WO2013084070A2 - Salmonella vaccine proteins - Google Patents

Abstract

Compositions and methods for the treatment or prevention of Gram negative bacterial, such as Salmonella spp. infection, in a vertebrate subject are provided. The methods provide administering a composition to the vertebrate subject in an amount effective to reduce or eliminate the Salmonella spp. bacterial infection and/or induce an immune response to the protein. Methods for the treatment or prevention of Salmonella spp. infection in a vertebrate are also provided. Other compositions and methods are also disclosed.

Description

SALMONELLA VACCINE PROTEINS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No.

61/567,526, filed December 6, 2011, the entire disclosure of which is hereby incorporated by reference in its entirety for all purposes.

[0002] This application is related to U.S. Provisional Application No. 61/376,619, filed August 24, 2010; and PCT/US2011/049009, filed August 24, 201 1; the entire disclosures of which are hereby incorporated by reference in their entirety for all purposes.

SEQUENCE LISTING

[0003] This application includes a Sequence Listing submitted electronically as a text file named X_US_sequencelisting.txt, created on X, with a size of X bytes. The sequence listing is incorporated by reference.

FIELD

[0004] Compositions and methods are disclosed for the treatment or prevention of Gram negative bacterial infection in a vertebrate subject such as Salmonella spp. bacterial infection. Methods are provided for administering a protein to the vertebrate subject in an amount effective to reduce or eliminate, e.g., the bacterial colonization and/or infection. Methods for the treatment or prevention of Salmonella spp. infection in an organism and other methods are also provided.

BACKGROUND

[0005] Salmonella enterica can cause significant morbidity and mortality worldwide. The predominant Salmonella enterica serovars causing disease in humans are Typhi, Paratyphi A and B, and Typhimurium. The Typhi and Paratyphi serovars are strict human pathogens and cause typhoid fever, while the non-typhoidal Salmonella enterica subspecies Typhimurium and Enteritidis results primarily in gastroenteritis in humans. Importantly, however, Typhimurium is not a strict human pathogen and can infect a number of animal species, with animal-human transmission being a common and well-recognized problem. Cross-species infection usually results from contamination of agricultural products such as chicken. These organisms can also cause significant agricultural disease, such as

gastroenteritis seen in cattle. [0006] Several non-typhoidal Salmonella vaccines have been developed for poultry and swine use. Most efforts have focused on single and double gene modification (or knockout) attenuated Salmonella vaccines. The gene(s) targeted for elimination are usually Salmonella Pathogenicity Island (SPI)-l and SPI-2, the PhoPQ regulatory operon, or aromatic amino acid biosynthetic pathways. Many Typhimurium-based live vaccines have been developed through the identification of mutations affecting metabolic functions or essential virulence factors. Clin. Microbiol. Rev. 5 (1992) 328-342. The most common strains attenuated metabolically are mutants deficient in the biosynthesis of aromatic amino acids, pyrimidines (aroA, aroC, aroD), purines {pur A), the production of adenylate cyclase (cya) or cyclic AMP (crp), two-component regulator system phoP/phoQ, or as previously mentioned, Salmonella pathogenicity island 2. Infect Immun. 67 (1999) 1093-1099. Many vaccines utilise attenuation through genetic modification of the salmonella pathogenicity island (SPI-1) to diminish pathogenicity. J Infect Dis. 2005 Aug l ;192(3):360-6. Infect Immun. 2002

Jul;70(7):3457-67.

[0007] There has also been significant effort in developing Salmonella vaccines, especially typhoidal vaccines for humans (Cheminay, C. and M. Hensel, Rational design of Salmonella recombinant vaccines. Int J Med Microbiol, 2007; Tacket, CO. and M.M.

Levine, CVD 908, CVD 908-htrA, and CVD 909 live oral typhoid vaccines: a logical progression. Clin Infect Dis, 2007. 45 Suppl 1 : p. S20-3; Lewis, G.K., Live-attenuated Salmonella as a prototype vaccine vector for passenger immunogens in humans: are we there yet? Expert Rev Vaccines, 2007. 6(3): p. 431-40; Kwon, Y.M., M.M. Cox, and L.N. Calhoun, Salmonella-based vaccines for infectious diseases. Expert Rev Vaccines, 2007. 6(2): p. 147- 52). There are two main approaches: to use live attenuated strains that are defective for either essential metabolic pathways or lacking virulence factors; or to use purified bacterial components. Some of the genes targeted for live attenuated vaccine strains include the adenylate cyclase pathway (cya/crp), a protease (degP), aromatic amino acid biosynthesis (aroA, aroC, aroD, pur), a regulator (ht ), Spi-1 and Spi-2 mutants (sipA, ssrA), sugar metabolism (galE), virulence regulators (phoP, rpoS), outer membrane proteins (ompC), LPS biosythesis, plus many others (Cheminay et al, supra). Most of these have been studied in S. Typhimurium in the murine model, with some being tested in the chicken model. Another major area of research has been to use live attenuated strains as potential vaccine delivery vehicles of heterologous cloned products and DNA (Lewis et al, supra). There has been less success using purified bacterial components, e.g., because Salmonellae modify their surface components such as LPS, as well as many proteins, once inside host cells such as macrophages (Eriksson, S., et al., Unravelling the biology of macrophage infection by gene expression profiling of intracellular Salmonella enterica. Mol Microbiol, 2003. 47: p. 103- 18; Prost, L.R., S. Sanowar, and S.I. Miller, Salmonella sensing of anti-microbial mechanisms to promote survival within macrophages. Immunol Rev, 2007. 219(1): p. 55-65).

[0008] There are also various commercially available Salmonella vaccines (Guzman, C.A., et al, Vaccines against typhoid fever. Vaccine, 2006. 24(18): p. 3804-1 1). There is currently no human vaccine to non-typhoidal Salmonella strains such as S. Typhimurium and 5^*. Enteritidis. Megan Health has received USDA regulatory approval for a live attenuated Salmonella spray for chicks to decrease bacterial load in poultry. Other researchers have been working on various potential Salmonella poultry vaccines (Barrow, P.A., Salmonella infections: immune and non-immune protection with vaccines. Avian Pathol, 2007. 36(1): p. 1-13).

[0009] Diseases resulting from Salmonella species (e.g., gastroenteritis and typhoid fever) remain a significant worldwide health issue. The current vaccine landscape is generally plagued by safety concerns and/or difficult regulatory approval hurdles. Thus, there remains an unmet need for an effective vaccine composition for treating or preventing infection by non-typhoid Salmonella species in, e.g., birds, pigs, cattle, and humans.

SUMMARY

[0010] Described herein is a composition comprising an effective amount of an isolated polypeptide to induce an immune response in a vertebrate subject to a Gram negative bacterial infection, wherein the isolated polypeptide comprises an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8 or wherein the isolated polypeptide comprises an amino acid sequence encoded by a polynucleotide sequence at least 80% identical to the polynucleotide sequence set forth in SEQ ID O:9, SEQ ID O: 10, SEQ ID O: l l, SEQ ID NO: 12, SEQ ID O: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16.

[0011] In some aspects, the isolated polypeptide comprises the amino acid sequence set forth in SEQ ID NO:7, wherein the Gram negative bacterial infection is a Salmonella spp. infection, and wherein the amount of the composition is an effective amount to reduce the Salmonella spp. colony forming units (CFUs) per gram (g) of cecum, spleen, and/or liver of the subject after Salmonella spp. infection compared to a control subject. In some aspects, the amount of the composition is an effective amount to reduce the Salmonella spp. colony forming units (CFUs) per gram (g) of cecum, spleen, and/or liver of the subject after

Salmonella spp. infection compared to a control subject.

[0012] In some aspects, the infection is a Salmonella spp. infection. In some aspects, the Salmonella spp. infection is a Salmonella Typhimurium infection or a Salmonella Enteritidis infection.

[0013] In some aspects, the amount of composition is effective in reducing or eliminating bacterial carriage and/or bacterial infection and/or bacterial shedding and/or bacterial colonization in the subject. In some aspects, the amount of composition is effective in reducing or eliminating bacterial carriage and/or bacterial infection and/or bacterial shedding and/or bacterial colonization in the subject after the subject has been contacted by the bacteria.

[0014] In some aspects, the composition further comprises a pharmaceutically acceptable carrier. In some aspects, the composition further comprises a pharmaceutically acceptable carrier comprising buffered saline and/or phosphate buffered saline.

[0015] In some aspects, the composition further comprises a pharmaceutically acceptable adjuvant. In some aspects, the composition further comprises a pharmaceutically acceptable adjuvant, and wherein the pharmaceutically acceptable adjuvant comprises Alum, RIBI, CpG, saponin, and/or TiterMax. In some aspects, the composition further comprises a

pharmaceutically acceptable adjuvant, and wherein the pharmaceutically acceptable adjuvant comprises an oil-in-water emulsion.

[0016] In some aspects, the vertebrate subject is a mammalian subject, a human subject, an avian subject, a bovine subject, or a porcine subject.

[0017] In some aspects, the isolated polypeptide is purified. In some aspects, the isolated polypeptide is recombinant.

[0018] In some aspects, the isolated polypeptide comprises an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO: 8. In some aspects, the isolated polypeptide comprises an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8. In some aspects, the isolated polypeptide comprises an amino acid sequence at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97 , 98, or 99% identical to the amino acid sequence set forth in SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8. In some aspects, the isolated polypeptide comprises the amino acid sequence set forth in SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8. In some aspects, the isolated polypeptide comprises an amino acid sequence encoded by a polynucleotide sequence at least 90% identical to the polynucleotide sequence set forth in SEQ ID O:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16. In some aspects, the isolated polypeptide comprises an amino acid sequence encoded by a polynucleotide sequence at least 95% identical to the polynucleotide sequence set forth in SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16. In some aspects, the isolated polypeptide comprises an amino acid sequence encoded by a polynucleotide sequence at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97 , 98, or 99% identical to the polynucleotide sequence set forth in SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16. In some aspects, the isolated polypeptide comprises an amino acid sequence encoded by a polynucleotide sequence the polynucleotide sequence set forth in SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16.

[0019] In some aspects, the composition comprises at least two or more isolated polypeptides. In some aspects, the composition comprises at least three or more isolated polypeptides. In some aspects, the composition comprises at least 2, 3, 4, 5, 6, 7, 8, or more isolated polypeptides. In some aspects, the composition comprises the amino acid sequences set forth in SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8. In some aspects, the composition does not comprise a FliF protein, a CsgA protein, CsgB protein, a PhoP protein, and/or

lipopolysaccharide (LPS). In some aspects, the composition further includes one or more recombinant or purified antigens and/or proteins. In some aspects, further includes one or more additional proteins selected from Table 3 or one or more additional proteins encoded by the nucleotide sequences shown in Table 4.

[0020] Also described herein is an isolated vector comprising a polynucleotide sequence at least 80% identical to the polynucleotide sequence set forth in SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16. In some aspects, the vector comprises a polynucleotide sequence at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97 , 98, or 99% identical to the polynucleotide sequence set forth in SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16. In some aspects, the polynucleotide sequence is operably linked to a promoter sequence.

[0021] Also described herein is a cell comprising an isolated polynucleotide sequence at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97 , 98, or 99% identical to the polynucleotide sequence set forth in SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16, or a vector described herein.

[0022] Also described herin is a cell culture comprising a culture medium and a cell described herein.

[0023] Also described herein is a composition comprising an effective immunizing amount of an isolated polynucleotide, wherein the composition is effective in a vertebrate subject to induce an immune response to a Gram negative bacterial infection, and wherein the isolated polynucleotide comprises a polynucleotide sequence at least 80% identical to the polynucleotide sequence set forth in SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16.

[0024] Also described herein is a method for preventing or treating a Gram negative bacterial infection in a vertebrate subject in need thereof comprising administering a composition described herein to the vertebrate subject in an amount effective to reduce or eliminate the bacterial infection. In some aspects, the amount of the composition is effective in the subject to reduce the Salmonella spp. colony forming units (CFUs) per gram (g) of cecum, spleen, and/or liver of the subject after Salmonella spp. infection compared to a control subject.

[0025] Also described herein is a method for inducing an immune response in a vertebrate subject against a Salmonella spp. bacterial infection comprising administering a composition described herein to the vertebrate subject in an amount effective to induce the immune response in the vertebrate subject.

[0026] Also described herein is a method for reducing colonization of Salmonella spp. bacteria in a vertebrate subject in need thereof comprising administering to the vertebrate subject a composition described herein in an amount effective to reduce Salmonella spp. colonization in the vertebrate subject. In some aspects, reducing colonization of Salmonella spp. bacteria in the vertebrate subject further comprises reducing a risk of infectious transfer from the vertebrate subject to a second distinct subject.

[0027] Also described herein is a method for reducing shedding of Salmonella spp.

bacteria in a vertebrate subject in need thereof comprising administering to the vertebrate subject a composition described herein in an amount effective to reduce Salmonella spp. shedding in the vertebrate subject. In some aspects, reducing shedding of Salmonella spp. bacteria in the vertebrate subject further comprises reducing a risk of infectious transfer from the vertebrate subject to a second distinct subject.

[0028] Also described herein is a method for reducing Salmonella spp. bacterial infection in a vertebrate subject in need thereof comprising administering to the vertebrate subject a composition described herein in an amount effective to reduce Salmonella spp. bacterial infection in the vertebrate subject.

[0029] Also described herein is a method for reducing carriage of Salmonella spp.

bacteria in a vertebrate subject in need thereof comprising administering to the vertebrate subject a composition described herein in an amount effective to reduce Salmonella spp. carriage in the vertebrate subject.

[0030] Also described herein is a method for reducing colonization of Salmonella spp. bacteria in a vertebrate subject in need thereof comprising administering to the vertebrate subject a composition described herein in an amount effective to reduce Salmonella spp. colonization in the vertebrate subject.

[0031] Also described herein is a method for reducing shedding of Salmonella spp. bacteria in a vertebrate subject in need thereof comprising administering to the vertebrate subject a composition described herein in an amount effective to reduce Salmonella spp. shedding in the vertebrate subject.

[0032] Also described herein is a method for reducing Salmonella spp. bacterial infection in a vertebrate subject in need thereof comprising administering to the vertebrate subject a composition described herein in an amount effective to reduce Salmonella spp. bacterial infection in the vertebrate subject.

[0033] Also described herein is a method for reducing carriage of Salmonella spp.

bacteria in a vertebrate subject in need thereof comprising administering to the vertebrate subject a composition described herein in an amount effective to reduce Salmonella spp. carriage in the vertebrate subject.

[0034] Also described herein is a method for preventing or treating a Gram negative bacterial infection in a vertebrate subject in need thereof comprising administering a composition described herein to the vertebrate subject in an amount effective to reduce or eliminate the bacterial infection.

[0035] Also described herein is a method for inducing an immune response in a vertebrate subject against a Salmonella spp. bacterial infection comprising administering a composition described herein to the vertebrate subject in an amount effective to induce the immune response in the vertebrate subject.

[0036] In some aspects, a method described herein, e.g., a method described above, further comprises administration of an adjuvant.

[0037] Also described herein is a method for producing a protein, comprising contacting a cell with a polynucleotide sequence at least 80% identical to the polynucleotide sequence set forth in SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 1 1, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16; and expressing a protein encoded by the polynucleotide sequence.

[0038] Also described herein is a method for producing a protein, comprising

synthesizing an isolated polypeptide comprising an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8 or an isolated polypeptide comprising an amino acid sequence encoded by a polynucleotide sequence at least 80% identical to the polynucleotide sequence set forth in SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16.

BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1. Supernatant of S. Typhimurium grown under SPI-2 conditions decreases bacterial load in systemic NTS. C57BL/6J mice (N=6-8 mice per group) were immunized subcutaneously with Salmonella supernatant and Titermax as adjuvant, challenged with 3 x 10⁶ cfu S. Typhimurium and euthanized 3 days later to obtain (A) cecal, (B) spleen and (C) liver Salmonella counts; (D) cecal weight; (E) survival curve where circles indicate control subjects and squares indicated supernatant test subjects. (F) Spleen

Salmonella counts of C57BL/6J, B6A29S2-IghmtmlCgn/J (B cells deficient), B6.129S2- Cd4tmlMaktt (CD4 cells deficient) or B6A29S2-Cd8atmlMak/J (CD 8 cells deficient) mice (N=5 per group) immunized against systemic salmonellosis; (G) specific IgG levels in the serum of C57BL/6J mice immunized against systemic salmonellosis; (H) specific IgA levels in the serum of C57BL/6J mice immunized against systemic salmonellosis. Ctrl: saline plus adjuvant control; Sup: supernatant from SL1344 plus adjuvant. Bars indicate median. Bars in ELISA graphs describe mean and SEM. *: p<0.05; **: p< 0.01 ; ***: p<0.001 ; ns: not statistically significant. [0040] Figure 2. Characterization of the protective molecule. C57BL/6J mice (N=4-13 mice per group) were immunized subcutaneous ly with different supernatants as described and the spleen was harvested 3 days post infection to determine Salmonella counts. (A) Spleen Salmonella counts of mice vaccinated with supernatant harvest from either WT 5^*.

Typhimurium (Sup), AssaR, AphoP or AfliF S. Typhimurium strains; (B) spleen Salmonella counts from mice vaccinated with Sup, LPS-free supernatant (LPS -); proteinase K and heat treated LPS free supernatant (ptnase K + heat + LPS-); supernatant contents that precipitated after 50% ammonium sulfate precipitation (50% pellet) and remaining supernatant (50% sup); supernatant pelleted with 50% ammonium sulfate and ultracentrifugated for outermembrane vesicles separation supernatant (50% pellet + ultra sup) and pellet (50% pellet OMV); (C) spleen counts of 50% ammonium sulfate precipitated supernatant and fractions after HPLC fractionation of the 50% pellet (F9, F13. F14, F 18, F19 and F22); (D) SDS-Page Gel; (E) western using the serum of immunized mice. Ctrl: saline plus adjuvant control; Sup: supernatant from SL1344 plus adjuvant. Bars indicate median; *: p< 0.05; **: p< 0.01; ***: p< 0.001; ns: not significant.

[0041] Figure 3. Supernatant is protective against S. Typhimurium gastrointestinal infection. C57BL/6J, 129Sl/SvImJ Nramp +/+ and Nramp -/- mice (N= 3-8 mice per group) were immunized orally with supernatant and CpG as adjuvant and challenged with 3 x 10⁶ cfu S. Typhimurium after streptomycin treatment. (A) Cecum Salmonella counts in

C57BL/6J mice after 3 days of infection; (B) intestinal pathology score of the cecum of C57B1/6J mice after infection, black bars represent pathology scores of the intestinal lumen, white bars represent scores of the surface epithelia, dark grey bars represent scores of the mucosa and light grey bars represent scores of the submucosa of the tissue; (C) cecum Salmonella counts in 129Sl/SvImJ Nramp +/+ and -/- mice; (D) intestinal pathology score of the cecum of Nramp +/+ and -/- after infection; (E) specific IgG levels in the serum of C57BL/6J mice immunized against gastroenteritis; (F) specific IgA levels in the serum of C57BL/6J mice immunized against gastroenteritis; (G) Specific slgA levels in feces of mice immunized against gastroenteritis. Ctrl: Saline plus adjuvant control; Sup: Supernatant from SL1344 plus adjuvant. Bars describe median. Bars in ELISA graphs describe mean and SEM. *: p<0.05; **: p<0.01 ; ***: pO.001 ; ns: not significant.

[0042] Figure 4. The general methodology used for administration of SL2251 and SL1780 is shown.

[0043] Figure 5. The bacterial load in the cecum, spleen, and liver as well as the cecal weight in mice administered SL2251 and SL1780 is shown. [0044] Figure 6. Purified SL2251 protein cross-reacts with serum from mice administered supernatant vaccine as described below.

[0045] Figure 7. HPLC of 50% (NH₄)₂S0₄ pellet (top) and each fraction. Fig. 7 also shows the western of selected fractions using the serum of previously vaccinated mice (bottom).

DETAILED DESCRIPTION

[0046] Disclosed herein are compositions and methods for the prevention or treatment of Gram negative bacterial infection and bacterial carriage such as Salmonella spp. bacterial infection and Salmonella spp. bacterial carriage, in a vertebrate subject. Methods for induction of an immune response to Salmonella spp. infection are provided. The methods provide administering a protein or agent to the vertebrate subject in need thereof in an amount effective to reduce, eliminate, or prevent the Gram negative bacterial infection or bacterial carriage (e.g., the Salmonella spp. bacterial infection or carriage or both).

[0047] Compositions and methods are provided for inducing an immune response to a Gram negative bacteria, such as a Salmonella spp. bacteria, in a subject comprising administering to the subject a composition comprising of an isolated polypeptide, such as SEQ ID NOs: 1-8, and an adjuvant in an amount effective to induce the immune response in the subject. The method can further include reducing a risk of infectious transfer from the subject to another subject, e.g., a human.

[0048] It is to be understood that the various aspects of the invention are not limited to particular methods, reagents, compounds, compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting. As used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural references unless the content clearly dictates otherwise. Thus, for example, reference to "a Salmonella spp. bacterium" includes a mixture of two or more such bacteria, and the like.

[0049] The term "about" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, ±5%, ±1%, and ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0050] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Any methods and materials similar or equivalent to those described herein can be used. [0051] "Vertebrate," "mammal," "mammalian subject," or " mammalian patient" are used interchangeably and refer to mammals such as human patients and non-human primates, as well as experimental animals such as rabbits, rats, and mice, and other animals. Animals include all vertebrates, e.g., mammals and non-mammals, such as mice, sheep, dogs, cows, avian species, ducks, geese, pigs, chickens, amphibians, and reptiles.

[0052] "Avian" and "avian subject" refers to males and females of any avian species, but is primarily intended to encompass poultry which are commercially raised for eggs, meat or as pets. Accordingly, the terms "avian" and "avian subject" are particularly intended to encompass chickens, turkeys, ducks, geese, quail, pheasant, parakeets, parrots, and the like. The avian subject can be a hatched bird, which term encompasses newly-hatched (i.e., about the first three days after hatch) as well as post-hatched birds such as, for example, adolescent, and adult birds. The avian subject can also be pre-hatch, i.e., in ovo.

