), (e), (f), (g) or (h).

[0040] In at least one embodiment, in one aspect, the at least one Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof may comprise any one of the amino acid sequences shown 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:60, SEQ.ID NO:61, SEQ.ID NO:62, SEQ.ID NO:63, SEQ.ID NO:64, SEQ.ID NO:65, SEQ.ID NO:66, SEQ.ID NO:67, SEQ.ID NO:68, SEQ.ID NO:69, SEQ.ID NO:70, SEQ.ID NO:71, SEQ.ID NO:72, SEQ.ID NO:73, SEQ.ID NO:74, SEQ.ID NO:75 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 immunogenic equivalent portions thereof, or a chimeric protein obtained by fusion of any one of the sequences or portions thereof.

[0041] In at least one embodiment, in one aspect, the food producing animal can be a ruminant species and the Pasteurella multocida infection causes respiratory disease.

[0042] In at least one embodiment, in one aspect, the food producing animal can be a bovine species and the Pasteurella multocida infection causes bovine respiratory disease (BRD) or hemorrhagic septicemia (HS).

[0043] In at least one embodiment, in one aspect, the food producing animal can be a Suidae species and the Pasteurella multocida infection causes porcine pneumonic pasteurellosis or porcine atrophic rhinitis (PAR).

[0044] In at least one embodiment, in one aspect, the food producing animal can be an avian species and the Pasteurella multocida infection causes fowl cholera.

[0045] In at least one embodiment, in one aspect, the food producing animal can be a ruminant species, wherein Pasteurella multocida infection causes respiratory disease, and wherein 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:60, SEQ.ID NO:61, SEQ.ID NO:62, SEQ.ID NO:63, SEQ.ID NO:64, SEQ.ID NO:65, SEQ.ID NO:66, SEQ.ID NO:67, SEQ.ID NO:68, SEQ.ID NO:69, SEQ.ID NO:70, SEQ.ID NO:71, SEQ.ID NO:72, SEQ.ID NO:73, SEQ.ID NO:74, SEQ.ID NO:75 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 immunogenic equivalent parts thereof, to prevent or improve Pasteurella multocida infection that causes respiratory diseases.

[0046] In at least one embodiment, in one aspect, the food producing animal can be a bovine species, wherein Pasteurella multocida infection causes BRD, and wherein 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:70, SEQ.ID NO:71, SEQ.ID NO:72, SEQ.ID NO:73, SEQ.ID NO:74, SEQ.ID NO:75 NO:71, SEQ.ID NO:73, SEQ.ID NO:77 or SEQ.ID NO:91, or an immunogenic equivalent thereof, to prevent or improve Pasteurella multocida infection that causes BRD.

[0047] In at least one embodiment, in one aspect, the food producing animal can be a bovine species, wherein Pasteurella multocida infection causes HS, and wherein 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 immunogenic equivalent portion thereof, to prevent or ameliorate Pasteurella multocida infection that causes HS.

[0048] In at least one embodiment, in one aspect, the food production animal can be a porcine species, and infection with Pasteurella multocida causes pneumonic pasteurellosis, and wherein 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 immunogenic equivalent portion thereof, to prevent or ameliorate infection with Pasteurella multocida that causes pneumonic pasteurellosis.

[0049] In at least one embodiment, in one aspect, the food production animal can be a porcine species, and Pasteurella multocida infection causes PAR, and wherein 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 immunogenic equivalent portion thereof, to prevent or ameliorate Pasteurella multocida infection that causes PAR.

[0050] In at least one embodiment, in one aspect, the food production animal can be an avian species, wherein infection with Pasteurella multocida causes fowl cholera, and wherein 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 immunogenic equivalent thereof, to prevent or ameliorate Pasteurella multocida infection that causes pneumonic pasteurellosis.

[0051] In at least one embodiment, in one aspect, the PmSLP protein or an immunogenic equivalent portion thereof can be recombinantly produced in a microbial host organism.

[0052] In at least one embodiment, in one aspect, the veterinary vaccine formulation can be a cross-protective vaccine formulation, which comprises a PmSLP protein obtained from a first strain of Pasteurella multocida or an immunogenic equivalent portion thereof, and which is administered to food-producing animals to prevent or ameliorate an infection caused by another strain of Pasteurella multocida.

[0053] In at least one embodiment, in one aspect, the vaccine formulation may be substantially free of other Pasteurella multocida components.

[0054] In at least one embodiment, in one aspect, the PmSLP protein or immunologically equivalent portion thereof can be a recombinantly produced protein, wherein the vaccine formulation is substantially free of host cell components.

[0055] In at least one embodiment, in one aspect, the vaccine formulation may further comprise a veterinary pharmaceutically acceptable adjuvant.

[0056] In at least one embodiment, in one aspect, the vaccine formulation may further comprise a veterinary pharmaceutically acceptable excipient, carrier or diluent.

[0057] In at least one embodiment, in one aspect, the vaccine formulation may comprise about 0.001% to about 20% by weight of a PmSLP protein or an immunogenic equivalent portion thereof, and about 0.1% to about 60% by weight or volume of a veterinary pharmaceutically acceptable adjuvant of the vaccine formulation.

[0058] In at least one embodiment, in one aspect, the vaccine formulation may comprise a second Pasteurella multocida PmSLP protein, or an immunologically equivalent portion thereof.

[0059] In at least one embodiment, in one aspect, a vaccine formulation may comprise a fusion polypeptide comprising a first and a second Pasteurella multocida PmSLP protein, or immunologically equivalent portions thereof.

[0060] In at least one embodiment, in one aspect, the second Pasteurella multocida PmSLP protein or an immunologically equivalent portion thereof may belong to the same or a different phylogenetic cluster as the first Pasteurella multocida PmSLP protein or an immunologically equivalent portion thereof.

[0061] In at least one embodiment, in one aspect, 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.

[0062] In at least one embodiment, in one aspect, the second Pasteurella multocida PmSLP protein or an immunologically equivalent portion thereof can be obtained from a Pasteurella multocida strain belonging to the same or a different serogroup as the first Pasteurella multocida PmSLP protein or an immunologically equivalent portion thereof.

[0063] On the other hand, according to the teachings herein, in at least one embodiment, the present disclosure provides a veterinary vaccine formulation for preventing, treating or ameliorating Pasteurella multocida infection in food-producing animals susceptible to infection with Pasteurella multocida, the vaccine formulation comprising an effective amount of at least one PmSLP protein, or an immunogenic equivalent portion thereof. In another embodiment, the present disclosure provides a veterinary vaccine formulation for preventing, treating or ameliorating Pasteurella multocida infection in food-producing animals susceptible to infection with Pasteurella multocida, the vaccine formulation comprising an effective amount of at least one PmSLP protein, or an immunogenic equivalent portion thereof.

[0064] In at least one embodiment, in one aspect, the vaccine formulation can 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 secretion pattern, (iv) cough pattern, (v) breathing pattern, and (vi) overall clinical health status, relative to an animal production animal not administered the veterinary vaccine formulation.

[0065] In at least one embodiment, in one aspect, the vaccine formulation is capable of eliciting an immune response in a food-producing animal, wherein anti-PmSLP antibodies are detectable in the serum of the food-producing animal for at least a period starting from 7 days and ending at 52 weeks from the date of administration of the vaccine formulation.

[0066] On the other hand, according to the teachings herein, in at least one embodiment, the present disclosure provides a method for preventing, treating or ameliorating Pasteurella multocida infection in a food-producing animal susceptible to Pasteurella multocida infection, the method comprising administering a veterinary vaccine preparation to the food-producing animal, the veterinary vaccine preparation comprising a Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof, wherein the vaccine preparation is administered in an effective amount to prevent, treat or ameliorate Pasteurella multocida infection.

[0067] In at least one embodiment, in one aspect, amelioration of a Pasteurella multocida infection can include reducing clinical signs of any disease caused by a Pasteurella multocida infection.

[0068] In at least one embodiment, in one aspect, the clinical signs can be selected from the group consisting of: (i) rectal temperature, (ii) animal behavior, (iii) nasal discharge pattern, (iv) coughing pattern, (v) breathing pattern, and (vi) overall clinical health status relative to an animal production animal not administered the veterinary vaccine formulation.

[0069] In at least one embodiment, in one aspect, the food producing animal susceptible to infection with Pasteurella multocida can be selected from the group consisting of ruminants, suis, and avian species.

[0070] In at least one embodiment, in one aspect, 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.

[0071] In at least one embodiment, in one aspect, the food producing animal can be a ruminant susceptible to infection with Pasteurella multocida, and wherein the veterinary vaccine formulation administered to the ruminant in an effective amount comprises at least one PmSLP protein from a strain of Pasteurella multocida that causes respiratory disease, or an immunogenic equivalent portion thereof.

[0072] In at least one embodiment, in one aspect, the food-producing animal susceptible to infection with Pasteurella multocida can be a bovine species, and wherein the veterinary vaccine formulation administered to the bovine species in an effective amount comprises at least one PmSLP protein from a strain of Pasteurella multocida that causes BRD, or an immunogenic equivalent portion thereof.

[0073] In at least one embodiment, in one aspect, the food-producing animal susceptible to infection with Pasteurella multocida can be a ruminant, and wherein the veterinary vaccine formulation administered to the ruminant in an effective amount comprises at least one PmSLP protein from a strain of Pasteurella multocida that causes HS, or an immunogenic equivalent portion thereof.

[0074] In at least one embodiment, in one aspect, the food production animal susceptible to infection with a Pasteurella multocida strain can be a porcine animal, and wherein the veterinary vaccine formulation administered to the porcine animal in an effective amount comprises at least one PmSLP protein from a Pasteurella multocida strain that causes porcine atrophic rhinitis (PAR), or an immunogenic equivalent portion thereof.

[0075] In at least one embodiment, in one aspect, the food production animal susceptible to infection with a Pasteurella multocida strain can be an avian animal, and wherein the veterinary vaccine formulation administered to the avian animal in an effective amount comprises at least one PmSLP protein from a Pasteurella multocida strain that causes fowl cholera, or an immunogenic equivalent portion thereof.

[0076] In at least one embodiment, in one aspect, a vaccine formulation administered to a food producing animal in an effective amount may comprise at least one Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof, wherein the PmSLP protein is selected from the phylogenetic cluster group consisting of PmSLP-1, PmSLP-2, PmSLP-3, PmSLP-4.1 and PmSLP-4.2.

[0077] In at least one embodiment, in one aspect, a veterinary vaccine formulation may comprise at least one Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof, which PmSLP protein or an immunogenic equivalent portion thereof confers homologous protection to food-producing animals susceptible to infection with Pasteurella multocida against Pasteurella multocida strains having PmSLP proteins from the same phylogenetic cluster.

[0078] In at least one embodiment, in one aspect, a veterinary vaccine formulation may comprise at least one Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof, which PmSLP protein or an immunogenic equivalent portion thereof confers heterologous protection to food production animals susceptible to infection with Pasteurella multocida against Pasteurella multocida strains having PmSLP proteins from different phylogenetic clusters.

[0079] In at least one embodiment, in one aspect, the vaccine formulation may comprise at least one Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof, wherein the PmSLP protein is selected from a protein encoded by a nucleic acid sequence selected from the group consisting of:

[0080] (a) SEQ.ID NO:1, SEQ.ID NO:3, SEQ.ID NO:5, SEQ.ID.NO:7, SEQ.ID NO:9, SEQ.IDNO: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.IDNO:54, SEQ.ID NO:56, SEQ.ID NO:58, SEQ.ID NO:60, SEQ.ID NO:62, SEQ.ID NO:64, SEQ.IDNO:66, SEQ.ID NO:68, SEQ.ID NO:70, SEQ.ID NO:72, SEQ.ID NO:74, SEQ.ID NO:76, SEQ.IDNO:78, SEQ.ID NO:80, SEQ.ID NO:82, SEQ.ID NO:84, SEQ.ID NO:86, SEQ.ID NO:88, SEQ.IDNO:90, SEQ.ID NO:92, SEQ.ID NO:94 or SEQ.ID NO:96;

[0081] (b) a nucleic acid sequence that is at least 70% identical to any of the nucleic acid sequences in (a);

[0082] (c) a nucleic acid sequence substantially identical to any of the nucleic acid sequences in (a) except for the degeneracy of the genetic code;

[0083] (d) a nucleic acid sequence complementary to any one of the nucleic acid sequences in (a);

[0084] (e) a chimeric nucleic acid obtained by fusing at least two nucleic acid sequences of (a), (b), (c) and (d) or parts thereof;

[0085] (f) a nucleic acid sequence complementary to any one of the nucleic acid sequences in (a);

[0086] (g) a nucleic acid sequence encoding a polypeptide having any of the amino acid sequences shown in the following, or an immunogenic equivalent portion thereof: 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:64, SEQ.ID NO:65 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;

[0087] (h) a nucleic acid sequence encoding a functional variant of any one of the amino acid sequences shown below, or an immunogenic portion thereof: 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:64, SEQ.ID NO:65 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

[0088] (i) A nucleic acid sequence that hybridizes under stringent conditions to any one of the nucleic acid sequences shown in (a), (b), (c), (d), (e), (f), (g) or (h).

[0089] In at least one embodiment, in one aspect, the vaccine formulation may comprise at least one Pasteurella multocida PmSLP protein or an immunogenic equivalent thereof, wherein the PmSLP protein has any one of the amino acid sequences shown in the following: 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:60, SEQ.ID NO:61, SEQ.ID NO:62, SEQ.ID NO:63, SEQ.ID NO:64, SEQ.ID NO:65, SEQ.ID NO:66, SEQ.ID NO:67, SEQ.ID NO:68, SEQ.ID NO:69, SEQ.ID NO:70, SEQ.ID NO:71, SEQ.ID NO:72, SEQ.ID NO:73, SEQ.ID NO:74, SEQ.ID NO:75 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 immunogenic equivalent portions thereof, or a chimeric protein obtained by fusion of any one of the sequences or portions thereof.

[0090] In at least one embodiment, in one aspect, the PmSLP protein or an immunogenic equivalent portion thereof can be recombinantly produced in a microbial host organism.

[0091] In at least one embodiment, in one aspect, the PmSLP protein or immunologically equivalent portion thereof can be substantially free of other Pasteurella multocida components.

[0092] In at least one embodiment, in one aspect, the PmSLP protein or immunologically equivalent portion thereof can be a recombinantly produced protein, wherein the PmSLP protein or immunologically equivalent portion thereof is substantially free of host cell components.

[0093] In at least one embodiment, in one aspect, the vaccine formulation may further comprise a veterinary pharmaceutically acceptable adjuvant.

[0094] In at least one embodiment, in one aspect, the vaccine formulation may comprise a veterinary pharmaceutically acceptable excipient, carrier or diluent.

[0095] On the other hand, according to the teachings herein, in at least one embodiment, the present disclosure provides a method for preparing a veterinary vaccine preparation for preventing or ameliorating Pasteurella multocida infection in food production animals susceptible to Pasteurella multocida infection, the method comprising:

[0096] (i) diagnosis of Pasteurella multocida infection in food-producing animals;

[0097] (ii) identifying a phylogenetic cluster to which the PmSLP protein present in the infected Pasteurella multocida belongs, wherein the phylogenetic cluster is selected from the group consisting of PmSLP-1, PmSLP-2, PmSLP-3, PmSLP-4.1 or PmSLP-4.2; and

[0098] (iii) preparing a vaccine preparation to form a veterinary vaccine preparation comprising an effective amount of a PmSLP protein of Pasteurella multocida or an immunogenic equivalent portion thereof, thereby treating food-producing animals susceptible to infection with Pasteurella multocida, wherein the vaccine preparation comprises a PmSLP protein of Pasteurella multocida belonging to the identified phylogenetic cluster or an immunogenic equivalent portion thereof, and a veterinary pharmaceutically acceptable adjuvant.

[0099] In at least one embodiment, in one aspect, the method can further include identifying a serogroup of the infectious Pasteurella multocida strain selected from the group consisting of serogroups A, B, D, E and F, and the vaccine is prepared using a Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof from the same or another Pasteurella multocida strain belonging to the selected serogroup.

[0100] On the other hand, according to the teachings herein, in at least one embodiment, the present disclosure provides a method for preparing a veterinary vaccine formulation comprising a Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof, the method comprising:

[0101] (a) providing a chimeric nucleic acid sequence comprising the following as operably linked components:

[0102] (i) a nucleic acid sequence encoding a Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof; and

[0103] (ii) one or more nucleic acid sequences capable of controlling the expression of a nucleic acid sequence encoding the PmSLP protein or an immunogenic equivalent portion thereof in a host cell;

[0104] (b) introducing the chimeric nucleic acid sequence into a host cell;

[0105] (c) growing the host cell to produce the Pasteurella multocida PmSLP protein or the immunogenic equivalent portion thereof; and

[0106] (d) recovering the Pasteurella multocida PmSLP protein or the immunogenic equivalent portion thereof; and

[0107] (e) formulating the Pasteurella multocida PmSLP protein or its immunogenic equivalent part with a veterinary pharmaceutically acceptable adjuvant to form a veterinary vaccine preparation for treating food-producing animals susceptible to Pasteurella infection, wherein the veterinary vaccine preparation comprises an effective amount of the Pasteurella multocida PmSLP protein or its immunogenic equivalent part.

[0108] In at least one embodiment, in one aspect, the nucleic acid sequence may be a nucleic acid sequence selected from the group consisting of:

[0109] (a) SEQ.ID NO:1, SEQ.ID NO:3, SEQ.ID NO:5, SEQ.ID.NO:7, SEQ.ID NO:9, SEQ.IDNO: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.IDNO:54, SEQ.ID NO:56, SEQ.ID NO:58, SEQ.ID NO:60, SEQ.ID NO:62, SEQ.ID NO:64, SEQ.IDNO:66, SEQ.ID NO:68, SEQ.ID NO:70, SEQ.ID NO:72, SEQ.ID NO:74, SEQ.ID NO:76, SEQ.IDNO:78, SEQ.ID NO:80, SEQ.ID NO:82, SEQ.ID NO:84, SEQ.ID NO:86, SEQ.ID NO:88, SEQ.IDNO:90, SEQ.ID NO:92, SEQ.ID NO:94 or SEQ.ID NO:96;

[0110] (b) a nucleic acid sequence that is at least 70% identical to any of the nucleic acid sequences in (a);

[0111] (c) a nucleic acid sequence substantially identical to any of the nucleic acid sequences in (a) except for the degeneracy of the genetic code;

[0112] (d) a nucleic acid sequence complementary to any one of the nucleic acid sequences in (a);

[0113] (e) a chimeric nucleic acid obtained by fusion of at least two nucleic acid sequences of (a), (b), (c) and (d) or parts thereof;

[0114] (f) a nucleic acid sequence complementary to any one of the nucleic acid sequences in (a);

[0115] (g) a nucleic acid sequence encoding a polypeptide having any of the amino acid sequences shown in the following, or an immunogenic equivalent portion thereof: 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:64, SEQ.ID NO:65 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;

[0116] (h) a nucleic acid sequence encoding a functional variant of any one of the amino acid sequences shown below, or an immunogenic equivalent thereof: 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:64, SEQ.ID NO:65 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

[0117] (i) A nucleic acid sequence that hybridizes under stringent conditions to any one of the nucleic acid sequences shown in (a), (b), (c), (d), (e), (f), (g) or (h).

[0118] On the other hand, according to the teachings herein, in at least one embodiment, the present disclosure provides an expression vector comprising:

[0119] (a) a nucleic acid sequence encoding a Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof; and

[0120] (b) a nucleic acid sequence capable of controlling the expression of a nucleic acid sequence encoding the Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof in a host cell.

[0121] In at least one embodiment, in one aspect, the nucleic acid sequence may be a nucleic acid sequence selected from the group consisting of:

[0122] (a) SEQ.ID NO:1, SEQ.ID NO:3, SEQ.ID NO:5, SEQ.ID.NO:7, SEQ.ID NO:9, SEQ.IDNO: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.IDNO:54, SEQ.ID NO:56, SEQ.ID NO:58, SEQ.ID NO:60, SEQ.ID NO:62, SEQ.ID NO:64, SEQ.IDNO:66, SEQ.ID NO:68, SEQ.ID NO:70, SEQ.ID NO:72, SEQ.ID NO:74, SEQ.ID NO:76, SEQ.IDNO:78, SEQ.ID NO:80, SEQ.ID NO:82, SEQ.ID NO:84, SEQ.ID NO:86, SEQ.ID NO:88, SEQ.IDNO:90, SEQ.ID NO:92, SEQ.ID NO:94 or SEQ.ID NO:96;

[0123] (b) a nucleic acid sequence that is at least 70% identical to any of the nucleic acid sequences in (a);

[0124] (c) a nucleic acid sequence substantially identical to any of the nucleic acid sequences in (a) except for the degeneracy of the genetic code;

[0125] (d) a nucleic acid sequence complementary to any one of the nucleic acid sequences in (a);

[0126] (e) a chimeric nucleic acid obtained by fusion of any nucleic acid sequence or part thereof in (a), (b), (c) and (d);

[0127] (f) a nucleic acid sequence complementary to any one of the nucleic acid sequences in (a);

[0128] (g) a nucleic acid sequence encoding a polypeptide having any of the amino acid sequences shown in the following, or an immunogenic equivalent portion thereof: 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:64, SEQ.ID NO:65 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;

[0129] (h) a nucleic acid sequence encoding a functional variant of any one of the amino acid sequences shown below, or an immunogenic equivalent thereof: 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:64, SEQ.ID NO:65 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

[0130] (i) A nucleic acid sequence that hybridizes under stringent conditions to any one of the nucleic acid sequences shown in (a), (b), (c), (d), (e), (f), (g) or (h).

[0131] In another aspect, according to the teachings herein, in at least one embodiment, the present disclosure provides a host cell comprising a chimeric nucleic acid comprising:

[0132] (a) a nucleic acid sequence encoding a Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof; and

[0133] (b) a nucleic acid sequence capable of controlling the expression of a nucleic acid sequence encoding the Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof in a host cell.

[0134] In at least one embodiment, in one aspect, the nucleic acid sequence can be a nucleic acid sequence selected from the group consisting of:

[0135] (a) SEQ.ID NO:1, SEQ.ID NO:3, SEQ.ID NO:5, SEQ.ID.NO:7, SEQ.ID NO:9, SEQ.IDNO: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.IDNO:54, SEQ.ID NO:56, SEQ.ID NO:58, SEQ.ID NO:60, SEQ.ID NO:62, SEQ.ID NO:64, SEQ.IDNO:66, SEQ.ID NO:68, SEQ.ID NO:70, SEQ.ID NO:72, SEQ.ID NO:74, SEQ.ID NO:76, SEQ.IDNO:78, SEQ.ID NO:80, SEQ.ID NO:82, SEQ.ID NO:84, SEQ.ID NO:86, SEQ.ID NO:88, SEQ.IDNO:90, SEQ.ID NO:92, SEQ.ID NO:94 or SEQ.ID NO:96;

[0136] (b) a nucleic acid sequence that is at least 70% identical to any of the nucleic acid sequences in (a);

[0137] (c) a nucleic acid sequence substantially identical to any of the nucleic acid sequences in (a) except for the degeneracy of the genetic code;

[0138] (d) a nucleic acid sequence complementary to any one of the nucleic acid sequences in (a);

[0139] (e) a chimeric nucleic acid obtained by fusion of any nucleic acid sequence or part thereof in (a), (b), (c) and (d);

[0140] (f) a nucleic acid sequence complementary to any one of the nucleic acid sequences in (a);

[0141] (g) a nucleic acid sequence encoding a polypeptide having any of the amino acid sequences shown in the following, or an immunogenic equivalent portion thereof: 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:64, SEQ.ID NO:65 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;

[0142] (h) a nucleic acid sequence encoding a functional variant of any one of the amino acid sequences shown below, or an immunogenic equivalent thereof: 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:64, SEQ.ID NO:65 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

[0143] (i) A nucleic acid sequence that hybridizes under stringent conditions to any one of the nucleic acid sequences shown in (a), (b), (c), (d), (e), (f), (g) or (h).

[0144] On the other hand, according to the teachings herein, in at least one embodiment, the present disclosure provides the use of Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof for preparing a veterinary vaccine preparation, wherein the veterinary vaccine preparation comprises the P protein or an immunogenic equivalent portion thereof and a veterinary pharmaceutically acceptable adjuvant.

[0145] Other features and advantages will become apparent from the following detailed description. However, it should be understood that although the detailed description indicates preferred embodiments of the present disclosure, they are given by way of example only, because various changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art based on the detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0146] The present disclosure is described in the paragraphs provided below by way of example in conjunction with the accompanying drawings. The drawings provided herein are for a better understanding of the exemplary embodiments and to more clearly show how the various embodiments can be implemented. These drawings are not intended to limit the present disclosure.

[0147] Figure 1A , 1B , 1C, and 1D depict certain aspects of an example production and purification workflow for recombinant PmSLP protein, as well as example analytical data and results obtained during different steps of performing an example method for purification of PmSLP-3. Figure 1A is a flow chart showing a schematic workflow for purification of recombinant PmSLP. Figure 1B is a photographic image of a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel showing sample material collected at different stages (1-6) of purification of his-tagged PmSLP-3 (SEQ. ID NO: 12) from E. coli lysate using nitrilotriacetic acid (NTA) nickel. Figure 1C It is a S75 gel filtration chromatogram obtained by size exclusion chromatography after removal of the poly-histidine tag of PmSLP-3 (SEQ. ID NO: 14). Figure 1D is a photographic image of an SDS-PAGE gel of purified PmSLP-3 following a polishing step using MonoQ. Note that after removal of the tag, PmSLP-3 migrates at approximately 35 kDa in the gel.

[0148] Figure 2A phylogenetic tree of PmSLP protein sequences collected from a local PmSLP sequence collection and sequences publicly available from online databases, as well as certain information related to the phylogenetic tree. Annotations regarding host species, confirmed disease status, geographic region, and serotype for each PmSLP variant are based on information obtained from the National Center for Biotechnology Information (NCBI) BioSample database, literature searches, and genome sequence analysis. Black circles on the phylogenetic branches of the tree represent sequences derived from bovine species; small light gray shaded dots represent other hosts. Four ring structures around the phylogenetic tree are also depicted, representing: (1) host (species), (2) disease, (3) (geographic) region, and (4) capsule. Each ring structure is composed of multiple rectangular blocks, each of which corresponds to a PmSLP variant in the phylogenetic tree. Thus, moving directly outward from a particular rectangular block selected from the innermost circular structure representing a particular PmSLP variant sequence are: (1) host species (a selected rectangular block in the inner circular structure, e.g., rectangular block (a)); (2) disease (a rectangular block moving directly outward and adjacent to the rectangular block selected under (1), e.g., rectangular block (b)); (3) geographic region (a rectangular block moving directly outward and adjacent to the block identified under (2), e.g., rectangular block (c)); and (4) capsule (a rectangular block moving directly outward and adjacent to the block identified under (3), e.g., rectangular block (d)). The grayscale indices of the rectangular blocks in the innermost circular structure are as follows, representing host species: black, bovine species (including rectangular block (a)); shaded, porcine species; intermediate gray shading, avian species; light gray shading, other species; and white, missing. For the circular structures representing diseases located at the periphery relative to the ring positioned at the center-most ring, the grayscale index of the rectangular blocks is as follows: black, bovine hemorrhagic septicemia (HS); intermediate gray shading, bovine respiratory disease (BRD) (including rectangular block (b)); white, disease status unconfirmed / unknown. For the circular structures representing the geographical regions where the samples were obtained located at the center relative to the ring positioned at the periphery, the grayscale index of the rectangular blocks is as follows: black, North America; intermediate gray shading, Asia; light gray shading, Europe (including rectangular block (c)); and white, unknown region. For the circular structures indicating the capsule serogroups of Pasteurella multocida (when known) located at the periphery, the grayscale scale is as follows: black, serogroup A (including rectangular block (d)); hatched pattern, serogroup B; intermediate gray shading, serogroup D; light gray shading, serogroup F; and white, cannot be classified.Thus, the example selected PmSLP sequences corresponding to the example rectangular blocks (a), (b), (c), (d) correspond to bovine PmSLP isolated from a strain of Pasteurella multocida causing BRD in Europe, which has serogroup A. Notably, it can be seen that the phylogenetic tree of PmSLP variants is divided into five phylogenetic clusters (PmSLP-1, PmSLP-2, PmSLP-3, PmSLP-4.1 and PmSLP-4.2).

[0149] Figure 3 is a graph depicting the results obtained when conducting an experiment, particularly an experiment to evaluate the antibody response of mice administered a PmSLP vaccine formulation. The change in endpoint IgG titer over time is plotted in the figure. The serum IgG titer of mice receiving 2 doses of the vaccine was measured in serum samples collected over a 26-week period. The data points in the figure represent the mean value, and the error bars depict the standard error. The dotted line at the endpoint IgG titer of 8,000 represents the detection limit, i.e., the lowest serum dilution measured. Throughout the experiment, the IgG titer remained stable, and none of the groups showed any obvious signs of weakening. The experimental results show that the use of a vaccine formulation containing PmSLP-1 (SEQ.ID NO: 14) can induce a long-term antibody response.

[0150] Figure 4A , 4B 4C, 4D and 4E are additional graphs depicting the results obtained when performing certain experiments, particularly experiments evaluating the immune response of mice administered with a PmSLP vaccine formulation. In this exemplary experiment, the efficacy of a PmSLP-1 (SEQ. ID NO: 14) vaccine formulation was evaluated against a bovine respiratory disease (BRD) isolate of Pasteurella multocida strain H246, which carries a pmSLP gene from the same phylogenetic cluster. Figure 4A The graph shown in depicts the percentage of survival over time, specifically the percentage of survival of mice after infection with Pasteurella multocida BRD strain H246 (the confirmed sequence of PmSLP in this strain is defined in SEQ. ID NO: 22). Mice vaccinated with the PmSLP-1 vaccine were 100% protected. Figure 4B The graphs shown in depict the changes in clinical scores over time, particularly for mice immunized with the PmSLP-1 vaccine, separated by individual animals. Figure 4C The graphs shown in depict the changes in clinical scores over time, particularly for mice immunized with adjuvant alone, separated by individual animals. Figure 4D The graphs shown in Figure 2 show the changes in clinical scores over time, particularly the average clinical scores of the two groups of mice; the error bars represent standard errors. Figure 4B , 4Cand 4D, a clinical score cutoff of 10 was considered a humane endpoint, at which time the animals were euthanized. Figure 4E The graphs shown in Figure 2 show the changes in bacterial recovery over time, specifically in tail vein blood during infection, separated by individual animals. The experimental results show that the PmSLP vaccine is effective against relevant bovine disease isolates of Pasteurella multocida in an acute mouse infection model.

[0151] Figure 5A and 5B are additional graphs depicting results obtained while performing certain experiments, particularly experiments evaluating the immune response of mice administered PmSLP vaccine formulations. Serum samples were obtained from mice immunized with two doses of PmSLP-1 (SEQ. ID NO: 14) vaccine or adjuvant prior to challenge. Figure 5A The graph shown in depicts α-PmSLP-1 IgG titers against purified antigen measured by protein ELISA. Figure 5B The graph shown in depicts the α-PmSLP-1 IgG titers against whole bacteria measured by heat-killed whole cell ELISA. Pasteurella multocida BRD strain H246 (containing the PmSLP defined in SEQ. ID NO: 22, which carries the pmSLP gene from the same phylogenetic cluster as the vaccine antigen) was used. Figure 5A and Figure 5B , individual points represent individual animals. Bars depict mean values ​​and error bars depict standard errors. The experimental results showed that α-PmSLP-1 antibodies produced by vaccinated mice could bind to purified antigens as well as antigens on the surface of bacteria.

[0152] Fig. 6A , 6B , 6C, 6D and 6E are additional graphs depicting the results obtained when performing certain experiments, particularly experiments evaluating the immune response of mice administered with 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. Fig. 6A The graph shown in depicts the evolution of the survival percentage over time, in particular the survival percentage of mice after infection with the swine Pasteurella multocida strain H229 (PmSLP containing the sequence defined in SEQ. ID NO: 24). Mice vaccinated with the PmSLP-3 vaccine were 100% protected. Figure 6B The graphs shown in depict the changes in clinical scores over time, particularly for mice immunized with the PmSLP-3 vaccine, separated by individual animals. Figure 6C The graphs shown in depict the changes in clinical scores over time, particularly for mice immunized with adjuvant alone, separated by individual animals. Fig.6D The graphs shown in Figure 2 depict the changes in clinical scores over time, specifically the mean clinical scores of the two groups of mice; the error bars represent the standard error. Figure 6B , 6C and 6D, a clinical score cutoff of 10 was considered a humane endpoint, at which time the animals were euthanized. Fig. 6E The graphs shown in Figure 2 depict bacterial recovery over time, specifically in tail vein blood during infection, separated by individual animals. The results of the experiments indicate that the PmSLP-containing vaccine is effective against relevant porcine Pasteurella multocida disease isolates in an acute mouse infection model.

[0153] Fig. 7A and 7B are additional graphs depicting results obtained while performing certain experiments, particularly experiments evaluating the immune response of mice administered PmSLP vaccine formulations. Serum samples were obtained from mice immunized with two doses of PmSLP-3 (SEQ. ID NO: 20) vaccine or adjuvant prior to challenge. Fig. 7A The graph shown in depicts α-PmSLP-3 IgG titers against purified antigen measured by protein ELISA. Figure 7B The graph shown in depicts the titer of α-PmSLP-3 IgG against whole bacteria measured by heat-killed whole cell ELISA. A porcine P. multocida isolate, P. multocida strain H229, was used, which carries the pmSLP gene from the same phylogenetic cluster as the vaccine antigen (the confirmed sequence of PmSLP in this strain is defined in SEQ. ID NO: 24). Fig. 7A and Figure 7B , individual points represent individual animals. Bars depict mean values ​​and error bars depict standard errors. The experimental results showed that α-PmSLP-3 antibodies produced by vaccinated mice could bind to purified antigens as well as antigens on the surface of bacteria.

[0154] Fig. 8A and 8B is an additional graph depicting the results obtained when performing certain experiments, particularly experiments evaluating the stability of the PmSLP polypeptide. The intrinsic fluorescence emitted by tryptophan and tyrosine residues (ratio 350nm / 330nm) ( Fig. 8A ) to generate a thermal curve and calculate the thermal inflection temperature (Ti) of the PmSLP-1 protein samples stored under specified storage conditions ( Figure 8B The results showed that the purified PmSLP-3 protein (SEQ.ID NO: 20) remained stable under various storage conditions after lyophilization for one year.

[0155] Fig.9A , 9B, 9C, 9D and 9E are additional graphs depicting results obtained while conducting certain experiments, particularly experiments evaluating the protection of mice administered the PmSLP vaccine formulation against lethal challenge. Fig.9A The graph shown in depicts the change in survival percentage over time, specifically the survival percentage of mice after infection with an antigenically matched porcine Pasteurella multocida strain H229 (the confirmed sequence of PmSLP of this strain is defined in SEQ.ID NO:24), where all mice receiving the PmSLP-3 vaccine formulation were fully protected from lethal challenge. Fig. 9B , 9C , 9D and 9E show the clinical scores of individual mice over time, particularly the clinical scores of mice immunized with adjuvant, vaccine 1 (freshly prepared vaccine whose protein was stored at -80°C until preparation), vaccine 2 (freshly prepared vaccine whose lyophilized protein was stored at 4°C until preparation) and vaccine 3 (prepared vaccine prepared before the first dose and stored at 4°C until the second dose). Each line depicts the clinical score of each mouse during the experimental challenge, where clinical monitoring was performed at multiple time points within 36 hours after infection. The dotted line at a clinical score of 10 depicts the clinical score cutoff, which is considered a humane endpoint, at which time the animal is euthanized. The experimental results show that PmSLP-3 (SEQ.ID NO: 20) vaccine preparations stored under different conditions can all work in the mouse model.

[0156] Fig.10 is an additional graph depicting the results obtained while conducting certain experiments, particularly experiments evaluating the immune response of ruminant (bovine) species to which PmSLP vaccine formulations were administered. In this example, zebu cattle were immunized subcutaneously with PmSLP-1 (SEQ. ID NO: 14) vaccine or adjuvant. Serum samples were obtained at baseline or 2-3 weeks after administration of the specified doses and assayed using a protein-based ELISA. The data points represent individual animals, the bars represent the mean α-PmSLP-1 IgG titer, and the error bars represent the standard error. The experimental results demonstrate that the PmSLP vaccine can be immunogenic in animal host species that are relevant to the food production industry and are affected by Pasteurella multocida infection.

[0157] Fig.11is an additional graph depicting the results obtained while conducting certain experiments, particularly experiments evaluating the immune response of ruminants (bovine species) to which PmSLP vaccine formulations were administered. In this example, beef cattle were immunized with PmSLP-1 (SEQ. ID NO: 14) vaccine or adjuvant by the intramuscular route. Serum samples were obtained at baseline or 2-3 weeks after administration of the specified doses and assayed using a protein-based ELISA. The data points represent individual animals, the bars represent the average α-PmSLP-1 IgG titer, and the error bars represent the standard error. The experimental results indicate that the PmSLP vaccine can be immunogenic in animal host species that are relevant to the food production industry and are affected by Pasteurella multocida infection.

[0158] Fig. 12A , 12B 12A and 12C are additional graphs depicting results obtained while conducting certain experiments, particularly experiments evaluating the immune response of ruminant (Bovidae) species administered with PmSLP vaccine formulations. In this example, zebu cattle were subcutaneously immunized with PmSLP-3 (SEQ. ID NO: 20) formulated in two different adjuvants in a prime-boost schedule. Fig. 12A The graphs shown in show serum samples obtained at baseline, before boost doses, and before challenge and assayed using a protein-based ELISA. Data points represent individual animals, bars represent mean α-PmSLP-3 IgG titers, and error bars represent standard errors. Fig. 12B The graphs shown in show the changes in animal survival over time, especially the survival of animals after lethal challenge. Fig. 12C The graphs shown in record any local reactogenicity assessed after administration of either vaccine dose. The example experimental results demonstrate that the PmSLP vaccines are immunogenic and safe in animal host species relevant to the food production industry and infected with P. multocida, and that these vaccine formulations protect against experimental challenge with a lethal dose of P. multocida.

[0159] Fig.13A , 13B , 13C, 13D, 13E, 13F, 13G and 13H are additional figures depicting the results obtained when conducting an experiment, particularly an experiment evaluating a PmSLP vaccine administered to ruminants (cattle). Specifically, the figures show the results of the assessment of multiple clinical parameters tracked during the experiment. Fig.13A The graph shown in depicts the change in rectal temperature over time in animals administered IVP 1 vaccine (containing PmSLP-2), autovaccine (positive control), and saline solution (negative control) following challenge with pathogenic Pasteurella multocida strains. Fig. 13BDepicted are bar graphs of animal behavior scores at different time points in the experiment (day 34 (D34) - day 42 (D42)) for animals administered IVP 1 vaccine (containing PmSLP-2), autovaccine (positive control), and saline solution (negative control) after challenge with pathogenic Pasteurella multocida strains. Fig. 13C Depicted are bar graphs of nasal secretion scores of animals administered IVP 1 vaccine (containing PmSLP-2), autovaccine (positive control), and saline solution (negative control) at different time points in the experiment (day 34 (D34) - day 42 (D42)) after challenge with pathogenic Pasteurella multocida strains. Fig.13D Depicted are bar graphs of cough scores at different time points in the experiment (Day 34 (D34) - Day 42 (D42)) in animals administered IVP 1 vaccine (containing PmSLP-2), autovaccine (positive control), and saline solution (negative control) after challenge with pathogenic Pasteurella multocida strains. Fig.13E Depicted are bar graphs of respiratory scores of animals administered IVP 1 vaccine (containing PmSLP-2), autovaccine (positive control), and saline solution (negative control) after challenge with pathogenic Pasteurella multocida strains at different time points in the experiment (day 34 (D34) - day 42 (D42)). Fig.13F Depicted are graphs showing changes in total clinical scores over time following challenge with pathogenic Pasteurella multocida strains in animals administered IVP 1 vaccine (containing PmSLP-2), autovaccine (positive control), and saline solution (negative control). Figure 13G Depicted are graphs depicting lung lesion scores of animals administered IVP 1 vaccine (containing PmSLP-2), autovaccine (positive control), and saline solution (negative control) following challenge with pathogenic Pasteurella multocida strains. Fig.13H A graph depicting antibody titers at two different experimental time points (Day 7 (D-7) (prior to vaccination) and Day 35 (D35) (challenge day)) of animals administered IVP 1 vaccine (containing PmSLP-2), autovaccine (positive control), and saline solution (negative control) after challenge with a pathogenic P. multocida strain. The example experimental results also demonstrate that the PmSLP vaccine is immunogenic and safe in animal host species associated with the food production industry and infected with P. multocida, and that these vaccine formulations can protect against experimental challenge with a dose of P. multocida.

[0160] These drawings and the following detailed description make apparent to those skilled in the art how to implement the disclosure in practice. DETAILED DESCRIPTION

[0161] Various compositions, methods or techniques will be described below to provide examples of the embodiments of each claimed subject matter. The embodiments described below do not limit any claimed subject matter, and any claimed subject matter can encompass a process, composition or method different from the process, composition or method described below. The claimed subject matter is not limited to a composition, method or technique with all the features of any composition, method or technique described below, or a feature common to multiple or all compositions, methods or techniques described below. The composition, method or technique described below may not be an embodiment of any claimed subject matter. Any subject matter disclosed in the composition, method or technique described below that is not claimed in this document can be used as the subject matter of another protection instrument such as a continuation patent application, and the applicant, inventor or holder are not intended to disclose any such subject matter in this document and abandon, deny or show such subject matter to the public.

[0162] Unless the context clearly dictates otherwise, as used herein and in the claims, singular forms such as "a", "an", and "the" include plural referents and vice versa. Throughout this specification, unless otherwise indicated, "comprise", "comprises", and "comprising" are used in an inclusive rather than exclusive sense, so that a stated integer or group of integers may include one or more other unstated integers or groups of integers. The term "or" is inclusive unless modified, for example, with "either".

[0163] When a range is used herein to describe a physical property (such as molecular weight) or a chemical property (such as a chemical formula), all combinations and subcombinations of the range and the specific embodiments thereof are intended to be included. Except in the operating examples, or in the case of additional indications, all numerals used herein to represent the amount of a component or reaction condition should be understood to be modified by the term "about" in all cases. It will be easily recognized according to the context that when a number or a numerical range is mentioned, the term "about" means that the number or numerical range mentioned is an approximate value within the range of experimental variability (or within the range of statistical experimental error), so the number or numerical range can vary between 1% and 15% of the stated number or numerical range. In addition, any range of values ​​described herein is intended to specifically include the limit values ​​of the range, as well as any intermediate value or subrange within a given range, and all these intermediate values ​​and subranges are individually and specifically disclosed (for example, a range of 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4 and 5). Similarly, as used herein, other terms of degree, such as "substantially" and "approximately" mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed to include such deviation if the deviation of the modified term would not negate the meaning of the modified term.

[0164] Unless otherwise defined, scientific and technical terms used in conjunction with the formulations described herein shall have the meanings commonly understood by those of ordinary skill in the art. The terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the present invention, which is defined only by the claims.

[0165] 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.

[0166] Terms and Definitions

[0167] 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) bonds. This term also includes modified or substituted sequences or portions thereof containing non-naturally occurring monomers. 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 these modified bases include aza and deazaadenine, guanine, cytosine, thymidine and uracil, as well as xanthine and hypoxanthine. A sequence of nucleotides or nucleoside monomers may be referred to as a polynucleotide sequence, a nucleic acid sequence, a nucleotide sequence, or a nucleoside sequence.

[0168] The terms "polypeptide" and "protein" used in conjunction with a reference SEQ. ID NO are used interchangeably herein to refer to any and all polypeptides and proteins comprising a sequence of amino acid residues that: (i) is substantially identical to the amino acid sequence that constitutes the polypeptide having such reference SEQ. ID NO; or (ii) is encoded by a nucleic acid sequence that is capable of hybridizing under conditions of at least moderate stringency to any nucleic acid sequence encoding a polypeptide having such reference SEQ. ID NO, but using synonymous codons. A sequence of amino acid residues may be referred to as an amino acid sequence or a polypeptide sequence.

[0169] The terms "nucleic acid sequence encoding a polypeptide" and "nucleic acid sequence encoding a protein" used herein in conjunction with a reference to a SEQ. ID NO refer to any and all nucleic acid sequences encoding a polypeptide or protein having such a reference SEQ. ID NO. In conjunction with a reference to a SEQ. ID NO, a nucleic acid sequence encoding a polypeptide also includes any and all nucleic acid sequences that (i) encode a polypeptide substantially identical to a polypeptide having such a reference SEQ. ID NO; or (ii) hybridize with any nucleic acid sequence encoding a polypeptide having such a reference SEQ. ID NO under at least moderate stringency hybridization conditions, or hybridize with any nucleic acid sequence encoding a polypeptide having such a reference SEQ. ID NO under at least moderate stringency conditions. However, synonymous codons are used.

[0170] The terms "nucleic acid sequence encoding PmSLP" and "nucleic acid sequence encoding a PmSLP polypeptide", "nucleic acid sequence encoding a PmSLP protein" are used interchangeably herein and refer to any and all nucleic acid sequences encoding PmSLP polypeptides, including, for example, SEQ.ID NO: 1. Nucleic acid sequences encoding PmSLP polypeptides also include any and all nucleic acid sequences that (i) encode a polypeptide substantially identical to the PmSLP polypeptide sequence set forth herein; or (ii) hybridize to any PmSLP nucleic acid sequence set forth herein under at least moderately stringent hybridization conditions, or hybridize to any PmSLP nucleic acid sequence set forth herein under at least moderately stringent conditions, but using synonymous codons.

[0171] The terms "PmSLP protein" or "PmSLP polypeptide" are used interchangeably herein to refer to any and all proteins comprising a sequence of amino acid residues that (i) is substantially identical to the amino acid sequence that constitutes any PmSLP polypeptide set forth herein, including, for example, SEQ. ID NO: 2; or (ii) is encoded by a nucleic acid sequence that is capable of hybridizing under at least moderate stringency conditions with any nucleic acid sequence encoding any PmSLP protein set forth herein, but using synonymous codons. PmSLP proteins may also be numbered to facilitate distinction between the different PmSLP proteins referred to herein, such as PmSLP-1, PmSLP-2, PmSLP-3, etc.

[0172] 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, such as 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical. In order to determine the percent identity between two amino acid sequences, the amino acid sequences of the two sequences are aligned using, for example, the alignment method of Needleman and Wunsch (J. Mol. Biol., 1970, 48: 443), as revised by Smith and Waterman (Adv. Appl. Math., 1981, 2: 482), thereby obtaining the highest order match between the two sequences and determining the number of identical amino acids between the two sequences. Methods for calculating the percent identity between two amino acid sequences are generally recognized in the art and include, for example, the methods described by Carillo and Lipton (SIAM J. Applied Math., 1988, 48: 1073) and the methods described in Computational Molecular Biology (Computational Molecular Biology; Lesk, ed., Oxford University Press, New York, 1988, Biocomputing: Informatics and Genomics Projects). In general, computer programs are used to perform these calculations. In this regard, computer programs that can be used 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) and the BLOSUM 62 scoring matrix (Henikoff S and 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, thereby obtaining the highest order match between the two sequences, wherein at least 50% of the total length of the two sequences is involved in the alignment.

[0173] "At least moderate stringency hybridization conditions" refers to conditions that are selected to promote selective hybridization between two complementary nucleic acid molecules in a solution. Hybridization can occur on all or part of a nucleic acid sequence molecule. The hybridization portion is usually at least 15 (e.g., 20, 25, 30, 40, or 50) nucleotides long. Those skilled in the art will recognize that the stability of a nucleic acid duplex or hybrid is determined by Tm, which is a function of sodium ion concentration and temperature in a sodium-containing buffer (Tm = 81.5°C -16.6 (Log10 [Na+]) + 0.41 (% (G+C) -600 / l), or similar formulas). Therefore, the parameters that determine hybrid stability in washing conditions are sodium ion concentration and temperature. In order to identify molecules that are similar but not identical to known nucleic acid molecules, it can be assumed that a 1% mismatch will reduce Tm by about 1°C, for example, if a nucleic acid molecule with > 95% identity is sought, the final washing temperature will be reduced by about 5°C. Based on these considerations, those skilled in the art will be able to easily select appropriate hybridization conditions. In a preferred embodiment, stringent hybridization conditions are selected. For example, stringent hybridization can be achieved using the following conditions: hybridization at a Tm of -5°C (based on the above formula) in 5× sodium chloride / sodium citrate (SSC) / 5×Denhardt's solution / 1.0% SDS, followed by washing with 0.2×SSC / 0.1% SDS at 60°C. Moderate stringency hybridization conditions include washing with 3×SSC at 42°C. However, it should be understood that alternative buffers, salts, and temperatures can be used to achieve equivalent stringency. Additional guidance on 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.