[0053] "Bovine" and "bovine subject" refers to males and females of any bovine species, but is primarily intended to encompass cows which are commercially raised for milk, meat or as pets. Accordingly, the terms "bovine" and "bovine subject" are particularly intended to encompass cattle, buffalo, and the like. The bovine subject can be a post-birth bovine, which term encompasses newly -birthed (i.e., about the first three days after birth) as well as post- birth bovine such as, for example, adolescent, and adult bovines. The bovine subject can also be pre-birth.

[0054] "Porcine" and "porcine subject" refers to males and females of any porcine species, but is primarily intended to encompass pigs which are commercially raised for meat or as pets. Accordingly, the terms "porcine" and "porcine subject" are particularly intended to encompass pigs and the like. The porcine subject can be a post-birth porcine, which term encompasses newly-birthed (i.e., about the first three days after birth) as well as post-birth porcine such as, for example, adolescent, and adult porcines. The porcine subject can also be pre-birth.

[0055] As used herein, "composition" refers to a composition that serves to stimulate an immune response to a Gram-negative bacterial antigen, e.g., a Salmonella spp. antigen, such as SEQ ID NOs 1-8 and 17-24, described herein. The immune response need not provide complete protection and/or treatment against Salmonella spp. infection or against colonization and shedding of Salmonella spp. Even partial protection against colonization and shedding of Salmonella spp. bacteria will find use herein as shedding and contaminated meat production will still be reduced. In some cases, a vaccine will include an adjuvant in order to enhance the immune response. [0056] The term "adjuvant" refers to an agent which acts in a nonspecific manner to increase an immune response to a particular antigen or combination of antigens, thus, e.g., reducing the quantity of antigen necessary in any given composition and/or the frequency of injection necessary to generate an adequate immune response to the antigen of interest. See, e.g., A. C. Allison J. Reticuloendothel. Soc. (1979) 26:619-630. Such adjuvants are described further below. The term "pharmaceutically acceptable adjuvant" refers to an adjuvant that can be safely administered to a subject and is acceptable for pharmaceutical use.

[0057] As used herein, "colonization" refers to the presence of Gram negative bacteria, e.g., Salmonella spp., in the intestinal tract of a mammal, such as a ruminant.

[0058] As used herein, "shedding" refers to the presence of Gram negative bacteria, e.g.,

Salmonella spp., in feces.

[0059] "Bacterial carriage" is the process by which bacteria such as Salmonella spp. and can thrive in a normal subject without causing the subject to get sick. Bacterial carriage is a very complex interaction of the environment, the host and the pathogen. Various factors dictate asymptomatic carriage versus disease. Therefore an aspect includes treating or preventing bacterial carriage.

[0060] "Treating" or "treatment" refers to either (i) the prevention of infection or reinfection, e.g., prophylaxis, or (ii) the reduction or elimination of symptoms of the disease of interest, e.g. , therapy. "Treating" or "treatment" can refer to the administration of a composition comprising a polypeptide of interest, e.g., SEQ ID NOs 1-8 and 17-24. Treating a subject with the composition can prevent or reduce the risk of infection to humans and/or induce an immune response to the polypeptide of interest. Treatment can be prophylactic (to prevent or delay the onset of the disease, or to prevent the manifestation of clinical or subclinical symptoms thereof) or therapeutic suppression or alleviation of symptoms after the manifestation of the disease.

[0061] "Preventing" or "prevention" refers to prophylactic administration or vaccination with antigen compositions. Preventing infection of Gram negative bacteria, e.g., Salmonella spp., refers to preventing colonization of a subject. Morbidity or mortality can result from infection or colonization of a subject.

[0062] "Therapeutically-effective amount" or "an amount effective to reduce or eliminate bacterial infection" or "an effective amount" refers to an amount of polypeptide that is sufficient to prevent Gram-negative bacterial infection or to alleviate (e.g., mitigate, decrease, reduce) at least one of the symptoms associated with Salmonella spp. bacterial infection or to induce an immune response to a Gram-negative antigen (e.g., SL2251). It is not necessary that the administration of the composition eliminate the symptoms of Salmonella spp.

bacterial infection, as long as the benefits of administration of compound outweigh the detriments. Likewise, the terms "treat" and "treating" in reference to Salmonella spp.

bacterial infection, as used herein, are not intended to mean that the subject is necessarily cured of infection or that all clinical signs thereof are eliminated, only that some alleviation or improvement in the condition of the subject is effected by administration of the composition.

[0063] "Protective immunity" or "protective immune responses," are intended to mean that the subject mounts an active immune response to a composition, such that upon subsequent exposure to the Gram-negative bacteria or bacterial challenge, the subject is able to combat the infection. Thus, a protective immune response will generally decrease the incidence of morbidity and mortality from subsequent exposure to the Gram-negative bacteria among subjects. A protective immune response will also generally decrease colonization by the Gram-negative bacteria in the subjects. In this manner, transmission of infectious Gram-negative bacteria from one subject to another will be decreased and controlled. As an example, those skilled in the art will understand that in a commercial poultry setting, the production of a protective immune response can be assessed by evaluating the effects of vaccination on the flock as a whole, e.g. , there can still be morbidity and mortality in individual vaccinated birds.

[0064] "Active immune response" refers to an immunogenic response of the subject to an antigen, e.g., a polypeptide of SEQ ID NOs 1-8 and 17-24. In particular, this term is intended to mean any level of protection from subsequent exposure to Gram-negative bacteria or bacterial antigens which is of some benefit in a population of subjects, whether in the form of decreased mortality, decreased lesions, improved feed conversion ratios, or the reduction of any other detrimental effect of the disease, and the like, regardless of whether the protection is partial or complete. An "active immune response" or "active immunity" is characterized by "participation of host tissues and cells after an encounter with the immunogen. It generally involves differentiation and proliferation of immunocompetent cells in lymphoreticular tissues, which lead to synthesis of antibody or the development cell-mediated reactivity, or both." Herbert B. Herscowitz, "Immunophysiology: Cell Function and Cellular Interactions in Antibody Formation," in Immunology: Basic Processes 1 11 (Joseph A. Bellanti ed., 1985). Alternatively stated, an active immune response is mounted by the host after exposure to immunogens by infection, or as in the present case, by administration of a composition. Active immunity can be contrasted with passive immunity, which is acquired through the "transfer or preformed substances (e.g., antibody, transfer factor, thymic graft, interleukin-2) from an actively immunized host to a non-immune host." Id.

SALMONELLA

[0065] Multiple Salmonella spp. organisms are known (for example, the S. enterica serotype which includes S. enteritidis, S. typhimurium, and S. typhi). The most common Salmonella serotypes include S. typhimurium, S. enteritides and S. typhi. Other serotypes include S. heidelberg, S. newport, S. javiana, S. oranienburg, S. muenchen, S. thompson, S. paratyphi B tartrate positive, S. infantis, S. braenderup, S. infantis, S. agona, S. montevideo, and S. saintpaul. In an aspect, the Salmonella spp. organism is a non-typhoidal Salmonella spp. organism. In an aspect, the Salmonella spp. organism is S. enteritidis. In an aspect, the Salmonella spp. organism is S. typhimurium. In an aspect, the Salmonella spp. organism is S. typhi, S. typhimurium, S. enteritidis, S. gallinarum, or S. pollorum.

PEPTIDES

[0066] The term "peptide" or "polypeptide" refers to a polymer of amino acids without regard to the length of the polymer; thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide. This term also does not specify or exclude post- expression modifications of polypeptides, for example, polypeptides which include the covalent attachment of glycosyl groups, acetyl groups, phosphate groups, lipid groups and the like are expressly encompassed by the term polypeptide. Also included within the definition are polypeptides which contain one or more analogs of an amino acid (including, for example, non-naturally occurring amino acids, amino acids which only occur naturally in an unrelated biological system, modified amino acids from mammalian systems etc.), polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.

[0067] The term "isolated protein," "isolated polypeptide," or "isolated peptide" is a protein, polypeptide or peptide that by virtue of its origin or source of derivation (1) is not associated with naturally associated components that accompany it in its native state, (2) is free of other proteins from the same species, (3) is expressed by a cell from a different species, or (4) does not occur in nature. Thus, a peptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be "isolated" from its naturally associated components. A protein may also be rendered substantially free of naturally associated components by isolation, using protein purification techniques well known in the art. [0068] In one aspect, the peptide consists of, consists essentially of, or comprises an amino acid sequence selected from the group consisting of SEQ ID Nos: 1 to 8 and functionally active variants thereof. In another aspect, said peptide consists of, consists essentially of, or comprises an amino acid sequence selected from the group consisting of SEQ ID Nos: 1 to 3 and functionally active variants thereof. In another aspect, said peptide consists of, consists essentially of, or comprises an amino acid sequence shown in SEQ ID No: 7 and functionally active variants thereof.

[0069] In an aspect, the peptide consists of, or consists essentially of, an amino acid sequence selected from the group consisting of SEQ ID Nos: 1, 2, 3, 4, 5, 6, 7, and/or 8. In an aspect, the polypeptide includes an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and/or SEQ ID NO:8. In an aspect, the polypeptide includes a contiguous amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the contiguous amino acid sequence set forth in SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and/or SEQ ID NO:8. In an aspect, the polypeptide includes an amino acid sequence having at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, or 500 (or any integer within these numbers) contiguous amino acids of the amino acid sequence set forth in SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and/or SEQ ID NO:8. Examples of proteins with 90-95% identity to one or more of SEQ ID NOs 1-8 are shown below in Table A.

NP 461224.1 , ZP 09726439.1 , YP 002216351.1, ZP 02654703.1,

ZP 02662354.1 , NP 456825.1 , ZP 02343483.1 , ZP 11744868.1,

YP 217272.1 , ZP 09765001.1 , YP 004730909.1, ZP 11689362.1,

SL2251 ZP 17128542.1 , ZP 13077862.1, ZP 13381741.1, YP 001569678.1 ,

ZP 12192543.1 , ZP 1 1859608.1 , ZP 03384594.1, ZP 12186827.1, ZP 03337231.1 , ZP 14873978.1 , ZP 16279188.1, ZP 04562759.1, ZP_10408796.1 , ZP_09336339.1, ZP_06352063.1, YP_001452130.1

NP 458856.1 , ZP 09728675.1 , ZP 02833142.1 , YP 002640061.1,

SL4369

ZP_02669013.1 , YP_004732648.1

ZP 03361758.1 , ZP 121 16635.1, NP 460095.1 , ZP 02662245.1,

ZP 12191091.1 , ZP 12158524.1, NP 455617.1 , ZP 12121971.1,

SL1061 ZP 12175052.1 , ZP 03221581.1 , YP 001588638.1, ZP 02833756.1,

ZP 17127145.1 , YP 0022431 15.1, YP 005213094.1, ZP 03361254.1 , ZP 03361758.1 , ZP 121 16635.1

NP 457904.1 , YP 003739665.1, YP 001906131.1 , NP 290632.1,

YP 006098648.1, YP 003518520.1, YP 001174954.1,

SL4109

YP 001338008.1, ZP 12363404.1 , YP 048365.1, ZP 12097417.1, YP 002410264.1, ZP 09386544.1 , ZP 07381 179.1 , ZP 10555020.1

NP_455930.1 , YP_004730252.1, YP_001570448.1 , ZP_17127621.1,

SL1492

AEY77413.1, ZP_03373231.1

[0070] In an aspect, the peptides are encoded by a nucleotide sequence, e.g., SEQ ID NOs 9 to 16. Nucleotide sequences can be useful for a number of applications, including: cloning, protein expression and purification, mutation introduction, DNA vaccination of a host in need therof, antibody generation for, e.g., passive immunization, PCR, primer and probe generation, siRNA design and generation (see, e.g., the Dharmacon siDesign website), and the like. In an aspect, the nucleotide sequence consists of, or consists essentially of, a nucleotide sequence selected from the group consisting of SEQ ID Nos: 9, 10, 1 1, 12, 13, 14, 15, and/or 16. In an aspect, the nucleotide sequence includes a nucleotide sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence set forth in SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and/or SEQ ID NO: 16. In an aspect, the nucleotide sequence includes a contiguous nucleotide sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the contiguous nucleotide sequence set forth in SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 1 1, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and/or SEQ ID NO: 16. In an aspect, the nucleotide sequence includes a nucleotide sequence having at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, or 500 (or any integer within these numbers) contiguous nucleotides of the nucleotide sequence set forth in SEQ ID NO:9, SEQ ID O: 10, SEQ ID NO: 11, SEQ ID O: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and/or SEQ ID NO: 16.

[0071] In an aspect, one or more of the peptides shown in SEQ ID NOs 1 to 8 can be combined with one or more of the peptides shown in Table 3. In an aspect, the polypeptide includes an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequences set forth in Table 3. In an aspect, the polypeptide includes a contiguous amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the contiguous amino acid sequences set forth in Table 3. In an aspect, the polypeptide includes an amino acid sequence having at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, or 500 (or any integer within these numbers) contiguous amino acids of the amino acid sequences set forth in Table 3. Further detail about the peptides shown in Table 3 is found in PCT US2011/049009, herein incorporated by reference in its entirety for all purposes.

[0072] In an aspect, the peptides shown in Table 3 are encoded by a nucleotide sequence, e.g., the sequences shown in Table 4. Nucleotide sequences can be useful for a number of applications, including: cloning, protein expression and purification, mutation introduction, DNA vaccination of a host in need therof, antibody generation for, e.g., passive

immunization, PCR, primer and probe generation, siRNA design and generation (see, e.g., the Dharmacon siDesign website), and the like. In an aspect, the nucleotide sequence includes a nucleotide sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequences set forth in Table 4. In an aspect, the nucleotide sequence includes a contiguous nucleotide sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the contiguous nucleotide sequences set forth in Table 4. In an aspect, the nucleotide sequence includes a nucleotide sequence having at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, or 500 (or any integer within these numbers) contiguous nucleotides of the nucleotide sequences set forth in Table 4. Further detail about the sequences shown in Table 4 is found in PCT7US2011/049009, herein incorporated by reference in its entirety for all purposes. [0073] The term "peptide" includes SEQ ID NOs 1 to 8 and 17 to 24, as well as their variants, analogs, orthologs, homologs and derivatives, and fragments thereof that exhibit a "peptide biological activity."

[0074] The term "peptide biological activity," when used herein, refers to the ability of the peptides to induce an immune response in a patient to the peptide. The putative peptide can be assayed to ascertain the immunogenicity of the construct, in that antisera raised by the putative peptide cross-react with the native protein of interest, and are also functional in inducing an immune response to the native protein of interest.

[0075] The peptides include an amino acid sequence derived from a portion of SEQ ID Nos: 1, 2, 3, 4, 5, 6, 7, 8, 17, 18, 19, 20, 21, 22, 23, and/or 24 such derived portion either corresponding to the amino acid sequence of a naturally occurring protein or corresponding to variant protein, i.e., the amino acid sequence of the naturally occurring protein in which a small number of amino acids have been substituted, added, or deleted but which retains essentially the same immunological properties. In addition, such derived portion can be further modified by amino acids, especially at the N- and C-terminal ends to allow the peptide to be conformationally constrained and/or to allow coupling of the peptide to an immunogenic carrier after appropriate chemistry has been carried out. The peptides encompass functionally active variant peptides derived from the amino acid sequence of SEQ ID Nos: 1, 2, 3, 4, 5, 6, 7, 8, 17, 18, 19, 20, 21, 22, 23, and/or 24 in which amino acids have been deleted, inserted, or substituted without essentially detracting from the immunological properties thereof, i.e. such functionally active variant peptides retain a substantial peptide biological activity. Typically, such functionally variant peptides have an amino acid sequence homologous to an amino acid sequence selected from the group consisting of SEQ ID Nos: 1 to 8 and 17 to 24.

[0076] In one aspect, such functionally active variant peptides exhibit at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence selected from the group consisting of SEQ ID Nos: 1 to 8 and 17 to 24. Sequence similarity for polypeptides, which is also referred to as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GCG contains programs such as "Gap" and "Bestfit" which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein thereof. See, e.g., GCG Version 6.1. Polypeptide sequences also can be compared using FASTA using default or recommended parameters, a program in GCG Version 6.1. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson, Methods Enzymol. 183:63-98 (1990); Pearson, Methods Mol. Biol. 132: 185-219 (2000)). An alternative algorithm when comparing a sequence to a database containing a large number of sequences from different organisms is the computer program BLAST, especially blastp or tblastn, using default parameters. See, e.g., Altschul et al, J. Mol. Biol. 215:403-410 (1990); Altschul et al, Nucleic Acids Res. 25:3389-402 (1997).

[0077] NCBI BLAST searches can be run by one of skill in the art to identify proteins that have 80% or more sequence identity to SEQ ID NOs 1 to 3. Similar BLAST searches can also be run on non-Enterobacteriales by one of skill in the art to identify proteins that have 80% or more sequence identity to SEQ ID NOs 1 to 3. In addition, similar BLAST searches can be run by one of skill in the art to identify proteins that have 80-90% or more sequence identity to SEQ ID NOs 4 to 8 and 17 to 24 in Enterobacteriales and non- Enterobacteriales. BLAST searches can also be run by one of skill in the art to identify nucleotides that have 80-90% or more sequence identity to SEQ ID NOs 9 to 16 and 25-32 in Enterobacteriales and non-Enterobacteriales. The nucleotide and amino acid sequences associated with each accession number identified in these searches are herein incorporated by reference in their entirety, for all purposes.

[0078] Functionally active variants comprise naturally occurring functionally active variants such as allelic variants and species variants and non-naturally occurring functionally active variants that can be produced by, for example, mutagenesis techniques or by direct synthesis.

[0079] A functionally active variant differs by about, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues from any of the peptides shown at SEQ ID Nos: 1 to 8 and 17 to 24, and yet retain a biological activity. Where this comparison requires alignment the sequences are aligned for maximum homology. The site of variation can occur anywhere in the peptide, as long as the biological activity is substantially similar to a peptide shown in SEQ ID Nos: 1 to 8. Guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie et al, Science, 247: 1306-1310 (1990), which teaches that there are two main strategies for studying the tolerance of an amino acid sequence to change. The first strategy exploits the tolerance of amino acid substitutions by natural selection during the process of evolution. By comparing amino acid sequences in different species, the amino acid positions which have been conserved between species can be identified. These conserved amino acids are likely important for protein function. In contrast, the amino acid positions in which substitutions have been tolerated by natural selection indicate positions which are not critical for protein function. Thus, positions tolerating amino acid substitution can be modified while still maintaining specific immunogenic activity of the modified peptide.

[0080] The second strategy uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene to identify regions critical for protein function. For example, site-directed mutagenesis or alanine-scanning mutagenesis can be used

(Cunningham et al, Science, 244: 1081-1085 (1989)). The resulting variant peptides can then be tested for specific biological activity.

[0081] According to Bowie et al, these two strategies have revealed that proteins are surprisingly tolerant of amino acid substitutions. The authors further indicate which amino acid changes are likely to be permissive at certain amino acid positions in the protein. For example, the most buried or interior (within the tertiary structure of the protein) amino acid residues require nonpolar side chains, whereas few features of surface or exterior side chains are generally conserved.

[0082] Methods of introducing a mutation into amino acids of a protein are well known to those skilled in the art. See, e.g., Ausubel (ed.), Current Protocols in Molecular Biology, John Wiley and Sons, Inc. (1994); T. Maniatis, E. F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor laboratory, Cold Spring Harbor, N.Y. (1989)).

[0083] Mutations can also be introduced using commercially available kits such as "QuikChange Site-Directed Mutagenesis Kit" (Stratagene) or directly by peptide synthesis. The generation of a functionally active variant to a peptide by replacing an amino acid which does not significantly influence the function of said peptide can be accomplished by one skilled in the art.

[0084] A type of amino acid substitution that may be made in one of the peptides is a conservative amino acid substitution. A "conservative amino acid substitution" is one in which an amino acid residue is substituted by another amino acid residue having a side chain R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well-known to those of skill in the art. See e.g. Pearson, Methods Mol. Biol. 243:307-31 (1994).

[0085] Examples of groups of amino acids that have side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains:

asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine and methionine. Some conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine- tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine.

[0086] Alternatively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al, Science 256: 1443-45 (1992). A "moderately conservative" replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix.

[0087] A functionally active variant peptide can also be isolated using a hybridization technique. Briefly, DNA having a high homology to the whole or part of a nucleic acid sequence encoding the peptide, polypeptide or protein of interest, e.g. SEQ ID Nos: 1 to 8 and 17 to 24 is used to prepare a functionally active peptide. Therefore, a peptide also includes peptides which are functionally equivalent to one or more of the peptide of SEQ ID Nos: 1 to 8 and 17 to 24 and which are encoded by a nucleic acid molecule which hybridizes with a nucleic acid encoding any one of SEQ ID Nos: 1 to 8 and 17 to 24 or a complement thereof. One of skill in the art can easily determine nucleic acid sequences that encode peptides using readily available codon tables. As such, these nucleic acid sequences are not presented herein.

[0088] Nucleic acid molecules encoding a functionally active variant can also be isolated by a gene amplification method such as PCR using a portion of a nucleic acid molecule DNA encoding a peptide, polypeptide or protein of interest, e.g. any one of the peptides shown SEQ ID Nos: 1 to 8 and 17 to 24, as the probe.

[0089] It can be considered that several peptides may be used in combination. All types of possible combinations can be envisioned. For example, a polypeptide comprising more than one peptide selected from SEQ ID Nos: 1 to 8 and 17 to 24, could be used, wherein the same peptide is used in several copies on the same polypeptide molecule, or wherein peptides of different amino acid sequences are used on the same polypeptide molecule; the different peptides or copies being directly fused to each other or spaced by appropriate linkers. As used herein the term "multimerized (poly)peptide" refers to both types of combination wherein peptides of either different or the same amino acid sequence are present on a single polypeptide molecule. From 2 to about 20 identical and/or different peptides, e.g., 2, 3, 4, 5, 6, or 7 peptides, can be thus present on a single multimerized polypeptide molecule.

[0090] In one aspect, a peptide, polypeptide or protein is derived from a natural source and isolated from a bacterial source. A peptide, polypeptide or protein can thus be isolated from sources using standard protein purification techniques.

[0091] Alternatively, peptides, polypeptides and proteins can be synthesized chemically or produced using recombinant DNA techniques. For example, a peptide, polypeptide or protein can be synthesized by solid phase procedures well known in the art. Suitable syntheses may be performed by utilising "T-boc" or "F-moc" procedures. Cyclic peptides can be synthesised by the solid phase procedure employing the well-known "F-moc" procedure and polyamide resin in the fully automated apparatus. Alternatively, those skilled in the art will know the necessary laboratory procedures to perform the process manually. Techniques and procedures for solid phase synthesis are described in ^"Solid Phase Peptide Synthesis: A Practical Approach^" by E. Atherton and R. C. Sheppard, published by IRL at Oxford

University Press (1989) and ^"Methods in Molecular Biology, Vol. 35: Peptide Synthesis Protocols (ed. M. W.Pennington and B. M. Dunn), chapter 7, pp 91-171 by D. Andreau et al.

[0092] Alternatively, a polynucleotide encoding a peptide, polypeptide or protein can be introduced into an expression vector that can be expressed in a suitable expression system using techniques well known in the art, followed by isolation or purification of the expressed peptide, polypeptide, or protein of interest. A variety of bacterial, yeast, plant, mammalian, and insect expression systems are available in the art and any such expression system can be used. Optionally, a polynucleotide encoding a peptide, polypeptide or protein can be translated in a cell-free translation system.

[0093] SEQ ID NOs 1 to 8 and 17 to 24 can also be used to design oligonucleotide probes and used to screen genomic or cDNA libraries for genes from other Salmonella spp.

serotypes. The basic strategies for preparing oligonucleotide probes and DNA libraries, as well as their screening by nucleic acid hybridization, are well known to those of ordinary skill in the art. See, e.g., DNA Cloning: Vol. I, supra; Nucleic Acid Hybridization, supra;

Oligonucleotide Synthesis, supra; Sambrook et al., supra. Once a clone from the screened library has been identified by positive hybridization, it can be confirmed by restriction enzyme analysis and DNA sequencing that the particular library insert contains a Salmonella spp. gene, or a homolog thereof. The genes can then be further isolated using standard techniques and, if desired, PCR approaches or restriction enzymes employed to delete portions of the full-length sequence.

[0094] Similarly, genes can be isolated directly from bacteria using known techniques, such as phenol extraction and the sequence further manipulated to produce any desired alterations. See, e.g., Sambrook et al, supra, for a description of techniques used to obtain and isolate DNA. Alternatively, DNA sequences encoding the proteins of interest can be prepared synthetically rather than cloned. The DNA sequences can be designed with the appropriate codons for the particular amino acid sequence. In general, one will select codons for the intended host if the sequence will be used for expression. The complete sequence is assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. See, e.g., Edge (1981) Nature 292: 756; Nambair et al. (1984) Science 223: 1299; Jay et al. (1984) J. Biol. Chem. 259: 631 1.

[0095] Once coding sequences for the desired proteins have been prepared or isolated, they can be cloned into any suitable vector or replicon. Numerous cloning vectors are known to those of skill in the art, and the selection of an appropriate cloning vector is a matter of choice. Examples of recombinant DNA vectors for cloning and host cells which they can transform include the bacteriophage λ (E. coll), pBR322 (E. coli), pACYC177 (E. coll), pKT230 (gram-negative bacteria), pGVl 106 (gram-negative bacteria), pLAFRl (gram- negative bacteria), pME290 (non-is. coli gram-negative bacteria), pHV14 (E. coli and Bacillus subtilis), pBD9 (Bacillus), pIJ61 (Streptomyces), pUC6 (Streptomyces), YIp5 (Saccharomyces), YCpl9 (Saccharomyces) and bovine papilloma virus (mammalian cells). See, Sambrook et al., supra; DNA Cloning, supra; B. Perbal, supra. The gene can be placed under the control of a promoter, ribosome binding site (for bacterial expression) and, optionally, an operator (collectively referred to herein as "control" elements), so that the DNA sequence encoding the desired protein is transcribed into RNA in the host cell transformed by a vector containing this expression construction. The coding sequence can or can not contain a signal peptide or leader sequence. Leader sequences can be removed by the host in post-translational processing. See, e.g., U.S. Patent Nos. 4,431,739; 4,425,437; 4,338,397.

[0096] Other regulatory sequences can also be desirable which allow for regulation of expression of the protein sequences relative to the growth of the host cell. Regulatory sequences are known to those of skill in the art, and examples include those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Other types of regulatory elements can also be present in the vector, for example, enhancer sequences.

[0097] The control sequences and other regulatory sequences can be ligated to the coding sequence prior to insertion into a vector, such as the cloning vectors described above.

Alternatively, the coding sequence can be cloned directly into an expression vector which already contains the control sequences and an appropriate restriction site.

[0098] In some cases it can be necessary to modify the coding sequence so that it can be attached to the control sequences with the appropriate orientation; i.e., to maintain the proper reading frame. It can also be desirable to produce mutants or analogs of the protein. Mutants or analogs can be prepared by the deletion of a portion of the sequence encoding the protein, by insertion of a sequence, and/or by substitution of one or more nucleotides within the sequence. Techniques for modifying nucleotide sequences, such as site-directed mutagenesis, are described in, e.g., Sambrook et ah, supra; DNA Cloning, supra; Nucleic Acid

Hybridization, supra.

[0099] The expression vector is then used to transform an appropriate host cell. A number of mammalian cell lines are known in the art and include immortalized cell lines available from the American Type Culture Collection (ATCC), such as, but not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), Madin-Darby bovine kidney ("MDBK") cells, as well as others. Similarly, bacterial hosts such as E. coli, Bacillus subtilis, and Streptococcus spp., will find use with the present expression constructs. Yeast hosts include, but are not limited to, Saccharomyces cerevisiae, Candida albicans, Candida maltosa, Hansenula polymorpha, Kluyveromyces fragilis, Kluyveromyces lactis, Pichia guillerimondii, Pichia pastoris, Schizosaccharomyces pombe and Yarrowia lipolytica. Insect cells for use with baculovirus expression vectors include, but are not limited to, Aedes aegypti, Autographa californica, Bombyx mori, Drosophila melanogaster, Spodoptera fmgiperda, and Trichoplusia ni.

[00100] Expression vectors having a polynucleotide of interest, e.g. SEQ ID NOs 9 to 16 and 25 to 32, can also be vectors normally used by one of skill in the art for DNA vaccination of a host in need thereof. DNA vaccination can be used in any manner, e.g., for the first host antigenic challenge and/or for a boost challenge with the antigen of interest. General characteristics of DNA vaccination and the associated techniques are well known in the art. Approriate dosages of DNA vectors can also be readily determined using well-defined techniques for measuring whether an immune response has been generated to the antigen(s) of interest and/or whether protection has been established in the host to bacterial challenge.

[00101] Depending on the expression system and host selected, the proteins are produced by culturing host cells transformed by an expression vector described above under conditions whereby the protein of interest is expressed. The protein is then isolated from the host cells and purified. The selection of the appropriate growth conditions and recovery methods are within the skill of the art.

[00102] Salmonella spp. protein sequences can also be produced by chemical synthesis such as solid phase peptide synthesis, using known amino acid sequences or amino acid sequences derived from the DNA sequence of the genes of interest. Such methods are known to those skilled in the art. See, e.g., J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co., Rockford, IL (1984) and G. Barany and R. B.

Merrifield, The Peptides: Analysis, Synthesis, Biology, editors E. Gross and J. Meienhofer, Vol. 2, Academic Press, New York, (1980), pp. 3-254, for solid phase peptide synthesis techniques; and M. Bodansky, Principles of Peptide Synthesis, Springer-Verlag, Berlin (1984) and E. Gross and J. Meienhofer, Eds., The Peptides: Analysis, Synthesis, Biology, supra, Vol. 1, for classical solution synthesis. Chemical synthesis of peptides can be used if a small fragment of the antigen in question is capable of raising an immunological response in the subject of interest.

[00103] Peptides can also comprise those that arise as a result of the existence of multiple genes, alternative transcription events, alternative RNA splicing events, and alternative translational and postranslational events. A peptide can be expressed in systems, e.g. cultured cells, which result in substantially the same postranslational modifications present as when the peptide is expressed in a native cell, or in systems that result in the alteration or omission of postranslational modifications, e.g. glycosylation or cleavage, present when expressed in a native cell.

[00104] A peptide, polypeptide or protein can be produced as a fusion protein that contains other distinct amino acid sequences that are not SEQ ID NOs 1 to 8 and 17 to 24, such as amino acid linkers or signal sequences or immunogenic carriers, as well as ligands useful in protein purification, such as glutathione-S-transferase, histidine tag, and staphylococcal protein A. More than one peptide can be present in a fusion protein. The heterologous polypeptide can be fused, for example, to the N- terminus or C-terminus of the peptide, polypeptide or protein. A peptide, polypeptide or protein can also be produced as fusion proteins comprising homologous amino acid sequences. [00105] The peptides might be linear or conformationally constrained. In some aspects, the peptide is conformationally constrained. As used herein in reference to a molecule, the term "conformationally constrained" means a molecule, such as a peptide, polypeptide or protein, in which the three-dimensional structure is maintained substantially in one spatial arrangement over time. Conformationally constrained molecules can have improved properties such as increased affinity, metabolic stability, membrane permeability or solubility.

ADJUVANTS

[00106] Compositions can include adjuvants to further increase the immunogenicity of one or more of the Salmonella spp. proteins. Such adjuvants include any compound or compounds that act to increase an immune response to peptides or combination of peptides, thus reducing the quantity of antigen necessary in the composition, and/or the frequency of injection necessary in order to generate an adequate immune response. Suitable adjuvants include those suitable for use in mammals, e.g., in humans. Examples of known suitable adjuvants that can be used in humans include, but are not necessarily limited to, alum, aluminum phosphate, aluminum hydroxide, MF59 (4.3% w/v squalene, 0.5% w/v polysorbate 80 (Tween 80), 0.5% w/v sorbitan trioleate (Span 85)), CpG-containing nucleic acid, QS21 (saponin adjuvant), MPL (Monophosphoryl Lipid A), 3DMPL (3-O-deacylated MPL), extracts from Aquilla, ISCOMS (see, e.g., Sjolander et al. (1998) J. Leukocyte Biol. 64:713; WO90/03184, W096/1 1711, WO 00/48630, W098/36772, WOOO/41720, WO06/134423 and WO07/026190), LT/CT mutants, poly(D,L-lactide-co-glycolide) (PLG) microparticles, Quil A, interleukins, and the like. For veterinary applications including but not limited to animal experimentation, one can use Freund's, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr- MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor- MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(l'-2'-dip- almitoyl-sn- glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835 A, referred to as MTP-PE), and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion.

[00107] In some aspects, the adjuvant is Alum. In some aspects, the adjuvant includes Alum.

[00108] In some aspects, the adjuvant is TiterMax®. TiterMax® is an adjuvant that forms a stable water- in-oil emulsion. TiterMax® includes: a proprietary block copolymer CRL- 8941, squalene, a metabolizable oil, and a unique microparticulate stabilizer. Like Freund's Complete Adjuvant, TiterMax® can be used with a wide variety of antigens because it can entrap any antigen in a water-in-oil emulsion. TiterMax® can aid in the antigens effective presentation to the immune system without the toxic effects of Freunds Complete Adjuvant.

[00109] In some aspects, the adjuvant is RIBI. Ribi adjuvants are oil-in-water emulsions where antigens can be mixed with small volumes of a metabolizable oil (squalene) which are then emulsified with saline containing the surfactant Tween 80. This system also contains refined mycobacterial products (cord factor, cell wall skeleton) as immunostimulants and bacterial monophosphoryl lipid A. Three different species oriented formulations of the adjuvant system are available. These adjuvants can interact with membranes of immune cells resulting in cytokine induction, which enhances antigen uptake, processing and presentation.

[00110] In some aspects the adjuvant can include an oligonucleotide. In some aspects, the adjuvant can include a phophodiester (see U.S. Pat. No. 7,371,734, incorporated herein by reference). In some aspects, the adjuvant can include a non-DNA base (see U.S. Ser. No. 12/900,674, herein incorporated by reference). In some aspects, the adjuvant can include one or more adjuvants described in U.S. Pub. No. 2008/0220022, herein incorporated by reference. In some aspects, the adjuvant can include a mycobacterial cell wall component (see U.S. Pat. No. 6,326,357 and PCT/IB201 1/054539, each of which is herein incorporated by reference). In some aspects, the adjuvant can include saponin. In some aspects, the adjuvant can include an oil-in-water emulsion, e.g., Emulsigen. In some aspects, the adjuvant can include a carbomer base.

[00111] Further exemplary adjuvants to enhance effectiveness of the composition include, but are not limited to: (1) oil-in-water emulsion formulations (with or without other specific immunostimulating agents such as muramyl peptides (see below) or bacterial cell wall components), such as for example (a) MF59 (WO90/14837; Chapter 10 in Vaccine design: the subunit and adjuvant approach, eds. Powell & Newman, Plenum Press 1995), containing 5% Squalene, 0.5% Tween 80 (polyoxyethylene sorbitan mono-oleate), and 0.5% Span 85 (sorbitan trioleate) (optionally containing muramyl tri-peptide covalently linked to dipalmitoyl phosphatidylethanolamine (MTP-PE)) formulated into submicron particles using a microfluidizer, (b) SAF, containing 10% Squalane, 0.4% Tween 80, 5% pluronic -blocked polymer L121, and thr-MDP either micro fluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion, and (c) RIBI adjuvant system (RAS), (Ribi

Immunochem, Hamilton, Mont.) containing 2% Squalene, 0.2% Tween 80, and one or more bacterial cell wall components such as monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), e.g., MPL+CWS (DETOX); (2) saponin adjuvants, such as QS21, STIMULON (Cambridge Bioscience, Worcester, Mass.), Abisco (Isconova, Sweden), or Iscomatrix (Commonwealth Serum Laboratories, Australia), may be used or particles generated therefrom such as ISCOMs (immunostimulating complexes), which ISCOMS may be devoid of additional detergent e.g. WO00/07621; (3) Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IF A); (4) cytokines, such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 (W099/44636), etc.), interferons (e.g. gamma interferon), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), etc.; (5) monophosphoryl lipid A (MPL) or 3-O-deacylated MPL (3dMPL) e.g. GB-2220221, EP -A -0689454, optionally in the substantial absence of alum when used with pneumococcal saccharides e.g. WO00/56358; (6) combinations of 3dMPL with, for example, QS21 and/or oil-in-water emulsions e.g. EP-A-0835318, EP-A-0735898, EP-A-0761231; (7)

oligonucleotides comprising CpG motifs [Krieg Vaccine 2000, 19, 618-622; Krieg Curr opin Mol Ther2001 3: 15-24; Roman et al, Nat. Med., 1997, 3, 849-854; Weiner et al, PNAS USA, 1997, 94, 10833-10837; Davis et al, J. Immunol, 1998, 160, 870-876; Chu et al, J. Exp. Med, 1997, 186, 1623-1631; Lipford et al, Ear. J. Immunol, 1997, 27, 2340-2344; Moldoveami e/ al, Vaccine, 1988, 16, 1216-1224, Krieg et al, Nature, 1995, 374, 546-549; Klinman et al, PNAS USA, 1996, 93, 2879-2883; Ballas et al, J. Immunol, 1996, 157, 1840- 1845; Cowdery et al, J. Immunol, 1996, 156, 4570-4575; Halpern et al, Cell Immunol, 1996, 167, 72-78; Yamamoto et al, Jpn. J. Cancer Res., 1988, 79, 866-873; Stacey et al, J.

Immunol, 1996, 157,2116-2122; Messina et al, J. Immunol, 1991, 147, 1759-1764; Yi et al, J. Immunol, 1996, 157,4918-4925; Yi et al, J. Immunol, 1996, 157, 5394-5402; Yi et al, J. Immunol, 1998, 160, 4755-4761; and Yi et al, J. Immunol, 1998, 160, 5898-5906;

International patent applications WO96/02555, W098/16247, WO98/18810, WO98/40100, W098/55495, W098/37919 and W098/52581] i.e. containing at least one CG dinucleotide, where the cytosine is unmethylated; (8) a polyoxyethylene ether or a polyoxyethylene ester e.g. W099/52549; (9) a polyoxyethylene sorbitan ester surfactant in combination with an octoxynol (WOO 1/21207) or a polyoxyethylene alkyl ether or ester surfactant in combination with at least one additional non-ionic surfactant such as an octoxynol (WO01/21152); (10) a saponin and an immunostimulatory oligonucleotide (e.g. a CpG oligonucleotide)

(WO00/62800); (11) an immunostimulant and a particle of metal salt e.g. WO00/23105; (12) a saponin and an oil-in-water emulsion e.g. W099/11241; (13) a saponin (e.g.

QS21)+3dMPL+IM2 (optionally+a sterol) e.g. W098/57659; (14) other substances that act as immunostimulating agents to enhance the efficacy of the composition, such as Muramyl peptides include N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-25 acetyl- normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D- isoglutarninyl-L-alanine-2-(r-2'-dipalmitoyl— sn-glycero-3-hydroxyphosphoryloxy)- ethylamine MTP-PE), (15) ligands for toll-like receptors (TLR), natural or synthesized (e.g. as described in Kanzler et al 2007, Nature Medicine 13, pl552-9), including TLR3 ligands such as polyl:C and similar compounds such as Hiltonol and Ampligen.

[00112] Adjuvants can also include for example, emulsifiers, muramyl dipeptides, avridine, aqueous adjuvants such as aluminum hydroxide, chitosan-based adjuvants, and any of the various saponins, oils, and other substances known in the art, such as Amphigen, LPS, bacterial cell wall extracts and complexes, bacterial DNA, synthetic oligonucleotides and combinations thereof (Schijns et al, Curr. Opi. Immunol. (2000) 12: 456). Other adjuvants and immunstimulants include mycobacterial cell wall based (US Patent No. 5,759,554, US Patent No. 6326357, PCT application No. PCT/IB201 1/054539), phosphodiester and non- DNA based oligonucleotides (US Patent No. 7371734, US Patent application No.

12/900674). For example, compounds which can serve as emulsifiers herein include natural and synthetic emulsifying agents, as well as anionic, cationic and nonionic compounds. Among the synthetic compounds, anionic emulsifying agents include, for example, the potassium, sodium and ammonium salts of lauric and oleic acid, the calcium, magnesium and aluminum salts of fatty acids (i.e., metallic soaps), and organic sulfonates such as sodium lauryl sulfate. Synthetic cationic agents include, for example, cetyltrhethylammonlum bromide, while synthetic nonionic agents are exemplified by glycerylesters (e.g., glyceryl monostearate), polyoxyethylene glycol esters and ethers, and the sorbitan fatty acid esters (e.g., sorbitan monopalmitate) and their polyoxyethylene derivatives (e.g., polyoxyethylene sorbitan monopalmitate). Natural emulsifying agents include acacia, gelatin, lecithin and cholesterol.

[00113] Other suitable adjuvants can be formed with an oil component, such as a single oil, a mixture of oils, a water-in-oil emulsion, or an oil-in-water emulsion. The oil can be a mineral oil, a vegetable oil, or animal oil. Mineral oil or oil-in-water emulsions in which the oil component is mineral oil are contemplated. In this regard, a "mineral oil" is defined herein as a mixture of liquid hydrocarbons obtained from petrolatum via a distillation technique; the term is synonymous with "liquid paraffin," "liquid petrolatum" and "white mineral oil." The term is also intended to include "light mineral oil," i.e., oil which is similarly obtained by distillation of petrolatum, but which has a slightly lower specific gravity than white mineral oil. See, e.g., Remington 's Pharmaceutical Sciences, supra. An oil component can be the oil- in-water emulsion sold under the trade name of EMULSIGEN PLUS (comprising a light mineral oil as well as 0.05% formalin, and 30 mcg/mL gentamicin as preservatives), available from MVP Laboratories, Ralston, Nebraska. Suitable animal oils include, for example, cod liver oil, halibut oil, menhaden oil, orange roughy oil and shark liver oil, all of which are available commercially. Suitable vegetable oils include, without limitation, canola oil, almond oil, cottonseed oil, corn oil, olive oil, peanut oil, safflower oil, sesame oil, soybean oil, and the like.

[00114] Alternatively, a number of aliphatic nitrogenous bases can be used as adjuvants with the vaccine formulations. For example, known immunologic adjuvants include mines, quaternary ammonium compounds, guanidines, benzamidines and thiouroniums (Gall, D. (1966) Immunology 11: 369-386). Specific compounds include

dimethyldioctadecylammoniumbromide (DDA) (available from Kodak) and N,N- dioctadecyl-N,N-bis(2-hydroxyethyl)propanediine ("avridine"). The use of DDA as an immunologic adjuvant has been described; see, e.g., the Kodak Laboratory Chemicals Bulletin 56(1): 1-5 (1986); Adv. Drug Deliv. Rev. 5(3): 163- 187 (1990); J. Controlled Release 7: 123-132 (1988); Clin. Exp. Immunol. 78(2): 256-262 (1989); J. Immunol. Methods 97(2): 159-164 (1987); Immunology 58(2): 245-250 (1986); and /«/. Arch. Allergy Appl. Immunol. 68(3): 201-208 (1982). Avridine is also a well-known adjuvant. See, e.g., U.S. Patent No. 4,310,550 to Wolff, III et al, which describes the use of Ν,Ν-higher alkyl-N',N'- bis(2-hydroxyethyl)propane diamines in general, and avridine in particular, as vaccine adjuvants. U.S. Patent No. 5, 151,267 to Babiuk, and Babiuk et al. (1986) Virology 159: 57- 66, also relate to the use of avridine as a vaccine adjuvant.