[0174] The term "functional variant" used herein with respect to a polynucleotide or polypeptide refers to a polynucleotide or polypeptide that is capable of performing the same function as a specified reference polynucleotide or polypeptide. Thus, for example, a functional variant of the polypeptide shown in SEQ.ID NO:2 refers to a polypeptide that is capable of performing the same function as the polypeptide shown in SEQ.ID NO:2. Functional variants include modified polypeptides, wherein the modification comprises substitution, deletion or addition of one or more amino acids relative to the specified reference polypeptide. In some embodiments, a substitution is a substitution that causes an amino acid to be replaced by an amino acid having similar characteristics. 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.

[0175] The term "chimeric" as used herein in the context of nucleic acids refers to at least two connected nucleic acids that are not naturally connected. Chimeric nucleic acids include nucleic acids of different natural origins that are connected. For example, a nucleic acid constituting a microbial promoter is connected to a nucleic acid encoding a plant polypeptide and is considered chimeric. Chimeric nucleic acids may also contain nucleic acids of the same natural origin, as long as they are not naturally connected. For example, a nucleic acid constituting a promoter obtained from a specific cell type can be connected to a nucleic acid encoding a polypeptide obtained from the same cell type, but is generally not connected to a nucleic acid constituting a promoter. Chimeric nucleic acids also include a class of nucleic acids that include any naturally occurring nucleic acid connected to any non-naturally occurring nucleic acid.

[0176] The term "phylogenetic cluster" refers to a group of evolutionarily related polypeptide sequences. In order to determine whether two polypeptides belong to the same phylogenetic cluster, a plurality of almost similar polypeptide sequences (e.g., preferably at least 25, at least 50, at least 100, or at least 1,000 polypeptide sequences) can be used to construct an evolutionary tree comprising a plurality of branches (e.g., at least 5, 7, 10, 15, or 20 branches). By examining the phylogenetic tree, the evolutionary relationships of the polypeptide sequences can be assessed. Polypeptides belonging to the same phylogenetic cluster are polypeptides located on a particular branch on the phylogenetic tree from a common ancestor. Those skilled in the art will appreciate software programs that facilitate 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), which can, for example, use the G-INS-I algorithm; evolutionary modeling software for identifying suitable evolutionary models, 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); and phylogenetic tree assembly software, such as PhyML (v3.3.20190909) (Guindon S. et al., 2010, Systematic Biology, 59(3):307-21, 2010) and RAxML (Stamakis, A. Bioinformatics, Vol. 30, No. 9, May 2014, pp. 1312-1313, doi.org / 10.1093 / bioinformatics / btu033). An exemplary phylogenetic tree of PmSLP polypeptide sequences showing exemplary phylogenetic clusters PmSLP-1, PmSLP-2, PmSLP-3, PmSLP-4.1, and PmSLP-4.2 includes Figure 2The phylogenetic tree shown in . An exemplary phylogenetic cluster PmSLP-1 includes, for example, at least a PmSLP polypeptide sequence having SEQ.ID NO:2. An exemplary phylogenetic cluster PmSLP-2 includes, for example, at least a PmSLP polypeptide sequence having SEQ.ID NO:4. An exemplary phylogenetic cluster PmSLP-3 includes, for example, at least a PmSLP polypeptide sequence having SEQ.ID NO:6. An exemplary phylogenetic cluster PmSLP-4.1 includes, for example, at least a PmSLP polypeptide sequence having SEQ.ID NO:8. An exemplary phylogenetic cluster PmSLP-4.2 includes, for example, at least a PmSLP polypeptide sequence having SEQ.ID NO:10.

[0177] As used herein, the term "animal" refers to all species belonging to the kingdom Animalia, excluding humans.

[0178] As used herein, the term "avian" refers to any animal species belonging to the class Aves, including, for example, poultry such as chickens, turkeys, geese, ducks, and quail.

[0179] As used herein, the term "bovine" refers to any animal species belonging to the subfamily Bovinae, including, for example, cows, oxen, bulls, bison, and buffalo.

[0180] As used herein, the term "suc" refers to any animal species belonging to the family Suidae, including, for example, pigs, hogs, and boars.

[0181] As used herein, the term "ruminant" refers to a herbivorous mammal capable of digesting plant material by a microbial fermentation process, the mammal having a digestive system including a multi-compartmental stomach system, typically including the rumen, reticulum, omasum, and abomasum. Ruminants include cows, cattle, bulls, goats, sheep, bison, and buffalo.

[0182] As used herein, the term "food-producing animals" refers to animals that are fed and raised by humans to produce human food. Food-producing animals include, but are not limited to, ruminant species, bovine species, porcine species, and avian species.

[0183] As used herein, the term "Pasteurella multocida" or "Pasteurella multocida" refers to any bacteria belonging to a taxonomically classified bacterial species and includes any subspecies thereof, including Pasteurella multocida subspecies, Pasteurella multocida subspecies, and Pasteurella multocida subspecies, and also includes any Pasteurella multocida strain, variant, serogroup (including serogroup A, B, D, E and F groups), serotype (including serotype 1-16 types) or genotype. It is noteworthy that Pasteurella multocida strains can be divided according to serogroups and serotypes. Thus, for example, a strain called Pasteurella multocida A:3 represents a Pasteurella multocida strain belonging to serogroup A group and serotype 3 type.

[0184] As used herein, the term "effective amount" refers to an amount of an active agent or veterinary pharmaceutical preparation (including veterinary vaccine preparation) sufficient to induce a desired biological or therapeutic effect (including a prophylactic effect). Such an effect may include an effect related to a sign, symptom or cause of a condition or disease, or any other desired change in a biological system. The effective amount may vary depending on, for example, the health of the animal being treated, the stage of injury, the stage of the condition or disease, the timing of administration, the mode of administration, the age of the animal, the size of the animal, etc., all of which can be determined by those skilled in the art.

[0185] As used herein, the term "immunologically equivalent" means that when a molecule is administered to a subject animal, the molecule is capable of eliciting a humoral immune response, i.e., producing natural polyclonal antibodies, wherein the binding specificity to the natural polyclonal antibodies is comparable to the specificity 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, will elicit a humoral immune response, i.e., produce natural antibodies with specificity to the PmSLP polypeptide, wherein the specificity is comparable to the binding specificity of the natural antibodies to the PmSLP polypeptide obtained when the reference full-length PmSLP is administered to the animal. An immunologically equivalent portion of a full-length PmSLP polypeptide may be of varying lengths and may, for example, include a polypeptide comprising or consisting of at least 10, at least or at most 15, at least or at most 20, at least or at most 30, at least or at most 50, or at least or at most 60 consecutive amino acid residues that are identical to a portion of a PmSLP polypeptide. In addition, immunologically equivalent portions of full-length PmSLP polypeptides 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. To compare the binding specificity between a reference molecule and an immunologically equivalent molecule, a radioimmunoassay (RIA) can be used and the extent of binding can 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.

[0186] As used herein, the term "cross protection" refers to the ability of a vaccine formulation to provide protection against infection by multiple pathogenic microbial organisms (e.g., 2, 3, 4, 5 or more strains), 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. In order to assess cross protection, a vaccine formulation comprising an immunogenic substance (e.g., PmSLP protein) obtained from a selected strain of a microbial organism can be used to immunize a test animal. The test animal can then be exposed to (challenged by) another strain of a pathogenic microbial organism, and the immune response and development of disease symptoms in the animal can be assessed. If the infection results in an improved immune response, or causes the disease symptoms of the infected animal to be milder or absent than the disease symptoms of an unvaccinated animal exposed to the same infection, the vaccine formulation can be considered to have a cross-protective effect.

[0187] As used herein, the term "veterinary pharmaceutically acceptable" refers to materials, including carriers, diluents or adjuvants, that are compatible with other materials in veterinary pharmaceutical preparations, including veterinary vaccine preparations, and are suitable for use in contact with animals without excessive toxicity, allergic response, irritation or other adverse reaction within the scope of sound medical judgment, and are commensurate with a reasonable risk / benefit ratio.

[0188] As used herein, the terms "treating" and "treatment" and the like are intended to mean obtaining a desired physiological, pharmacological or biological effect. The effect may result in prevention (i.e., prophylactic treatment), inhibition, improvement, attenuation, reversal of signs, symptoms or causes of the condition or disease attributable to the condition or disease. Clinical evidence of treatment may vary depending on the condition or disease, the animal, and the selected treatment. In the case of therapeutic indications, physiological effects may include, for example, improvement in breathing capacity or lung function, reduced bleeding, reduced mucous nasal or oral secretions, improved rectal temperature, improved animal behavior, reduced coughing, or reduced lung lesions.

[0189] As used herein, the term "respiratory disease" refers to the generally accepted veterinary definition of respiratory disease and includes any disease involving the invasion and colonization of the respiratory tract (including the upper and lower respiratory tracts of an animal) by Pasteurella multocida bacteria and includes, for example, BRD and HS.

[0190] As used herein, the term "bovine respiratory disease" or "BRD" refers to the generally accepted veterinary definition of bovine respiratory disease, and generally includes respiratory disease states in bovines caused by infection with the pathogenic Pasteurella multocida.

[0191] As used herein, the term "hemorrhagic septicemia" or "HS" refers to the generally accepted veterinary definition of hemorrhagic septicemia and generally includes respiratory disease states in bovines caused by infection with the pathogenic Pasteurella multocida.

[0192] As used herein, the term "porcine atrophic rhinitis" or "PAR" refers to the generally accepted veterinary definition of porcine atrophic rhinitis and generally encompasses a respiratory disease state in swine caused by infection with the pathogenic Pasteurella multocida.

[0193] As used herein, the term "pneumonic pasteurellosis" refers to the generally accepted veterinary definition of pneumonic pasteurellosis and generally includes respiratory disease states in swine caused by infection with the pathogenic Pasteurella multocida.

[0194] As used herein, the term "fowl cholera" refers to the generally accepted veterinary definition of avian cholera and generally includes a respiratory disease state in avian animals caused by infection with the pathogenic Pasteurella multocida.

[0195] As used herein, the terms "vaccine", "vaccine formulation", "veterinary vaccine" and "veterinary vaccine formulation" refer to veterinary pharmaceutically acceptable formulations that can be administered to animals to induce a humoral immune response (including eliciting a soluble antibody response) and / or a cell-mediated immune response (including eliciting a cytotoxic T lymphocyte (CTL) response).

[0196] As used herein, the term "homologous protection" refers to the protection conferred by a vaccine against infection by a strain of a microbial species, wherein the infecting strain has an antigen, such as a PmSLP protein antigen, that belongs to the same phylogenetic cluster as the antigen, such as a PmSLP protein antigen or an immunologically equivalent portion thereof, included in the vaccine formulation.

[0197] As used herein, the term "heterologous protection" refers to protection conferred by a vaccine against infection by a strain of a microbial species, wherein the infecting strain has an antigen, such as a PmSLP protein antigen, that belongs to a different phylogenetic cluster than the antigen, such as a PmSLP protein antigen or an immunologically equivalent portion thereof, included in the vaccine formulation.

[0198] The terms "substantially pure" and "isolated" are used interchangeably herein to describe a compound, such as a polypeptide, that has been separated from its naturally associated components. Typically, a compound is substantially pure when at least 60%, more preferably at least 75%, more preferably at least 90%, 95%, 96%, 97% or 98%, and most preferably at least 99% (by volume, wet weight or dry weight, or in mole percent or mole fraction) of the total material in a sample is the compound of interest. Purity can be measured by any appropriate method, such as in the case of a polypeptide, by chromatography, gel electrophoresis or HPLC analysis.

[0199] General implementation plan

[0200] As mentioned above, the present disclosure relates to veterinary vaccine formulations. In general, the methods and compositions provided herein can be used to prevent or ameliorate Pasteurella multocida infections in food-producing animals, including ruminant species, bovine species, swine species, and avian species. In this regard, in particular, the methods and compositions of the present disclosure can be used to treat bovine respiratory disease or hemorrhagic septicemia in bovine species; pneumonic pasteurellosis or progressive atrophic rhinitis in pigs; or fowl cholera in poultry. The veterinary vaccine formulations of the present disclosure can provide long-term, effective protection against Pasteurella multocida infections.

[0201] The inventors of the present case have found that the veterinary vaccine formulations of the present disclosure have cross-protective effects and can provide protection for food-producing animals against infection by multiple strains of Pasteurella multocida. Surprisingly, the veterinary vaccine formulations of the present disclosure can exert cross-protective effects using a single immunogenic active agent.

[0202] Furthermore, the vaccine formulations of the present disclosure can provide long-term protection, wherein antibodies to the immunizing agent can be detected in the serum of the subject food producing animals for at least 26 weeks from administration of the vaccine formulation, for example.

[0203] Furthermore, the vaccine formulations of the present disclosure involve the use of polypeptide-based immunogenic active agents, and therefore, the compositions and methods of the present disclosure do not involve the use of attenuated live microbial species, thereby avoiding the infection risks associated with the use of live vaccines.

[0204] In addition, the vaccine formulations of the present disclosure can limit the administration of antibiotics to food-producing animals and thereby limit the development of antibiotic-resistant strains of microorganisms. In addition, some consumers prefer to eat food made from animals that have not been treated with antibiotics.

[0205] In addition, the polypeptide-based immunogenic active agent included in the vaccine formulation of the present disclosure can be prepared using a convenient recombinant production system, and the immunogenic active agent can be stably stored.

[0206] Specifically, the inventors of the present invention have discovered that proteins selected from a class of proteins known as PmSLP proteins can be used as immunogenic active agents in veterinary vaccine formulations to prevent or ameliorate Pasteurella multocida infections in food-producing animals. The PmSLP protein can be selected and obtained from a strain of Pasteurella multocida. The Pasteurella multocida strain from which the PmSLP protein is selected is preferably a Pasteurella multocida strain that belongs to the same phylogenetic cluster as the Pasteurella multocida strain causing the infection in the food-producing animal. Therefore, the PmSLP protein does not need to be selected from the same Pasteurella multocida strain as the Pasteurella multocida infection strain, however, the prerequisite is that the other strain belongs to the same phylogenetic cluster.

[0207] Described below are illustrative embodiments of the compositions and methods of the present disclosure.

[0208] Therefore, in at least one aspect, and in at least one embodiment, the present disclosure provides a veterinary vaccine formulation for preventing or ameliorating Pasteurella multocida infection in food-producing animals susceptible to Pasteurella multocida infection, the vaccine formulation comprising an effective amount of PmSLP protein or an immunogenic equivalent portion thereof.

[0209] In another aspect, and in at least one other embodiment, the present disclosure also provides a method for treating a food-producing animal susceptible to infection with Pasteurella multocida, the method comprising administering to the food-producing animal a veterinary vaccine formulation comprising a PmSLP protein or an immunogenic equivalent portion thereof, wherein the vaccine formulation is administered in an effective amount to prevent or ameliorate Pasteurella multocida infection.

[0210] In general, according to one aspect, veterinary vaccine formulations comprising a selected PmSLP protein or an immunogenic equivalent portion thereof are prepared or obtained. These formulations are administered to food-producing animals in need thereof in an effective amount. Therefore, suitable formulations comprising a selected PmSLP protein or an immunogenic equivalent portion thereof, and methods for preparing a selected PmSLP protein or an immunogenic equivalent portion thereof, will be described below. Thereafter, veterinary vaccine formulations comprising a selected PmSLP protein or an immunogenic equivalent portion thereof, and methods for preparing and administering veterinary vaccine formulations to food-producing animals in need thereof will be described.

[0211] Thus, initially, considering PmSLP protein preparations, in one aspect, a preparation containing a selected PmSLP protein or an immunogenic equivalent portion thereof can be prepared by biosynthesis using a host cell system. In this regard, an isolated nucleic acid encoding an amino acid sequence corresponding to a PmSLP protein or an immunogenic equivalent portion thereof can be introduced into a host cell and expressed therein.

[0212] In general, any PmSLP protein can be used according to this article. The PmSLP protein can be obtained from Pasteurella multocida bacteria, including any strain containing PmSLP thereof, including any Pasteurella multocida strain containing PmSLP belonging to any serogroup or serotype. In this regard, it is worth noting that, as known to those skilled in the art, Pasteurella multocida strains can be classified as belonging to different serogroups and / or serotypes. In this regard, serogroups include serogroups A, B, D, E and F groups, and serotypes include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and 16 types. When referring to different Pasteurella multocida strains, serogroups and serotypes can be mentioned accordingly. For example, Pasteurella multocida strain A:3 can be classified as belonging to serogroup A group and serotype 3 type, Pasteurella multocida strain E:2 can be classified as belonging to serogroup E group and serotype 2 type, etc. Thus, according to the present invention, the PmSLP protein may be a PmSLP protein obtained from a Pasteurella multocida strain selected from serogroups A, B, D, E and F. In addition, the PmSLP protein may be a PmSLP protein obtained from a Pasteurella multocida strain selected from serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and 16. Thus, it should be understood that according to the present invention, the PmSLP protein may be selected from the PmSLP protein obtainable or obtained from a Pasteurella multocida strain belonging to any serogroup or any serotype or any combination thereof.

[0213] According to one aspect, in an exemplary embodiment, a nucleic acid sequence encoding a PmSLP protein can be selected, wherein such nucleic acid includes 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: 61, SEQ.ID NO: 62, SEQ.ID NO: 63, SEQ.ID NO: 64, SEQ.ID NO: 65, SEQ.ID NO: 66, SEQ.ID NO: 67, SEQ.ID NO: 68, SEQ.ID NO: 69, SEQ.ID NO: 70, SEQ.ID NO: 71, SEQ.ID NO: 72, SEQ.ID NO: 73, SEQ.ID NO: 74, SEQ.ID NO: 75 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.IDNO: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. Selected example 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, the protein is a polypeptide encoded by the following sequences, respectively: 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:16, SEQ.ID NO:17, SEQ.ID NO:18, SEQ.ID NO:19, SEQ.ID NO:20, SEQ.ID NO:21, SEQ.ID NO:22, SEQ.ID NO:23, SEQ.ID NO:24, SEQ.ID NO:25, SEQ.ID NO:26, SEQ.ID NO:27, SEQ.ID NO:28, SEQ.ID NO:29, SEQ.ID NO:30 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.IDNO:54, SEQ.ID NO:56, SEQ.ID NO:58, SEQ.ID NO:60, SEQ.ID NO:62, SEQ.ID NO:64, SEQ.IDNO: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.IDNO:90, SEQ.IDNO:92, SEQ.IDNO:94 or SEQ.IDNO:96. .

[0214] In some embodiments, the PmSLP protein may be a protein expressed by a nucleic acid sequence selected from the group consisting of:

[0215] (a) SEQ.ID NO:1, SEQ.ID NO:3, SEQ.ID NO:5, SEQ.ID.NO:7, SEQ.ID NO:9, SEQ.IDNO: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.IDNO:54, SEQ.ID NO:56, SEQ.ID NO:58, SEQ.ID NO:60, SEQ.ID NO:62, SEQ.ID NO:64, SEQ.IDNO:66, SEQ.ID NO:68, SEQ.ID NO:70, SEQ.ID NO:72, SEQ.ID NO:74, SEQ.ID NO:76, SEQ.IDNO:78, SEQ.ID NO:80, SEQ.ID NO:82, SEQ.ID NO:84, SEQ.ID NO:86, SEQ.ID NO:88, SEQ.IDNO:90, SEQ.ID NO:92, SEQ.ID NO:94 or SEQ.ID NO:96;

[0216] (b) a nucleic acid sequence substantially identical to any of the nucleic acid sequences in (a);

[0217] (c) a nucleic acid sequence substantially identical to any of the nucleic acid sequences in (a) except for the degeneracy of the genetic code;

[0218] (d) a nucleic acid sequence complementary to any one of the nucleic acid sequences in (a);

[0219] (e) a nucleic acid sequence encoding a polypeptide having any of the amino acid sequences shown in the following, or an immunogenic equivalent portion thereof: 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:64, SEQ.ID NO:65 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;

[0220] (f) a nucleic acid sequence encoding a functional variant of any one of the amino acid sequences shown below, or an immunogenic equivalent thereof: 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:64, SEQ.ID NO:65 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

[0221] (g) A nucleic acid sequence that hybridizes to any one of the nucleic acid sequences shown in (a), (b), (c), (d), (e) or (f) under stringent conditions.

[0222] The PmSLP proteins that can be used according to the present invention include naturally occurring PmSLP proteins, as well as PmSLP proteins that can be modified. In this regard, modifications include modifications to the amino acid sequence of the PmSLP polypeptide, including, for example, modifications to one or more specific single amino acids, which can be referred to as "site-directed mutations", such as PmSLP proteins having SEQ.ID NO: 65 and SEQ.ID NO: 67.

[0223] Other modifications that may be made to the PmSLP protein include modifications that reduce the binding of the PmSLP protein to a natural host protein (i.e., a protein in a food-producing animal susceptible to infection with Pasteurella multocida). Such reduced binding can generally be assessed by evaluating the affinity of the PmSLP protein for the natural host protein. Affinity can be quantitatively assessed by experimentally determining the dissociation constant (Kd) between the PmSLP protein and the natural host protein. Thus, for example, in this regard, the Kd between a natural PmSLP protein and a natural host protein (e.g., the Kd between PmSLP-1 and bovine complement factor I) can be compared to the Kd between a modified PmSLP protein and the same natural host protein. In general, the higher the Kd value, the weaker the affinity of the PmSLP protein for the natural host protein. In some embodiments, the Kd between the modified PmSLP and the native host protein can be at least 2x, at least 5x, at least 10x, at least 25x, at least 50x, at least 100x, at least 250x, or at least 500x higher than the Kd between the native, unmodified PmSLP and the native host protein. 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 bio-layer interferometry (see further, for example, 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).

[0224] In this regard, examples of PmSLP polypeptides that can be used 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, the PmSLP polypeptides having SEQ.ID NO: 65 and SEQ.ID NO: 67 encode a PmSLP-1 polypeptide (SEQ.ID NO: 2) comprising a V214D mutation (valine amino acid residue 214 is substituted with an aspartic acid amino acid residue) and an E240A mutation (glutamic acid amino acid residue 240 is substituted with an alanine amino acid residue). The Kd between the modified PmSLP having SEQ.ID NO: 65 and SEQ.ID NO: 67, respectively, and the natural host protein was determined to be less than 2,000 nM, wherein the Kd between the wild-type PmSLP-1 protein (SEQ.ID NO: 2) and the natural host protein is 30 nM. +10 nM. Therefore, the Kd between these two exemplary modified PmSLP proteins and the native host protein is at least 500x higher than the Kd between the native unmodified PmSLP and the native host protein.

[0225] According to one aspect, suitable nucleic acid sequences include nucleic acid sequences encoding immunogenic equivalent portions of PmSLP polypeptides, in particular, portions of PmSLP polypeptides that are at least immunologically equivalent to full-length PmSLP polypeptides, including those shown 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:62, SEQ.ID NO:63, SEQ.ID NO:64, SEQ.ID NO:65, SEQ.ID NO:66, SEQ.ID NO:67, SEQ.ID NO:68, SEQ.ID NO:69, SEQ.ID NO:70, SEQ.ID NO:71, SEQ.ID NO:72, SEQ.ID NO:73, SEQ.ID NO:74, SEQ.ID NO:75 NO:61, SEQ.ID NO:63, SEQ.ID NO:65, SEQ.ID NO:67, SEQ.ID NO:69, SEQ.IDNO:71, SEQ.ID NO:73, SEQ.ID NO:75, SEQ.ID NO:77, SEQ.ID NO:79, SEQ.ID NO:81, SEQ.IDNO: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.Thus, other exemplary nucleic acid sequences that can be used in selected embodiments include nucleic acid sequences encoding an amino acid sequence that is at least immunologically equivalent to a PmSLP polypeptide, the amino acid sequence corresponding to at least 10 contiguous amino acids and up to 150 amino acids of a full-length PmSLP polypeptide, including 10, 20, 30, 40, 50, 55, 60, 70, 80, 90, 100, 110, 120, 130, 140 or 150 amino acids, including the PmSLP polypeptide 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:29, SEQ.ID.NO:30, SEQ.ID.NO:31, SEQ.ID.NO:32, SEQ.ID.NO:33, SEQ.ID.NO:34, SEQ.ID.NO:35 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.IDNO:77, SEQ.ID NO:79, SEQ.ID NO:81, SEQ.ID NO:83, SEQ.ID NO:85, SEQ.ID NO:87, SEQ.IDNO:89, SEQ.ID NO:91, SEQ.ID NO:93, SEQ.ID NO:95 or SEQ.ID NO:97.

[0226] In another embodiment, the PmSLP protein or an immunogenic equivalent portion thereof can be linked to another polypeptide to form a hybrid polypeptide, including, for example, a carrier protein or extension for facilitating detection or purification, such as a polyhistidine extension (HIS tag) or a FLAG tag peptide extension, or another immunogenic polypeptide, and also includes another PmSLP or an immunogenic equivalent portion thereof.