[00115] An adjuvant for use with the vaccine is "VSA3" which is a modified form of the EMULSIGEN PLUS™ adjuvant which includes DDA (see, U.S. Patent No. 5,951,988, incorporated herein by reference in its entirety).

[00116] Compositions including one or more of peptides can be prepared by uniformly and intimately bringing into association the composition preparations and the adjuvant using techniques well known to those skilled in the art including, but not limited to, mixing, sonication and microfluidation. The adjuvant can comprise about 10 to 50% (v/v) of the composition, about 20 to 40% (v/v) and about 20 to 30% or 35% (v/v), or any integer within these ranges. PHARMACEUTICAL COMPOSITIONS

[00117] An aspect provides a composition comprising an effective immunizing amount of of an isolated peptide and a pharmaceutically acceptable carrier, wherein the composition is effective in a vertebrate subject to reduce or eliminate Gram-negative bacterial infection. A further aspect provides pharmaceutical compositions comprising nucleic acids (e.g. nucleic acids that encode SEQ ID NOs 1 to 8 and 17 to 24), polypeptides (including antibodies that target SEQ ID NOs 1 to 8 and 17 to 24), a peptidomimetic, a small non-nucleic acid organic molecule, or a small inorganic molecule.

[00118] The compositions are normally prepared as injectables, either as liquid solutions or suspensions, or as solid forms which are suitable for solution or suspension in liquid vehicles prior to injection. The preparation can also be prepared in solid form, emulsified or the active ingredient encapsulated in liposome vehicles or other particulate carriers used for sustained delivery. For example, the vaccine can be in the form of an oil emulsion, water in oil emulsion, water-in-oil-in-water emulsion, site-specific emulsion, long-residence emulsion, sticky emulsion, microemulsion, nanoemulsion, liposome, microparticle, microsphere, nanosphere, nanoparticle and various natural or synthetic polymers, such as nonresorbable impermeable polymers such as ethylenevinyl acetate copolymers and Hytrel® copolymers, swellable polymers such as hydrogels, or resorbable polymers such as collagen and certain polyacids or polyesters such as those used to make resorbable sutures, that allow for sustained release of the vaccine.

[00119] Peptides are formulated into compositions for delivery to a mammalian subject. The composition is administered alone, and/or mixed with a pharmaceutically acceptable vehicle or excipient. Suitable vehicles are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. In addition, the vehicle can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants in the case of compositions, which enhance the effectiveness of the composition. Suitable adjuvants are described above. The compositions can also include ancillary substances, such as pharmacological agents, cytokines, or other biological response modifiers.

[00120] Furthermore, the compositions including, for example, one or more of SEQ ID NOs 1 to 8 and 17 to 24 can be formulated into compositions in either neutral or salt forms. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the active polypeptides) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric, mandelic, and the like. Salts formed from free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

[00121] Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in the art. See, e.g., Remington 's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, current edition.

[00122] The composition is formulated to contain an effective amount of a protein, the exact amount being readily determined by one skilled in the art, wherein the amount depends on the animal to be treated and the capacity of the animal's immune system to synthesize antibodies. The composition or formulation to be administered will contain a quantity of one or more secreted proteins adequate to achieve the desired state in the subject being treated. A therapeutically effective amount of a composition comprising a protein, can contain about 0.05 to 1500 μg protein, about 10 to 1000 μg protein, about 30 to 500 μg and about 40 to 300 pg, or any integer between these values. For example, peptides can be administered to a subject at a dose of about 0.1 μg to about 200 mg, e.g., from about 0.1 μg to about 5 μg, from about 5 μg to about 10 μg, from about 10 μg to about 25 μg, from about 25 μg to about 50 μg, from about 50 μg to about 100 μg, from about 100 μg to about 500 μg, from about 500 μg to about 1 mg, from about 1 mg to about 2 mg, with optional boosters given at, for example, 1 week, 2 weeks, 3 weeks, 4 weeks, two months, three months, 6 months and/or a year later. For prophylaxis purposes, the amount of peptide in each dose is selected as an amount which induces an immunoprotective response without significant adverse side effects in typical vaccinees. Following an initial vaccination, subjects may receive one or several booster immunisations adequately spaced. It is understood that the specific dose level for any particular patient depends upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the particular disease undergoing therapy.

[00123] Routes of administration include, but are not limited to, oral, topical,

subcutaneous, intramuscular, intravenous, subcutaneous, intradermal, transdermal and subdermal. Depending on the route of administration, the volume per dose is about 0.001 to 10 ml, about 0.01 to 5 ml, and about 0.1 to 3 ml. Compositions can be administered in a single dose treatment or in multiple dose treatments (boosts) on a schedule and over a time period appropriate to the age, weight and condition of the subject, the particular vaccine formulation used, and the route of administration.

[00124] In some aspects, a single dose of peptide or pharmaceutical composition is administered. In other aspects, multiple doses of a peptide or pharmaceutical composition are administered. The frequency of administration can vary depending on any of a variety of factors, e.g., severity of the symptoms, degree of immunoprotection desired, whether the composition is used for prophylactic or curative purposes, etc. For example, in some aspects, a peptide or pharmaceutical composition is administered once per month, twice per month, three times per month, every other week (qow), once per week (qw), twice per week (biw), three times per week (tiw), four times per week, five times per week, six times per week, every other day (qod), daily (qd), twice a day (qid), or three times a day (tid). When the composition is used for prophylaxis purposes, they will be generally administered for both priming and boosting doses. It is expected that the boosting doses will be adequately spaced, or given yearly or at such times where the levels of circulating antibody fall below a desired level. Boosting doses may consist of the peptide in the absence of the original immunogenic carrier molecule. Such booster constructs may comprise an alternative immunogenic carrier or may be in the absence of any carrier. Such booster compositions may be formulated either with or without adjuvant.

[00125] The duration of administration of a peptide, e.g., the period of time over which a peptide is administered, can vary, depending on any of a variety of factors, e.g., patient response, etc. For example, a peptide can be administered over a period of time ranging from about one day to about one week, from about two weeks to about four weeks, from about one month to about two months, from about two months to about four months, from about four months to about six months, from about six months to about eight months, from about eight months to about 1 year, from about 1 year to about 2 years, or from about 2 years to about 4 years, or more.

[00126] Any suitable pharmaceutical delivery means can be employed to deliver the compositions to the vertebrate subject, e.g., an avian subject or mammalian subject. For example, conventional needle syringes, spring or compressed gas (air) injectors (U.S. Patent Nos. 1,605,763 to Smoot; 3,788,315 to Laurens; 3,853,125 to Clark et al ; 4,596,556 to Morrow et ah ; and 5,062,830 to Dunlap), liquid jet injectors (U.S. Patent Nos. 2,754,818 to Scherer; 3,330,276 to Gordon; and 4,518,385 to Lindcaner et al), and particle injectors (U.S. Patent Nos. 5, 149,655 to McCabe et al. and 5,204,253 to Sanford et al.) are all appropriate for delivery of the compositions.

[00127] If ajet injector is used, a single jet of the liquid vaccine composition is ejected under high pressure and velocity, e.g., 1200-1400 PSI, thereby creating an opening in the skin and penetrating to depths suitable for immunization.

[00128] The compositions, or nucleic acids, or polypeptides can be combined with a pharmaceutically acceptable carrier (excipient) to form a pharmacological composition. Pharmaceutically acceptable carriers can contain a physiologically acceptable compound that acts to, e.g., stabilize, or increase or decrease the absorption or clearance rates of the pharmaceutical compositions. Physiologically acceptable compounds can include, e.g., carbohydrates, such as glucose, sucrose, or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, compositions that reduce the clearance or hydrolysis of the peptides or polypeptides, or excipients or other stabilizers and/or buffers. Detergents can also used to stabilize or to increase or decrease the absorption of the pharmaceutical composition, including liposomal carriers. Pharmaceutically acceptable carriers and formulations for peptides and polypeptide are known to the skilled artisan and are described in detail in the scientific and patent literature, see e.g., the latest edition of Remington's Pharmaceutical Science, Mack Publishing Company, Easton, Pa.

("Remington's").

[00129] Other physiologically acceptable compounds include wetting agents, emulsifying agents, dispersing agents or preservatives which are particularly useful for preventing the growth or action of microorganisms. Various preservatives are well known and include, e.g., phenol and ascorbic acid. One skilled in the art would appreciate that the choice of a pharmaceutically acceptable carrier including a physiologically acceptable compound depends, for example, on the route of administration of the peptide or polypeptide and on its particular physio-chemical characteristics.

[00130] In one aspect, a solution of the composition or nucleic acids, peptides or polypeptides are dissolved in a pharmaceutically acceptable carrier, e.g., an aqueous carrier if the composition is water-soluble. Examples of aqueous solutions that can be used in formulations for enteral, parenteral or transmucosal drug delivery include, e.g., water, saline, phosphate buffered saline, Hank's solution, Ringer's solution, dextrose/saline, glucose solutions and the like. The formulations can contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as buffering agents, tonicity adjusting agents, wetting agents, detergents and the like. Additives can also include additional active ingredients such as bactericidal agents, or stabilizers. For example, the solution can contain sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, or triethanolamine oleate. These compositions can be sterilized by conventional, well-known sterilization techniques, or can be sterile filtered. The resulting aqueous solutions can be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration. The concentration of peptide in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the patient's needs.

[00131] Solid formulations can be used for enteral (oral) administration. They can be formulated as, e.g., pills, tablets, powders or capsules. For solid compositions, conventional nontoxic solid carriers can be used which include, e.g., pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. For oral administration, a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10% to 95% of active ingredient (e.g., peptide). A non-solid formulation can also be used for enteral administration. The carrier can be selected from various oils including those of petroleum, animal, vegetable or synthetic origin, e.g., peanut oil, soybean oil, mineral oil, sesame oil, and the like. Suitable

pharmaceutical excipients include e.g., starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol.

[00132] Compositions or nucleic acids, polypeptides, or small chemical molecules, when administered orally, can be protected from digestion. This can be accomplished either by complexing the nucleic acid, peptide or polypeptide with a composition to render it resistant to acidic and enzymatic hydrolysis or by packaging the nucleic acid, peptide or polypeptide in an appropriately resistant carrier such as a liposome. Means of protecting compounds from digestion are well known in the art, see, e.g., Fix, Pharm Res. 13: 1760-1764, 1996;

Samanen, J. Pharm. Pharmacol. 48: 119-135, 1996; U.S. Pat. No. 5,391,377, describing lipid compositions for oral delivery of therapeutic agents (liposomal delivery is discussed in further detail, infra).

[00133] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated can be used in the formulation. Such penetrants are generally known in the art, and include, e.g., for transmucosal administration, bile salts and fusidic acid derivatives. In addition, detergents can be used to facilitate permeation. Transmucosal administration can be through nasal sprays or using suppositories. See, e.g., Sayani, Crit. Rev. Ther. Drug Carrier Syst. 13: 85-184, 1996. For topical, transdermal administration, the agents are formulated into ointments, creams, salves, powders and gels. Transdermal delivery systems can also include, e.g., patches.

[00134] Compositions or nucleic acids, polypeptides, or small chemical molecule can also be administered in sustained delivery or sustained release mechanisms, which can deliver the formulation internally. For example, biodegradeable microspheres or capsules or other biodegradeable polymer configurations capable of sustained delivery of a peptide can be included in the formulations (see, e.g., Putney, Nat. Biotechnol. 16: 153-157, 1998).

[00135] For inhalation, compositions or nucleic acids, nucleic acids, polypeptides, or small chemical molecule as aspects can be delivered using any system known in the art, including dry powder aerosols, liquids delivery systems, air jet nebulizers, propellant systems, and the like. See, e.g., Patton, Biotechniques 16: 141-143, 1998; product and inhalation delivery systems for polypeptide macromolecules by, e.g., Dura Pharmaceuticals (San Diego, Calif), Aradigrn (Hayward, Calif), Aerogen (Santa Clara, Calif), Inhale Therapeutic Systems (San Carlos, Calif), and the like. For example, the pharmaceutical formulation can be

administered in the form of an aerosol or mist. For aerosol administration, the formulation can be supplied in finely divided form along with a surfactant and propellant. In another aspect, the device for delivering the formulation to respiratory tissue is an inhaler in which the formulation vaporizes. Other liquid delivery systems include, e.g., air jet nebulizers.

[00136] Avian subjects can be administered the vaccine compositions or nucleic acids, polypeptides, or small chemical molecule as aspects by any suitable means. Exemplary means are oral administration (e.g., in the feed or drinking water), intramuscular injection, subcutaneous injection, intravenous injection, intra-abdominal injection, eye drop, or nasal spray. Avian subjects can also be administered the compounds in a spray cabinet, i.e., a cabinet in which the birds are placed and exposed to a vapor containing vaccine, or by coarse spray. When administering the compounds described herein to birds post-hatch,

administration by subcutaneous injection or spray cabinet are commonly used techniques.

[00137] The compositions or nucleic acids, polypeptides, or small chemical molecule as aspects can also be administered in ovo. The in ovo administration of the compounds involves the administration of the compounds to the avian embryo while contained in the egg. The compounds can be administered to any suitable compartment of the egg (e.g., allantois, yolk sac, amnion, air cell, or into the avian embryo itself), as would be apparent to one skilled in the art. Eggs administered the compounds can be fertile eggs which in the last half, and/or the last quarter, of incubation. Chicken eggs can be treated on about day 18 of incubation, although other time periods can be employed. Those skilled in the art will appreciate that the various aspects of the present invention can be carried out at various predetermined times in ovo.

[00138] Eggs can be administered the vaccine compositions or nucleic acids, polypeptides, or small chemical molecule by any means which transports the compound through the shell. A common method of administration is, however, by injection. For example, the compound can be injected into an extraembryonic compartment of the egg (e.g., yolk sac, amnion, allantois, air cell) or into the embryo itself. As an example, the site of injection can be within the region defined by the amnion, including the amniotic fluid and the embryo itself. By the beginning of the fourth quarter of incubation, the amnion is sufficiently enlarged that penetration thereof is assured nearly all of the time when the injection is made from the center of the large end of the egg along the longitudinal axis.

[00139] The mechanism of egg injection is not critical, but it is contemplated that the methods not unduly damage the tissues and organs of the embryo or the extraembryonic membranes surrounding it so that the treatment will not decrease hatch rate. The size of the needle and the length of penetration can be determined by one skilled in the art. A pilot hole can be punched or drilled through the shell prior to insertion of the needle to prevent damaging or dulling of the needle. If desired, the egg can be sealed with a substantially bacteria-impermeable sealing material such as wax or the like to prevent subsequent entry of undesirable bacteria.

[00140] In preparing pharmaceuticals a variety of formulation modifications can be used and manipulated to alter pharmacokinetics and biodistribution. A number of methods for altering pharmacokinetics and biodistribution are known to one of ordinary skill in the art. Examples of such methods include protection of the compositions in vesicles composed of substances such as proteins, lipids (for example, liposomes, see below), carbohydrates, or synthetic polymers (discussed above). For a general discussion of pharmacokinetics, see, e.g., Remington's, Chapters 37-39.

[00141] Compositions or nucleic acids, polypeptides, or small chemical molecule can be delivered alone or as pharmaceutical compositions by any means known in the art, e.g., systemically, regionally, or locally (e.g., directly into, or directed to, a tumor); by

intraarterial, intrathecal (IT), intravenous (IV), parenteral, intra-pleural cavity, topical, oral, or local administration, as subcutaneous, intra-tracheal (e.g., by aerosol) or transmucosal (e.g., buccal, bladder, vaginal, uterine, rectal, nasal mucosa). Actual methods for preparing administrable compositions will be known or apparent to those skilled in the art and are described in detail in the scientific and patent literature, see e.g., Remington's. For a

"regional effect," e.g., to focus on a specific organ, one mode of administration includes intra-arterial or intrathecal (IT) injections, e.g., to focus on a specific organ, e.g., brain and CNS (see e.g., Gurun, Anesth Analg. 85: 317-323, 1997). For example, intra-carotid artery injection where it is desired to deliver a nucleic acid, peptide or polypeptide directly to the brain. Parenteral administration is a route of delivery if a high systemic dosage is needed. Actual methods for preparing parenterally administrable compositions will be known or apparent to those skilled in the art and are described in detail, in e.g., Remington's, See also, Bai, J. Neuroimmunol. 80: 65-75, 1997; Warren, J. Neurol. Sci. 152: 31-38, 1997; Tonegawa, J. Exp. Med. 186: 507-515, 1997.

[00142] In one aspect, the pharmaceutical formulations comprising compositions or nucleic acids, polypeptides, or small chemical molecule are incorporated in lipid monolayers or bilayers, e.g., liposomes, see, e.g., U.S. Pat. Nos. 6, 110,490; 6,096,716; 5,283, 185;

5,279,833. Aspects also provide formulations in which water soluble nucleic acids, peptides or polypeptides have been attached to the surface of the monolayer or bilayer. For example, peptides can be attached to hydrazide-PEG-(distearoylphosphatidyl) ethanolamine-containing liposomes (see, e.g., Zalipsky, Bioconjug. Chem. 6: 705-708, 1995). Liposomes or any form of lipid membrane, such as planar lipid membranes or the cell membrane of an intact cell, e.g., a red blood cell, can be used. Liposomal formulations can be by any means, including administration intravenously, transdermally (see, e.g., Vutla, J. Pharm. Sci. 85: 5-8, 1996), transmucosally, or orally. Also provided are pharmaceutical preparations in which the nucleic acid, peptides and/or polypeptides are incorporated within micelles and/or liposomes (see, e.g., Suntres, J. Pharm. Pharmacol. 46: 23-28, 1994; Woodle, Pharm. Res. 9: 260-265, 1992). Liposomes and liposomal formulations can be prepared according to standard methods and are also well known in the art, see, e.g., Remington's; Akimaru, Cytokines Mol. Ther. 1: 197-210, 1995; Alving, Immunol. Rev. 145: 5-31, 1995; Szoka, Ann. Rev. Biophys. Bioeng. 9: 467, 1980, U.S. Pat. Nos. 4, 235,871, 4,501,728 and 4,837,028.

[00143] In one aspect, the compositions are prepared with carriers that will protect the protein against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,81 1.

[00144] It is advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[00145] Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD5₀/ED5₀. Compounds that exhibit high therapeutic indices are contemplated. While compounds that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

[00146] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies within a range of circulating concentrations that include the ED5₀ with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models, e.g., of inflammation or disorders involving undesirable inflammation, to achieve a circulating plasma concentration range that includes the IC₅₀ (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography, generally of a labeled agent. Animal models useful in studies, e.g., preclinical protocols, are known in the art, for example, animal models for inflammatory disorders such as those described in Sonderstrup (Springer, Sem. Immunopathol. 25: 35-45, 2003) and Nikula et al, Inhal. Toxicol. 4(12): 123-53, 2000), and those known in the art, e.g., for Gram-negative bacterial infection, e.g., Salmonella spp. infection.

[00147] As defined herein, a therapeutically effective amount of compositions, protein or polypeptide such as an antibody (i.e., an effective dosage) can range from about 0.001 to 30 to 500 mg/kg body weight, for example, about 0.01 to 25 mg/kg body weight, about 0.1 to 20 mg/kg body weight, or about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. The protein or polypeptide can be administered one or several times per day or per week for between about 1 to 10 weeks, for example, between 2 to 8 weeks, between about 3 to 7 weeks, or about 4, 5, or 6 weeks. In some instances the dosage can be required over several months or more. The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including, but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of an agent such as a protein or polypeptide (including an antibody) can include a single treatment or can include a series of treatments.

[00148] For antibodies, the dosage is generally 0.1 mg/kg of body weight (for example, 10 mg/kg to 20 mg/kg). Partially human antibodies and fully human antibodies generally have a longer half-life within the human body than other antibodies. Accordingly, lower dosages and less frequent administration is often possible. Modifications such as lipidation can be used to stabilize antibodies and to enhance uptake and tissue penetration (e.g., into the brain). A method for lipidation of antibodies is described by Cruikshank et ah, J. Acquired Immune Deficiency Syndromes and Human Retrovirology , 14: 193, 1997).

[00149] Also encompassed is a composition comprising an effective immunizing amount of an isolated Salmonella spp. protein and a pharmaceutically acceptable carrier, wherein said composition is effective in a vertebrate subject to reduce or eliminate Gram-negative bacterial infection.

[00150] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[00151] Compounds as described herein can be used for the preparation of a medicament for use in any of the methods of treatment described herein.

[00152] The pharmaceutical compositions are generally formulated as sterile, substantially isotonic and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration. TREATMENT REGIMENS: PHARMACOKINETICS

[00153] The pharmaceutical composition aspects can be administered in a variety of unit dosage forms depending upon the method of administration. Dosages for typical vaccine compositions or nucleic acids, peptide and polypeptide pharmaceutical compositions are well known to those of skill in the art. Such dosages are typically advisory in nature and are adjusted depending on the particular therapeutic context or patient tolerance. The amount of nucleic acid, peptide or polypeptide adequate to accomplish this is defined as a

"therapeutically effective dose." The dosage schedule and amounts effective for this use, i.e., the "dosing regimen," will depend upon a variety of factors, including the stage of the disease or condition, the severity of the disease or condition, the general state of the patient's health, the patient's physical status, age, pharmaceutical formulation and concentration of active agent, and the like. In calculating the dosage regimen for a patient, the mode of

administration also is taken into consideration. The dosage regimen must also take into consideration the pharmacokinetics, i.e., the pharmaceutical composition's rate of absorption, bioavailability, metabolism, clearance, and the like. See, e.g., the latest Remington's;

Egleton, Peptides 18: 1431-1439, 1997; Langer, Science 249: 1527-1533, 1990.

[0001] In therapeutic applications, compositions are administered to a patient at risk for Gram negative bacterial infection such as Salmonella spp. bacterial infection or suffering from Gram negative bacterial infection such as Salmonella spp. , in an amount sufficient to at least partially arrest or prevent the condition or a disease and/or its complications.