[0227] Other examples of hybrid polypeptides are hybrid polypeptides comprising a first and a second PmSLP polypeptide, such as PmSLP-1 and PmSLP-3, and PmSLP-1 and PmSLP-2, as shown 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 can be encoded by the nucleic acid sequences shown in SEQ.ID NO:50, SEQ.ID NO:52, SEQ.ID NO:54 and SEQ.ID NO:55, respectively.

[0228] Still other examples of hybrid polypeptides are hybrid polypeptides comprising the first, second and third PmSLP polypeptides, such as PmSLP-1, PmSLP-2 and PmSLP-3, and PmSLP-1, PmSLP-2 and PmSLP-4, as shown in the following: SEQ.ID NO:53 (PmSLP-1, PmSLP-2 and PmSLP-3 hybrid polypeptide) and SEQ.ID NO:55 (PmSLP-1, PmSLP-2 and PmSLP-3 hybrid polypeptide), which can be encoded by the nucleic acid sequences shown in the following: SEQ.ID.NO:52 and SEQ.ID NO:54, respectively.

[0229] As known to those skilled in the art, expression of nucleic acids in host cells can be achieved by providing one or more nucleic acids capable of controlling expression in host cells and operably linking the one or more nucleic acids capable of controlling expression in host cells to the nucleic acids desired to be expressed, thereby producing proteins through biosynthesis. Such operable linking of nucleic acids that control expression generally involves linking the nucleic acids capable of controlling expression in host cells to the nucleic acids desired to be expressed (i.e., in the context of the present disclosure, the PmSLP protein) in the 5' to 3' expression direction. Nucleic acid sequences capable of controlling expression in host cells that can be used herein include any transcription promoter capable of controlling expression of a polypeptide in a host cell. Generally speaking, when bacterial host cells are selected, promoters obtained from bacterial cells will be used, while when yeast host cells are selected, yeast promoters will be used; when plant cells are selected, plant promoters will be used, and so on. The nucleic acids obtained comprising a promoter and a nucleic acid expressing a PmSLP protein are generally chimeric nucleic acids. Other nucleic acid elements capable of controlling expression in host cells include transcription terminators, enhancers, and the like, all of which can be included in the chimeric nucleic acid sequences of the present disclosure.

[0230] According to the disclosure, the chimeric nucleic acid sequence can be integrated into the recombinant expression vector to ensure good expression in the host cell, wherein 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 connected to the genetic elements required for expression in the cell. As described, this genetic element can include a transcription promoter, a terminator, and an enhancer, etc. Other genetic elements that may be included in the expression vector are one or more nucleic acid sequences encoding marker genes, and one or more replication origins. In some embodiments, the expression vector can be freely replicated in the host cell. In other embodiments, the chimeric nucleic acid can be integrated into the genomic DNA of the host cell. In some embodiments, the expression vector may also include a vector or part thereof that is integrated into the genetic elements required for the host cell genome, for example, if a plant host cell is used, the T-DNA left border sequence and the right border sequence that are conducive to integration into the nuclear genome of the plant may be included in the vector.

[0231] The marker gene that can be used according to the present disclosure includes all genes that can distinguish transformed cells from non-transformed cells, including all selectable and screenable marker genes. The marker gene can be a resistance marker, such as an antibiotic resistance marker for, for example, kanamycin, chloramphenicol, methotrexate or ampicillin. In other cases, the marker gene can be a gene that allows the cell to produce essential nutrients such as amino acids.

[0232] Therefore, in one aspect, the present disclosure provides, in an exemplary embodiment, an expression vector comprising:

[0233] (a) a nucleic acid sequence encoding a PmSLP protein or an immunogenic equivalent portion thereof; and

[0234] (b) a nucleic acid sequence capable of controlling the expression of a nucleic acid sequence encoding the PmSLP protein or an immunogenic equivalent portion thereof in a host cell.

[0235] In an exemplary embodiment, the 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 immunogenic equivalent portion thereof.

[0236] Turning now to host cells, it is first noted that any host cell that expresses a chimeric nucleic acid when cultured can be selected and used in accordance with the present disclosure. In this regard, suitable host cells include, for example, microbial cells, such as bacterial cells, yeast cells, and algae cells or plant cells. There are a variety of techniques and methods for manipulating host cells to introduce nucleic acid sequences into cells and achieve expression, and these techniques and methods 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; gene gun method; and glass bead-based methods. As is known to those skilled in the art, depending on the selected host cell, the method of introducing nucleic acid material into the host cell may vary, and the method can be optimized for the uptake of nucleic acid material by the host cell, such as by comparing the uptake of 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 Edition. It should be noted that chimeric nucleic acids are non-naturally occurring chimeric nucleic acid sequences and can be considered to be heterologous to the host cell.

[0237] An exemplary host cell suitable for use is an Escherichia coli cell. The preparation of Escherichia coli vectors can be completed by using commonly known techniques, such as restriction digestion, connection, gel electrophoresis, DNA sequencing, polymerase chain reaction (PCR) and other methods. There are a variety of cloning vectors that can be used to perform the necessary steps required for preparing recombinant expression vectors. There are vectors such as pBR322, pUC series vectors, M13 mp series vectors, and pBluescript in the vectors with a replication system that works in Escherichia coli. Promoter sequences suitable for use in Escherichia coli include, for example, T7 promoters, T5 promoters, tryptophan (trp) promoters, lactose (lac) promoters, tryptophan / lactose (tac) promoters, lipoprotein (Ipp) promoters, and lambda phage PL promoters. Typically, cloning vectors contain markers, such as antibiotic resistance markers, such as ampicillin or kanamycin resistance markers, so that transformed cells can be selected. Nucleic acid sequences can be introduced into these vectors, and vectors can be introduced into Escherichia coli by preparing competent cells, electroporation, or using other methods well known to those skilled in the art. E. coli can be grown in an appropriate medium, such as Luria-Broth medium, and harvested. After harvesting and lysing the cells, the recombinant expression vector can be easily recovered from the cells.

[0238] Another exemplary host cell suitable for use is a yeast cell. An exemplary yeast host cell that can be used is a yeast cell belonging to Candida, Kluyverom yces, Saccharomyces, Schizosaccharomyces, Pichia, Hansenula and Yarrowia. In specific exemplary embodiments, the yeast cell can be a Saccharomyces cerevisiae cell, a Yarrowia lipolytica cell or a Pichia pastoris cell.

[0239] There are many 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 2 μm plasmids. These vectors are known in the art and are described, for example, in Cregg et al., Mol Biotechnol. (2000) 16 (1): 23-52. Promoter sequences suitable for yeast host cells are also known and 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 promoters of glycolytic enzymes, such as triosephosphate isomerase (TPI), phosphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH or GAP) and variants thereof, lactase (LAC) and galactosidase (GAL), Pichia pastoris glucose-6-phosphate isomerase promoter (PPGI), 3-phosphoglycerate kinase promoter (PPGK), glyceraldehyde phosphate dehydrogenase promoter (PGAP), translation elongation factor promoter (PTEF), Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase / 3-phosphoglyceraldehyde dehydrogenase (ADH1, ADH2 / GAP), Saccharomyces cerevisiae triosephosphate isomerase (TPI), Saccharomyces cerevisiae metallothionein (CUP1), Saccharomyces cerevisiae 3-phosphoglycerate kinase (PGK) and maltase gene promoter (MAL). Marker genes suitable for use in yeast host cells are also known in the art. Therefore, antibiotic resistance markers, such as ampicillin resistance markers, and marker genes that provide essential nutrient genetic functions, such as leucine (LEU2), tryptophan (TRP1 and TRP2), uracil (URA3, URA5, URA6), histidine (HIS3), etc., can be used in yeast. Methods for introducing vectors into yeast host cells can be found, for example, in S. Kawai et al., 2010, Bioeng. Bugs 1(6): 395-403.

[0240] Other exemplary host cells that can be used herein are plant cells. Methods for introducing nucleic acids into plant cells are known to those skilled in the art. Methods for plant cell transformation mediated by Agrobacterium are described in, for example, Microbiol.Mol.Biol.Rev., 2003, 67(1): 16-37 by Gelvin S., and physical transformation methods for plant cells are described in Rivera AL et al., 2012, Phys.Life Rev.9(3): 308-345. Plant selective 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 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 beta-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) and the parsley ubiquitin promoter (Kawalleck, P. et al., 1993, Plant Mol. Biol. 21:673-684); and organ-specific promoters, such as seed-specific promoters, for example, the phaseolin promoter (Sengupta-Gopalan et al., 1985, Proc. Natl. Acad. Sci. USA 82:3320-3324) or the oleosin promoter (U.S. Pat. No. 5,792,922).

[0241] In addition, guidance on the preparation of expression vectors and their introduction into host cells (including E. coli cells, yeast cells and other host cells) can be found in, for example: Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press, 2012, 4th edition.

[0242] Thus, in another aspect, the present disclosure provides an exemplary embodiment, namely, a host cell comprising a chimeric nucleic acid comprising:

[0243] (a) a nucleic acid sequence encoding a PmSLP protein or an immunogenic equivalent portion thereof; and

[0244] (b) a nucleic acid sequence capable of controlling the expression of a nucleic acid sequence encoding the PmSLP protein or an immunogenic equivalent portion thereof in a host cell.

[0245] In an exemplary embodiment, the 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 immunogenic equivalent portion thereof.

[0246] Therefore, in brief summary, a host cell comprising a chimeric nucleic acid can be prepared according to the present disclosure, wherein the chimeric nucleic acid comprises (i) a nucleic acid sequence encoding a PmSLP protein or an immunogenic equivalent portion thereof; and (ii) a nucleic acid sequence capable of controlling the expression of the nucleic acid sequence encoding a PmSLP protein or an immunogenic equivalent portion thereof in the host cell.

[0247] Accordingly, the host cell is grown and propagated and the chimeric nucleic acid is expressed. The expression of the chimeric nucleic acid causes the biosynthesis of the PmSLP protein or its immunogenic equivalent in the host cell. It should be readily understood by those skilled in the art that the growth medium and growth conditions may vary depending on the selected host cell. The growth medium typically contains a carbon source; one or more nitrogen sources; essential salts, including potassium salts, sodium salts, magnesium salts, phosphates and sulfates; trace metals; water-soluble vitamins; and processing aids, including but not limited to defoamers, protease inhibitors, stabilizers, ligands and inducers. Typical carbon sources are, for example, monosaccharides or disaccharides. Typical nitrogen sources are, for example, ammonia, urea, amino acids, yeast extract, corn steep liquor and completely or partially hydrolyzed proteins. Typical trace metals are Fe, Zn, Mn, Cu, Mo and H3BO3. Typical water-soluble vitamins are, for example, biotin, pantothenic acid, niacin, thiamine, p-aminobenzoic acid, choline, pyridoxine, folic acid, riboflavin and ascorbic acid. In addition, specific exemplary culture media include liquid culture media for yeast cell and bacterial cell growth, including Luria-Bertani (LB) broth for bacterial cell culture, and yeast extract peptone dextrose (YEPD or YPD) for yeast cell culture. For more culture media and growth conditions, see Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press, 2012, 4th edition.

[0248] After the host cells produce the PmSLP protein or its immunogenic equivalent portion, the PmSLP protein or its immunogenic equivalent portion can be recovered from the host cells and separated from other components such as cell debris or culture medium components. Separation techniques are well 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, etc. Further general guidance on protein purification can be found, for example, in Cutler, P. Protein Purification Protocols, Humana Press, 2004, 2nd edition. In this way, a substantially pure preparation of the PmSLP protein or its immunogenic equivalent portion can be obtained. The recovered PmSLP protein can be obtained in a substantially pure form, for example, a preparation 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) of PmSLP protein or an immunogenic equivalent portion thereof can be obtained.

[0249] In addition, it is noteworthy that recombinant production of PmSLP protein or its immunogenic equivalent portion in a host cell system allows the production of PmSLP protein or its immunogenic equivalent portion in a manner such that the PmSLP protein or its immunogenic equivalent portion is substantially free of other Pasteurella multocida constituent materials that are naturally associated with the PmSLP protein, such as other Pasteurella multocida proteins, membrane materials, lipopolysaccharide, etc.

[0250] It is worth noting that in some embodiments, the cells can secrete a portion of the produced PmSLP protein or its immunogenic equivalent portion in the cell growth medium, so a portion of the produced PmSLP protein or its immunogenic equivalent portion can be recovered from the cells, and another portion of the PmSLP protein or its immunogenic equivalent portion can be recovered from the growth medium.

[0251] It should also be noted that the veterinary vaccine formulations of the present disclosure may contain a substantially pure form of a PmSLP protein or an immunogenic equivalent portion thereof. Thus, according to the present invention, a substantially pure PmSLP protein or an immunogenic equivalent portion thereof may be obtained and used to prepare a veterinary vaccine formulation. Thus, for example, in some embodiments, the PmSLP protein may be substantially free of other host cell constituent materials, such as host cell proteins, membrane materials, lipopolysaccharides, and the like. In other embodiments, a relatively crude preparation comprising a PmSLP protein or an immunogenic equivalent portion thereof may be obtained and used to prepare a vaccine formulation. Thus, for example, in such an embodiment, a host cell, a host cell lysate, or a host cell fraction comprising a PmSLP protein or an immunogenic equivalent portion thereof may be used to prepare a vaccine formulation.

[0252] It is noteworthy that any PmSLP or its immunogenic equivalent portion can be used to formulate the veterinary vaccine formulation of the present disclosure, including a PmSLP protein having any of the amino acid sequences shown below: 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:56, SEQ.ID NO:57, SEQ.ID NO:58, SEQ.ID NO:59, SEQ.ID NO:60, SEQ.ID NO:61, SEQ.ID NO:62, SEQ.ID NO:63, SEQ.ID NO:64, SEQ.ID NO:65, SEQ.ID NO:66, SEQ.ID NO:67, SEQ.ID NO:68, SEQ.ID NO:69, SEQ.ID NO:70, SEQ.ID NO:71, SEQ.ID NO:72, SEQ.ID NO:73, SEQ.ID NO:74, SEQ.ID NO:75 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 immunogenic equivalent thereof.

[0253] Furthermore, in one aspect of the present disclosure, in preparing a vaccine formulation, when selecting a PmSLP protein, it is preferred to consider Pasteurella multocida strains that cause infection in food-producing animals.

[0254] Specifically, when preparing a vaccine formulation to treat an animal infected with a Pasteurella multocida strain having a PmSLP protein belonging to a particular phylogenetic cluster, the vaccine is preferably prepared by selecting a PmSLP polypeptide belonging to the same phylogenetic cluster, including the same PmSLP polypeptide or another PmSLP polypeptide belonging to the same phylogenetic cluster, and including the PmSLP polypeptide thus selected in the vaccine formulation. Thus, for example, see Figure 2 When preparing a vaccine preparation, preferably, when treating an animal infected with a Pasteurella 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. Thus, for example, if the animal is infected with a Pasteurella multocida strain having a PmSLP polypeptide belonging to the phylogenetic cluster PmSLP-1 (e.g., SEQ.ID NO: 2), the vaccine preparation is preferably prepared by selecting a PmSLP polypeptide belonging to the phylogenetic cluster PmSLP-1 (e.g., SEQ.ID NO: 2) or an immunogenic equivalent portion thereof, or another PmSLP polypeptide belonging to the phylogenetic cluster PmSLP-1, and including it in the vaccine preparation; and by way of another example, if the animal is infected with a Pasteurella multocida strain having a PmSLP protein belonging to the phylogenetic cluster PmSLP-3 (e.g., SEQ.ID NO: 6), the vaccine preparation is preferably prepared by selecting a PmSLP polypeptide belonging to the phylogenetic cluster PmSLP-3 (e.g., SEQ.ID NO: 6, e.g., SEQ.ID NO: 6) or an immunogenic equivalent portion thereof, or another PmSLP polypeptide belonging to the phylogenetic cluster PmSLP-3, and including it in the vaccine preparation. The strain from which the PmSLP protein is selected can be the same Pasteurella multocida strain, or another strain different from the Pasteurella multocida infecting strain, however, as long as the other strain belongs to the same phylogenetic cluster. Therefore, the PmSLP protein does not need to be selected from the same Pasteurella multocida strain as the infecting Pasteurella multocida infecting strain, however, as long as the other strain belongs to the same phylogenetic cluster.

[0255] In addition, preferably, when selecting a PmSLP polypeptide, the host and the clinical indication are considered. Specifically, when preparing a vaccine formulation to treat an animal host infected with a strain of Pasteurella multocida that causes a specific indication, the vaccine is preferably prepared by selecting a PmSLP polypeptide from a strain of Pasteurella multocida that is capable of causing the indication in the host, including the same PmSLP polypeptide or another PmSLP polypeptide isolated from a strain of Pasteurella multocida that is capable of causing the indication in the host, and including the PmSLP polypeptide thus selected in the vaccine formulation. Thus, for example, again referring to Figure 2 In the preparation of a vaccine formulation, preferably, to treat an animal infected with a Pasteurella 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 for the preparation of the vaccine, wherein in addition, the PmSLP can be obtained from a Pasteurella multocida strain known to cause the indication to be treated in the host species to be treated. Thus, for example, if a bovine animal is diagnosed with BRD caused by infection with a Pasteurella multocida strain having a PmSLP polypeptide belonging to the phylogenetic cluster PmSLP-2 (e.g., SEQ. ID NO: 4), the vaccine formulation is preferably prepared by selecting a PmSLP polypeptide belonging to the phylogenetic cluster PmSLP-2 (e.g., SEQ. ID NO: 4) or an immunogenic equivalent portion thereof or another PmSLP polypeptide belonging to the phylogenetic cluster PmSLP-2, wherein in addition, the PmSLP can be obtained from a Pasteurella multocida strain known to cause the indication to be treated in the host species to be treated. Figure 2 It is understood from the literature that the PmSLP-2 polypeptide is also preferably obtainable from a Pasteurella multocida strain known to cause BRD in bovine animals, rather than, for example, from porcine animals also infected with a Pasteurella multocida strain containing PmSLP-2.

[0256] In an exemplary embodiment, the food-producing animal may be a ruminant susceptible to infection with a strain of Pasteurella multocida that causes respiratory disease, and the vaccine formulation is prepared to contain at least one Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof, wherein the PmSLP protein is selected from the group of phylogenetic clusters consisting of PmSLP-1, PmSLP-2, PmSLP-3 and PmSLP-4.2, wherein the selected Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof belongs to the same phylogenetic cluster as the PmSLP protein present in the infecting Pasteurella multocida strain.

[0257] In an exemplary embodiment, the food-producing animal can be a bovine susceptible to infection with a strain of Pasteurella multocida that causes BRD, and the vaccine formulation is prepared to contain at least one Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof, wherein the PmSLP protein is selected from the group of phylogenetic clusters consisting of PmSLP-1, PmSLP2 and PmSLP-4.2, wherein the selected Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof belongs to the same phylogenetic cluster as the PmSLP protein present in the infecting Pasteurella multocida strain.

[0258] In an exemplary embodiment, the food-producing animal can be a bovine susceptible to infection with a strain of Pasteurella multocida that causes HS, and the vaccine formulation is prepared to contain at least one Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof, wherein the PmSLP protein is selected from the group of phylogenetic clusters consisting of PmSLP-3, wherein the selected Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof belongs to the same phylogenetic cluster as the PmSLP protein present in the infecting Pasteurella multocida strain.

[0259] In an exemplary embodiment, the food-producing animal may be a porcine animal susceptible to infection with a strain of Pasteurella multocida that causes porcine atrophic rhinitis (PAR), and the vaccine formulation is prepared to contain at least one Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof, wherein the PmSLP protein is selected from the group of phylogenetic clusters consisting of PmSLP-2, PmSLP-4.1 and PmSLP-4.2, wherein the selected Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof belongs to the same phylogenetic cluster as the PmSLP protein present in the infecting Pasteurella multocida strain.

[0260] In an exemplary embodiment, the food-producing animal may be a porcine animal susceptible to infection with a strain of Pasteurella multocida that causes pneumonic pasteurellosis, and the vaccine formulation is prepared to contain at least one Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof, wherein the PmSLP protein is selected from the group of phylogenetic clusters consisting of PmSLP-2, PmSLP-4.1 and PmSLP-4.2, wherein the selected Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof belongs to the same phylogenetic cluster as the PmSLP protein present in the infecting Pasteurella multocida strain.

[0261] In an exemplary embodiment, the food-producing animal may be an avian animal susceptible to infection with a strain of Pasteurella multocida that causes avian cholera, and the vaccine formulation is prepared to contain at least one Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof, wherein the PmSLP protein is selected from the group of phylogenetic clusters consisting of PmSLP-3 and PmSLP-4.2, wherein the selected Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof belongs to the same phylogenetic cluster as the PmSLP protein present in the infecting Pasteurella multocida strain.

[0262] It is noteworthy that the Pasteurella multocida strain can be isolated from infected food production animals and cultured to identify the Pasteurella multocida strain, and the sequence of the PmSLP polypeptide present therein can be determined using techniques known to those of ordinary skill in the art, see, for example, Pasteurellaceae: Biology, Genomics and Molecular Aspects. Publisher: Caister Academic Press Editors: Peter Kuhnert (1) and Henrik Christensen (2). Institute of Veterinary Bacteriology, Vetsuisse Faculty University of Bern, 122, 3001 Bern, Switzerland (1) and Department of Veterinary Pathobiology, Faculty of Life Science, Copenhagen University, Dyrlaegevej 88, 1870 Frederiksberg, Denmark (2).

[0263] Furthermore, in an exemplary embodiment, when preparing a vaccine formulation for treating an animal infected with a Pasteurella multocida strain belonging to a particular serogroup, the vaccine is preferably prepared using a PmSLP polypeptide obtained from a Pasteurella multocida strain belonging to the same serogroup. Thus, for example, when preparing a vaccine formulation, preferably to treat an animal infected with a Pasteurella multocida strain belonging to serogroups A, B, C, D, E or F, a vaccine formulation comprising a PmSLP polypeptide obtained from a Pasteurella multocida strain of the same serogroup is selected for inclusion in the vaccine. Thus, for example, if the animal is infected with a Pasteurella multocida strain of serogroup A, the vaccine formulation is preferably prepared by selecting and including in the formulation a PmSLP polypeptide obtained from a Pasteurella multocida strain of serogroup A, or an immunogenic equivalent portion thereof; and, for another example, if the animal is infected with a Pasteurella multocida strain belonging to serogroup F, the vaccine formulation is preferably prepared by selecting and including in the formulation a PmSLP polypeptide obtained from a Pasteurella multocida strain belonging to serogroup F, or an immunogenic equivalent portion thereof. The strain may be the same strain, or another strain, as long as the other strain belongs to the same serogroup.

[0264] In another preferred embodiment, both phylogenetic clusters and serogroups are considered. Thus, preferably, when preparing a vaccine formulation to treat an animal infected with a Pasteurella multocida strain containing a PmSLP polypeptide belonging to a specific phylogenetic cluster and a specific serogroup, the vaccine formulation is preferably prepared by selecting and using a PmSLP polypeptide belonging to the same phylogenetic cluster as the infecting strain and a strain belonging to the same serogroup. Thus, again referring to Figure 2For example, when preparing a vaccine formulation, preferably to treat an animal infected with a Pasteurella multocida strain containing a PmSLP protein of a Pasteurella multocida strain belonging to the phylogenetic cluster PmSLP-1, PmSLP-2, PmSLP-3, PmSLP-4.1 or PmSLP-4.2 and belonging to serogroup 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 the same as the PmSLP contained in the infecting strain or may be different, provided that the PmSLP polypeptide belongs to the same phylogenetic cluster as the infecting strain and is obtained from a strain of the same serogroup as the infecting strain. Thus, for example, if an animal is infected with a Pasteurella multocida strain containing a PmSLP polypeptide belonging to the phylogenetic cluster PmSLP-2, and the infecting strain belongs to serogroup A, the vaccine preparation is preferably prepared to include a PmSLP polypeptide or an immunogenic equivalent portion thereof, which is obtained from a Pasteurella multocida strain containing a PmSLP polypeptide belonging to the phylogenetic cluster PmSLP-2 and a strain belonging to serogroup A; if an animal is infected with a Pasteurella multocida strain containing a PmSLP polypeptide belonging to the phylogenetic cluster PmSLP-3 and a strain belonging to serogroup F, the vaccine preparation is preferably prepared to include a PmSLP polypeptide or an immunogenic equivalent portion thereof, which is obtained from a Pasteurella multocida strain containing a PmSLP polypeptide belonging to the phylogenetic cluster PmSLP-3 and a strain belonging to serogroup F. The strain may be the same strain or another strain, as long as the other strain belongs to the same phylogenetic cluster and the same serogroup.