Compositions are administered to a patient at risk for Gram negative bacterial carriage such as Salmonella spp. bacterial carriage or suffering from Gram negative bacterial carriage such as Salmonella spp. For example, in one aspect, vaccine composition comprising a soluble peptide pharmaceutical composition dosage for intravenous (IV) administration would be about 0.01 mg/hr to about 1.0 mg/hr administered over several hours (typically 1, 3, or 6 hours), which can be repeated for weeks with intermittent cycles. Considerably higher dosages (e.g., ranging up to about 10 mg/ml) can be used, particularly when the drug is administered to a secluded site and not into the blood stream, such as into a body cavity or into a lumen of an organ, e.g., the cerebrospinal fluid (CSF).

METHODS OF TREATMENT

[00154] Also described herein are both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or a method of preventing or treating a Gram negative bacterial infection, e.g., Salmonella spp. infection by administering a composition. PROPHYLACTIC METHODS

[00155] An aspect relates to methods for preventing or treating in a subject a Salmonella spp. bacterial infection or bacterial carriage or both by administering a composition comprising an effective immunizing amount a protein and a pharmaceutically acceptable carrier, wherein the composition is effective in a vertebrate subject to reduce or eliminate Gram-negative bacterial infection. The composition can also include an antagonist of Type III secretion systems administered to the vertebrate subject in an amount effective to reduce or eliminate the bacterial infection or bacterial carriage. Subjects at risk for a disorder or undesirable symptoms that are caused or contributed to by Salmonella spp. bacterial infection and bacterial carriage can be identified by, for example, any of a combination of diagnostic or prognostic assays as described herein or is known in the art. In general, such disorders involve undesirable activation of the innate immune system, e.g., as a result of Salmonella spp. bacterial infection or bacterial carriage. Administration of the agent as a prophylactic agent can occur prior to the manifestation of symptoms, such that the symptoms are prevented, delayed, or diminished compared to symptoms in the absence of the agent.

THERAPEUTIC METHODS

[00156] An aspect relates to methods for preventing or treating in a subject a Salmonella spp. bacterial infection or bacterial carriage by administering a composition comprising an effective immunizing amount of a protein and a pharmaceutically acceptable carrier, wherein the composition is effective in a vertebrate subject to reduce or eliminate Gram-negative bacterial infection.

[00157] Also provided are methods for preventing or treating an individual affected by a disease or disorder, e.g., Gram negative bacterial infection and more specifically, a

Salmonella spp. bacterial infection or bacterial carriage. In one aspect, the method involves administering a composition comprising an effective immunizing amount of an isolated protein and a pharmaceutically acceptable carrierConditions that can be treated by compositions include those in which a subject is treated for Gram negative bacterial infection, e.g., Salmonella spp. infection.

KITS

[00158] Also provided are kits comprising one or more compositions described herein, e.g., nucleic acids, expression cassettes, vectors, cells, and polypeptides. The kits also can contain instructional material teaching the methodologies and uses, as described herein.

THERAPEUTIC APPLICATIONS [00159] The compositions identified by the methods can be used in a variety of methods of treatment. Thus, compositions and methods for treating Gram negative bacterial infection such as Salmonella spp. infection, and Gram negative bacterial carriage, such as Salmonella spp. carriage are disclosed.

[00160] Exemplary infectious disease, include but are not limited to, bacterial diseases. The polypeptide or polynucleotide can be used to treat or detect infectious agents. For example, by increasing the immune response, particularly increasing the proliferation and differentiation of B and/or T cells, infectious diseases can be treated. The immune response can be increased by either enhancing an existing immune response, or by initiating a new immune response. Alternatively, the polypeptide or polynucleotide can also directly inhibit the infectious agent, without necessarily eliciting an immune response.

[00161] Exemplary infectious disease, include but are not limited to, Gram negative infections. Gram-negative bacterial agents that can cause disease or symptoms and that can be treated or detected by a polynucleotide or polypeptide include, but not limited to, the following Gram-negative bacterial families. Bacteremia can be caused by Gram-negative bacteria. Gram-negative bacteria have thin walled cell membranes consisting of a single layer of peptidoglycan and an outer layer of lipopolysacchacide, lipoprotein, and phospholipid. Exemplary Gram-negative organisms include, but are not limited to, Enterobacteriacea consisting of Campylobacter, Escherichia, Shigella, Edwardsiella, Salmonella, Citrobacter, Klebsiella, Enterobacter, Hafnia, Serratia, Proteus, Morganella, Providencia, Yersinia, Erwinia, Buttlauxella, Cedecea, Ewingella, Kluyvera, Tatumella and Rahnella. Other exemplary Gram-negative organisms not in the family Enterobacteriacea include, but are not limited to, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Burkholderia, Cepacia, Gardenerella, Vaginalis, and Acinetobacter species.

[00162] Treatment using a composition or a polypeptide or polynucleotide could either be by administering an effective amount of a polypeptide to the patient having a Gram negative bacterial infection or at risk for a Gram negative bacterial infection (for example, Salmonella spp. bacterial infection). Moreover, the polypeptide or polynucleotide can be used as an antigen in a composition to raise an immune response against Gram negative bacterial infectious disease, e.g., a Salmonella spp. infectious disease. In such cases, antibodies can be harvested from the subject for use in passive immunity for the same subject at a later date or in a distinct subject. [00163] The following examples of specific aspects for carrying out some aspects of the present invention are offered for illustrative purposes only, and are not intended to limit the scope of the aspects in any way.

EXAMPLES

Materials and Methods

[00164] Bacterial strains and supernatant preparations.

[00165] We harvested supernatants from S. Typhimurium (SL1344) grown under conditions that selectively induce for SPI-2 secretion (27). Briefly, S. Typhimurium were grown overnight in LB broth, washed twice in low phosphate, low magnesium-containing medium (LPM) and then inoculated at a 1 :50 dilution in 3 ml of LPM medium at pH 5.8. The composition of LPM medium was 5 mM KC1, 7.5 mM (NH4)₂S0₄, 0.5 mM K₂S0₄, 38 mM glycerol (0.3% v/v), 0.1% casamino acids, 8 M MgCl₂, 337 M P0₄, 80 mM MES (for titration to pH 5.8). Cultures were grown at 37 °C with shaking for 4-6 h after which the optical density at 600 nm was measured. Bacteria were collected by centrifugation for 2 min at 12,000 rpm (4 °C). The supernatant was passed through a 0.22-μιη filter. To obtain a highly concentrated supernatant, S. Typhimurium was grown at the same media described above in the 30 L MBR fermenter a at the National Research Council (NRC). The culture was allowed to grow for approximately 23 hours. The culture was then concentrated using a Millipore Pellicon Tangential Flow unit (0.22 μιη) powered by a peristaltic pump, which also minimizes cell lysis. The filtrate collected was further concentrated using the same device with a 5 KDa membrane (Biomax-5, Milipore) to approximately 100 ml. A 50 ml Amicon air-pressure filtration device (5 KDa) was used to concentrate to approximately 5 to 10 ml.

[00166] Generation of chromosomal mutations.

[00167] Genetic chromosomal mutations were performed as previously described in the literature. The ssaR (40) and fliF mutant was generated by allelic exchange using a counter- selectable suicide vector containing the levansucrase-encoding sacB gene (41). The csgAB mutation was generated by lambda red recombination (42). The PhoP mutant was constructed by P22 transduction from 14028s from phoP::Tnl0d Tc (43).

[00168] Mice.

[00169] Six to 8 week-old Sa/mo«e//a-susceptible C57BL/6, B6.129S2-IghmtmlCgn/J (B cells deficient), B6A29S2-Cd4tmlMak/J (CD4 cells deficient), B6A29S2-Cd8atmlMak/J (CDS cells deficient) (Jackson Laboratories) mice were used in the immunization experiments. Also 6-8 week old 129Sl/SvImJ Nramp +/+ and Nramp -/- mice were bred at CDM and used in the immunization experiments. Mice were housed in sterilized, filter-top cages under specific pathogen- free conditions at the University of British Columbia Animal Facility. The protocols used in the experiments were in accordance with animal care guidelines as outlined by the University of British Columbia's Animal care Committee and the Canadian Council on the use of Laboratory Animals.

[00170] Immunization of mice against systemic salmonellosis.

[00171] Between 4 to 8 C57BL/6 (Jackson Laboratory, Bar Harbor, ME) female age- matched (6-8 weeks old) mice per group at each time the experiment was performed were injected subcutaneously (s.c.) with supernatant containing 40 μg of total protein and 1 volume of TiterMax Gold Adjuvant (Sigma, Missouri, USA). Thirty days later mice were boosted s.c. with supernatant containing 25 μg of total protein and 1 volume of TiterMax (Sigma). Mice were challenged 30 days after the booster, with 3xl0⁶ colony forming units (cfu) of S.

Typhimurium (SL1344) orally. The strain was streaked from a frozen lab stock in Luria- Bertani (LB, Difco, Maryland, USA) agar plates containing 100 μg/ml of streptomycin (Sigma) and grown overnight at 37°C. Three days after the bacterial challenge, Salmonella cfu was determined for cecum, spleen, and liver in the mice in LB plates supplemented with 100 μg/ml of streptomycin.

[00172] Immunization of mice against Salmonella gastroenteritis infection.

[00173] Mice were administered 90 μg of supernatant with 1 μg CpG (Invivogen,

California, USA) orally. The mice were boosted 21 days later with 45 μg of supernatant with 1 ug CpG orally. Thirteen days later the mice were treated with 100 μΐ of streptomycin (Sigma, 20mg) orally and the day after the mice were challenged with 3xl0⁶ cfu of S.

Typhimurium orally, as described before. Three days later the cfu/g was determined for the cecum, liver, and spleen as described above.

[00174] Lipopolysaccharide (LPS) studies.

[00175] The supernatant was run over a polymyxin B column (Detoxy-Gel AffinityPack pre-packed columns, Pierce, Illinois, USA) to remove LPS. The removal of LPS was verified by an LAL assay (Limilus Amebocyte Lysate Pyrogen Plus, Cambrex, New Jersey, USA) following manufacturer's instructions.

[00176] To extract LPS overnight bacterial cultures were inoculated 1 :50 in 250 ml LB (SPI-1) or LPM (SPI-2) and grown up to OD₆₀₀0.4. The LPS extraction kit (Intron

Biotechnology, Korea) was used to extract the LPS from the cultures. The LPS concentration was determined using the LAL assay (Cambrex) and the same concentration determined to be present in the wild type supernatant was used to vaccinate the mice (184.6 μg for the prime and 92.3 μg for the booster).

[00177] Supernatant characterization.

[00178] For the proteinase K treatment, LPS-free supernatant was digested with proteinase K (Sigma, 0.2 mg/ml) at 55°C overnight, following incubation in boiling water for 30 min. The supernatant was then centrifuged at 13,000 rpm for 1 min and the supernatant was collected and used to vaccinate the mice. For the ammonium sulfate precipitation we added ammonium sulfate (Sigma) at 20 % final concentration to the supernatant and incubated at 4°C shaking for 1 hour. The mixture was then centrifuged at 13,000 rpm for 15 min and the supernatant and pellet collected as separated fractions. Ammonium sulfate at 50% final concentration was added to the supernatant fraction from the 20% precipitation and the same procedure described above was performed. The pellet of the 20% and 50% fractions and the supernatant of the 50% fraction were dialyzed in PBS and used to vaccinate the mice. To separate the outer membrane vesicles (OMV) fraction from the bacterial supernatant samples were centrifuge (in a Beckman TLA 100 Ultracentrifuge with a TLA100.3 rotor) for 30 min at 100,000 rcf to pellet the membranes. The supernatant was collected and the pellet was washed once with 2 ml of saline and then resuspended in 1 ml (same volume as above) buffer. This fraction was referred as OMV.

[00179] Proteomic analysis of the fractions after 50% ammonium sulfate

precipitation.

[00180] The pH of both the pellet and supernatant was adjusted to 8.5 by using 3 M NaOH. Prior to digestion, proteins in both samples were reduced with 2 mM DTT and alkylated with 4 mM iodoacetamide. A total of 4 μΙ_, trypsin (0.5 μg/μL) was added to the pellet fraction and 3 μϊ_^ to the supernatant fraction and both samples were incubated overnight at 37 °C. After digestion, both samples were desalted as described above and reconstituted in 40 μϊ_^ 0.1 M acetic acid in water and analyzed by mass spectrometry.

[00181] RP-HPLC fractionation of the 50% ammonium sulfate pellet.

[00182] Reversed phase protein separation was performed using a Zorbax 300SB-C3 analytical column (4.6 mm i.d., 50 mm length, 3.5 μιη, Agilent Technologies), a Rheodyne 7725i injection valve and an Agilent 1 100/1200 HPLC system consisting of a G1376A capillary pump operated in normal flow mode, a G1315C diode array detector and a G1364C analytical scale fraction collector. The pellet fraction of the ammonium sulfate precipitation was dried completely, resuspended in 50 μΐ., 0.1 % TFA in water and injected onto the column at a flow rate of 1 mL/min in 100 % solvent A (0.1 % TFA in water). Proteins were separated in a 15 minute gradient from 10 % to 80 % solvent B (0.1 % TFA in 8/2 (v/v) acetonitrile/water) and a total of 30 fractions were collected (30 s each).

[00183] Nanoflow HPLC-MS of HPLC fractions.

[00184] All samples were analyzed by nanoflow liquid chromatography using an Agilent 1200 HPLC system (Agilent Technologies) coupled online to an LTQ-Orbitrap XL mass spectrometer (Thermo Electron). The liquid chromatography part of the system was operated in a setup essentially as described previously (2). Aqua C₁₈, 5 μιη, (Phenomenex) resin was used for the trap column and ReproSil-Pur Cis-AQ, 3 μιη, (Dr. Maisch GmbH) resin was used for the analytical column. Peptides were trapped at 5 μί/ιηϊη in 100 % solvent A (0.1 M acetic acid in water) on a 2 cm trap column (100 μιη i.d., packed in-house) and eluted to a 20 cm analytical column (50 μιη i.d., packed in-house) at ~ 100 nL/min in a 90 min gradient from 10 to 40 % solvent B (0.1 M acetic acid in 8/2 (v/v) acetonitrile/water). The eluent was sprayed via in-house made emitter tips, butt-connected to the analytical column. The mass spectrometer was operated in data-dependent mode, automatically switching between MS and MS/MS. Full scan MS spectra (from m/z 300 to 1600) were acquired in the Orbitrap with a resolution of 60000 at m/z 400 after accumulation to a target value of 1000000. The five most intense ions at a threshold above 500 were selected for collision-induced fragmentation in the linear ion trap at normalized collision energy of 35 % after accumulation to a target value of 10000.

[00185] Database searching and label-free quantitation of HPLC fractions.

[00186] All MS2 spectra were converted to single dta files using DTASuperCharge (44)and MS File Reader 2.2 (Thermo) and merged into a Mascot generic format file which was searched using an in-house licensed Mascot v2.3.01 search engine (Matrix Science) against a concatenated Salmonella database (containing 9472 forward and reversed sequences). Carbamidomethyl cysteine was set as a fixed modification and oxidized methionine as a variable modification. Trypsin was specified as the proteolytic enzyme and one missed cleavage was allowed. In the case of the Proteinase K experiment no enzyme was defined. The mass tolerance of the precursor ion was set to 50 ppm and that of fragment ions was set to 0.6 Da. A peptide false-positive discovery rate (FDR) of <1% was estimated (45) and accomplished by adjusting the mass tolerance cut-off, the peptide score cut-off and the peptide length. A minimum of two peptides per protein and a protein cut-off score of 60 led to a protein FDR of <1%. Label-free relative quantitation was performed by calculating the exponentially modified protein abundance index (emPAI) (46) and protein ratios were obtained by comparing their emPAI value between different fractions. [00187] Enzyme-linked immunosorbent assay (ELISA).

[00188] Vaccine specific IgG and IgA levels were measured from serum and specific secretory IgA levels were measured from the feces of 4 immunized and 4 control mice from each model of vaccination by antibody ELISA.

[00189] Flat bottom 96 well micro-titre plates (Corning-Costar) were coated with lOOuL of 0.5 ug/mL vaccine supernatant, diluted in PBS, at 4°C overnight. The wells were washed three times with PBS/0.05% Tween 20 (PBS/T) prior to blocking with lOOuL PBS/2% bovine serum albumin (BSA)/0.05% Tween 20 (PBS/BSA/T) for 1 hr at room temperature. Dilutions of mouse serum in PBS/0.5% BSA were added to the plates (lOOuL) and incubated at 37°C for 2 h prior to washing three times with PBS/T. Negative controls of normal mouse serum were included in every assay. To detect total immunoglobulin G (IgG) or immunoglobulin A (IgA), lOOuL of horseradish peroxidase (HRP) labelled goat anti-mouse IgG or IgA (1 : 1000) (Santa Cruz) was added to each well and the plates were incubated 1 h at 37 °C. After three washes in PBS/T, the reaction was developed with ΙΟΟμΙ of 3,3',5,5'-Tetramethylbenzidine substrate solution (BD Biosciences) for 20 min and stopped 50 μΐ 3 M H2SO4. Optical density (OD) was measured at 450 nm.

[00190] Statistical Analysis.

[00191] Statistical significance was calculated using the Mann- Whitney test with a 95% confidence interval. All analyses were performed using GraphPad Prism version 4.0

(GraphPad Software, San Diego, CA). The results were expressed as mean values with standard errors of the means.

Example 1: Supernatant Administration, Fractionation, and

Characterization

[00192] 5^*. Typhimurium is a pathogen that can modify its surface components such as LPS, as well as many proteins, once inside host cells such as macrophages (25, 26). S.

Typhimurium contains two type three secretion systems; one involved in initial invasion into non-phagocytic host cells (Salmonella Pathogenicity Island- 1, or SPI-1) and the other is critical for survival in phagocytic cells (SPI-2). These systems are used to secrete proteins important for virulence into the host cell. In the laboratory, different media conditions can be used to activate secretion of these two systems (27). By harvesting supernatants from S. Typhimurium grown under conditions that selectively induce SPI-2 secretion many of the modifications that occur when Salmonella is inside host cells will occur.

[00193] We found that mice immunized with culture supernatant from 5^*. Typhimurium grown under SPI-2-inducing conditions, but not SPI-1 conditions, dramatically protected mice from subsequent heterologous challenge, significantly decreasing the bacterial load in the cecum, spleen, and liver (Fig. 1 A, B, and C). Moreover, both supernatants provided protection against disease, preventing a decrease in cecal weight, a hallmark of infection, and markedly decreasing histological scores of disease (Fig. ID) (28). The protection in these mice was able to significantly increase their survival rate compared to control mice immunized with saline and adjuvant (Fig. IE). We analyzed the immune response responsible for the supernatant-elicited protection. Mice deficient in B cells, CD4⁺ and CD8⁺ T-cells were immunized and the effects on Salmonella colonization of the spleen recorded (Fig. IF). Immunization with the supernatant did not confer protection in either B cell or CD8⁺ T cell knockout mice. CD4⁺ knockout mice showed a significant decrease in 5^*. Typhimurium colonization. These results indicate that B cells and CD8⁺ T cells are important for the immune response elicited by the supernatant. Antibody responses were also measured from serum of immunized mice and compared to control mice (Fig. 1G and H). Both specific IgG and IgA were significantly increased in immunized mice, confirming the immune response activation.

[00194] To characterize the protective antigen(s) present in the supernatant and responsible for the protection observed in mice, we performed studies using both genetic mutants as well as biochemical approaches. We first examined whether the SPI-2 secreted proteins were responsible for the protection effect. Surprisingly, we found that although the supernatant was grown under SPI-2 inducing conditions, mice immunized with supernatant of a mutant incapable of secreting SPI-2 secreted proteins (ssaR), grown under the same conditions, provided the same level of protection as the WT supernatant. Dramatic decreases were seen in bacterial counts in the spleen, as well as cecum and liver, when mice were immunized with either supernatant (Fig. 2A). Since the growth conditions that were used might also affect the expression of PhoP, a global virulence regulator, we tested whether it was needed for protection. PhoP is a transcriptional regulator that is activated by PhoQ under low extracytoplasmic Mg²⁺ concentrations (29) or acidic pH (26), similar to the conditions used to obtain the supernatant in our study, regulating the transcription of more than 50 proteins (30). However, the supernatant from a phoP mutant found to still be protective, decreasing 5^*. Typhimurium colonization at similar levels as immunization with the WT supernatant (Fig. 2A). PhoPQ expression also regulates modifications of the lipid A component of S. Typhimurium lipopolysaccharide (LPS). LPS is known to elicit an immune response, we therefore tested whether the LPS present in the supernatant was responsible for the protection seen in mice. LPS was then removed from the supernatant and used to immunize the mice. The LPS-free supernatant retained the protection observed in mice (Fig. 2B). In addition, when LPS was purified under SPI-2 inducing conditions and used as a vaccine, no protection was observed further indicating that LPS is not responsible for the protection seen in this study. Another major secreted antigen in Salmonella is flagellin, which is known to induce an immune response against Salmonella (22). Therefore, we examined whether a genetic mutant incapable of secreting flagella (AfliF) was able to provide protection. We found that immunization with a supernatant obtained from a fliF mutant gave similar protection as the WT supernatant (Fig. 2A), indicating that flagella is not likely responsible for the protection of mice against Salmonella infection.

[00195] To further characterize the immune-activating compound(s) present in the supernatant we performed heat and proteinase K treatments, as well as ammonium sulfate precipitation. We found that mice immunized with supernatant that was boiled for 20 minutes and then digested with proteinase K exhibited higher colonization of S. Typhimurium compared to mice immunized with WT supernatant but still lower counts compared to the control mice (Fig. 2B). We also found that the protective component(s) precipitated after 50% ammonium sulfate treatment (Fig. 2B), whereas the remaining solution (50% sup) failed to confer protection to S. Typhimurium systemic infection.

[00196] Outer-membrane vesicles (OMV) have been previously shown to stimulate protective immunity against 5^*. Typhimurium (31). Therefore we tested the hypothesis that the OMV present in high levels in the supernatant are responsible for the protection effect observed in mice. Supernatant components after 50% ammonium sulfate precipitation were separated by ultracentrifugation and both fractions (OMV and ultra sup) were tested for their immunization ability. Both fractions protected mice against Salmonella infection to the same extent (Fig. 2B), indicating that the component(s) responsible for protection in our supernatant are likely present in both fractions.