[0265] Therefore, it is now apparent from one embodiment that the vaccine formulation may comprise a Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof, wherein the PmSLP protein is 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 Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof belongs to the same phylogenetic cluster as the PmSLP protein present in the infecting Pasteurella multocida strain, wherein the selected Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof is also a PmSLP protein of a Pasteurella 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 Pasteurella multocida strain.

[0266] In addition, the PmSLP protein can be a PmSLP protein obtained from a Pasteurella multocida strain selected from serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and 16 of Pasteurella multocida strains, and the food production animal is susceptible to infection with the selected serotype of Pasteurella multocida strain. Therefore, in one embodiment, when preparing a vaccine formulation to treat an animal infected with a Pasteurella multocida strain containing a PmSLP protein belonging to a specific phylogenetic cluster, a specific serogroup and a specific 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, when the Pasteurella 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, when the Pasteurella multocida infection causes progressive atrophic rhinitis in porcine species and the strain belongs to serogroup D, the PmSLP protein can be obtained from a Pasteurella multocida strain belonging to serogroup D, etc. It is noteworthy that a particular Pasteurella multocida strain can be obtained (e.g., from a microbial species collection, such as the American Type Culture Collection (ATCC)) or isolated from, for example, an infected food production animal, and the serogroup and / or serotype of the strain can be determined using methods known to those skilled in the art (see, e.g., Wilson, M. et al., 1992, J. Clin. Microbiol, 1518-1524; Arumugam, N. et al., 2011, Tropical Biomed. 28(1) 55-63).

[0267] In another exemplary embodiment, the selected food-producing animal can be a ruminant species, and the prepared vaccine formulation can be administered to the ruminant species, wherein Pasteurella multocida infection causes respiratory disease, and wherein 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.IDNO:36, SEQ.ID NO:38, SEQ.ID NO:40, SEQ.ID NO:51, SEQ.ID NO:53, SEQ.ID NO:55, SEQ.IDNO:57, SEQ.ID NO:59, SEQ.ID NO:61, SEQ.ID NO:65, SEQ.ID NO:67, SEQ.ID NO:69, SEQ.IDNO: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 immunogenic equivalent part thereof, to prevent or improve Pasteurella multocida infection that causes respiratory diseases.

[0268] In another exemplary embodiment, the selected food-producing animal can be a bovine species and the prepared vaccine formulation can be administered to the bovine species, wherein Pasteurella multocida infection causes BRD, and wherein 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:70, SEQ.ID NO:71, SEQ.ID NO:72, SEQ.ID NO:73, SEQ.ID NO:74, SEQ.ID NO:75 NO:71, SEQ.ID NO:73, SEQ.ID NO:77 or SEQ.ID NO:91, or an immunogenic equivalent thereof, to prevent or improve Pasteurella multocida infection that causes BRD.

[0269] In another exemplary embodiment, the selected food-producing animal can be a bovine species, and the prepared vaccine formulation can be administered to the bovine species, wherein Pasteurella multocida infection causes HS, and wherein 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 immunogenic equivalent portion thereof, to prevent or ameliorate Pasteurella multocida infection that causes HS.

[0270] In another exemplary embodiment, the selected food-producing animal can be a porcine species, and the prepared vaccine formulation can be administered to the porcine species, wherein infection with Pasteurella multocida causes pneumonic pasteurellosis, and wherein 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 immunogenic equivalent portion thereof, to prevent or ameliorate infection with Pasteurella multocida that causes pneumonic pasteurellosis.

[0271] In another exemplary embodiment, the selected food-producing animal can be a porcine species, and the prepared vaccine formulation can be administered to the porcine species, wherein Pasteurella multocida infection causes PAR, and wherein 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 immunogenic equivalent portion thereof, to prevent or ameliorate Pasteurella multocida infection that causes PAR.

[0272] In yet another exemplary embodiment, the food-producing animal can be an avian species, and the prepared vaccine formulation can be administered to the avian species, wherein infection with Pasteurella multocida causes fowl cholera, and wherein 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 immunogenic equivalent portion thereof, to prevent or ameliorate infection with Pasteurella multocida that causes pneumonic pasteurellosis.

[0273] However, it is noteworthy that the vaccine formulations of the present disclosure can be used to ameliorate or prevent infection with a strain of Pasteurella multocida that is not the strain from which the PmSLP protein included in the veterinary vaccine formulation was selected. Therefore, surprisingly, the vaccine formulations of the present disclosure do not necessarily need to include PmSLPs from multiple strains of Pasteurella multocida to be used to treat food-producing animals, even if the food-producing animals may be exposed to or have been exposed to multiple strains of Pasteurella multocida, or may be or have been infected by multiple strains of Pasteurella multocida. Thus, for example, a vaccine formulation comprising a PmSLP protein obtained from a first serotype of a Pasteurella multocida strain can be used to treat an infection in a food-producing animal caused by another serotype of a Pasteurella multocida strain. In this regard, a veterinary vaccine formulation can be considered cross-protective even if it includes a single PmSLP or an immunogenic equivalent portion thereof, and can confer homologous or heterologous protection.

[0274] Notwithstanding the foregoing, in some embodiments, a vaccine formulation may comprise two or more PmSLP proteins. Thus, in exemplary embodiments, a vaccine formulation may comprise PmSLP proteins obtained from two or more strains of Pasteurella multocida, each strain belonging to a single serogroup selected from serogroups A, B, D, E, or F, or a veterinary vaccine formulation may comprise PmSLP proteins obtained from two strains of Pasteurella multocida, the two strains of Pasteurella multocida belonging to two or more serogroups selected from groups A, B, D, E, or F. In such embodiments, the vaccine may prevent or ameliorate infection by two or more strains of Pasteurella multocida, and such vaccines may be considered cross-protective.

[0275] In other exemplary embodiments, the vaccine formulation may include two or more PmSLP proteins belonging to two or more strains, each strain belonging to a single phylogenetic cluster PmSLP-1, PmSLP-2, PmSLP-3, PmSLP-4.1 and PmSLP-4.2, or the veterinary vaccine formulation may include PmSLP proteins obtained from two or more Pasteurella multocida strains belonging to two or more phylogenetic clusters PmSLP-1, PmSLP-2, PmSLP-3, PmSLP-4.1 and PmSLP-4.2. In these embodiments, the vaccine can prevent or ameliorate infection by two or more Pasteurella multocida strains, and these vaccines can be considered to be cross-protective.

[0276] In further exemplary embodiments, the vaccine formulation may comprise two or more PmSLP proteins obtained from two strains of Pasteurella multocida, 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 may comprise PmSLP proteins obtained from two or more strains of Pasteurella multocida, each of which belongs to two or more serotypes selected from 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 two or more strains of Pasteurella multocida, and such vaccines can be considered cross-protective.

[0277] In further exemplary embodiments, the vaccine formulation may comprise 2 or at least 2, 3 or at least 3, 4 or at least 4, 5 or at least 5 PmSLP proteins having any of the amino acid sequences shown 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:54, SEQ.ID NO:55, SEQ.ID NO:56, SEQ.ID NO:57, SEQ.ID NO:58, SEQ.ID NO:59, SEQ.ID NO:60, SEQ.ID NO:61, SEQ.ID NO:62, SEQ.ID NO:63, SEQ.ID NO:64, SEQ.ID NO:65, SEQ.ID NO:66, SEQ.ID NO:67, SEQ.ID NO:68, SEQ.ID NO:69, SEQ.ID NO:70, SEQ.ID NO:71, SEQ.ID NO:72, SEQ.ID NO:73, SEQ.ID NO:74, SEQ.ID NO:75 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 immunogenic equivalent thereof.

[0278] Therefore, in brief summary, according to the present disclosure, a Pasteurella multocida PmSLP polypeptide or an immunogenic equivalent portion thereof can be selected for inclusion in a veterinary vaccine formulation to treat food-producing animals infected with Pasteurella multocida. Preferably, the selected Pasteurella multocida PmSLP polypeptide belongs to the same phylogenetic cluster as the PmSLP polypeptide contained in the infected Pasteurella multocida strain.

[0279] Next, turning to the preparation of vaccine formulations, in one aspect of the present disclosure, in order to prepare a vaccine formulation, a formulation comprising a PmSLP protein or an immunogenic equivalent portion thereof can be combined with at least one other veterinary pharmaceutically acceptable ingredient, wherein the veterinary pharmaceutically acceptable ingredient includes but is not limited to a diluent, an excipient, a carrier, an adjuvant or a mixture thereof, wherein the PmSLP protein or an immunogenic equivalent portion thereof and the at least one other ingredient are mixed together or blended or homogenized or otherwise prepared until a vaccine formulation is formed.

[0280] The amount of PmSLP protein or its immunogenic equivalent portion in the veterinary vaccine formulation may vary. In general, the dose administered to the animal is considered. The dose of PmSLP protein or its immunogenic equivalent portion can be formulated to include PmSLP protein or its immunogenic equivalent portion in an amount ranging from about 1 pg / kg animal body weight to about 0.25 mg / kg animal body weight, preferably about 1 μg / kg animal body weight to about 100 μg / kg animal body weight. In addition, the vaccine formulation is preferably formulated so that the dose contains at least about 0.001%, at least 0.025% or about 0.025%, at least 0.05% or about 0.05%, at least 0.1% or about 0.1%, at least 0.5% or about 0.5%, at least 1% or about 1%, at least 5% or about 5%, at least 10% or about 10%, at least 15% or about 15%, at least 20% or about 20%, or at least 25% or about 25% by weight or volume. PmSLP protein (by weight) 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 (by weight or volume), respectively. However, the exact amount required will vary depending on the species, age and general condition of the animal being treated, the severity of the condition being treated, the specific formulation being delivered, the site of administration, the biological species of the subject, and other factors. In this regard, depending on the species of food-producing animal to which the vaccine formulation is administered, veterinary vaccine formulations may vary, particularly in the amount of PmSLP protein or its immunogenic equivalent portion included in the dose. A suitable effective amount can be readily determined by one skilled in the art. Thus, the therapeutically effective amount of a PmSLP protein or immunogenic equivalent portion to be included in the veterinary vaccine formulations of the present disclosure will be an amount sufficient to cause amelioration or prevention of disease or disorder symptoms and will be within a relatively wide range that can be determined by routine experimentation.

[0281] Veterinary vaccine formulations comprising the PmSLP protein of the present disclosure or its immunogenic equivalent portion thereof are preferably additionally prepared by combining the PmSLP protein or its immunogenic equivalent portion with, for example, carriers, excipients, diluents and auxiliary substances, such as wetting agents or emulsifiers, pH buffer substances, and the like. These carriers, excipients, diluents and auxiliary substances are all veterinary pharmaceutically acceptable ingredients. Veterinary pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, polyethylene glycol, hyaluronic acid, glycerol, and ethanol. Veterinary pharmaceutically acceptable salts, such as mineral acid salts, such as hydrochlorides, phosphates, sulfates, and the like, and organic acid salts, such as acetates, propionates, benzoates, and the like, may also be included in the formulation. Although not required, it is also preferred that the vaccine formulation will contain a veterinary pharmaceutically acceptable carrier that acts as a stabilizer, particularly for stabilizing the polypeptide of the present disclosure. Examples of suitable carriers that also act as stabilizers for peptides include, but are not limited to, veterinary grade glucose, sucrose, lactose, sorbitol, inositol, dextran, and the like. Other suitable carriers include, but are not limited to, starch, cellulose, sodium or calcium phosphate, citric acid, glycine, polyethylene glycol (PEG), and combinations thereof.The carrier, excipient, diluent may comprise about 10% to about 95% of the weight or volume of the vaccine formulation.

[0282] In addition, the vaccine formulation of the present disclosure may also include adjuvants, such as freeze-drying stabilizers, wetting agents or emulsifiers, pH buffers, gelling agents or viscosity-enhancing additives, and preservatives. The vaccine formulation generally contains less than about 5% of these adjuvants by weight.

[0283] In order to enhance the immune response of animals, the veterinary vaccine formulations provided herein also preferably include one or more adjuvants, such as pharmacological agents, cytokines, etc. Suitable adjuvants include any substance that enhances the immune response of recipient animals to the immunogenic PmSLP protein disclosed herein or its immunogenic equivalent portion. Non-limiting examples of adjuvants include cytokines, such as IL-1, IL-2, IL-12, IL-6, and also include inorganic salts, such as aluminum hydroxide, aluminum phosphate and calcium phosphate; oil emulsions, such as mineral oil, MF59, QS-21, Montanide Tm ISA51, Montanide Tm ISA61, Montanide Tm ISA 61VG、Montanide Tm Gel 02, Montanide Tm ISA-720 or Isocoms, such as ISCOMATRIX; microbial derivatives, such as monophosphoryl lipid A (MPLA), macrophage activation protein-2, virosomes, LT / CT, CpG; natural polymers, such as polysaccharides; and synthetic polymers, such as polyanhydrides and polyesters; or nucleic acid analogs, such as poly I:C. The adjuvant can be administered, for example, as a protein or other macromolecule, at the same time as (e.g., by inclusion in a vaccine formulation), before or after administration of the polypeptide antigen. When included in a vaccine formulation, the adjuvant can account for, for example, 0.1% or about 0.1% to 50% or about 50%, 0.1% or about 0.1% to 20% or about 20%, or 1% or about 1% to 10% or about 10%, by weight or volume of the vaccine formulation.

[0284] In view of the above, it should be understood that the present disclosure provides a method for preparing a vaccine formulation in another aspect. In this regard, in an exemplary embodiment, the present disclosure provides a method for preparing a veterinary vaccine formulation to prevent or improve Pasteurella multocida infection in food production animals susceptible to Pasteurella multocida infection, the method comprising:

[0285] (i) diagnosis of Pasteurella multocida infection in food-producing animals;

[0286] (ii) identifying the phylogenetic cluster to which the PmSLP protein contained in the infecting Pasteurella multocida belongs, wherein the phylogenetic cluster is selected from the group consisting of PmSLP-1, PmSLP-2, PmSLP-3, PmSLP-4.1 or PmSLP-4.2; and

[0287] (iii) preparing a vaccine preparation to form a veterinary vaccine preparation comprising an effective amount of a Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof, thereby treating food-producing animals susceptible to infection with Pasteurella multocida, the vaccine preparation comprising a Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof belonging to the identified phylogenetic cluster, and a veterinary pharmaceutically acceptable adjuvant.

[0288] In one embodiment, in one aspect, the method may further include identifying a serogroup of the infecting Pasteurella multocida strain selected from the group consisting of serogroups A, B, D, E and F, and the vaccine is prepared using a Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof from the same or another Pasteurella multocida strain belonging to the selected serogroup.

[0289] In another exemplary embodiment, the present disclosure provides a method for preparing a veterinary vaccine formulation, the veterinary vaccine formulation comprising a PmSLP protein or an immunogenic equivalent portion thereof, the method comprising:

[0290] (a) providing a chimeric nucleic acid sequence comprising the following as operably linked components:

[0291] (i) a nucleic acid sequence encoding a PmSLP protein or an immunogenic equivalent portion thereof; and

[0292] (ii) one or more nucleic acid sequences capable of controlling expression in a host cell;

[0293] (b) introducing the chimeric nucleic acid sequence into a host cell;

[0294] (c) growing the host cell to produce the PmSLP protein or the immunogenic equivalent portion thereof; and

[0295] (d) recovering the PmSLP protein or an immunogenic equivalent portion thereof; and

[0296] (e) formulating the PmSLP protein or its immunogenic equivalent part with an adjuvant to form a veterinary vaccine preparation for treating food-producing animals susceptible to infection with Pasteurella multocida, wherein the veterinary vaccine preparation comprises an effective amount of the PmSLP protein or its immunogenic equivalent part.

[0297] The vaccine formulations of the present disclosure can be used to prevent infection or disease caused by pathogenic infectious Pasteurella multocida in food-producing animals. The vaccine formulations can be used to immunize any food-producing animal, including any bovine, porcine, or avian species.

[0298] The veterinary vaccine preparations of the present invention can be administered to food producing animals using any convenient mode of administration. Thus, for example, the veterinary vaccine preparations can be injected, such as intramuscularly or subcutaneously, or the vaccine preparations can be administered orally to the animal, such as as a feed supplement. It should be understood that, in this regard, the mode of administration and technique, such as the specification of the injection needle in the case of injection, may vary from animal to animal. The dosage of the veterinary vaccine preparation will depend on the disease, route of administration, animal species, body weight and other standard factors. In this regard, a person of ordinary skill in the art can easily adjust the appropriate dosage of the effective amount and select a suitable method of administration.

[0299] It should also be noted that the veterinary vaccine formulations of the present disclosure may be administered prophylactically, ie, to prevent Pasteurella multocida infection in food-producing animals, or to ameliorate symptoms associated with Pasteurella multocida infection after infection has occurred in food-producing animals.

[0300] Administration of the veterinary vaccine formulations of the present disclosure generally induces an immune response in the subject food production animal. Thus, the food production animal can form antibodies against the PmSLP protein included in the vaccine formulation. In some embodiments, anti-PmSLP antibodies can be detected in the serum of the food production 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.

[0301] In view of the above, it should now be understood that, on the other hand, the present disclosure provides, in an exemplary embodiment, the use of PmSLP protein or an immunogenic equivalent portion thereof for preparing a veterinary vaccine preparation, wherein the veterinary vaccine preparation comprises PmSLP protein or an immunogenic equivalent portion thereof and a veterinary pharmaceutically acceptable adjuvant.

[0302] In view of the above, it should now be understood that, in another aspect, the present disclosure provides, in an exemplary embodiment, the use of a veterinary vaccine preparation to improve or prevent Pasteurella multocida infection in food-producing animals susceptible to Pasteurella multocida infection, wherein the veterinary vaccine preparation comprises a PmSLP protein or an immunogenic equivalent portion thereof and a veterinary pharmaceutically acceptable adjuvant.

[0303] It is now understood that a veterinary vaccine formulation comprising a PmSLP protein or an immunogenic equivalent portion thereof can be prepared. The veterinary vaccine formulation can be administered to food-producing animals to ameliorate or prevent Pasteurella multocida infection in the animals.

[0304] Of course, the above exemplary embodiments of the present disclosure are intended to be illustrative and not restrictive. These embodiments are susceptible to many modifications of composition, details, and order of operation. The present invention and the disclosure are intended to encompass all such modifications within the scope defined by the claims, which should be given a broad interpretation consistent with the entire description.

[0305] Sequence Overview

[0306] SEQ.ID NO: 1 and SEQ.ID NO: 2 show the polynucleotide sequence and deduced amino acid of mature PmSLP-1 of Pasteurella multocida (belonging to phylogenetic cluster 1), respectively.

[0307] SEQ.ID NO: 3 and SEQ.ID NO: 4 show the polynucleotide sequence and deduced amino acid of mature PmSLP-2 of Pasteurella multocida (belonging to phylogenetic cluster 2), respectively.

[0308] SEQ.ID NO: 5 and SEQ.ID NO: 6 show the polynucleotide sequence and deduced amino acid of mature PmSLP-3 of Pasteurella multocida (belonging to phylogenetic cluster 3), respectively.

[0309] SEQ.ID NO: 7 and SEQ.ID NO: 8 show the polynucleotide sequence and deduced amino acid of mature PmSLP-4 of Pasteurella multocida (belonging to phylogenetic cluster 4.1), respectively.

[0310] SEQ.ID NO: 9 and SEQ.ID NO: 10 show the polynucleotide sequence and deduced amino acid of mature PmSLP-4 of Pasteurella multocida (belonging to phylogenetic cluster 4.2), respectively.

[0311] SEQ.ID NO: 11 and SEQ.ID NO: 12 show the polynucleotide sequence and deduced amino acid of PmSLP-1 of Pasteurella multocida (belonging to phylogenetic cluster 1), respectively, which has a 14-residue anchor truncation of PmSLP, a thrombin cleavage site and a C-terminal polyhistidine tag.

[0312] SEQ. ID NO: 13 and SEQ. ID NO: 14 show the polynucleotide sequence and the deduced amino acid, respectively, of PmSLP-1 of Pasteurella multocida (belonging to phylogenetic cluster 1) with a 14-residue anchor truncation.

[0313] SEQ.ID NO: 15 and SEQ.ID NO: 16 show the polynucleotide sequence and deduced amino acid of PmSLP-1 of Pasteurella multocida (belonging to phylogenetic cluster 1), respectively, having an anchor truncation of 126 residues, a thrombin cleavage site and a C-terminal polyhistidine tag.

[0314] SEQ.ID NO: 17 and SEQ.ID NO: 18 show the polynucleotide sequence and deduced amino acid of PmSLP-1 of Pasteurella multocida (belonging to phylogenetic cluster 1), respectively, with a 94-residue anchor truncation, an introduced SERp1 mutation, a thrombin cleavage site and a C-terminal polyhistidine tag.

[0315] SEQ.ID NO: 19 and SEQ.ID NO: 20 show the polynucleotide sequence and deduced amino acid of PmSLP-3 of Pasteurella multocida (belonging to phylogenetic cluster 3), respectively, having a 15-residue anchor truncation and a mutation of residue 16 from valine to methionine.

[0316] SEQ.ID NO:21 and SEQ.ID NO:22 show the polynucleotide sequence and deduced amino acid of PmSLP-1 from Pasteurella multocida challenge strain H246 (belonging to phylogenetic cluster 1), respectively.

[0317] SEQ.ID NO: 23 and SEQ.ID NO: 24 show the polynucleotide sequence and deduced amino acid of PmSLP-3 from Pasteurella multocida challenge strain H229 (belonging to phylogenetic cluster 3), respectively.

[0318] SEQ.ID NO: 25 and SEQ.ID NO: 26 show the polynucleotide sequence and deduced amino acid of PmSLP-1 with a FLAG tag from Pasteurella multocida (belonging to phylogenetic cluster 1), respectively.

[0319] SEQ.ID NO: 27 and SEQ.ID NO: 28 show the polynucleotide sequence and deduced amino acid, respectively, of PmSLP-1 from Pasteurella multocida (belonging to phylogenetic cluster 1) having a 14-residue anchor truncation, a thrombin cleavage site and a C-terminal polyhistidine tag.

[0320] SEQ.ID NO:29 and SEQ.ID NO:30 show the polynucleotide sequence and deduced amino acid of PmSLP-3 from Pasteurella multocida (belonging to phylogenetic cluster 3), respectively, having a 15-residue anchor truncation, a mutation of residue 16 from valine to methionine, and a C-terminal polyhistidine tag.

[0321] SEQ.ID NO:31 and SEQ.ID NO:32 show the polynucleotide sequence and deduced amino acid of a fusion protein of PmSLP-1 with a 14-residue anchor truncation from Pasteurella multocida (belonging to phylogenetic cluster 1) and PmSLP-3 with a 15-residue anchor truncation and a mutation from valine to methionine at residue 16 (belonging to phylogenetic cluster 3), respectively. This construct lacks a signal peptide and contains a thrombin cleavage site between the two PmSLP proteins and a second thrombin cleavage site at the C-terminus followed by a polyhistidine tag.

[0322] SEQ.ID NO:33 and SEQ.ID NO:34 show the polynucleotide sequence and deduced amino acid of a fusion protein of PmSLP-1 with a 14-residue anchor truncation from Pasteurella multocida (belonging to phylogenetic cluster 1) and PmSLP-3 with a 15-residue anchor truncation and a mutation from valine to methionine at residue 16 (belonging to phylogenetic cluster 3), respectively. This construct lacks a signal peptide, contains a serine-alanine linker peptide between the two PmSLP proteins, a C-terminal thrombin cleavage site, followed by a polyhistidine tag.

[0323] SEQ.ID NO: 35 and SEQ.ID NO: 36 show the polynucleotide sequence and deduced amino acid of a fusion protein of a full-length PmSLP-1 protein including an endogenous signal peptide from Pasteurella multocida (belonging to phylogenetic cluster 1) and PmSLP-3 (belonging to phylogenetic cluster 3) with a 15-residue anchor truncation and a mutation from valine to methionine at residue 16. This construct contains a serine-alanine linker peptide between the two PmSLP proteins, a thrombin cleavage site at the C-terminus, followed by a polyhistidine tag.

[0324] SEQ. ID NO: 37 and SEQ. ID NO: 38 show the polynucleotide sequence and deduced amino acid, respectively, of the mature PmSLP from Pasteurella multocida strain 1500E (belonging to phylogenetic cluster 1).

[0325] SEQ.ID NO:39 and SEQ.ID NO:40 show the polynucleotide sequence and deduced amino acid of PmSLP-4 from Pasteurella multocida strain HS-Canada1 (belonging to phylogenetic cluster 4.2), respectively.

[0326] SEQ.ID NO:41 shows a thrombin cleavage site that occurs 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.

[0327] SEQ.ID NO:42 shows the endogenous signal sequence contained in SEQ.ID NO:36.

[0328] 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.

[0329] SEQ.ID NO:44 shows the first nucleic acid primer sequence.

[0330] SEQ.ID NO:45 shows the second nucleic acid primer sequence.

[0331] SEQ.ID NO:46 shows the third nucleic acid primer sequence.

[0332] SEQ.ID NO:47 shows the fourth nucleic acid primer sequence.

[0333] SEQ.ID NO:48 shows the fifth nucleic acid primer sequence.

[0334] SEQ.ID NO:49 shows the sixth nucleic acid primer sequence.

[0335] SEQ.ID NO: 50 and SEQ.ID NO: 51 show the polynucleotide sequence and deduced amino acid of a fusion protein of PmSLP-1 with a 14-residue anchor truncation from Pasteurella multocida (belonging to phylogenetic cluster 1) and PmSLP-2 with a 14-residue anchor truncation and a mutation of residue 15 from lysine to methionine (belonging to phylogenetic cluster 2), respectively. This construct lacks a signal peptide, contains a serine-alanine linker peptide between the two PmSLP proteins, a C-terminal thrombin cleavage site, followed by a polyhistidine tag.

[0336] SEQ.ID NO: 52 and SEQ.ID NO: 53 show the polynucleotide sequences and deduced amino acids of a fusion protein of PmSLP-1 with a 14-residue anchor truncation from Pasteurella multocida (belonging to phylogenetic cluster 1) with a 14-residue anchor truncation and a mutation of residue 15 from lysine to methionine PmSLP-2 (belonging to phylogenetic cluster 2) and with a 15-residue anchor truncation and a mutation of residue 16 from valine to methionine PmSLP-3 (belonging to phylogenetic cluster 3), respectively. This construct contains a serine-alanine linker peptide between the two PmSLP proteins, each of the three PmSLP proteins, a C-terminal thrombin cleavage site, followed by a polyhistidine tag.

[0337] SEQ.ID NO: 54 and SEQ.ID NO: 55 show the polynucleotide sequence and deduced amino acid of PmSLP-1 from Pasteurella multocida (belonging to phylogenetic cluster 1) with a 14-residue anchor truncation and a fusion protein of PmSLP-2 (belonging to phylogenetic cluster 2) with a 14-residue anchor truncation and a mutation of residue 15 from lysine to methionine, and a fusion protein of PmSLP-4 (belonging to phylogenetic cluster 4.2) with a 41-residue N-terminal truncation and a mutation of residue 42 from alanine to methionine, respectively. This construct contains a serine-alanine linker peptide between each PmSLP protein, a C-terminal thrombin cleavage site, followed by a polyhistidine tag.

[0338] SEQ.ID NO: 56 and SEQ.ID NO: 57 show the polynucleotide sequence and deduced amino acid of PmSLP-2 from Pasteurella multocida (belonging to phylogenetic cluster 2) and Pasteurella multocida with a 14-residue anchor truncation and a mutation from lysine to methionine at residue 15, respectively. This construct contains a C-terminal thrombin cleavage site followed by a polyhistidine tag.

[0339] SEQ.ID NO:58 and SEQ.ID NO:59 show the polynucleotide sequence and deduced amino acid, respectively, of PmSLP-2 from Pasteurella multocida (belonging to phylogenetic cluster 2) having a 14-residue anchor truncation and a mutation of residue 15 from lysine to methionine.

[0340] SEQ.ID NO: 60 and SEQ.ID NO: 61 show the polynucleotide sequence and deduced amino acid, respectively, of PmSLP-4 from Pasteurella multocida (belonging to phylogenetic cluster 4.2) with a 15-residue anchor truncation.

[0341] SEQ.ID NO: 62 and SEQ.ID NO: 63 show the polynucleotide sequence and deduced amino acid, respectively, of PmSLP-4 from Pasteurella multocida (belonging to phylogenetic cluster 4.1) with a 15-residue anchor truncation.

[0342] SEQ.ID NO:64 and SEQ.ID NO:65 show the polynucleotide sequence and deduced amino acid of PmSLP-1 from Pasteurella multocida (belonging to phylogenetic cluster 1), respectively, having a 14-residue anchor truncation, a mutation changing residue 214 from valine to aspartic acid, a C-terminal thrombin cleavage site, followed by a polyhistidine tag.

[0343] SEQ.ID NO:66 and SEQ.ID NO:67 show the polynucleotide sequence and deduced amino acid of PmSLP-1 from Pasteurella multocida (belonging to phylogenetic cluster 1), respectively, having a 14-residue anchor truncation, a mutation of residue 240 from glutamic acid to alanine, a C-terminal thrombin cleavage site, followed by a polyhistidine tag.

[0344] SEQ.ID NO: 68 and SEQ.ID NO: 69 show the polynucleotide sequence and deduced amino acid, respectively, of mature PmSLP-4 from Pasteurella multocida (belonging to an unclassified phylogenetic cluster).

[0345] SEQ.ID NO: 70 and SEQ.ID NO: 71 show the polynucleotide sequence and deduced amino acid, respectively, of mature PmSLP-1 from Pasteurella multocida (belonging to phylogenetic cluster 1).

[0346] SEQ.ID NO: 72 and SEQ.ID NO: 73 show the polynucleotide sequence and deduced amino acid, respectively, of mature PmSLP-1 from Pasteurella multocida (belonging to phylogenetic cluster 1).

[0347] SEQ.ID NO: 74 and SEQ.ID NO: 75 show the polynucleotide sequence and deduced amino acid, respectively, of mature PmSLP-4 from Pasteurella multocida (belonging to phylogenetic cluster 4.1).

[0348] SEQ.ID NO: 76 and SEQ.ID NO: 77 show the polynucleotide sequence and deduced amino acid, respectively, of mature PmSLP-4 from Pasteurella multocida (belonging to phylogenetic cluster 4.2).

[0349] SEQ.ID NO: 78 and SEQ.ID NO: 79 show the polynucleotide sequence and deduced amino acid, respectively, of mature PmSLP-3 from Pasteurella multocida (belonging to phylogenetic cluster 3).

[0350] SEQ.ID NO:80 and SEQ.ID NO:81 show the polynucleotide sequence and deduced amino acid, respectively, of mature PmSLP-3 from Pasteurella multocida (belonging to phylogenetic cluster 3).

[0351] SEQ.ID NO:82 and SEQ.ID NO:83 show the polynucleotide sequence and deduced amino acid, respectively, of mature PmSLP-3 from Pasteurella multocida (belonging to phylogenetic cluster 3).

[0352] SEQ.ID NO:84 and SEQ.ID NO:85 show the polynucleotide sequence and deduced amino acid, respectively, of mature PmSLP-3 from Pasteurella multocida (belonging to phylogenetic cluster 3).

[0353] SEQ.ID NO:86 and SEQ.ID NO:87 show the polynucleotide sequence and deduced amino acid, respectively, of mature PmSLP-3 from Pasteurella multocida (belonging to phylogenetic cluster 3).

[0354] SEQ.ID NO:88 and SEQ.ID NO:89 show the polynucleotide sequence and deduced amino acid, respectively, of mature PmSLP-3 from Pasteurella multocida (belonging to phylogenetic cluster 3).

[0355] SEQ.ID NO:90 and SEQ.ID NO:91 show the polynucleotide sequence and deduced amino acid, respectively, of mature PmSLP-2 from Pasteurella multocida (belonging to phylogenetic cluster 2).

[0356] SEQ.ID NO:92 and SEQ.ID NO:93 show the polynucleotide sequence and deduced amino acid of PmSLP-1 from Pasteurella multocida (belonging to phylogenetic cluster 1), respectively.

[0357] SEQ.ID NO:94 and SEQ.ID NO:95 show the polynucleotide sequence and deduced amino acid, respectively, of mature PmSLP-2 from Pasteurella multocida (belonging to phylogenetic cluster 2).

[0358] SEQ.ID NO:96 and SEQ.ID NO:97 show the polynucleotide sequence and deduced amino acid, respectively, of mature PmSLP-4 from Pasteurella multocida (belonging to phylogenetic cluster 4.2).

[0359] Provided below are examples of specific implementations for carrying out the methods of the present disclosure, as well as implementations representing compositions of the present disclosure. The examples are provided for illustrative purposes only, and are not intended to limit the scope of the present disclosure in any way.

[0360] Example

[0361] Example 1 - Gene cloning, protein production and purification of PmSLP.

[0362] This example illustrates the general workflow involved in producing recombinant PmSLP protein in E. coli.

[0363] Cloning and site-directed mutagenesis

[0364] Based on the key structural residues predicted by XtalPred (Slabinski et al., 2007), the amino-terminal truncated portion of PmSLP (SEQ. ID NO: 12) was designed for protein expression and purification and cloned by unrestricted cloning (van den Ent and 2006). All primers were synthesized by Sigma Aldrich and Taq DNA polymerase (Thermo Fisher Scientific) was used in PCR reactions according to the manufacturer's recommended conditions. A megaprimer containing PmSLP with a carboxy-terminal thrombin cleavage site and a decapsidine tag was amplified from pET52b using forward primers (a 15 amino acid truncated portion amplified with primer (SEQ. ID NO: 44), a 95 amino acid truncated portion amplified with primer (SEQ. ID NO: 45), and a 121 amino acid truncated portion amplified with primer (SEQ. ID NO: 46), which are complementary to the amino terminus of the truncation) and a reverse primer (reverse primer (SEQ. ID NO: 47)) in the vector plasmid pET52b. Subsequently, the megaprimer was purified by gel electrophoresis and excision according to the manufacturer's instructions (Geneaid). The purified gene fragment was subjected to a linear amplification reaction on an empty pET52b plasmid template. The reaction was incubated with 1U Dpn1 (Thermo Fisher Scientific) at 37°C for 1 hour to degrade the methylated template. 5 μL of the reaction was used to transform E. coli MM294 cells by heat shock recovery in LB at 37°C with shaking for 1 hour. Transformants were selected on LB agar containing 100 μg / mL ampicillin and grown overnight at 37°C. 5 mL of LB culture was inoculated with a single colony and incubated with shaking at 37°C overnight. Subsequently, the plasmid was purified using the MiniPrep kit according to the manufacturer's instructions (Geneaid). The charged residues that mutated in PmSLP were predicted using the UCLAMBI SERp server (Goldschmidt, Cooper, Derewenda, & Eisenberg, 2007). A large primer with a mutation was amplified in the vector using a forward primer (SEQ. ID NO: 48) and a reverse primer (SEQ. ID NO: 49) with ≥10 nt complementarity on both sides of the mutation. A secondary PCR was performed using the purified megaprimer and the pET52b template containing the construct. The construct was transformed into E. coli MM294 and propagated therein and extracted using the 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 shown in SEQ.ID NO:17.

[0365] Expression and purification of recombinant PmSLP

[0366] The plasmid containing the PmSLP construct was transformed into E. coli T7 express by 45 sec heat shock and 1 hr recovery in LB with shaking at 37°C. Transformants were selected on LB agar containing 100 μg / mL ampicillin. Several colonies were used to inoculate starter cultures in 20 mL LB containing 100 μg / mL ampicillin and grown for 16 hrs at 37°C with shaking. These overnight cultures were centrifuged at 4,500 x g for 4 min and the pelleted cells were used to inoculate 2 L of LB and the larger culture was grown for approximately 3 hrs at 37°C with shaking or until the OD 600 =0.5. Protein expression was induced by adding isopropyl β-d-1-thiogalactopyranoside (IPTG) to a final concentration of 5 mM, and the cells were allowed to continue growing overnight at 20°C with shaking. The cells were pelleted at 4500 x g and suspended 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, 0.03 mg / mL deoxyribonuclease I (DNase I). The cells were lysed by sonication (Branson) for 2.5 minutes and centrifuged at 30,000 x g for 45 minutes to remove cell debris. The supernatant was passed through a 0.45 μm syringe filter and incubated overnight at 4°C with 2 mL of HisPur Ni-NTA resin (ThermoFisher Scientific) with shaking. The beads were pelleted at 700 x g for 2 minutes and loaded onto a gravity flow column. Bio Rad) and washed with 100mL cold wash buffer (lysate buffer containing 20mM imidazole). Eluted protein in 12mL cold elution buffer (lysate buffer containing 400mM imidazole), and dialyzed overnight for 500mL of 25mM Tris-HCl [pH 8.0] and 100mM NaCl at 4 ° C with 2U bovine thrombin (Sigma Aldrich, catalog number T4648). The dialyzed sample was incubated with 100 μL HisPur Ni-NTA resin (Thermo Fisher Scientific) and 100 μL p-aminobenzamidine-agarose (Sigma Aldrich) at 22 ° C for 1 hour in shaking. The protein after cutting was filtered through a 0.22 μm needle filter, concentrated to 20mg / mL using a 10K MWCO concentrator (Thermo Fisher Scientific), and purified by size exclusion chromatography (Superdex 7510 / 300GL, GE Healthcare). For antigenic studies, the PmSLP protein was further purified on a strong anion exchange chromatography column (MonoQ 5 / 50GL, GE Healthcare) to remove endotoxin (workflow, see Figure 1A ).

[0367] Expression and purification of PmSLP incorporated with selenomethionine was performed as described above, but with significant differences in the growth medium. The starter culture was inoculated into 50 mL of minimal medium (M9, final 0.2% glucose, 1 mM MgSO4, 1 mM thiamine, 0.02 mg / mL essential L-amino acids) supplemented with 0.02 mg / ml methionine and containing 100 μg / mL ampicillin, then allowed to settle and in 2 L of minimal medium supplemented with 0.02 mg / ml selenomethionine (ACROS, Thermo Fisher Scientific).

[0368] Figure 1 shows the workflow for the production and purification of PmSLP proteins and an example gel of purified PmSLP-3. Figure 1A A flow chart showing a schematic workflow for purifying recombinant PmSLP is depicted. Figure 1B Depicted is an SDS PAGE gel showing samples collected at different stages of purification of his-tagged PmSLP-3 (SEQ. ID NO: 12) from E. coli lysate using Ni-NTA (lane 1: whole cell lysate; lane 2: cell pellet; lane 3: soluble fraction; lane 4 flow-through fraction; lane 5: wash fraction; lane 6: elution fraction). Figure 1CShown is a S75 gel filtration chromatogram obtained by size exclusion chromatography after removal of the poly-histidine tag of PmSLP-3 (SEQ. ID NO: 14). Figure 1D SDS-PAGE gel showing different fractions (B9, B10, B11, B12, C1, C2) of purified PmSLP-3 after a polishing step using a MonoQ column. After removal of the tag, PmSLP-3 migrated at approximately 35 kDa in the gel.

[0369] Example 2 - Phylogenetic analysis of PmSLP clusters and associated disease types.

[0370] This example illustrates the variation in PmSLP sequences in isolates of Pasteurella multocida from bovine respiratory disease, bovine hemorrhagic septicemia, swine pasteurellosis, and avian cholera infection, as well as other host species, by constructing a phylogenetic tree based on multiple PmSLP sequences. By evaluating the phylogenetic tree, PmSLP polypeptides or immunogenic fragments can be selected for inclusion in veterinary vaccine formulations.

[0371] The pmSLP gene was obtained from 263 publicly available assembled Pasteurella multocida genomes obtained from public repositories on June 24, 2020, 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). Where available, information on the host animals from which the Pasteurella multocida strains were obtained was included, including cattle (n=87), bison / buffalo (n=17), alpacas (n=2), chickens (n=10), ducks (n=11), turkeys (n=10), dogs (n=1), humans (n=12), sheep (n=4), goats (n=2), rabbits (n=20), rodents (n=2), wolves (n=2), cats (n=2), horses (n=2), wild boars (n=2), pigs (n=59), and geese / anards / poultry (n=8). For sequences derived from bovine samples, sequences were stratified according to available disease (BRD vs. HS) as well as annotated capsular serotype.

[0372] The PmSLP protein sequence was aligned with MAFFT (v7.450) using the G-INS-I algorithm. The most appropriate evolutionary model was identified using ProtTest (v3.4.2), which was found to be WAG+I+G+F. A phylogenetic tree was generated using PhyML (v3.3.20190909) and is shown in Figure 2 middle.

[0373] See also Figure 2 , it can be seen that the phylogenetic tree of PmSLP variants is divided into five unrelated clusters (PmSLP-1, PmSLP-2, PmSLP-3, PmSLP-4.1 and PmSLP-4.2). On the phylogenetic tree, variants derived from bovine species are shown with larger black circles. In addition, Figure 2 The four ring structures in the figure indicate, from the inner to the outer ring structure: (1) host species, (2) disease, (3) geographic region, and (4) capsule type. It can be seen that the BRD-PmSLP variants represent two of the four major clusters (PmSLP-1 and PmSLP-2), while the HS-PmSLP variants represent a single cluster (PmSLP-3). The BRD-PmSLP variants were mainly isolated from samples originating from North America or Europe, while the 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 has at least 99.7% identity with the cluster PmSLP-1 sequence. PmSLP-2 has at least 99.7% identity with the cluster PmSLP-2 sequence and has an average of 100.0% identity with the cluster PmSLP-2 sequence. PmSLP-3 has at least 93.9% identity with cluster PmSLP-3 sequences and an average of 99.4% identity with cluster PmSLP-3 sequences. PmSLP-4.1 has at least 99.7% identity with cluster PmSLP-4.1 sequences and an average of 99.8% identity with cluster PmSLP-4.1 sequences. PmSLP-4.2 has at least 96.8% identity with cluster PmSLP-4.2 sequences and an average of 97.2% identity with cluster PmSLP-4.2 sequences.

[0374] Example 3 - Evaluation of IgG responses and duration of immunogenicity in mice using vaccine formulations containing various PmSLP-1.

[0375] This example demonstrates that PmSLP protein is immunogenic in mammalian hosts and can be formulated with various types of vaccine adjuvants to elicit robust, long-lasting antibody responses.

[0376] 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 had a total volume of 100 μL per dose and consisted of 20 μg of PmSLP-1 protein formulated with: (i) 20% (v / v) Montanide Tm Gel 02 (Seppic) + 3μg poly (I:C) (Invivogen); (ii) 20% (v / v) Montanide Tm Gel 02 (Seppic); (iii) 100 μg aluminum hydroxide (Invivogen); (iv) 20% (v / v) (MVPAdjuvants); (v)50% (v / v)Montanide Tm ISA61 (Seppic). Two doses of each vaccine were administered via subcutaneous injection, 21 days apart. Serum samples were collected 20 days after the first dose, 2 weeks after the second dose, and regularly up to 26 weeks after the second dose.

[0377] Serum antibody titers against PmSLP-1 protein were measured using an enzyme-linked immunosorbent assay (ELISA). Total anti-PmSLP-1 IgG as well as anti-PmSLP-1 IgG subclasses (IgG1, IgG2b, IgG2c) were measured in samples collected after administration of one and two doses of the vaccine (Figure 9). All five vaccine formulations were able to elicit PmSLP-1-specific IgG in serum after only one dose, and IgG titers increased further after the second dose. No PmSLP-1-specific IgG was detected in unvaccinated naive control mice. This suggests that the PmSLP-1 antigen is immunogenic in vivo and can be used in combination with a variety of adjuvants. Antibody subclass analysis showed that the major subclass induced in mice was IgG1, followed by IgG2b, while in mice receiving vaccines containing Montanide Tm Gel02+Poly(I:C) or Montanide Tm Only IgG2c antibodies were consistently induced in the ISA61-adjuvanted vaccine group. Since different IgG subclasses have different effector functions, this suggests that the type of immune response elicited by PmSLP-1 vaccines can be further modulated by adjuvant selection.

[0378] The duration of immunogenicity after two doses of five different PmSLP-1 formulations was assessed for up to 26 weeks ( Figure 3). The PmSLP-1-specific IgG titers in the serum of all groups remained stable over the duration of the assessment without any obvious signs of weakening. This suggests that the PmSLP-1-based vaccine can elicit a long-lasting antibody response.

[0379] Example 4 - Evaluation of the efficacy of PmSLP-1 against an antigenically matched bovine Pasteurella multocida isolate in a mouse model of invasive infection.

[0380] This example illustrates the efficacy of a vaccine formulation comprising PmSLP-1. Mice were immunized with the vaccine formulation and then challenged with a Pasteurella multocida isolate carrying a different PmSLP gene but from the same phylogenetic cluster as the PmSLP-1 protein included in the vaccine. In this example, a bovine respiratory disease (BRD) Pasteurella multocida isolate was used for challenge. This example also illustrates that a vaccine formulation can be prepared that cross-protects against Pasteurella multocida infections caused by different strains.

[0381] Use 20 μg protein with Montanide Tm PmSLP-1 (SEQ. ID NO: 14) was formulated as a vaccine with a mixture of Gel 02 (Seppic) and poly (I:C) (Invivogen) in a final volume of 100 μl per dose. Two doses of the vaccine were given to male C57Bl / 6 mice (Charles River) aged 4-6 weeks via subcutaneous injection three weeks apart. Pre-challenge blood was collected before infection.

[0382] Two weeks after the second dose, approximately 10 4 Anesthetized animals were challenged with logarithmic phase CFU of Pasteurella multocida strain H246 (antigen-matched BRD isolate, the confirmed sequence of PmSLP in this strain is defined by SEQ.ID NO:22. Strain H246 is a serogroup A strain.). Notably, SEQ.ID NO:14 and SEQ.ID NO:22 show 100% sequence identity in the antigenic portion contained in the immunizing protein. After infection, mice were monitored every 6-12 hours for clinical symptoms, including lethargy, respiration, movement, dehydration, diarrhea, posture, and weight loss. A composite clinical score of 10 or higher was considered a clinical endpoint, at which time the mice were humanely euthanized ( Figure 4B , 4C At 3, 24, 48, and 72 hours post-infection, or at clinical endpoint, tail vein blood was collected and bacteria were counted by plating on selective media ( Figure 4EAll mice that received the PmSLP-1 vaccine survived infection, compared with all animals that received the adjuvant alone, which reached endpoint within 24 to 32 hours after infection ( Figure 4A ). Mice immunized with adjuvant alone developed severe bacteremia, whereas mice receiving PmSLP-1 either never developed detectable bacteremia or were able to clear any bacteremia by 72 hours post-infection.

[0383] Purified PmSLP-1 ( Figure 5A ) or heat-killed whole Pasteurella multocida strain H246 ( Figure 5B ) Pre-challenge serum samples were evaluated. All mice that received the PmSLP-1 vaccine had high levels of anti-PmSLP-1 and anti-Pa. multocida serum IgG before challenge, whereas mice that received only the adjuvant had lower levels of serum IgG against the protein antigens or whole bacteria.

[0384] These results indicate that the purified PmSLP-1 protein is a highly effective vaccine antigen against P. multocida strains harboring PmSLP genes from the same phylogenetic cluster.

[0385] Example 5 - Protective effect of PmSLP-3 against an antigenically matched porcine Pasteurella multocida isolate in an acute mouse infection model.

[0386] This example demonstrates the efficacy of a vaccine formulation comprising PmSLP-3. Mice were immunized with the vaccine formulation and then challenged with an isolate of P. multocida that carried a different PmSLP gene but was from the same phylogenetic cluster as the PmSLP-3 protein included in the vaccine. In this example, a porcine isolate of P. multocida was used for the challenge. This example also demonstrates that a vaccine formulation can be prepared that is cross-protective against infection with different strains of P. multocida. 20 μg of protein was used with Montanide Tm PmSLP-3 (SEQ. ID NO: 20) was formulated into a vaccine using a combination of Gel 02 (Seppic) and poly (I:C) (Invivogen) in a final volume of 100 μl per dose. Two doses of the vaccine were given via subcutaneous injection to male C57Bl / 6 mice (Charles River) aged 4-6 weeks at an interval of three weeks. Pre-challenge blood was collected prior to infection.

[0387] Two weeks after the second dose, approximately 10 4Anesthetized animals were challenged with CFU of the logarithmic phase Pasteurella multocida strain H229 (antigen-matched porcine isolate, the confirmed sequence of PmSLP in this strain is defined by SEQ.ID NO:24. H229 is a serogroup A strain). Notably, SEQ.ID NO:20 and SEQ.ID NO:24 show 99.4% sequence identity in the antigenic portion contained in the immunizing protein. After infection, mice were monitored every 6-12 hours for clinical symptoms, including lethargy, respiration, movement, dehydration, diarrhea, posture, and weight loss. A composite clinical score of 10 or higher was considered a clinical endpoint, at which time the mice were humanely euthanized ( Figure 6B , 6C At 3, 24, 48, 78, 100, and 124 hours post-infection, or at clinical endpoint, tail vein blood was collected and bacteria were counted by plating on selective media ( Fig.13E All mice that received the PmSLP-3 vaccine survived infection, compared with all animals that received the adjuvant alone, which reached endpoint within 24 to 32 hours after infection ( Fig. 6A Mice immunized with adjuvant alone developed severe bacteremia, whereas mice receiving the PmSLP-3 vaccine either never developed detectable bacteremia or only developed bacteremia without overt clinical signs.