[00197] In an effort to identify specific antigens responsible for the protection conferred by the supernatant, we separated the precipitated components after 50% ammonium sulfate using high-performance liquid chromatography (HPLC) (Fig. 7). A total of 30 fractions were collected and tested by western analysis using the serum of the immunized mice (Fig. 7). We observed several proteins that reacted with serum in fractions F13 to F30. Fractions F9, F 13, F14, F18, F 19 and F22 were chosen to immunize mice based on the unique set of bands they exhibited. Four fractions (F 13, F14, F18 and F 19) conferred similar protection as the supernatant after 50% ammonium sulfate precipitation as measured by S. Typhimurium colonization (Fig. 2C). However, fractions F9 and F22 showed no protection compared to control mice. We analyzed the proteins present in all six fractions by mass spectrometry and found 10 peptides corresponding to 10 proteins that were present in one or more of the protective fractions (Tables 1, 2, and 5). Three of these peptides have been previously described to be an antibody target in serum of Salmonella infected mice and/or humans (32- 35). These peptides were SL4489 (OsmY), SL1010 (OmpA) and SL0731 (Pal - peptidoglycan-associated lipoprotein). The others are SL0866 (Artl), SL1780 (hypothetical protein), SL2251 (GlpQ), SL4363 (CybC), SL1061 (putative secreted protein), SL4109 (Hup A) and SL1492 (HdeB). This data also attests that the compounds are very stable as they were partially resistant to proteinase K treatment and able to confer protection even after extended heating time.

[00198] We next sought to determine if the vaccine could also protect animals from Salmonella-induced gastroenteritis. To do so, mice were immunized with WT supernatant and infected according to the S. Typhimurium gastroenteritis model (37). In mice, S.

Typhimurium does not cause gastroenteritis as seen in humans. To model the disease, mice were pre-treated with streptomycin 24 h prior to the infection with Salmonella and bacterial counts and histopathology changes in the cecum of the animals were observed similar to gastroenteritis. Oral delivery of the supernatant of Salmonella grown under SPI-2 inducing conditions with a CpG adjuvant was able to significantly protect against gastroenteritis in C57BL/6J mice (Fig. 3 A). Intestinal pathology decreased with immunization of these mice (Fig. 3B). C57B1/6J mice are highly susceptible to S. Typhimurium infection, displaying high colonization and sever intestinal pathology that cannot be overcome by the mouse immune system leading to the death (38). In a more resistant mouse model, 129Sl/SvImJ mice, which are NRAMP +/+, display significantly lower colonization and are generally able to recover from infection, most similar to what happens in humans with Salmonella gastroenteritis (38). We therefore tested whether the oral immunization would confer protection in this resistant mouse model, as well as in the 129S l/SvImJ NRAMP -/- mice, which are more susceptible to infection. Remarkably, significant protection was observed in both mouse strains, with NRAMP +/+ showing no colonization by S. Typhimurium (sterility) when immunized with the supernatant (Fig. 3C). Intestinal pathology was also significantly decreased in both mouse strains (Fig. 3D). Overall, we showed that immunization with WT supernatant was able to strongly decrease, and even eliminate (as in the case of 129Sl/SvImJ Nramp +/+), colonization of S. Typhimurium and prevent intestinal pathology, a well-known symptom of Salmonella-induced colitis (37). Similarly to the systemic model, the antibody levels were also examined in mice immunized orally (gastrointestinal model) (Fig. 3E-G). Significantly higher specific IgG and IgA titers were detected on serum of immunized mice compared to control mice and higher secretory IgA levels were also detected on feces of immunized mice, indicating that the immunization elicited the expected antibody response to the vaccine.

Example 2: Administration of SL2251 and SL1780

[00199] SL2251 and SL1780 (Tables 1, 2, and 5) were cloned into a pET vector with a HIS tag. The vectors were expressed in E. coli BL21 (DE3) overnight. The cells were then collected, pelleted, and sonicated. The proteins were then purified using Ni-beads. The general methodologies used for protein expression and purification are described in:

Gruenheid et al, Molecular Microbiology (2004) 51(5), 1233-49; and Deng et al, Infection and Immunity (2005) 73(4):2135.

[00200] Between 4 to 8 C57BL/6 (Jackson Laboratory, Bar Harbor, ME) female age- matched (6-8 weeks old) mice per group at each time the experiment was performed were injected subcutaneously (s.c.) with SL2251 or SL1780 protein containing 20 μg of total protein and 1 volume of TiterMax Gold Adjuvant (Sigma, Missouri, USA). Thirty days later mice were boosted s.c. with supernatant containing 12.5 μg of total protein and 1 volume of TiterMax (Sigma). Mice were challenged 30 days after the booster, with 3xl0⁶ colony forming units (cfu) of S. Typhimurium (SL1344) orally. The strain was streaked from a frozen lab stock in Luria-Bertani (LB, Difco, Maryland, USA) agar plates containing 100 μg/ml of streptomycin (Sigma) and grown overnight at 37°C. Four days after the bacterial challenge, Salmonella cfu was determined for cecum, spleen, and liver in the mice in LB plates supplemented with 100 μg/ml of streptomycin. The general methodology used is shown in Figure 4.

[00201] Mice immunized with SL2251 were dramatically protected from subsequent heterologous challenge, significantly decreasing the bacterial load (cfu/g) in the cecum, spleen, and liver (Fig. 5). Moreover, SL2251 provided protection against disease, preventing a decrease in cecal weight (g), a hallmark of infection (Fig. 5) (28).

[00202] The serum of mice immunized with supernatant (as described above) was collected and found to cross-react with purified SL2251 via western blot (Fig. 6); indicating that supernatant administration to mice results in a specific immune response against SL2251.

Example 3: Administration of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, and/or 8

[00203] SEQ ID NOs 1-8 (Tables 1, 2, and 5) are cloned into a pET vector with a HIS tag. The vectors are expressed in E. coli BL21 (DE3) overnight. The cells are then collected, pelleted, and sonicated. The proteins are then purified using Ni-beads. The general methodologies used for protein expression and purification are described in: Gruenheid et al, Molecular Microbiology (2004) 51(5), 1233-49; and Deng et al, Infection and Immunity (2005) 73(4):2135.

[00204] Between 4 to 8 C57BL/6 (Jackson Laboratory, Bar Harbor, ME) female age- matched (6-8 weeks old) mice per group at each time the experiment is performed are injected subcutaneously (s.c.) with protein containing 20 μg of total protein and 1 volume of TiterMax Gold Adjuvant (Sigma, Missouri, USA). Thirty days later mice are boosted s.c. with supernatant containing 12.5 μg of total protein and 1 volume of TiterMax (Sigma). Mice are challenged 30 days after the booster, with 3xl0⁶ colony forming units (cfu) of S.

Typhimurium (SL1344) orally. The strain is streaked from a frozen lab stock in Luria-Bertani (LB, Difco, Maryland, USA) agar plates containing 100 μg/ml of streptomycin (Sigma) and grown overnight at 37°C. Four days after the bacterial challenge, Salmonella cfu is determined for cecum, spleen, and liver in the mice in LB plates supplemented with 100 μg/ml of streptomycin. Cecal weight is also measured. The general methodology used is shown in Figure 4.

[00205] The serum of mice immunized with supernatant (as described above) is collected and tested for cross-reaction with purified protein via western blot (Fig. 6); indicating that supernatant administration to mice results in a specific immune response against the respective protein.

Example 4: Immunization of mice in a Thyphoid Model

[00206] 8 proteins (SEQ ID NOs 1-8) are tested in a thyphoid mouse model. See Fig. 2A of PCT US2011/049009 for one example of a methodology used for this model. In some instances, one or more of the 8 proteins shown in SEQ ID NOs 1-8 is combined with one or more of the proteins shown in Table 3 (SEQ ID NOs 17-24). Briefly, mice are injected subcutaneously (s.c.) with an appropriate dose of one or more proteins (see detailed description above) with or without an appropriate dose of an adjuvant (see detailed description above). 30 days later mice are boosted s.c. with appropriate doses of the same protein(s) with or without an adjuvant. 30 days after the boost, mice are challenged with 3xl0⁶ colony forming units (cfu) of S. Typhimurium orally. 3 days after the bacterial challenge, cfu/g is determined for cecum, spleen, and liver in the mice. One of skill in the art will understand that the above noted dosage schedules, dosages, challenge schedules, concentrations, etc. can be adjusted as appropriate to optimize the experimental design, e.g., to obtained statistically significant results. [00207] A decrease in counts is observed in the cecum and liver and a decrease in splenic counts is observed. An immune response to the administered protein(s) is also detected via standard means, e.g., ELISPOT, FACS, and/or ELISA.

Example 5: Immunization of mice in a Gastroenteritis Model

[00208] 8 proteins (SEQ ID NOs 1-8) are tested in the murine Salmonella gastroenteritis model (see Fig. 8 of PCT/US2011/049009 for one aspect of a general model description), which is a model for the human non-typhoidal salmonella disease. In some instances, one or more of the 8 proteins shown in SEQ ID NOs 1-8 is combined with one or more of the proteins shown in Table 3 (SEQ ID NOs 17-24). Briefly, mice are administered an appropriate dose of one or more proteins (see detailed description above) with or without an appropriate dose adjuvant (see detailed description above) via intranasal or oral injection. 21 days later the mice are boosted with appropriate doses of the same protein(s) with or without an adjuvant via intranasal or oral administration. 13 days later the mice are administered streptomycin (20mg) orally and the day after the mice are challenged with 3xl0⁶ cfu of S. Typhimurium orally. 3 days later the cfu/g is determined for the cecum, liver, and spleen. One of skill in the art will understand that the above noted dosage schedules, dosages, challenge schedules, concentrations, etc. can be adjusted as appropriate to optimize the experimental design, e.g., to obtained statistically significant results.

[00209] A decrease in counts is observed in the cecum and liver and a decrease in splenic counts is observed. An immune response to the administered protein(s) is also detected via standard means, e.g., ELISPOT, FACS, and/or ELISA.

Example 6: Treatment of mice in a Thyphoid Model

[00210] 8 proteins (SEQ ID NOs 1-8) are tested in a thyphoid mouse model. In some instances, one or more of the 8 proteins shown in SEQ ID NOs 1-8 is combined with one or more of the proteins shown in Table 3 (SEQ ID NOs 17-24). Briefly, mice are challenged with 0.1 to 3xl0⁶ colony forming units (cfu) of S. Typhimurium orally. Mice are then injected 1 to 30 days later subcutaneously (s.c.) with an appropriate dose of one or more proteins (see detailed description above) with or without an appropriate dose of an adjuvant (see detailed description above). 1 to 30 days later mice are boosted s.c. with appropriate doses of the same protein(s) with or without an adjuvant. 1 to 10 days after the boost, cfu/g is determined for cecum, spleen, and liver in the mice. One of skill in the art will understand that the above noted dosage schedules, dosages, challenge schedules, concentrations, etc. can be adjusted as appropriate to optimize the experimental design, e.g., to obtained statistically significant results.

[00211] A decrease in counts is observed in the cecum and liver and a decrease in splenic counts is observed. An immune response to the administered protein(s) is also detected via standard means, e.g., ELISPOT, FACS, and/or ELISA. The infection is treated via the composition admininistration.

Example 7: Treatment of mice in a Gastroenteritis Model

[00212] 8 proteins (SEQ ID NOs 1-8) are tested in the murine Salmonella gastroenteritis model, which is a model for the human non-typhoidal salmonella disease. In some instances, one or more of the 8 proteins shown in SEQ ID NOs 1-8 is combined with one or more of the proteins shown in Table 3 (SEQ ID NOs 17-24). Briefly, mice are administered streptomycin (20mg) oraly and challenged 1 day after with 0.1 to 3xl0⁶ colony forming units (cfu) of S. Typhimurium orally. Mice are then administered, 1 to 30 days later, an appropriate dose of one or more proteins (see detailed description above) with or without an appropriate dose adjuvant (see detailed description above) via intranasal or oral injection. 1 to 21 days later the mice are boosted with appropriate doses of the same protein(s) with or without an adjuvant via intranasal or oral administration. 1 to 10 days after the boost the cfu/g is determined for the cecum, liver, and spleen. One of skill in the art will understand that the above noted dosage schedules, dosages, challenge schedules, concentrations, etc. can be adjusted as appropriate to optimize the experimental design, e.g., to obtained statistically significant results.

[00213] A decrease in counts is observed in the cecum and liver and a decrease in splenic counts is observed. An immune response to the administered protein(s) is also detected via standard means, e.g., ELISPOT, FACS, and/or ELISA. The infection is treated via the composition admininistration.

Example 8: Immunization of mice with proteins

[00214] Isolated polypeptides including an amino acid sequence at least 80-90% identical to the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8 are tested alone or in combination in a murine model of Gram-negative bacteria infection. In some instances, one or more of the 8 proteins shown in SEQ ID NOs 1-8 is combined with one or more of the proteins shown in Table 3 (SEQ ID NOs 17-24). Briefly, mice are administered an appropriate dose of one or more proteins (see above) with or without an appropriate dose adjuvant (see above) via s.c, intranasal, or oral injection. 1 to 21 days later the mice are boosted with appropriate doses of the same protein(s) with or without an adjuvant via s.c, intranasal, or oral administration. 13 days later the mice are administered streptomycin (20mg) orally and 1 day later mice are challenged with lxlO⁵ to 3xl0⁶ cfu of an appropriate Gram-negative bacteria, e.g., via oral adminstration. 1 or more days later the cfu/g is determined from the appropriate tissues and/or cell types, e.g., cecum, liver, blood, and spleen. One of skill in the art will understand that the above noted dosage schedules, dosages, challenge schedules, concentrations, etc. can be adjusted as appropriate to optimize the experimental design, e.g., to obtained statistically significant results.

[00215] A decrease in counts is observed in the appropriate tissues. An immune response to the administered protein(s) is also detected via standard means, e.g., ELISPOT, FACS, and/or ELISA.

Example 9: Treatment of mice with proteins

[00216] Isolated polypeptides including an amino acid sequence at least 80-90% identical to the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8 are tested alone or in combination in a murine model of Gram-negative bacteria infection. In some instances, one or more of the 8 proteins shown in SEQ ID NOs 1-8 is combined with one or more of the proteins shown in Table 3 (SEQ ID NOs 17-24). One day before the challenge the mice are administered streptomycin (20mg) orally. Mice are challenged with lxlO⁵ to 3xl0⁶ cfu of an appropriate Gram-negative bacteria, e.g., via oral administration. Mice are then adminsitered 1 to 30 days later subcutaneously (s.c), orally, or intranasally with an appropriate dose of one or more proteins (see above) with or without an appropriate dose of an adjuvant (see above). 1 to 30 days later mice are boosted s.c, orally, or intranasally with appropriate doses of the same protein(s) with or without an adjuvant. 1 to 10 days after the boost, cfu/g is determined from the appropriate tissues and/or cell types, e.g., cecum, liver, blood, and spleen. One of skill in the art will understand that the above noted dosage schedules, dosages, challenge schedules, concentrations, etc. can be adjusted as appropriate to optimize the experimental design, e.g., to obtained statistically significant results.

[00217] A decrease in counts is observed in the appropriate tissues. An immune response to the administered protein(s) is also detected via standard means, e.g., ELISPOT, FACS, and/or ELISA. The infection is treated via the composition admininistration. Example 10: Immunization of humans with proteins

[00218] Isolated polypeptides including an amino acid sequence at least 80-90% identical to the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8 are tested alone or in combination in humans. In some instances, one or more of the 8 proteins shown in SEQ ID NOs 1-8 is combined with one or more of the proteins shown in Table 3 (SEQ ID NOs 17- 24). Briefly, humans are administered an appropriate dose of one or more proteins (see above) with or without an appropriate dose adjuvant (see above) via s.c, intranasal, or oral injection. 1 to 21 days later the humans are boosted with appropriate doses of the same protein(s) with or without an adjuvant via s.c, intranasal, or oral administration. 1 or more days later the immune response is determined from the appropriate tissues and/or cell types, e.g., cecum, liver, blood, and spleen. One of skill in the art will understand that the above noted dosage schedules, dosages, challenge schedules, concentrations, etc. can be adjusted as appropriate to optimize the experimental design, e.g., to obtained statistically significant results.

[00219] An immune response to the administered protein(s) is detected via standard means, e.g., ELISPOT, FACS, and/or ELISA.

Example 11: Immunization of pigs with proteins

[00220] Isolated polypeptides including an amino acid sequence at least 80-90% identical to the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8 are tested alone or in combination in pigs. In some instances, one or more of the 8 proteins shown in SEQ ID NOs 1-8 is combined with one or more of the proteins shown in Table 3 (SEQ ID NOs 17-24). Briefly, pigs are administered an appropriate dose of one or more proteins (see above) with or without an appropriate dose adjuvant (see above) via s.c, intranasal, or oral injection. 1 to 21 days later the pigs are boosted with appropriate doses of the same protein(s) with or without an adjuvant via s.c, intranasal, or oral administration. 1 or more days later the immune response is determined from the appropriate tissues and/or cell types, e.g., cecum, liver, blood, and spleen. One of skill in the art will understand that the above noted dosage schedules, dosages, challenge schedules, concentrations, etc. can be adjusted as appropriate to optimize the experimental design, e.g., to obtained statistically significant results.

[00221] An immune response to the administered protein(s) is detected via standard means, e.g., ELISPOT, FACS, and/or ELISA. Example 12: Immunization of chickens with proteins

[00222] Isolated polypeptides including an amino acid sequence at least 80-90% identical to the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8 are tested alone or in combination in chickens. In some instances, one or more of the 8 proteins shown in SEQ ID NOs 1-8 is combined with one or more of the proteins shown in Table 3 (SEQ ID NOs 17- 24). Briefly, chickens are administered an appropriate dose of one or more proteins (see above) with or without an appropriate dose adjuvant (see above) via s.c, intranasal, or oral injection. 1 to 21 days later the chickens are boosted with appropriate doses of the same protein(s) with or without an adjuvant via s.c, intranasal, or oral administration. 1 or more days later the immune response is determined from the appropriate tissues and/or cell types, e.g., cecum, liver, blood, and spleen. One of skill in the art will understand that the above noted dosage schedules, dosages, challenge schedules, concentrations, etc. can be adjusted as appropriate to optimize the experimental design, e.g., to obtained statistically significant results.

[00223] An immune response to the administered protein(s) is detected via standard means, e.g., ELISPOT, FACS, and/or ELISA.

[00224] While specific aspects of the invention have been described and illustrated, such aspects should be considered illustrative of the invention only and not as limiting the invention as construed in accordance with the accompanying claims.