[0388] Purified PmSLP-3 ( Fig. 7A ) or against heat-killed Pasteurella multocida strain H229 ( Figure 7B ) Pre-challenge serum samples were evaluated. All mice that received the PmSLP-3 vaccine had high levels of anti-PmSLP-3 and anti-Pa. multocida serum IgG before challenge, whereas mice that received only the adjuvant had lower levels of serum IgG against the protein antigens or whole bacteria.

[0389] These results indicate that the purified PmSLP-3 protein is a highly effective vaccine antigen against P. multocida strains harboring PmSLP genes from the same phylogenetic cluster.

[0390] Example 6 - Stability and efficacy of PmSLP antigen under different storage conditions.

[0391] This example considers practical aspects of utilizing PmSLP as a vaccine antigen and examines the effects of different storage conditions and lyophilization of the protein. The stability and efficacy of the PmSLP antigen were evaluated using biophysical methods as well as protection studies in a mouse model.

[0392] For the biophysical method, the stability of the purified PmSLP-3 protein (SEQ.ID NO: 20) stored for one year after freeze-drying at 4°C or room temperature was evaluated by thermal denaturation using Tycho (NanoTemper) and compared with the reference antigen. This method involves measuring the intrinsic fluorescence (detected at 350nm and 330nm) emitted by tryptophan and tyrosine residues when a thermal gradient is applied to the sample and the protein begins to unfold. The change in the fluorescence signal (ratio 350nm / 330nm) indicates the transition of the protein folding state, and the temperature at which the transition occurs is called the inflection point temperature (T i ). Comparison of thermal denaturation curves and Ti of samples stored under different conditions will provide a rapid biophysical assessment of the structural integrity of the protein.

[0393] PmSLP-3 proteins that were lyophilized and stored at 4°C or room temperature (RT) for one year were compared to reference PmSLP-3 proteins that were freshly prepared at a concentration of 100 μg / mL in buffer (phosphate buffer solution, pH 7.4). The lyophilized proteins were reconstituted in the same buffer at 100 μg / mL before analysis. When evaluating the fluorescence signal, the thermal denaturation curves and T were compared after storage at RT or 4°C for one year. i s showed no significant changes ( Fig. 8A ; Figure 8B ), indicating that the freeze-dried protein maintained structural integrity during this period.

[0394] The effect of antigen lyophilization and storage at 4°C in vivo was further evaluated in a mouse infection model. 20 μg of protein was used with Montanide Tm PmSLP-3 protein (SEQ.ID NO: 20) was formulated into a vaccine with a mixture of Gel 02 (Seppic) and poly (I:C) (Invivogen) in a final volume of 100 μl per dose. Two doses of vaccine were given to male C57Bl / 6 mice (Charles River) aged 4-6 weeks via subcutaneous injection three weeks apart. Vaccine Group 1 received a freshly prepared formulation using aliquots of PmSLP-3 protein stored at -80°C before each administration. Vaccine Group 2 received a freshly prepared formulation using aliquots of PmSLP-3 protein that had been lyophilized and stored at 4°C before each administration. Vaccine Group 3 received a freshly prepared formulation using PmSLP-3 protein stored at -80°C before the first administration; then, the mixed formulation was stored at 4°C for three weeks and re-administered at the second administration.

[0395] Six weeks after the second dose, approximately 10 4Anesthetized animals were challenged with 100 CFU of logarithmic-phase Pasteurella multocida strain H229 (antigen-matched porcine isolate, the confirmed sequence of PmSLP in this strain is defined as SEQ. ID NO: 24). After infection, mice were monitored every 6-12 hours for clinical signs, including lethargy, respiration, movement, dehydration, diarrhea, posture, and weight loss. A composite clinical score of 10 or higher was considered the clinical endpoint, at which point the mice were humanely euthanized (Figure 9). All mice that received any of the PmSLP-3 vaccines were completely protected from infection, in contrast, animals that received only the adjuvant reached the endpoint at 21 hours post-infection ( Fig.9A ). Adjuvant only ( Fig. 9B ), Vaccine 1 (a freshly prepared vaccine containing protein, the protein was stored at -80°C until just before each administration; Fig. 9C ), Vaccine 2 (a freshly prepared vaccine containing protein that was lyophilized and stored at 4°C until just before each administration; Fig.9D ) or vaccine 3 (vaccine formulated with proteins stored at −80°C prior to the first dose, and then the remaining vaccine stored at 4°C for three weeks until the second dose; Fig.9E ) of individual mice.

[0396] Example 7 - Immunogenicity of PmSLP-1 in cattle.

[0397] This example illustrates the immunogenicity of the PmSLP vaccine in food production animals affected by Pasteurella multocida infection. In this example, two different breeds of cattle were used to evaluate the immunogenicity of the PmSLP-1 vaccine. The PmSLP-1 vaccine was chosen for the bovine host in this example because this variant is expressed in the vast majority of Pasteurella multocida isolates that cause bovine respiratory disease (BRD).

[0398] Healthy zebu cattle (4-6 months old) seronegative to all serotypes of Pasteurella multocida were randomly divided into two groups. Directly before vaccination, 200 μg of lyophilized PmSLP-1 protein (SEQ. ID NO: 14) was reconstituted with PBS and mixed with 30 μg of poly(I:C) (Invivogen) and 20% v / v Montanide Tm Gel 02 (Seppic) was formulated with a final volume of 2 mL per dose. The animals were randomly divided into groups and three doses of PmSLP-1 vaccine or adjuvant alone were administered by subcutaneous injection at intervals of three weeks. Blood was collected before vaccination (baseline) and approximately 2-3 weeks after the first, second and third doses. The presence of a-PmSLP-1 IgG in serum samples was evaluated using ELISA (enzyme-linked immunosorbent assay). Fig.10Endpoint titers for cattle immunized with PmSLP-1 vaccine or adjuvant are shown. As shown, there was a detectable increase in PmSLP-1 specific antibodies after one dose and a significant increase after two doses. The boosting effect of the third dose was minimal.

[0399] The second cattle immunization was performed using healthy beef cattle (about 10 months old) that were seronegative to all serotypes of Pasteurella multocida. The animals were randomly divided into two groups of 9 animals each. The animals received three doses of PmSLP-1 (SEQ.ID NO: 14) vaccine or adjuvant. Before each immunization, 200 μg of PmSLP-1 protein stored at -80°C was mixed with 30 μg of poly (I:C) (Invivogen) and 20% v / v Montanide. Tm The vaccine was prepared by mixing with Gel 02 (Seppic) in a final volume of 2 mL per dose. Animals were vaccinated intramuscularly every three weeks, and blood was collected before the first immunization (baseline) and 2-3 weeks after the first, second and third doses. The presence of a-PmSLP-1 IgG in serum was evaluated. Fig.11 Endpoint titers for cattle immunized with PmSLP-1 vaccine or adjuvant at baseline and after one, two, or three doses of vaccine are shown. As shown, there was a detectable increase in specific antibodies after one dose, a significant increase after two doses, and a very small boost from the third dose.

[0400] Overall, the PmSLP-1 vaccine was immunogenic in both cattle species, with peak titers achieved after 2 doses of the vaccine with this formulation administered intramuscularly or subcutaneously.

[0401] Example 8 - PmSLP-3 protects cattle from infection with serogroup B strains of Pasteurella multocida.

[0402] This example demonstrates the immunogenicity, safety and protective efficacy of PmSLP vaccines in food production animals affected by Pasteurella multocida infection. In this example, cattle of the Zebu breed were used to evaluate the immunogenicity and safety of two PmSLP-3 vaccine formulations, as well as to evaluate the protective efficacy of this vaccine against lethal challenge with serogroup B Pasteurella multocida. The PmSLP-3 vaccine was chosen for the bovine host in this example because this variant is expressed by all Pasteurella multocida isolates known to cause hemorrhagic septicemia (HS).

[0403] Healthy zebu cattle (4-6 months old) seronegative to all serotypes of Pasteurella multocida were randomly divided into three groups. Prior to vaccination, 200 μg of PmSLP-3 (SEQ. ID NO 20) was reconstituted directly in PBS and mixed with 1 mg of aluminum hydroxide (Alhydrogel, Sigma-Aldrich) or with 30 μg of poly(I:C) (Invivogen) and 20% v / v Montanide. Tm Gel 02 (Seppic) was formulated with a final volume of 2 mL per dose. A control group receiving only adjuvant was used as a negative control group. Animals were vaccinated subcutaneously twice at intervals of three weeks, and blood was collected before the first immunization (baseline), before the second immunization (after the first dose), and before the challenge (after the second dose). The presence of a-PmSLP-1 IgG in the serum was evaluated. After vaccination, local reactions at the injection site were monitored. Fourteen days after the booster dose, 4.4×10 4 Cattle were challenged subcutaneously with serogroup B Pasteurella multocida at 10 CFU / mL and monitored for 8 days post infection.

[0404] Fig. 12A Endpoint titers are shown for cattle immunized with PmSLP-3 formulated with aluminum hydroxide, PmSLP-3 formulated with Montanide Gel 02 + poly(I:C), or adjuvant alone. As shown, serum IgG against PmSLP-3 was detectable after two vaccinations with both PmSLP-3 vaccine formulations, while PmSLP-3 formulated with aluminum hydroxide elicited detectable serum IgG against PmSLP-3 after the first dose. Fig. 12B Shown is the survival of cattle challenged with serogroup B Pasteurella multocida. As shown, zebu cattle receiving adjuvant alone were fully susceptible to infection and died one day after infection. In contrast, 87.5% (7 / 8) of cattle vaccinated with PmSLP-3 vaccine formulated with aluminum hydroxide survived the challenge, while 75% (6 / 8) of cattle vaccinated with PmSLP-3 vaccine formulated with Montanide Gel 02 + poly (I:C) survived the challenge. Fig. 12C A table containing reactogenicity after one or two doses of the vaccine is shown. As shown, PmSLP-3 formulated with aluminum hydroxide did not cause local reactions after either dose, while PmSLP-3 formulated with Montanide Gel 02 + poly (I; C) or with Montanide Gel 02 alone caused local swelling in 50% of the animals after the first dose.

[0405] Overall, PmSLP-3 vaccines formulated with aluminum hydroxide or with Montanide Gel 02 + poly(I:C) were safe, immunogenic, and protective in zebu cattle after two vaccination doses when delivered subcutaneously.

[0406] Example 9 - Evaluation of the efficacy of vaccines containing PmSLP-2 in ruminants.

[0407] This example illustrates the efficacy of a vaccine formulation comprising PmSLP-2. Ruminants were immunized with the vaccine formulation and then challenged with a Pasteurella multocida isolate. In this example, a bovine respiratory disease (BRD) Pasteurella multocida isolate was used for challenge.

[0408] Vaccine and control preparations

[0409] 200 μg protein was added to phosphate buffered saline (PBS) and Montanide Tm PmSLP-2 (SEQ. ID NO: 4) was formulated into a vaccine with a mixture of ISA61VG (60% v / v) in a final volume of 2,000 μl per dose and a final antigen concentration of 100 μg / mL. The vaccine formulation containing PmSLP-2 is otherwise referred to herein as IVP 1.

[0410] The negative and positive controls were saline solution formulated with adjuvant VAP#07 and an autovaccine corresponding to the challenge strain Pasteurella multocida A:3 strain 671 / 90 (Lainson F. et al., Genome Announc. 2013 Oct 3; 1(5):e00803-13. doi: 10.1128 / genomeA.00803-13), respectively. Pasteurella multocida A:3 strain 671 / 90 contained the same PmSLP-2 polypeptide as that included in the IVP 1 formulation.

[0411] Ruminants and vaccination

[0412] A total of 30 healthy calves three to four weeks old from commercial animal farms, seronegative or weakly positive for P. multocida antibodies and not previously vaccinated against P. multocida, were included in the assessment.

[0413] The calves were divided into three groups: the first group of 10 calves received the IVP1 vaccine formulation, the second group of 10 calves received the autovaccine, and the third group of 10 calves received the saline solution. All product administrations were performed by intramuscular injection on the left side of the animal's neck. Each product (i.e., IVP 1, or autovaccine, or saline solution) was administered to the animals twice, the first time at the beginning of the evaluation (D0) and the second time twenty-one days later (D21). It is worth noting that for the purpose of this evaluation, the time when the product was first administered to the animals was referred to as day 0 (D0). Similarly, other time points may be referred to herein, such as D10 or D35, which means the 10th day or the 35th day after the first administration, respectively.

[0414] Attack Test

[0415] On D35, all calves were challenged by depositing 300 ml of a dilute broth culture of Pasteurella multocida A:3 (strain 671 / 90) into the trachea at the bifurcation of the main bronchus using a fiberoptic bronchoscope, with the expected challenge dose concentration being 1 × 10 9 colony forming units (CFU) (the acceptable range is 5×10 8 Up to 5×10 9 CFU).

[0416] Safety Assessment

[0417] Injection site assessments were performed on all animals prior to dosing on D0 and D21 and then once daily for four consecutive days (D1 to D4 and from D22 to D25).

[0418] The injection site area was inspected and scored for the presence of swelling (yes / no), and if present, the length, width, and height were measured using a calibrated ruler. All detected swellings were also rated for pain, warmth, and firmness. No substantial swelling was identified in any animal that excluded the animal from further evaluation.

[0419] Clinical assessment

[0420] Clinical observations were conducted on D34, before challenge on D35, then twice at approximately 4 and 9 hours (± 1 hour) post-challenge on D35, twice daily from D36 to D41, and once on D42.

[0421] Each clinical observation included an assessment of behavior, nasal discharge, cough and respiration, and rectal temperature (°C) according to the scoring system listed in Table 1 .

[0422] Table 1 - Clinical observation parameters

[0423]

[0424] Animals were euthanized by lethal injection on D42. Lungs were removed from each animal and the percentage of lung lesions (based on the presence of lesions) was recorded for each lung lobe.

[0425] In order to be able to quantitatively assess the clinical severity of the development of pathology in each animal, the clinical parameters observed at each monitoring session were converted into a score. These individual scores for each clinical symptom were added and recorded to give a total clinical score for each animal at each observation. This allowed the temporal progression of the pathology to be assessed.

[0426] Clinical observation parameters were scored throughout the evaluation period according to Table 1, and the clinical parameter scores were statistically evaluated and used to draw bar graphs, box plots, and charts showing the clinical parameter scores in the time period just before challenge (D34) and after challenge (D35-D42), as described below.

[0427] Clinical Assessment Outcomes – Rectal Temperature

[0428] Fig.13A is a graph showing the results of rectal temperature measurements of animals after administration of three products, ie, IVP 1, autovaccine, and saline solution, from D34 to D42 and administration of the challenge strain to the animals on D35.

[0429] from Fig.13A As can be seen in the figure, rectal temperature began to increase immediately after the administration of the pathogenic challenge strain, and then the average rectal temperature showed a clear downward trend in animals administered with the autovaccine and IVP 1, gradually returning to the average or near-average rectal temperature recorded before the administration of the challenge strain by D42. In contrast, the rectal temperature of the unvaccinated animals (i.e., animals administered with saline solution) was always higher than the average rectal temperature recorded before the administration of the challenge strain. It is worth noting that the rectal temperature of animals administered with IVP-1 returned to the baseline (i.e., the temperature before the challenge) faster than the recovery rate of animals administered with the autovaccine.

[0430] Clinical Assessment Outcomes – Behavior

[0431] Fig. 13B is a graph showing the results of rectal temperature measurements of animals after administration of three products, ie, IVP 1, autovaccine, and saline solution, from D34 to D42, and administration of the challenge strain to the animals on D35.

[0432] from Fig. 13B As can be seen in the graph, all animals initially showed a depressive response immediately after administration of the pathogenic challenge strain, after which the average animal behavior in all animal groups returned to relatively normal. However, the animal behavior of animals administered the IVP 1 vaccine formulation recovered slightly faster than that of the unvaccinated animals (i.e., animals administered the saline solution).

[0433] Clinical Assessment Findings – Nasal Discharge

[0434] Fig. 13C is a bar graph showing the results of nasal secretion scoring after administration of three products, ie, IVP 1, autovaccine, and saline solution, from D34 to D42, and administration of the challenge strain to the animals on D35.

[0435] from Fig. 13C As can be seen in the Figures, all animals initially had a nasal discharge response immediately after administration of the pathogenic challenge strain, after which the average animal nasal discharge in all animal groups returned to relatively normal. However, the nasal discharge of animals administered the IVP 1 vaccine formulation recovered slightly faster than that of unvaccinated animals (i.e., animals administered the saline solution), which showed lingering nasal discharge effects at D42.

[0436] Clinical Assessment Outcomes – Cough

[0437] Fig.13D is a bar graph showing the results of cough scores after administration of three products, ie, IVP 1, autovaccine and saline solution, from D34 to D42, and administration of the challenge strain to the animals on D35.

[0438] from Fig.13D As can be seen in the Figures, by D42, all animal groups continued to have at least occasional dry coughs, however, cough scores were always slightly higher in the non-vaccinated animals during the period of clinical observation.

[0439] Clinical Assessment Outcomes – Respiratory

[0440] Fig.13E is a bar graph showing the results of respiratory scoring after administration of three products, ie, IVP 1, autovaccine and saline solution, from D34 to D42, and administration of the challenge strain to the animals on D35.

[0441] from Fig.13E As can be seen in the Figures, all animals initially showed a marked moderate increase and / or mild abnormality in respiratory scores immediately after administration of the pathogenic challenge strain, after which respiration in any animal group recovered during the period of clinical observation, but never reached a completely normal score. However, the respiration of animals administered with the IVP 1 vaccine formulation or the autovaccine formulation appeared to return to normal respiration more quickly than unvaccinated animals (i.e., animals administered with saline solution), wherein the animal groups receiving the saline formulation still showed moderate or markedly increased respiratory scores at D42, whereas these deviations from normal levels were not observed in animals receiving the IVP 1 vaccine formulation or the autovaccine formulation.

[0442] Clinical Assessment Results – Total Clinical Score

[0443] Fig.13F is a graph showing the results of total clinical scores after administration of three products, ie, IVP 1, autovaccine and saline solution, from D34 to D42, and administration of the challenge strain to the animals on D35.

[0444] from Fig.13F As can be seen in the Figures, all animals initially experienced a sharp increase immediately after administration of the pathogenic challenge strain, after which the total clinical scores progressed to almost completely normal scores (i.e., score 1) in animals administered the autovaccine and IVP1. However, animals receiving the saline formulation still showed moderately or significantly increased total clinical scores at D42. Notably, the total clinical scores of animals administered IVP 1 decreased faster than those of animals administered the autovaccine.

[0445] Clinical Assessment Outcomes – Total Lesion Score

[0446] Figure 13G is a box plot showing the results of total lung lesion scores after administration of three products, namely IVP 1, autovaccine and saline solution, from D34 to D42, and the challenge strain was administered to the animals on D35.

[0447] from Figure 13G As can be seen, after challenge with a pathogenic P. multocida strain, at D42, statistically significantly higher lung consolidation scores were observed in animals administered saline solution (mean % [SD] = 30.90% [10.24]) than in animals administered autovaccine (mean % [SD] = 23.25% [8.70]; p = 0.049) or IVP 1 (mean % [SD] = 13.29% [11.44]).

[0448] Serological results

[0449] Fig.13H Shown are group average results for PmSLP-2 ELISA of serum samples collected before the first vaccination (D7) and on the day of challenge (D35). Compared with the average results of the IVP 1 group at D7 and the results of the saline-vaccinated group and the autovaccine group at D7 and D35, a significant increase in serum titers against PmSLP-2 was observed only in the IVP 1 group at D35.

Claims

1. A veterinary vaccine preparation for preventing, treating or ameliorating Pasteurella multocida infection in food-producing animals susceptible to Pasteurella multocida infection, the vaccine preparation comprising an effective amount of at least one PmSLP protein, or an immunogenic equivalent portion thereof.

2. The veterinary vaccine formulation according to claim 1, wherein the vaccine formulation comprises at least one Pasteurella multocida PmSLP protein, or an immunogenic equivalent portion thereof, wherein the PmSLP protein is selected from the same phylogenetic cluster as the PmSLP protein present in the infecting Pasteurella multocida strain.

3. The veterinary vaccine formulation according to claim 1, wherein the vaccine formulation comprises a Pasteurella multocida PmSLP protein, or an immunogenic equivalent portion thereof, wherein the PmSLP protein is 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 Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof belongs to the same phylogenetic cluster as the PmSLP protein present in the infecting Pasteurella multocida strain.

4. The veterinary vaccine formulation according to any one of claims 1 to 3, wherein the food-producing animal is a ruminant susceptible to infection with a strain of Pasteurella multocida that causes respiratory disease, and the vaccine formulation comprises a Pasteurella multocida PmSLP protein from a strain of Pasteurella multocida that causes respiratory disease, or an immunogenic equivalent portion thereof, wherein the PmSLP protein is selected from the group of phylogenetic clusters consisting of PmSLP-1, PmSLP-2, PmSLP-3 and PmSLP-4.2, wherein the selected Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof belongs to the same phylogenetic cluster as the PmSLP protein present in the infecting Pasteurella multocida strain.

5. The veterinary vaccine formulation according to any one of claims 1 to 3, wherein the food-producing animal is a bovine susceptible to infection with a strain of Pasteurella multocida that causes BRD, and the vaccine formulation comprises a Pasteurella multocida PmSLP protein from the strain of Pasteurella multocida that causes BRD, or an immunogenic equivalent portion thereof, wherein the PmSLP protein is selected from the group of phylogenetic clusters consisting of PmSLP-1, PmSLP-2 and PmSLP-4.2, wherein the selected Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof belongs to the same phylogenetic cluster as the PmSLP protein present in the infecting Pasteurella multocida strain.

6. The veterinary vaccine formulation according to any one of claims 1 to 3, wherein the food-producing animal is a bovine susceptible to infection with a strain of Pasteurella multocida that causes HS, and the vaccine formulation comprises a Pasteurella multocida PmSLP protein from the strain of Pasteurella multocida that causes HS, or an immunogenic equivalent portion thereof, wherein the PmSLP protein is selected from the phylogenetic cluster PmSLP-3, wherein the selected Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof belongs to the same phylogenetic cluster as the PmSLP protein present in the infecting Pasteurella multocida strain.

7. A veterinary vaccine formulation according to any one of claims 1 to 3, wherein the food-producing animal is a porcine animal susceptible to infection with a Pasteurella multocida strain that causes porcine atrophic rhinitis (PAR), and the vaccine formulation comprises a Pasteurella multocida PmSLP protein from the Pasteurella multocida strain that causes PAR, or an immunogenic equivalent portion thereof, wherein the PmSLP protein is selected from the group of phylogenetic clusters consisting of PmSLP-2, PmSLP-4.1 and PmSLP-4.2, wherein the selected Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof belongs to the same phylogenetic cluster as the PmSLP protein present in the infecting Pasteurella multocida strain.

8. The veterinary vaccine formulation according to any one of claims 1 to 3, wherein the food-producing animal is a porcine animal susceptible to infection with a strain of Pasteurella multocida that causes pneumonic pasteurellosis, and the vaccine formulation comprises a Pasteurella multocida PmSLP protein from a strain of Pasteurella multocida that causes pneumonic pasteurellosis, or an immunogenic equivalent portion thereof, wherein the PmSLP protein is selected from the group of phylogenetic clusters consisting of PmSLP-2, PmSLP-4.1 and PmSLP-4.2, wherein the selected Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof belongs to the same phylogenetic cluster as the PmSLP protein present in the infecting Pasteurella multocida strain.

9. The veterinary vaccine formulation according to any one of claims 1 to 3, wherein the food-producing animal is an avian animal susceptible to infection with a Pasteurella multocida strain that causes fowl cholera, and the vaccine formulation comprises a Pasteurella multocida PmSLP protein from a Pasteurella multocida strain that causes fowl cholera, or an immunogenic equivalent portion thereof, wherein the PmSLP protein is selected from the group of phylogenetic clusters consisting of PmSLP-3 and PmSLP-4.2, wherein the selected Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof belongs to the same phylogenetic cluster as the PmSLP protein present in the infecting Pasteurella multocida strain.