Tables

Table 1

SEQ ID NO Name Amino acid sequence

1 SL0731 MQLNKVLKGLMIALPVMAIAACSSNKNASNDGSEGGMLNGAGTGMDANGN

GNMSSEEQARLQMQQLQQNNIVYFDLDKYDIRSDFAAMLDAHANFLRSNP SYKVTVEGHADERGTPEYNISLGERRANAVKMYLQGKGVSADQISIVSYG KEKPAVLGHDEAAYAKNRRAVLVY

2 SL1061 MKRIFLTCAALLISSQALADECASASTQLEMNRCAAAQYQAADKKLNETY

QSALKRAQPPQRELLQKAQVAWIALRDADCALIRSGTEGGSVQPMIASQC LTDKTNEREAFLASLLQCEEGDLSCPLPPAG

3 SL1780 MMKTSVRIGAFEIDDAELHGESPGERTLTIPCKSDPDLCMQLDAWDAETS

VPAILNGEHSVLFRTHYDPKSDAWVMRLA

4 SL4109 MNKTQLIDVIADKAELSKTQAKAALESTLAAITESLKEGDAVQLVGFGTF

KVNHRAERTGRNPQTGKEIKIAAANVPAFVSGKALKDAVK

5 SL0866 MKKVLIAALIAGFSLSATAAQTIRFATEASYPPFESMDANNKIVGFDVDL

ANALCKEIDASCTFTNQAFDSLIPSLKFRRFDAVMAGMDITPEREKQVLF TTPYYDNSALFVGQQGKYTSVDQLKGKKVGVQNGTTHQKFIMDKYPEITT VPYDSYQNAKLDLQNGRIDAVFGDTAWTEWLKANPKLAPVGDKVTDKDY FGTGLGIAVRQGNTELQQKFNTALEKVKKDGTYETIYNKWFQK

6 SL1492 MNKFSLATAGI IVAALVTSVSVNAATDTTKTNVTPKGMSCQEFVDLNPQT

MAPVAFWVLNEDEDFKGGDYVDFQETETTAVPLAVELCKKNPQSELSKIK DEIKKELSK

7 SL2251 MKTTLKNLSVALMLAGMTIGSGAVAAEKWIAHRGASGYLPEHTLPAKAM

AYAQGADYLEQDLVMTKDDHLVVLHDHYLDRVTDVADRFPDRARKDGRYY AIDFTLDEIKSLKFTEGFDIENGKKVQTYPGRFPMGKSDFRIHTFEEEIE Table 1

SEQ ID NO Name Amino acid sequence

FVQGLNHSTGKNIGIYPEIKAPWFHHQEGKDIAAKTLEVLKKYGYTGKQD NVYLQCFDVAELKRIKNELEPKMGMDLNLVQLIAYTDWNETQQKQPDGRW VNYNYDWMFKPGAMKQVAEYADGIGPDYHMLVAEGSTKGNIKLTGMVQDA HQNKMVVHPYTVRADQLPDYATDVNQLYDILYNKAGVDGLFTDFPDKAVM FLQKND

8 SL4369 MRKSLLAILAVSSLVFGSAVFAADLEDNMDILNDNLKWEKTDSAPELKA

ALTKMRAAALDAQKATPPKLEDKAPDSPEMKDFRHGFDILVGQIDGALKL ANEGNVKEAKAAAEALKTTRNTYHKKYR

Table 2

SEQ ID NO Name Nucleotide sequence

9 SL0731 TGCAACTGAACAAAGTGCTGAAGGGCCTGATGATTGCCCTGCCTGTTATG

GCAATCGCGGCATGTTCTTCCAACAAGAACGCCAGCAATGACGGTAGCGA AGGCGGTATGCTGAACGGCGCCGGCACTGGTATGGACGCTAACGGCAACG GCAACATGTCATCTGAAGAGCAAGCGCGTCTGCAGATGCAGCAGCTGCAG CAGAACAACATCGTTTACTTCGATCTCGACAAGTACGATATCCGTTCTGA CTTCGCGGCAATGCTGGATGCGCACGCTAACTTCCTGCGTAGCAACCCGT CTTACAAAGTCACCGTAGAAGGTCACGCGGACGAACGCGGTACTCCGGAG TACAACATCTCCCTGGGTGAGCGTCGTGCTAACGCCGTTAAAATGTACCT GCAGGGTAAAGGCGTTTCCGCTGACCAGATCTCCATCGTTTCTTACGGTA AAGAAAAACCTGCCGTACTGGGCCACGACGAAGCGGCTTACGCTAAGAAC CGTCGCGCTGTACTGGTTTACTAA

10 SL1061 ATGAAACGAATTTTCCTTACCTGCGCGGCGTTGTTGATCAGCAGTCAGGC

GTTGGCCGATGAGTGTGCCAGCGCCAGTACGCAGCTGGAAATGAATCGCT GCGCCGCCGCGCAATACCAGGCGGCAGATAAAAAGCTGAACGAAACCTAT CAAAGCGCGCTTAAGCGTGCGCAACCGCCGCAGCGCGAGCTGTTACAAAA AGCACAGGTGGCATGGATTGCCCTGCGCGACGCCGATTGCGCGCTGATTC GCTCAGGTACGGAGGGCGGCAGCGTGCAACCCATGATCGCCAGCCAGTGC CTGACCGATAAAACGAACGAACGCGAAGCGTTTTTAGCCTCGCTGCTGCA ATGTGAAGAGGGGGATTTGAGCTGCCCACTGCCGCCAGCCGGTTAA

1 1 SL1780 TGATGAAAACCAGTGTGCGCATTGGCGCTTTTGAAATCGACGATGCCGAA

TTACATGGCGAATCGCCGGGGGAGCGAACGTTAACAATCCCCTGTAAATC CGATCCGGATTTATGTATGCAACTGGACGCCTGGGATGCCGAAACCAGCG TTCCCGCCATTCTCAACGGGGAACATTCCGTTTTGTTCCGTACTCATTAC GATCCTAAATCTGATGCCTGGGTCATGCGTCTTGCCTGA

12 SL4109 ATGAACAAGACTCAACTGATTGATGTAATTGCAGACAAAGCAGAACTGTC

CAAAACCCAGGCTAAAGCTGCTCTGGAATCCACTCTGGCTGCTATTACTG AGTCTCTGAAAGAAGGCGATGCTGTACAACTGGTTGGTTTCGGTACCTTC AAAGTGAACCACCGTGCTGAGCGCACTGGCCGTAACCCACAGACCGGTAA AGAAATCAAAATCGCCGCCGCTAACGTACCGGCGTTTGTTTCTGGTAAAG CTCTGAAAGACGCAGTTAAGTAA

13 SL0866 ATGAAAAAAGTTCTGATTGCCGCGCTAATTGCAGGCTTTAGCCTTTCCGC

TACAGCAGCCCAGACCATTCGTTTTGCGACCGAAGCGTCCTATCCGCCGT TCGAATCGATGGATGCTAATAACAAGATTGTCGGCTTTGACGTCGACCTG GCCAACGCGCTATGTAAAGAGATCGACGCCTCCTGTACCTTTACCAATCA GGCGTTCGACAGCCTGATCCCCAGCCTGAAATTCCGCCGCTTCGACGCTG TAATGGCGGGAATGGATATCACGCCGGAACGTGAAAAGCAGGTGCTGTTT ACCACGCCATATTACGACAACTCCGCGCTGTTCGTGGGTCAGCAGGGCAA ATACACCAGCGTTGATCAACTGAAAGGCAAGAAAGTCGGCGTACAGAACG GTACGACGCACCAGAAATTCATCATGGATAAGTATCCGGAAATCACCACC GTACCGTATGACAGCTATCAGAACGCGAAGCTGGATCTACAAAATGGCCG TATCGACGCTGTTTTCGGCGACACGGCGGTCGTGACCGAATGGCTGAAAG CCAATCCTAAGCTGGCGCCGGTCGGCGATAAAGTCACCGATAAAGATTAT TTCGGCACCGGCCTGGGTATCGCGGTACGCCAGGGCAACACCGAGCTGCA GCAGAAATTCAATACTGCGCTGGAAAAAGTGAAGAAAGATGGGACTTACG AGACCATCTATAACAAATGGTTCCAGAAGTAA

14 SL1492 ATGAATAAATTCTCCCTTGCTACAGCAGGTATTATCGTGGCAGCGCTGGT

AACCAGTGTTAGCGTGAATGCGGCAACAGATACTACTAAAACAAACGTTA CGCCTAAAGGTATGAGCTGCCAGGAGTTTGTTGACCTCAATCCGCAGACG ATGGCGCCAGTCGCTTTCTGGGTGCTGAATGAAGATGAAGATTTTAAAGG CGGGGACTACGTAGATTTCCAGGAAACTGAGACGACAGCAGTGCCGCTAG CCGTTGAGCTTTGTAAGAAAAACCCGCAGAGTGAATTAAGCAAAATAAAA GACGAAATCAAGAAAGAACTCTCAAAATAA

15 SL2251 ATGAAAACCACACTGAAAAACCTTAGCGTGGCGTTAATGTTGGCAGGAAT Table 2

SEQ ID NO Name Nucleotide sequence

GACCATCGGAAGCGGCGCCGTGGCGGCTGAAAAAGTCGTTATCGCCCACC GTGGTGCCAGCGGTTATCTGCCGGAGCATACGCTACCGGCGAAAGCGATG GCGTATGCGCAAGGGGCAGATTATCTGGAACAGGATTTAGTGATGACAAA GGACGACCATCTGGTCGTCCTCCATGACCACTACCTTGACCGGGTGACTG ACGTCGCTGACCGTTTTCCGGACCGGGCGCGTAAAGATGGGCGCTATTAC GCTATCGATTTTACGCTGGATGAAATTAAATCCCTGAAGTTTACCGAAGG GTTTGATATTGAAAACGGCAAAAAAGTACAAACTTATCCGGGCCGTTTCC CGATGGGAAAATCTGATTTCCGCATTCATACCTTTGAAGAGGAAATTGAA TTCGTTCAGGGATTAAATCACTCTACCGGTAAAAATATCGGTATTTATCC GGAAATTAAAGCGCCGTGGTTCCATCATCAGGAAGGGAAAGATATTGCCG CGAAAACGCTGGAAGTGCTGAAGAAGTATGGTTACACCGGCAAGCAGGAC AATGTCTATTTGCAGTGTTTTGATGTCGCTGAGCTGAAACGTATTAAGAA TGAACTGGAACCCAAAATGGGGATGGATCTCAATCTGGTTCAACTTATTG CGTATACCGACTGGAATGAAACACAGCAGAAACAGCCGGACGGTCGTTGG GTAAATTACAACTACGACTGGATGTTTAAGCCGGGCGCTATGAAGCAGGT GGCGGAATATGCGGACGGTATTGGTCCGGATTACCATATGCTGGTTGCGG AAGGCTCAACGAAAGGGAATATCAAGCTGACTGGAATGGTGCAAGACGCG CATCAGAATAAGATGGTAGTGCATCCTTACACTGTGCGTGCCGATCAATT GCCGGACTATGCCACAGACGTCAATCAGTTGTACGACATCCTGTATAACA AGGCGGGCGTCGACGGGCTGTTCACTGACTTCCCGGATAAGGCGGTCATG TTCCTGCAAAAAAATGACTAA

16 SL4369 ATGCGTAAAAGCCTGTTAGCTATTCTTGCTGTGTCATCGCTGGTATTCGG

TTCGGCTGTTTTTGCTGCCGATCTTGAAGATAATATGGATATTCTCAATG ATAACCTGAAAGTTGTTGAGAAAACGGATAGTGCCCCTGAACTGAAAGCG GCATTAACCAAAATGCGCGCGGCCGCGCTGGATGCTCAAAAAGCTACGCC GCCAAAGCTGGAAGATAAAGCGCCGGATAGCCCGGAAATGAAGGATTTTC GTCACGGTTTTGACATTCTGGTCGGCCAAATTGATGGCGCGCTGAAGCTG GCGAACGAAGGTAACGTTAAAGAAGCGAAAGCGGCGGCGGAAGCGCTAAA AACCACCCGCAATACATATCATAAGAAGTATCGTTAA

Table 3

SEQ Accession Amino acid Sequence

ID Number

NO

17 SL0808 MKKIACLSALAAVLAFSAGTAVAATSTVTGGYAQSDAQGVANKMSGFNLKYRYEQDD

NPLGVIGSFTYTEKDRTNGAGDYNKGQYYGITAGPAYRLNDWASIYGVVGVGYGKFQ TTDYPTYKHDTSDYGFSYGAGLQFNPMENVALDFSYEQSRIRSVDVGTWIAGVGYRF

18 SL3596 VAMTVAASVQAKTLVYCSEGSPEGFNPQLFTSGTTYDASSVPIYNRLVEFKTGTTEV

IPGLAEKWDISEDGKTYTFHLRKGVKWQSSKDFKPTRELNADDVVFSFDRQKNEQNP YHKVSGGSYEYFEGMGLPDLISEVKKVDDHTVQFVLTRPEAPFLADLAMDFASILSK EYADNMLKAGTPEKVDLNPVGTGPFQLVQYQKDSRILYKAFDGYWGTKPQIDRLVFS ITPDASVRYAKLQKNECQVMPYPNPADIARMKEDKNINLMEQAGLNVGYLSYNVQKK PLDDVKVRQALTYAVNKEAIIKAVYQGAGVAAKNLIPPTMWGYNDDIKDYGYDPEKA KALLKEAGLEKGFTIDLWAMPVQRPYNPNARRMAEMIQADWAKIGVQAKIVTYEWGE YLKRAKDGEHQTVMMGWTGDNGDPDNFFATLFSCDAAQQGSNYSKWCYKPFEDLIQP ARATDDHNKRIELYKQAQVVMHDQAPALI IAHSTVYEPVRKEVKGYWDPLGKHHFE NVSVE

19 SL1 184 VGYAQSKVQDFKNIRGVNVKYRYEDDSPVSFISSLSYLYGDRQASGSVEPEGIHYHD

KFEVKYGSLMVGPAYRLSDNFSLYALAGVGTVKATFKEHSTQDGDSFSNKISSRKTG FAWGAGVQMNPLENIWDVGYEGSNISSTKINGFNVGVGYRF

20 SL2756 MAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTAN

IKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQA EITQRLNEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLD SLNVQKAYDVKDTAVTTKAYANNGTTLDVSGLDDAAIKAATGGTNGTASVTGGAVKF DADNNKYFVTIGGFTGADAAKNGDYEVNVATDGTVTLAAGATKTTMPAGATTKTEVQ ELKDTPAWSADAKNALIAGGVDATDANGAELVKMSYTDKNGKTIEGGYALKAGDKY YAADYDEATGAIKAKTTSYTAADGTTKTAANQLGGVDGKTEVVTIDGKTYNASKAAG HDFKAQPELAEAAAKTTENPLQKIDAALAQVDALRSDLGAVQNRFNSAITNLGNTVN NLSEARSRIEDSDYATEVSNMSRAQILQQAGTSVLAQANQVPQNVLSLLR

21 SL1010 MKKTAIAIAVALAGFATVAQAAPKDNTWYAGAKLGWSQYHDTGFIHNDGPTHENQLG

AGAFGGYQVNPYVGFEMGYDWLGRMPYKGDNINGAYKAQGVQLTAKLGYPITDDLDV YTRLGGMVWRADTKSNVPGGPSTKDHDTGVSPVFAGGIEYAITPEIATRLEYQWTNN IGDANTIGTRPDNGLLSVGVSYRFGQQEAAPWAPAPAPAPEVQTKHFTLKSDVLFN FNKSTLKPEGQQALDQLYSQLSNLDPKDGSVWLGFTDRIGSDAYNQGLSEKRAQSV Table 3

SEQ Accession Amino acid Sequence

ID Number

NO

VDYLI SKGI PSDKI SARGMGESNPVTGNTCDNVKPRAALIDCLAPDRRVE IEVKGVK DWTQPQA

22 SL2237 MRIGFKGETQVNDQLTGYGQWEYQI QGNQTEGSNDSWTRVAFAGLKFADAGS FDYGR

NYGVTYDVTSWTDVLPEFGGDTYGADNFMQQRGNGYATYRNTDFFGLVDGLDFALQY QGKNGSVSGENTNGRSLLNQNGDGYGGSLTYAIGEGFSVGGAITTSKRTADQNNTAN ARLYGNGDRATVYTGGLKYDANNIYLAAQYSQTYNATRFGTSNGSNPSTSYGFANKA QNFEVVAQYQFDFGLRPSVAYLQSKGKDI SNGYGASYGDQDIVKYVDVGATYYFNKN MSTYVDYKI NLLDKNDFTRDAGI NTDD I VALGLVYQF

23 SL1133 MSTIEERVKKI IGEQLGVKQEEVTNNASFVEDLGADSLDTVELVMALEEEFDTE I PD

EEAEKITTVQAAIDYINGHQA

24 SL4489 MTMTRLKI SKTLLAVMLTSAVATGSAFAENATTDKAQSGTETAGQKVDSSMNKVGNF

MDDSAITAKVKAALVDHDNIKSTDI SVETNQKVVTLSGFVESQAQAEAAVKVAKGVE GVTSVSDKLHVRDNKEGSVKGYAGDTATTSEVKAKLLADDLVPSRKVKVETTDGWQ LSGTVETQEQSDRAES IAKAVDGVKSVKNDLKVQ

Table 4

SEQ Accession Nucleotide Sequence

ID Number

NO

2 5 SL08 08 ATGAAAAAAATTGCATGTCTTTCAGCACTGGCCGCTGTTCTGGCTTTTTCCGCAGGT

ACTGCAGTAGCTGCTACTTCTACCGTTACCGGTGGTTACGCTCAGAGCGACGCGCAG GGCGTAGCGAATAAAATGAGCGGTTTCAACCTGAAGTATCGTTACGAGCAGGACGAC AACCCGCTGGGCGTAATCGGTTCCTTCACCTACACCGAAAAAGATCGTACTAACGGT GCGGGCGATTACAACAAAGGTCAGTACTACGGCATCACTGCCGGCCCGGCTTACCGT CTGAACGATTGGGCAAGCATCTACGGTGTAGTCGGTGTGGGTTACGGTAAATTCCAG ACGACCGATTACCCAACCTACAAACATGACACCAGCGATTATGGCTTCTCCTATGGC GCTGGTCTGCAATTCAACCCGATGGAAAATGTTGCTCTGGACTTCTCTTACGAGCAG AGCCGTATTCGTAGCGTTGACGTTGGCACCTGGATTGCTGGCGTAGGTTACCGCTTC TAA

2 6 SL3 596 GTGGCCATGACCGTTGCAGCAAGCGTGCAGGCCAAAACCCTGGTTTATTGTTCAGAA

GGCTCGCCGGAAGGCTTTAACCCACAGCTCTTTACGTCTGGCACCACCTATGATGCC AGCTCCGTACCTATCTATAACCGTCTGGTTGAATTCAAAACCGGCACCACGGAAGTG ATCCCGGGTCTTGCTGAGAAGTGGGATATCAGCGAAGACGGTAAAACCTATACGTTC CACCTACGTAAAGGGGTGAAATGGCAATCCAGCAAGGATTTCAAACCCACGCGCGAG CTGAACGCCGATGATGTCGTGTTCTCTTTTGACCGGCAGAAAAACGAGCAGAACCCG TACCATAAAGTGTCTGGCGGCAGCTATGAATACTTTGAAGGCATGGGGCTGCCGGAT CTGATTAGCGAAGTGAAGAAGGTCGACGATCACACGGTGCAGTTTGTGCTGACGCGT CCGGAAGCGCCGTTCCTTGCCGATTTAGCCATGGACTTTGCCTCTATTCTTTCCAAA GAATATGCTGACAACATGCTGAAAGCCGGTACGCCGGAAAAAGTGGATCTGAACCCG GTCGGCACTGGCCCGTTCCAACTGGTGCAATATCAGAAAGACTCCCGCATTCTCTAC AAAGCCTTTGACGGCTACTGGGGCACGAAGCCGCAGATTGACCGTCTGGTCTTCTCC ATCACGCCTGACGCCTCTGTGCGTTACGCCAAACTGCAGAAGAACGAATGTCAGGTG ATGCCGTATCCGAACCCGGCGGATATTGCGCGCATGAAAGAAGATAAAAACATCAAC CTGATGGAGCAGGCCGGTCTGAACGTGGGTTATCTCTCCTATAACGTGCAGAAAAAA CCGCTGGATGATGTCAAAGTTCGCCAGGCGTTGACCTATGCCGTGAATAAAGAGGCC ATCATCAAAGCCGTTTATCAGGGCGCGGGCGTTGCGGCGAAAAACCTGATCCCGCCG ACAATGTGGGGCTACAACGACGATATTAAAGACTATGGCTACGATCCGGAAAAAGCG AAGGCGCTGCTGAAAGAAGCCGGTCTGGAAAAAGGCTTCACCATCGATCTGTGGGCG ATGCCGGTACAGCGTCCCTATAACCCGAATGCGCGTCGTATGGCGGAAATGATCCAG GCGGATTGGGCGAAGATTGGCGTTCAGGCCAAAATTGTCACCTATGAATGGGGCGAA TACCTCAAGCGCGCTAAAGATGGCGAGCACCAGACGGTGATGATGGGCTGGACCGGC GATAATGGCGATCCGGATAACTTCTTCGCCACGCTGTTCAGCTGCGATGCCGCCCAG CAAGGCTCCAACTATTCAAAATGGTGTTATAAGCCGTTTGAAGACCTGATTCAGCCT GCGCGTGCGACCGATGACCACAACAAGCGTATTGAGCTCTATAAACAGGCCCAGGTT GTGATGCATGACCAGGCGCCAGCGCTGATCATCGCTCACTCCACGGTTTATGAGCCA GTGCGTAAAGAAGTTAAAGGCTATGTGGTTGATCCATTAGGCAAACATCACTTCGAA AACGT CTCTGT CGAATAA

2 7 SL1184 GTGGGGTATGCACAAAGTAAAGTTCAGGATTTCAAAAATATCCGAGGGGTAAATGTG

AAATACCGTTATGAGGATGACTCTCCGGTAAGTTTTATTTCCTCGCTAAGTTACTTA TATGGAGACAGACAGGCTTCCGGGTCTGTTGAGCCTGAAGGTATTCATTACCATGAC AAGTTTGAGGTGAAGTACGGTTCTTTAATGGTTGGGCCAGCCTATCGATTGTCTGAC Table 4

SEQ Accession Nucleotide Sequence

ID Number

NO

AATTTTTCGTTATACGCGCTGGCGGGTGTCGGCACGGTAAAGGCGACATTTAAAGAA CATTCCACTCAGGATGGCGATTCTTTTTCTAACAAAATTTCCTCAAGGAAAACGGGA TTTGCCTGGGGCGCGGGTGTACAGATGAATCCGCTGGAGAATATCGTCGTCGATGTT GGGTATGAAGGAAGCAACATCTCCTCTACAAAAATAAACGGCTTCAACGTCGGGGTT GGATACCGTTTCTGA

28 SL2756 ATGGCACAAGTAATCAACACTAACAGTCTGTCGCTGCTGACCCAGAATAACCTGAAC

AAATCCCAGTCCGCACTGGGCACCGCTATCGAGCGTCTGTCTTCTGGTCTGCGTATC AACAGCGCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTCACCGCGAAC ATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAG ACCACTGAAGGCGCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTG GCGGTTCAGTCTGCTAACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCT GAAATCACCCAGCGCCTGAACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAAC GGCGTGAAAGTCCTGGCGCAGGACAACACCCTGACCATCCAGGTTGGCGCCAACGAC GGTGAAACTATCGATATCGATCTGAAGCAGATCAACTCTCAGACCCTGGGTCTGGAC TCACTGAACGTGCAGAAAGCGTATGATGTGAAAGATACAGCAGTAACAACGAAAGCT TATGCCAATAATGGTACTACACTGGATGTATCGGGTCTTGATGATGCAGCTATTAAA GCGGCTACGGGTGGTACGAATGGTACGGCTTCTGTAACCGGTGGTGCGGTTAAATTT GACGCAGATAATAACAAGTACTTTGTTACTATTGGTGGCTTTACTGGTGCTGATGCC GCCAAAAATGGCGATTATGAAGTTAACGTTGCTACTGACGGTACAGTAACCCTTGCG GCTGGCGCAACTAAAACCACAATGCCTGCTGGTGCGACAACTAAAACAGAAGTACAG GAGTTAAAAGATACACCGGCAGTTGTTTCAGCAGATGCTAAAAATGCCTTAATTGCT GGCGGCGTTGACGCTACCGATGCTAATGGCGCTGAGTTGGTCAAAATGTCTTATACC GATAAAAATGGTAAGACAATTGAAGGCGGTTATGCGCTTAAAGCTGGCGATAAGTAT TACGCCGCAGATTACGATGAAGCGACAGGAGCAATTAAAGCTAAAACCACAAGTTAT ACTGCTGCTGACGGCACTACCAAAACAGCGGCTAACCAACTGGGTGGCGTAGACGGT AAAACCGAAGTCGTTACTATCGACGGTAAAACCTACAATGCCAGCAAAGCCGCTGGT CATGATTTCAAAGCACAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAAC CCGCTGCAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTG GGTGCGGTACAAAACCGTTTCAACTCTGCTATCACCAACCTGGGCAATACCGTAAAC AATCTGTCTGAAGCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCC AACATGTCTCGCGCGCAGATTCTGCAGCAGGCCGGTACTTCCGTTCTGGCGCAGGCT AACCAGGTCCCGCAGAACGTGCTGTCTCTGTTACGTTAA

29 SL1010 ATGAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTTCGCTACCGTAGCG

CAGGCCGCTCCGAAAGATAACACCTGGTACGCTGGTGCTAAACTGGGCTGGTCTCAG TACCATGACACCGGCTTCATTCACAATGATGGCCCGACTCATGAAAACCAACTGGGC GCAGGTGCTTTTGGTGGTTACCAGGTTAACCCGTATGTTGGCTTTGAAATGGGCTAC GACTGGTTAGGCCGTATGCCGTACAAAGGCGACAACATCAATGGCGCTTATAAAGCT CAGGGCGTTCAGTTGACCGCTAAACTGGGTTATCCAATCACTGACGATCTGGACGTT TATACCCGTCTGGGTGGTATGGTATGGCGTGCAGACACCAAGTCTAACGTCCCTGGC GGCCCGTCTACTAAAGACCACGACACCGGCGTTTCCCCGGTATTCGCGGGCGGTATC GAGTATGCTATCACCCCTGAAATCGCAACCCGTCTGGAATACCAGTGGACTAACAAC ATCGGTGATGCCAACACCATCGGCACCCGTCCGGACAACGGCCTGCTGAGCGTAGGT GTTTCCTACCGTTTCGGCCAGCAAGAAGCTGCTCCGGTAGTAGCTCCGGCACCAGCT CCGGCTCCGGAAGTACAGACCAAGCACTTCACTCTGAAGTCTGACGTACTGTTCAAC TTCAACAAATCTACCCTGAAGCCGGAAGGCCAGCAGGCTCTGGATCAGCTGTACAGC CAGCTGAGCAACCTGGATCCGAAAGACGGTTCCGTTGTCGTTCTGGGCTTCACTGAC CGTATCGGTTCTGACGCTTACAACCAGGGTCTGTCCGAGAAACGTGCTCAGTCTGTT GTTGATTACCTGATCTCCAAAGGTATTCCGTCTGACAAAATCTCCGCACGTGGTATG GGCGAATCTAACCCGGTTACCGGCAACACCTGTGACAACGTGAAACCTCGCGCTGCC CTGATCGATTGCCTGGCTCCGGATCGTCGCGTAGAGATCGAAGTTAAAGGCGTTAAA GACGTGGTAACTCAGCCGCAGGCTTAA

30 SL2237 ATGCGTATCGGCTTCAAAGGCGAAACGCAGGTTAACGATCAGCTGACCGGTTATGGC

CAGTGGGAATATCAGATTCAGGGCAACCAGACTGAAGGCAGCAACGACTCCTGGACG CGTGTGGCGTTTGCGGGTCTGAAATTCGCTGATGCAGGTTCCTTCGATTATGGTCGT AACTACGGCGTAACCTATGACGTGACCTCCTGGACCGACGTTCTGCCGGAGTTCGGC GGCGACACCTACGGCGCTGACAACTTTATGCAGCAGCGTGGTAACGGCTATGCTACC TACCGTAACACCGACTTCTTCGGCCTGGTGGATGGTCTGGACTTCGCGTTACAGTAT CAGGGCAAAAACGGCAGCGTGAGCGGTGAAAACACCAACGGTCGCAGCCTGCTGAAC CAGAACGGCGACGGTTACGGCGGATCGCTGACTTATGCAATCGGCGAAGGCTTCTCT GTCGGTGGCGCTATCACCACGTCTAAACGTACTGCCGATCAGAACAACACCGCTAAC GCTCGCCTGTATGGTAACGGCGATCGCGCCACGGTTTACACCGGCGGCCTGAAATAC GATGCGAACAACATCTATCTGGCAGCGCAGTATTCTCAGACCTATAACGCAACCCGT TTTGGTACCTCTAACGGTAGCAACCCGTCCACCTCTTACGGTTTTGCCAACAAAGCG CAGAACTTTGAAGTGGTTGCTCAGTACCAGTTCGACTTTGGTCTGCGTCCGTCTGTG Table 4

SEQ Accession Nucleotide Sequence

ID Number

NO

GCTTACCTGCAGTCTAAAGGTAAGGACATCAGCAACGGTTACGGCGCCAGCTATGGC GACCAGGACATCGTAAAATACGTTGATGTCGGCGCGACTTACTACTTCAACAAAAAC ATGTCCACCTATGTTGATTACAAAATCAACCTGCTGGATAAAAACGACTTTACCCGC GATGCGGGCATCAACACCGACGACATCGTAGCGCTGGGTCTGGTTTACCAGTTCTAA

3 1 SL1133 ATGAGCACTATCGAAGAACGCGTTAAGAAAATTATCGGCGAACAGCTGGGCGTTAAG

CAGGAAGAAGTAACCAACAATGCTTCTTTCGTTGAAGACCTGGGCGCAGATTCTCTT GACACCGTTGAGCTGGTAATGGCTCTGGAAGAAGAGTTTGATACTGAGATTCCGGAC GAAGAAGCTGAGAAAATCACCACCGTTCAGGCTGCCATTGATTACATCAACGGCCAC CAGGCGTAA

3 2 SL44 89 ATGACTATGACAAGACTGAAGATTTCTAAAACTCTGCTGGCCGTAATGTTGACCTCT

GCTGTTGCGACAGGTTCTGCCTTTGCAGAAAACGCAACAACGGACAAGGCGCAAAGC GGAACCGAAACCGCAGGGCAAAAAGTCGATAGCTCTATGAATAAAGTCGGTAACTTC ATGGATGACAGCGCCATCACTGCGAAAGTAAAAGCTGCGCTGGTAGACCATGACAAT ATT AAAAGCAC CGATATTT CTGT CGAAAC CAAT CAGAAAGTTGT CACC CTGAGCGGC TTTGTAGAAAGCCAGGCGCAGGCTGAAGCCGCCGTGAAAGTGGCGAAAGGCGTAGAA GGCGTGACCTCCGTTAGCGACAAACTTCACGTTCGCGACAATAAAGAAGGTTCCGTG AAAGGTTATGC CGGCGATACGGC CACGAC CAGTGAAGT CAAAGC CAAGTTGCTGGCG GACGATCTCGTCCCTTCCCGTAAAGTGAAAGTGGAAACGACCGATGGCGTCGTACAG CTCTCCGGTACCGTTGAAACTCAGGAACAAAGCGACCGCGCTGAAAGCATCGCGAAA GCCGTTGATGGCGTAAAAAGTGTTAAAAACGATCTGAAAGTTCAGTAA

Table 5

Fraction presence/amount

Peptides Gene Location / Function F9 F13 F14 F18 F19 F22

SL4489 osmY Outer membrane ptn/ + + + +

protection against osmotic

shock

SL0866 artl Periplasmic/ arginine- < + + + + < binding protein

SL1010 ompA Outer membrane ptn/ < + + + < < structural

SL0731 pal Outer membrane ptn/ < + + + + +

peptidoglycan associated

lipoprotein

SL1780 Unknown - + + + + +

SL2251 gipQ Periplasmic/ + + + +

gly ceropho sphodie ster

phosphodiesterase

SL4369 cybC Periplasmic/ soluble < + + < < < cytochrome b562-electron

carrier

SL1061 Putative Unknown - + - - - - lipoptn

SL4109 hupA Cytoplasmic/ DNA binding +

transcriptional regulator SL1492 hdeB Periplasmic /acid resistance < + + + + +

chaperone

SL0808 ompX Outer membrane ptn/

bacterial adherence,

resistance to complement

SL3596 dppA Periplasmic/ dipeptide

transport protein

SL2237 ompC Outer membrane protein/

structural

+: absence of peptide; - absence of peptide; < : lower amount of peptide; ptn = protein

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[00225] All publications and patent applications cited in this specification are herein incorporated by reference in their entirety for all purposes as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference for all purposes.

[00226] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

Claims

Claims:

1. A composition comprising an effective amount of an isolated polypeptide to induce an immune response in a vertebrate subject to a Gram negative bacterial infection, wherein the isolated polypeptide comprises an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8 or wherein the isolated polypeptide comprises an amino acid sequence encoded by a polynucleotide sequence at least 80% identical to the polynucleotide sequence set forth in SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID

NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16.

2. The composition of claim 1, wherein the isolated polypeptide comprises the amino acid sequence set forth in SEQ ID NO:7, wherein the Gram negative bacterial infection is a Salmonella spp. infection, and wherein the amount of the composition is an effective amount to reduce the Salmonella spp. colony forming units (CFUs) per gram (g) of cecum, spleen, and/or liver of the subject after Salmonella spp. infection compared to a control subject.

3. The composition of any one of claims 1-2, wherein the amount of the composition is an effective amount to reduce the Salmonella spp. colony forming units (CFUs) per gram (g) of cecum, spleen, and/or liver of the subject after Salmonella spp. infection compared to a control subject.

4. The composition of any one of claims 1-3, wherein the infection is a Salmonella spp. infection.

5. The composition of any one of claims 1-4, wherein the Salmonella spp. infection is a Salmonella Typhimurium infection or a Salmonella Enteritidis infection.

6. The composition of any one of claims 1-5, wherein the amount of composition is

effective in reducing or eliminating bacterial carriage and/or bacterial infection and/or bacterial shedding and/or bacterial colonization in the subject.

7. The composition of any one of claims 1-6, wherein the amount of composition is

effective in reducing or eliminating bacterial carriage and/or bacterial infection and/or bacterial shedding and/or bacterial colonization in the subject after the subject has been contacted by the bacteria.

8. The composition of any one of claims 1-7, wherein the composition further comprises a pharmaceutically acceptable carrier.

9. The composition of any one of claims 1-8, wherein the composition further comprises a pharmaceutically acceptable carrier comprising buffered saline and/or phosphate buffered saline.

10. The composition of any one of claims 1-9, wherein the composition further comprises a pharmaceutically acceptable adjuvant.

11. The composition of any one of claims 1-10, wherein the composition further comprises a pharmaceutically acceptable adjuvant, and wherein the pharmaceutically acceptable adjuvant comprises Alum, RIBI, CpG, saponin, a phosphodiester, a non-DNA-based oligonucleotide, and/or TiterMax.

12. The composition of any one of claims 1-11, wherein the composition further comprises a pharmaceutically acceptable adjuvant, and wherein the pharmaceutically acceptable adjuvant comprises an oil-in-water emulsion.

13. The composition of any one of claims 1-12, wherein the vertebrate subject is a

mammalian subject, a human subject, an avian subject, a bovine subject, or a porcine subject.

14. The composition of any one of claims 1-13, wherein the isolated polypeptide is purified.

15. The composition of any one of claims 1-14, wherein the isolated polypeptide is

recombinant.

16. The composition of any one of claims 1-15, wherein the isolated polypeptide comprises an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: l, SEQ ID NO:2, SEQ ID O:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8.

17. The composition of any one of claims 1-16, wherein the isolated polypeptide comprises an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8.

18. The composition of any one of claims 1-17, wherein the isolated polypeptide comprises an amino acid sequence at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97 , 98, or 99% identical to the amino acid sequence set forth in SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID N0:4, SEQ ID N0:5, SEQ ID N0:6, SEQ ID N0:7, or SEQ ID O:8.

19. The composition of any one of claims 1-18, wherein the isolated polypeptide comprises the amino acid sequence set forth in SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8.

20. The composition of any one of claims 1-19, wherein the isolated polypeptide comprises an amino acid sequence encoded by a polynucleotide sequence at least 90% identical to the polynucleotide sequence set forth in SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16.

21. The composition of any one of claims 1-20, wherein the isolated polypeptide comprises an amino acid sequence encoded by a polynucleotide sequence at least 95% identical to the polynucleotide sequence set forth in SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16.

22. The composition of any one of claims 1-21, wherein the isolated polypeptide comprises an amino acid sequence encoded by a polynucleotide sequence at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97 , 98, or 99% identical to the

polynucleotide sequence set forth in SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16.

23. The composition of any one of claims 1-22, wherein the isolated polypeptide comprises an amino acid sequence encoded by a polynucleotide sequence the polynucleotide sequence set forth in SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:l 1, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16.

24. The composition of any one of claims 1-23, wherein the composition comprises at least two or more isolated polypeptides.

25. The composition of any one of claims 1-24, wherein the composition comprises at least three or more isolated polypeptides.

26. The composition of any one of claims 1-25, wherein the composition comprises at least 2, 3, 4, 5, 6, 7, 8, or more isolated polypeptides.

27. The composition of any one of claims 1-26, wherein the composition comprises the amino acid sequences set forth in SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8.

28. The composition of any one of claims 1-27, wherein the composition does not comprise a FliF protein, a CsgA protein, CsgB protein, a PhoP protein, and/or lipopolysaccharide (LPS).

29. The composition of any one of claims 1-28, further comprising one or more recombinant or purified antigens and/or proteins.

30. The composition of any one of claims 1-29, further comprising one or more additional proteins selected from Table 3 or one or more additional proteins encoded by the nucleotide sequences shown in Table 4.

31. An isolated vector comprising a polynucleotide sequence at least 80% identical to the polynucleotide sequence set forth in SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16.

32. The vector of claim 31, wherein the vector comprises a polynucleotide sequence at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97 , 98, or 99% identical to the polynucleotide sequence set forth in SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 1 1, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16.

33. The vector of any one of claims 31-32 wherein the polynucleotide sequence is operably linked to a promoter sequence.

34. A cell comprising an isolated polynucleotide sequence at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97 , 98, or 99% identical to the polynucleotide sequence set forth in SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: l 1, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16, or the vector of any one of claims 31-33.

35. A cell culture comprising a culture medium and the cell of claim 34.

36. A composition comprising an effective immunizing amount of an isolated

polynucleotide, wherein the composition is effective in a vertebrate subject to induce an immune response to a Gram negative bacterial infection, and wherein the isolated polynucleotide comprises a polynucleotide sequence at least 80% identical to the polynucleotide sequence set forth in SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16.

37. The composition of claim 36, wherein the isolated polynucleotide comprises the amino acid sequence set forth in SEQ ID NO: 15, wherein the Gram negative bacterial infection is a Salmonella spp. infection, and wherein the amount of the composition is an effective amount to reduce the Salmonella spp. colony forming units (CFUs) per gram (g) of cecum, spleen, and/or liver of the subject after Salmonella spp. infection compared to a control subject.

38. The composition of any one of claims 36-37, wherein the amount of the composition is an effective amount to reduce the Salmonella spp. colony forming units (CFUs) per gram (g) of cecum, spleen, and/or liver of the subject after Salmonella spp. infection compared to a control subject.

39. The composition of any one of claims 36-38, wherein the infection is a Salmonella spp. infection.

40. The composition of any one of claims 36-39, wherein the Salmonella spp. infection is a Salmonella Typhimurium infection or a Salmonella Enteritidis infection.

41. The composition of any one of claims 36-40, wherein the amount of composition is effective in reducing or eliminating bacterial carriage and/or bacterial infection and/or bacterial shedding and/or bacterial colonization in the subject.

42. The composition of any one of claims 36-41, wherein the amount of composition is effective in reducing or eliminating bacterial carriage and/or bacterial infection and/or bacterial shedding and/or bacterial colonization in the subject after the subject has been contacted by the bacteria.

43. The composition of any one of claims 36-42, wherein the composition further comprises a pharmaceutically acceptable carrier.

44. The composition of any one of claims 36-43, wherein the composition further comprises a pharmaceutically acceptable carrier comprising buffered saline and/or phosphate buffered saline.

45. The composition of any one of claims 36-44, wherein the composition further comprises a pharmaceutically acceptable adjuvant.

46. The composition of any one of claims 36-45, wherein the composition further comprises a pharmaceutically acceptable adjuvant, and wherein the pharmaceutically acceptable adjuvant comprises RIBI, CpG, saponin, a phosphodiester, a non-DNA-based oligonucleotide, and/or TiterMax.

47. The composition of any one of claims 36-46, wherein the composition further comprises a pharmaceutically acceptable adjuvant, and wherein the pharmaceutically acceptable adjuvant comprises an oil-in-water emulsion.

48. The composition of any one of claims 36-47, wherein the vertebrate subject is a

mammalian subject, a human subject, an avian subject, a bovine subject, or a porcine subject.

49. The composition of any one of claims 36-48, wherein the isolated polynucleotide is purified.

50. The composition of any one of claims 36-49, wherein the isolated polynucleotide is recombinant.

51. The composition of any one of claims 36-50, wherein the isolated polynucleotide

comprises a nucleotide sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16.

52. The composition of any one of claims 36-51, wherein the isolated polynucleotide

comprises a nucleotide sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16.

53. The composition of any one of claims 36-52, wherein the isolated polynucleotide

comprises a nucleotide sequence at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97 , 98, or 99% identical to the amino acid sequence set forth in SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID

NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16.

54. The composition of any one of claims 36-53, wherein the isolated polynucleotide

comprises a nucleotide sequence set forth in SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 1 1, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16.

55. The composition of any one of claims 36-54, wherein the composition comprises at least two or more isolated polynucleotides.

56. The composition of any one of claims 36-55, wherein the composition comprises at least three or more isolated polynucleotides.

57. The composition of any one of claims 36-56, wherein the composition comprises at least 2, 3, 4, 5, 6, 7, 8, or more isolated polynucleotides.

58. The composition of any one of claims 36-57, wherein the composition comprises the nucleotide sequences set forth in SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16.

59. The composition of any one of claims 36-58, wherein the composition does not comprise a FliF protein, a CsgA protein, CsgB protein, a PhoP protein, and/or lipopolysaccharide (LPS).

60. The composition of any one of claims 36-59, further comprising one or more

recombinant or purified antigens and/or proteins and/or nucleotides.

61. The composition of any one of claims 36-60, further comprising one or more additional nucleotide sequences shown in Table 4.

62. A method for preventing or treating a Gram negative bacterial infection in a vertebrate subject in need thereof comprising administering the composition of any one of claims 1- 61 to the vertebrate subject in an amount effective to reduce or eliminate the bacterial infection.

63. The method of claim 62, wherein the amount of the composition is effective in the

subject to reduce the Salmonella spp. colony forming units (CFUs) per gram (g) of cecum, spleen, and/or liver of the subject after Salmonella spp. infection compared to a control subject.

64. A method for inducing an immune response in a vertebrate subject against a Salmonella spp. bacterial infection comprising administering the composition of any one of claims 1-61 to the vertebrate subject in an amount effective to induce the immune response in the vertebrate subject.

65. A method for reducing colonization of Salmonella spp. bacteria in a vertebrate subject in need thereof comprising administering to the vertebrate subject the composition of any one of claims 1-61 and a pharmaceutically acceptable adjuvant in an amount effective to reduce Salmonella spp. colonization in the vertebrate subject.

66. The method of claim 65, wherein reducing colonization of Salmonella spp. bacteria in the vertebrate subject further comprises reducing a risk of infectious transfer from the vertebrate subject to a second distinct subject.

67. A method for reducing shedding of Salmonella spp. bacteria in a vertebrate subject in need thereof comprising administering to the vertebrate subject the composition of any one of claims 1-61 and a pharmaceutically acceptable adjuvant in an amount effective to reduce Salmonella spp. shedding in the vertebrate subject.

68. The method of claim 67, wherein reducing shedding of Salmonella spp. bacteria in the vertebrate subject further comprises reducing a risk of infectious transfer from the vertebrate subject to a second distinct subject.

69. A method for reducing Salmonella spp. bacterial infection in a vertebrate subject in need thereof comprising administering to the vertebrate subject the composition of any one of claims 1-61 and a pharmaceutically acceptable adjuvant in an amount effective to reduce Salmonella spp. bacterial infection in the vertebrate subject.

70. A method for reducing carriage of Salmonella spp. bacteria in a vertebrate subject in need thereof comprising administering to the vertebrate subject the composition of any one of claims 1-61 and a pharmaceutically acceptable adjuvant in an amount effective to reduce Salmonella spp. carriage in the vertebrate subject.

71. A method for reducing colonization of Salmonella spp. bacteria in a vertebrate subject in need thereof comprising administering to the vertebrate subject the composition of any one of claims 1-61 in an amount effective to reduce Salmonella spp. colonization in the vertebrate subject.

72. A method for reducing shedding of Salmonella spp. bacteria in a vertebrate subject in need thereof comprising administering to the vertebrate subject the composition of any one of claims 1-61 in an amount effective to reduce Salmonella spp. shedding in the vertebrate subject.

73. A method for reducing Salmonella spp. bacterial infection in a vertebrate subject in need thereof comprising administering to the vertebrate subject the composition of any one of claims 1-61 in an amount effective to reduce Salmonella spp. bacterial infection in the vertebrate subject.

74. A method for reducing carriage of Salmonella spp. bacteria in a vertebrate subject in need thereof comprising administering to the vertebrate subject the composition of any one of claims 1-61 in an amount effective to reduce Salmonella spp. carriage in the vertebrate subject.

75. A method for preventing or treating a Gram negative bacterial infection in a vertebrate subject in need thereof comprising administering the composition of any one of claims 1- 61 and a pharmaceutically acceptable adjuvant to the vertebrate subject in an amount effective to reduce or eliminate the bacterial infection.

76. A method for inducing an immune response in a vertebrate subject against a Salmonella spp. bacterial infection comprising administering the composition of any one of claims 1-61 and a pharmaceutically acceptable adjuvant to the vertebrate subject in an amount effective to induce the immune response in the vertebrate subject.

77. A method for producing a protein, comprising contacting a cell with a polynucleotide sequence at least 80% identical to the polynucleotide sequence set forth in SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16; and expressing a protein encoded by the polynucleotide sequence.

78. A method for producing a protein, comprising synthesizing an isolated polypeptide

comprising an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8 or an isolated polypeptide comprising an amino acid sequence encoded by a polynucleotide sequence at least 80% identical to the polynucleotide sequence set forth in SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16.