10. The veterinary vaccine formulation according to any one of claims 1 to 3, wherein the vaccine formulation comprises a Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof, wherein the PmSLP protein is 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 Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof belongs to the same phylogenetic cluster as the PmSLP protein present in the infecting Pasteurella multocida strain, and wherein the selected Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof is also a PmSLP protein of a Pasteurella 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 Pasteurella multocida strain.

11. The veterinary vaccine formulation according to any one of claims 1 to 3, wherein the at least one PmSLP protein or an immunologically equivalent portion thereof is a protein expressed by a nucleic acid sequence selected from the group consisting of: (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.IDNO:54, SEQ.ID NO:56, SEQ.ID NO:58, SEQ.ID NO:60, SEQ.ID NO:62, SEQ.ID NO:64, SEQ.IDNO:66, SEQ.ID NO:68, SEQ.ID NO:70, SEQ.ID NO:72, SEQ.ID NO:74, SEQ.ID NO:76, SEQ.IDNO:78, SEQ.ID NO:80, SEQ.ID NO:82, SEQ.ID NO:84, SEQ.ID NO:86, SEQ.ID NO:88, SEQ.IDNO:90, SEQ.ID NO:92, SEQ.ID NO:94 or SEQ.ID NO:96; (b) a nucleic acid sequence that is at least 70% identical to any of the nucleic acid sequences in (a); (c) a nucleic acid sequence substantially identical to any of the nucleic acid sequences in (a) except for the degeneracy of the genetic code; (d) a nucleic acid sequence complementary to any one of the nucleic acid sequences in (a); (e) a chimeric nucleic acid obtained by fusion of at least two nucleic acid sequences of (a), (b), (c) and (d) or parts thereof; (f) a nucleic acid sequence complementary to any one of the nucleic acid sequences in (a); (g) a nucleic acid sequence encoding a polypeptide having any of the amino acid sequences shown in the following, or an immunogenic equivalent portion thereof: 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:64, SEQ.ID NO:65 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 a functional variant of any one of the amino acid sequences shown below, or an immunogenic equivalent thereof: 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:64, SEQ.ID NO:65 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 stringent conditions to any one of the nucleic acid sequences shown in (a), (b), (c), (d), (e), (f), (g) or (h).

12. The veterinary vaccine formulation according to any one of claims 1 to 3, wherein the at least one Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof comprises any one of the amino acid sequences shown in the following: 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:60, SEQ.ID NO:61, SEQ.ID NO:62, SEQ.ID NO:63, SEQ.ID NO:64, SEQ.ID NO:65, SEQ.ID NO:66, SEQ.ID NO:67, SEQ.ID NO:68, SEQ.ID NO:69, SEQ.ID NO:70, SEQ.ID NO:71, SEQ.ID NO:72 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 immunogenic equivalent portions thereof, or a chimeric protein obtained by fusion of any one of the sequences or portions thereof.

13. The veterinary vaccine formulation according to any one of claims 1 to 3, wherein the food producing animal is a ruminant species and the Pasteurella multocida infection causes respiratory disease.

14. The veterinary vaccine formulation according to any one of claims 1 to 3, wherein the food producing animal is a bovine species and the Pasteurella multocida infection causes bovine respiratory disease (BRD) or hemorrhagic septicemia (HS).

15. The veterinary vaccine formulation according to any one of claims 1 to 3, wherein the food producing animal is a Suidae species and the Pasteurella multocida infection causes porcine pneumonic pasteurellosis or porcine atrophic rhinitis (PAR).

16. The veterinary vaccine formulation according to any one of claims 1 to 3, wherein the food producing animal is an avian species and the Pasteurella multocida infection causes avian cholera.

17. The veterinary vaccine formulation according to any one of claims 1 to 3, wherein the food-producing animal is a ruminant species, wherein the Pasteurella multocida infection causes respiratory disease, and wherein 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:60, SEQ.ID NO:61, SEQ.ID NO:62, SEQ.ID NO:63, SEQ.ID NO:64, SEQ.ID NO:65, SEQ.ID NO:66, SEQ.ID NO:67, SEQ.ID NO:68, SEQ.ID NO:69, SEQ.ID NO:70, SEQ.ID NO:71, SEQ.ID NO:72, SEQ.ID NO:73, SEQ.ID NO:74 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 immunogenic equivalent parts thereof, to prevent or improve Pasteurella multocida infection that causes the respiratory disease.

18. The veterinary vaccine formulation of any one of claims 1 to 3, wherein the food-producing animal is a bovine species, wherein the Pasteurella multocida infection causes BRD, and wherein 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:68, SEQ.ID NO:69, SEQ.ID NO:70, SEQ.ID NO:71, SEQ.ID NO:72, SEQ.ID NO:73, SEQ.ID NO:74, SEQ.ID NO:75 NO:69, SEQ.ID NO:71, SEQ.ID NO:73, SEQ.ID NO:77 or SEQ.ID NO:91, or an immunogenic equivalent thereof, to prevent or improve Pasteurella multocida infection that causes BRD.

19. The veterinary vaccine formulation of any one of claims 1 to 3, wherein the food-producing animal is a bovine species, wherein the Pasteurella multocida infection causes HS, and wherein 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 immunogenic equivalent thereof, to prevent or ameliorate Pasteurella multocida infection that causes HS.

20. The veterinary vaccine formulation of any one of claims 1 to 3, wherein the food-producing animal is a porcine species and the Pasteurella multocida infection causes pneumonic pasteurellosis, and wherein 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 immunogenic equivalent thereof, to prevent or ameliorate Pasteurella multocida infection that causes pneumonic pasteurellosis.

21. A veterinary vaccine formulation according to any one of claims 1 to 3, wherein the food-producing animal is a porcine species and the Pasteurella multocida infection causes PAR, and wherein 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 immunogenic equivalent thereof, to prevent or ameliorate Pasteurella multocida infection that causes PAR.

22. The veterinary vaccine formulation of any one of claims 1 to 3, wherein the food-producing animal is an avian species, wherein the Pasteurella multocida infection causes fowl cholera, and wherein 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 immunogenic equivalent thereof, to prevent or ameliorate Pasteurella multocida infection that causes pneumonic pasteurellosis.

23. The veterinary vaccine formulation according to any one of claims 1 to 22, wherein the PmSLP protein or an immunogenic equivalent portion thereof is recombinantly produced in a microbial host organism.

24. The veterinary vaccine formulation according to any one of claims 1 to 22, wherein the veterinary vaccine formulation is a cross-protective vaccine formulation, the vaccine formulation comprising a PmSLP protein obtained from a first Pasteurella multocida strain or an immunogenic equivalent portion thereof, and the vaccine formulation is a formulation administered to the food-producing animal to prevent or ameliorate an infection caused by another Pasteurella multocida strain.

25. The veterinary vaccine formulation according to any one of claims 1 to 24, wherein the vaccine formulation is substantially free of other Pasteurella multocida components.

26. The veterinary vaccine formulation according to any one of claims 1 to 25, wherein the PmSLP protein or an immunologically equivalent portion thereof is a recombinantly produced protein, wherein the vaccine formulation is substantially free of host cell components.

27. The veterinary vaccine formulation according to any one of claims 1 to 26, wherein the vaccine formulation further comprises a veterinary pharmaceutically acceptable adjuvant.

28. The veterinary vaccine formulation according to claim 27, wherein the vaccine formulation further comprises a veterinary pharmaceutically acceptable excipient, carrier or diluent.

29. The veterinary vaccine formulation according to any one of claims 1 to 28, wherein the vaccine formulation comprises about 0.001% to about 20% by weight of the PmSLP protein or the immunogenic equivalent portion thereof, and about 0.1% to about 60% by weight or volume of a veterinary pharmaceutically acceptable adjuvant of the vaccine formulation.

30. The veterinary vaccine formulation according to any one of claims 1 to 29, wherein the vaccine formulation comprises a second Pasteurella multocida PmSLP protein, or an immunologically equivalent portion thereof.

31. The veterinary vaccine formulation of claim 30, wherein the vaccine formulation comprises a fusion polypeptide comprising the first Pasteurella multocida PmSLP protein and the second Pasteurella multocida PmSLP protein, or immunologically equivalent portions thereof.

32. The veterinary vaccine formulation according to claim 30 or 31, wherein the second Pasteurella multocida PmSLP protein or an immunologically equivalent portion thereof belongs to the same or a different phylogenetic cluster as the first Pasteurella multocida PmSLP protein or an immunologically equivalent portion thereof.

33. The veterinary vaccine formulation according to claim 31, wherein the fusion polypeptide comprises 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.

34. The veterinary vaccine formulation according to claim 30 or 31, wherein the second Pasteurella multocida PmSLP protein or an immunologically equivalent portion thereof is obtained from a Pasteurella multocida strain belonging to the same or a different serogroup as the first Pasteurella multocida PmSLP protein or an immunologically equivalent portion thereof.

35. Use of a veterinary vaccine formulation according to any one of claims 1 to 33, for preventing, treating or ameliorating Pasteurella multocida infection in food-producing animals susceptible to Pasteurella multocida infection.

36. The use of claim 34, wherein the treatment of the Pasteurella multocida infection comprises an improvement toward normalization of one or more clinical parameters selected from the group consisting of: (i) rectal temperature, (ii) animal behavior, (iii) nasal discharge pattern, (iv) cough pattern, (v) breathing pattern, and (vi) overall clinical health status, relative to an animal production animal not administered the veterinary vaccine formulation.

37. The use according to claim 34 or 35, wherein the vaccine formulation is capable of eliciting an immune response in the food-producing animal, wherein anti-PmSLP antibodies are detectable in the serum of the food-producing animal at least within a period starting from 7 days and ending at 52 weeks from the date of use of the vaccine formulation.

38. A method for preventing, treating or ameliorating Pasteurella multocida infection in a food-producing animal susceptible to infection with Pasteurella multocida, the method comprising administering a veterinary vaccine preparation to the food-producing animal, the veterinary vaccine preparation comprising a Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof, wherein the vaccine preparation is administered in an effective amount to prevent, treat or ameliorate the Pasteurella multocida infection.

39. The method of claim 38, wherein the amelioration of the Pasteurella multocida infection comprises a reduction in clinical signs of any disease caused by the Pasteurella multocida infection.

40. The method of claim 39, wherein the clinical signs are selected from the group consisting of: (i) rectal temperature, (ii) animal behavior, (iii) nasal discharge pattern, (iv) coughing pattern, (v) breathing pattern, and (vi) overall clinical health status relative to an animal production animal not administered the veterinary vaccine formulation.

41. The method according to any one of claims 39 to 40, wherein the food producing animal susceptible to infection with Pasteurella multocida is selected from the group consisting of ruminants, suis and avian species.

42. The method according to any one of claims 38 to 41, wherein the disease caused by infection with Pasteurella multocida is selected from the group consisting of respiratory disease, bovine respiratory disease (BRD), hemorrhagic septicemia (HS), porcine atrophic rhinitis (PAR) and fowl cholera.

43. The method of any one of claims 38 to 42, wherein the food producing animal is a ruminant susceptible to infection with Pasteurella multocida, and wherein the veterinary vaccine formulation administered to the ruminant in an effective amount comprises at least one PmSLP protein from a strain of Pasteurella multocida that causes respiratory disease, or an immunogenic equivalent portion thereof.

44. The method of any one of claims 38 to 42, wherein the food-producing animal susceptible to infection with Pasteurella multocida is a bovine species, and wherein the veterinary vaccine formulation administered to the bovine species in an effective amount comprises at least one PmSLP protein from a strain of Pasteurella multocida that causes BRD, or an immunogenic equivalent portion thereof.

45. The method of any one of claims 38 to 42, wherein the food-producing animal susceptible to infection with Pasteurella multocida is a ruminant, and wherein the veterinary vaccine formulation administered to the ruminant in an effective amount comprises at least one PmSLP protein from a strain of Pasteurella multocida that causes HS, or an immunogenic equivalent portion thereof.

46. ​​The method of any one of claims 38 to 42, wherein the food producing animal susceptible to infection with a strain of Pasteurella multocida is a porcine animal, and wherein the veterinary vaccine formulation administered to the porcine animal in an effective amount comprises at least one PmSLP protein from Pasteurella multocida that causes porcine atrophic rhinitis (PAR), or an immunogenic equivalent portion thereof.

47. The method of any one of claims 38 to 42, wherein the food-producing animal susceptible to infection with a Pasteurella multocida strain is an avian animal, and wherein the veterinary vaccine formulation administered to the avian animal in an effective amount comprises at least one PmSLP protein from a Pasteurella multocida strain that causes fowl cholera, or an immunogenic equivalent portion thereof.

48. The method of any one of claims 38 to 42, wherein the vaccine formulation administered to the food producing animal in an effective amount comprises at least one Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof, the PmSLP protein being selected from the group of phylogenetic clusters consisting of PmSLP-1, PmSLP-2, PmSLP-3, PmSLP-4.1 and PmSLP-4.

2.

49. The method of claim 48, wherein the veterinary vaccine formulation comprises at least one Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof, which PmSLP protein or an immunogenic equivalent portion thereof confers homologous protection to the food-producing animal susceptible to infection with Pasteurella multocida against Pasteurella multocida strains having PmSLP proteins from the same phylogenetic cluster.

50. The method of claim 48, wherein the veterinary vaccine formulation comprises at least one Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof, which PmSLP protein or an immunogenic equivalent portion thereof confers heterologous protection to the food-producing animal susceptible to infection with Pasteurella multocida against Pasteurella multocida strains having PmSLP proteins from different phylogenetic clusters.

51. The method according to any one of claims 38 to 50, wherein the vaccine formulation comprises at least one Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof, wherein the PmSLP protein is selected from the group consisting of proteins encoded by a nucleic acid sequence selected from the group consisting of: (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.IDNO:54, SEQ.ID NO:56, SEQ.ID NO:58, SEQ.ID NO:60, SEQ.ID NO:62, SEQ.ID NO:64, SEQ.IDNO:66, SEQ.ID NO:68, SEQ.ID NO:70, SEQ.ID NO:72, SEQ.ID NO:74, SEQ.ID NO:76, SEQ.IDNO:78, SEQ.ID NO:80, SEQ.ID NO:82, SEQ.ID NO:84, SEQ.ID NO:86, SEQ.ID NO:88, SEQ.IDNO:90, SEQ.ID NO:92, SEQ.ID NO:94 or SEQ.ID NO:96; (b) a nucleic acid sequence that is at least 70% identical to any of the nucleic acid sequences in (a); (c) a nucleic acid sequence substantially identical to any of the nucleic acid sequences in (a) except for the degeneracy of the genetic code; (d) a nucleic acid sequence complementary to any one of the nucleic acid sequences in (a); (e) a chimeric nucleic acid obtained by fusion of at least two nucleic acid sequences of (a), (b), (c) and (d) or parts thereof; (f) a nucleic acid sequence complementary to any one of the nucleic acid sequences in (a); (g) a nucleic acid sequence encoding a polypeptide having any of the amino acid sequences shown in the following, or an immunogenic equivalent portion thereof: 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:64, SEQ.ID NO:65 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 a functional variant of any one of the amino acid sequences shown below, or an immunogenic equivalent thereof: 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:64, SEQ.ID NO:65 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 stringent conditions to any one of the nucleic acid sequences shown in (a), (b), (c), (d), (e), (f), (g) or (h).

52. The method of any one of claims 38 to 51, wherein the vaccine formulation comprises at least one Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof, wherein the PmSLP protein or an immunogenic equivalent portion thereof has any one of the amino acid sequences shown 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:56, SEQ.ID NO:57, SEQ.ID NO:58, SEQ.ID NO:59, SEQ.ID NO:60, SEQ.ID NO:61, SEQ.ID NO:62, SEQ.ID NO:63, SEQ.ID NO:64, SEQ.ID NO:65, SEQ.ID NO:66, SEQ.ID NO:67, SEQ.ID NO:68, SEQ.ID NO:69, SEQ.ID NO:70, SEQ.ID NO:71, SEQ.ID NO:72, SEQ.ID NO:73, SEQ.ID NO:74 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 immunogenic equivalent portions thereof, or a chimeric protein obtained by fusion of any one of the sequences or portions thereof.

53. The method of any one of claims 38 to 52, wherein the PmSLP protein or an immunogenic equivalent portion thereof is recombinantly produced in a microbial host organism.

54. The method of any one of claims 38 to 53, wherein the PmSLP protein or immunologically equivalent portion thereof is substantially free of other Pasteurella multocida components.

55. The method of any one of claims 53, wherein the PmSLP protein or immunologically equivalent portion thereof is a recombinantly produced protein, wherein the PmSLP protein or immunologically equivalent portion thereof is substantially free of host cell components.

56. The method according to any one of claims 38 to 55, wherein the vaccine formulation further comprises a veterinary pharmaceutically acceptable adjuvant.

57. The method of claim 56, wherein the vaccine formulation comprises a veterinarily acceptable excipient, carrier or diluent.

58. A method for preparing a veterinary vaccine formulation for preventing, treating or ameliorating Pasteurella multocida infection in food-producing animals susceptible to Pasteurella multocida infection, the method comprising: (i) diagnosis of Pasteurella multocida infection in food-producing animals; (ii) identifying a phylogenetic cluster to which the PmSLP protein present in the infected Pasteurella multocida belongs, wherein the phylogenetic cluster is selected from the group consisting of PmSLP-1, PmSLP-2, PmSLP-3, PmSLP-4.1 or PmSLP-4.2; and (iii) preparing a vaccine formulation comprising the following: a Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof belonging to the identified phylogenetic cluster, and a veterinary pharmaceutically acceptable adjuvant, to form a veterinary vaccine formulation comprising an effective amount of a Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof, thereby treating food-producing animals susceptible to infection with Pasteurella multocida.

59. The method of claim 58, wherein the method further comprises identifying the serogroup of the infecting Pasteurella multocida strain, the serogroup being selected from the group consisting of serogroups A, B, D, E and F, and the vaccine is prepared using a Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof from the same or another Pasteurella multocida strain belonging to the selected serogroup.

60. A method for preparing a veterinary vaccine formulation comprising a Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof, the method comprising: (a) providing a chimeric nucleic acid sequence comprising the following as operably linked components: (i) a nucleic acid sequence encoding a Pasteurella multocida PmSLP protein or an immunogenic 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 immunogenic equivalent portion thereof in a host cell; (b) introducing the chimeric nucleic acid sequence into a host cell; (c) growing the host cell to produce the Pasteurella multocida PmSLP protein or the immunogenic equivalent portion thereof; and (d) recovering the Pasteurella multocida PmSLP protein or the immunogenic equivalent portion thereof; and (e) formulating the Pasteurella multocida PmSLP protein or the immunogenic equivalent part thereof with a veterinary pharmaceutically acceptable adjuvant to form a veterinary vaccine preparation, thereby treating food-producing animals susceptible to infection with Pasteurella multocida, wherein the veterinary vaccine preparation comprises an effective amount of the Pasteurella multocida PmSLP protein or the immunogenic equivalent part thereof.

61. The method of claim 60, wherein the nucleic acid sequence is a nucleic acid sequence selected from the group consisting of: (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.IDNO:54, SEQ.ID NO:56, SEQ.ID NO:58, SEQ.ID NO:60, SEQ.ID NO:62, SEQ.ID NO:64, SEQ.IDNO:66, SEQ.ID NO:68, SEQ.ID NO:70, SEQ.ID NO:72, SEQ.ID NO:74, SEQ.ID NO:76, SEQ.IDNO:78, SEQ.ID NO:80, SEQ.ID NO:82, SEQ.ID NO:84, SEQ.ID NO:86, SEQ.ID NO:88, SEQ.IDNO:90, SEQ.ID NO:92, SEQ.ID NO:94 or SEQ.ID NO:96; (b) a nucleic acid sequence that is at least 70% identical to any of the nucleic acid sequences in (a); (c) a nucleic acid sequence substantially identical to any of the nucleic acid sequences in (a) except for the degeneracy of the genetic code; (d) a nucleic acid sequence complementary to any one of the nucleic acid sequences in (a); (e) a chimeric nucleic acid obtained by fusion of at least two nucleic acid sequences of (a), (b), (c) and (d) or parts thereof; (f) a nucleic acid sequence complementary to any one of the nucleic acid sequences in (a); (g) a nucleic acid sequence encoding a polypeptide having any of the amino acid sequences shown in the following, or an immunogenic equivalent portion thereof: 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:64, SEQ.ID NO:65 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 a functional variant of any one of the amino acid sequences shown below, or an immunogenic equivalent thereof: 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:64, SEQ.ID NO:65 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 stringent conditions to any one of the nucleic acid sequences shown in (a), (b), (c), (d), (e), (f), (g) or (h).

62. A recombinant expression vector comprising: (a) a nucleic acid sequence encoding a Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof; and (b) a nucleic acid sequence capable of controlling the expression of the nucleic acid sequence encoding the Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof in a host cell.

63. The expression vector according to claim 62, wherein the nucleic acid sequence is a nucleic acid sequence selected from the group consisting of: (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.IDNO:54, SEQ.ID NO:56, SEQ.ID NO:58, SEQ.ID NO:60, SEQ.ID NO:62, SEQ.ID NO:64, SEQ.IDNO:66, SEQ.ID NO:68, SEQ.ID NO:70, SEQ.ID NO:72, SEQ.ID NO:74, SEQ.ID NO:76, SEQ.IDNO:78, SEQ.ID NO:80, SEQ.ID NO:82, SEQ.ID NO:84, SEQ.ID NO:86, SEQ.ID NO:88, SEQ.IDNO:90, SEQ.ID NO:92, SEQ.ID NO:94 or SEQ.ID NO:96; (b) a nucleic acid sequence that is at least 70% identical to any of the nucleic acid sequences in (a); (c) a nucleic acid sequence substantially identical to any of the nucleic acid sequences in (a) except for the degeneracy of the genetic code; (d) a nucleic acid sequence complementary to any one of the nucleic acid sequences in (a); (e) a chimeric nucleic acid obtained by fusion of at least two nucleic acid sequences of (a), (b), (c) and (d) or parts thereof; (f) a nucleic acid sequence complementary to any one of the nucleic acid sequences in (a); (g) a nucleic acid sequence encoding a polypeptide having any of the amino acid sequences shown in the following, or an immunogenic equivalent portion thereof: 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:64, SEQ.ID NO:65 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 a functional variant of any one of the amino acid sequences shown below, or an immunogenic equivalent thereof: 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:64, SEQ.ID NO:65 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 stringent conditions to any one of the nucleic acid sequences shown in (a), (b), (c), (d), (e), (f), (g) or (h).

64. A host cell comprising a chimeric nucleic acid, the chimeric nucleic acid comprising: (a) a nucleic acid sequence encoding a Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof; and (b) a nucleic acid sequence capable of controlling the expression of the nucleic acid sequence encoding the Pasteurella multocida PmSLP protein or an immunogenic equivalent portion thereof in a host cell.

65. according to the host cell described in claim 64, wherein said nucleic acid sequence is a nucleic acid sequence selected from the nucleic acid sequence consisting of: (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.IDNO:54, SEQ.ID NO:56, SEQ.ID NO:58, SEQ.ID NO:60, SEQ.ID NO:62, SEQ.ID NO:64, SEQ.IDNO:66, SEQ.ID NO:68, SEQ.ID NO:70, SEQ.ID NO:72, SEQ.ID NO:74, SEQ.ID NO:76, SEQ.IDNO:78, SEQ.ID NO:80, SEQ.ID NO:82, SEQ.ID NO:84, SEQ.ID NO:86, SEQ.ID NO:88, SEQ.IDNO:90, SEQ.ID NO:92, SEQ.ID NO:94 or SEQ.ID NO:96; (b) a nucleic acid sequence that is at least 70% identical to any of the nucleic acid sequences in (a); (c) a nucleic acid sequence substantially identical to any of the nucleic acid sequences in (a) except for the degeneracy of the genetic code; (d) a nucleic acid sequence complementary to any one of the nucleic acid sequences in (a); (e) a chimeric nucleic acid obtained by fusion of at least two nucleic acid sequences of (a), (b), (c) and (d) or parts thereof; (f) a nucleic acid sequence complementary to any one of the nucleic acid sequences in (a); (g) a nucleic acid sequence encoding a polypeptide having any of the amino acid sequences shown in the following, or an immunogenic equivalent portion thereof: 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:64, SEQ.ID NO:65 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 a functional variant of any one of the amino acid sequences shown below, or an immunogenic equivalent thereof: 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:64, SEQ.ID NO:65 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 stringent conditions to any one of the nucleic acid sequences shown in (a), (b), (c), (d), (e), (f), (g) or (h).

66. A use of a Pasteurella multocida PmSLP protein or an immunogenic equivalent part thereof for preparing a veterinary vaccine preparation, the veterinary vaccine preparation comprising the P protein or an immunogenic equivalent part thereof, and a veterinary pharmaceutically acceptable adjuvant.

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