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Product USA. M. No. 1

PATENT NUMBER This data is not available for free

PATENT GRANT DATE June 4, 2002

PATENT TITLE Live attenuated salmonella vaccines to control avian pathogens

PATENT ABSTRACT A vaccine for protecting birds against infection by avian pathogenic gram negative microbes is disclosed. The vaccine is a recombinant Salmonella strain expressing O-antigen of an avian pathogenic gram negative microbe such as an E. coli strain that is pathogenic in poultry. The recombinant Salmonella strain also does not express Salmonella O-antigen. Methods of using the vaccine to immunize birds are also disclosed

PATENT INVENTORS This data is not available for free

PATENT ASSIGNEE This data is not available for free

PATENT FILE DATE July 24, 1998

PATENT REFERENCES CITED Falt et al. (1996) Microb. Path., vol. 20, 11-30, 1996.*
Viret et al. (1993) Mol. Micro., vol. 7 (2), 239-252, 1993.*
Patent Cooperation Treaty International Search Report for PCT/US99/15842, Feb. 24, 2000.
Roland et al., Abstracts in Immunology, Entry E-85, XP-000867749, Construction and Characterization of a Salmonella typhimurium vaccine strain expressing Escherichia coli O78 lipopolysacchande, p. 255, 1997.
Bastin et al., Molecular cloning and expression in Escherichia coli K-12 of the rfb gene cluster determining the O antigen of an E. coli O111 strain, Mol. Microbiol. 5:2223-2231 (1991).
Black et al., Prevention of Shigellosis by a Salmonella typhi-Shigella sonnei Bivalent Vaccine, J. Infect. Dis. 155:1260-1265 (1987).
Collins et al. Molecular Cloning, Characterization, and Nucleotide Sequence of the rfc Gene, Which Encodes an O-Antigen Polymerase of Salmonella typhimurium, J. Bacteriol. 173:2521-2529 (1991).
Formal et al., Construction of a Potential Bivalent Vaccine Strain: Introduction of Shigella sonnei Form I antigen Genes into the galE Salmonella typhi Ty21a Typhoid Vaccine Strain, Infect. Immun. 34:746-750 (1981).
Galan et al., Cloning and characterization of the asd gene of Salmoennal typhimurium: use in stable maintenance of recombinant plasmids in Salmonella strains, Gene 94:29-35 (1990).
Haraguchi et al., Molecular cloning and expression of the 04 polysaccharide gene cluster from Escherichia coli, Microb. Pathol. 6:123-132 (1989).
Haraguchi et al., Genetic characterization of the 04 polysaccharide gene cluster from Escherichia coli, Microb. Pathol. 10:351-361 (1991).
Hassan et al. Development and Evaluation of an Experimental Vaccination Program Using a Live Avirulent Salmonella typhimurium Strain To Protect Immunized Chickens against Challenge with Homologous and Heterologous Salmonella Serotypes, Infect. Immun. 62:5519-5527 (1994).
Hassan et al., Control of Colonization by Virulent Salmonella Typhimurium by Oral Immunization of Chickens with Avirulent .DELTA. cya , .DELTA. crp S. Typhimurium, Res. Microbiol. 141:839-850 (1990).
Heuzenroeder et al., Molecular cloning and expression in Escherichia coli K-12 of the 0101 rfb region from E. coli B41 (0101:K99/F41) and the genetic relationship to other 0101 rfb loci, Mol. Microbiol. 3:295-302 (1989).
Hone et al., A chromosomal integration system for stabilization of heterologous genes in Samonella based vaccine strains, Microb. Pathog. 5:407-418 (1988).
Nakayama et al., Construction of an ASD+ Expression-Cloning Vector: Stable Maintenance and High Level Expression of Cloned Genes in a Salmonella Vaccine Strain, Bio/Technology 6:693-694 (1995).
Neal et al., Molecular cloning and expression in Escherichia coli K-12 of chromosomal genes determining the O antigen of an E. coli 02:K1 strain, FEMS Microbiol. Lett. 82:345-352 (1991).
Pier et al., Clearance of Pseudomonas aeruginosa from the Murine Gastrointestinal Tract Is Effectively Mediated by O-Antigen-Specific Circulating Antibodies, Infect. Immun. 63:2818-2825 (1995).
Porter et al., Virulence of Salmonella typhimurium Mutants for White Leghorn Chicks, Avian Dis. 37:265-273 (1993).
Strugnell et al., Stable expression of foreign antigens from the chromosome of Salmonella typhimurium. vaccine strains, Gene 88:57-63 (1990).
Sugiyama et al., Expression of the Cloned Escherichia coli 09 rfb Gene in Various Mutant Strains of Salmonella typhimurium, J. Bacteriol. 173:55-58 (1991).
Valvano et al., Molecular Cloning and Expression in Escherichia coli K-12 of Chromosal Genes Determining the 07 Lipopolysaccharide Antigen of a Human Invasive Strain of E. coli 07:K1, Infect. Immun. 57:937-943 (1989).

PATENT CLAIMS What is claimed is:

1. A composition for stimulating an immune response in birds against an avian pathogenic gram-negative (AP.sub.G-N) microbe selected from the group consisting of Escherichia coli serotypes O1, O2, O3, O6, O8, O15, O18, O35, O71, O74, or O78; Salmonella group C strains; Salmonella group D strains; and avian pathogenic species from Bordetella, Haemophilus, Pasturella, Klebsiella, Pseudomonas, or Ornithobacterium, comprising live cells of a recombinant Salmonella strain expressing an O-antigen of the AP.sub.G-N microbe against which the immune response is directed, the recombinant Salmonella strain having an rfb/rfc gene cluster of the AP.sub.G-N microbe stably integrated into the Salmonella chromosome and having a mutation in the Salmonella rfb gene cluster or in the Salmonella rfc gene which inactivates expression of Salmonella O-antigen, wherein the recombinant Salmonella strain is an attenuated mutant of a virulent Salmonella strain which is capable of colonizing birds.

2. The composition of claim 1, wherein the AP.sub.G-N microbe is an avian pathogenic Escherichia coli (APEC) strain.

3. The composition of claim 2, wherein the integrated APEC rfb/rfc gene cluster comprises an attenuating mutation in a Salmonella gene selected from the group consisting of pab, pur, aro, asd, dap, nadA, pncB, galE, pmi, fur, rpsL, ompR, htrA, hemA, cdt, cya, crp, phoP, phoQ, rfc, poxA, and galU.

4. The composition of claim 3, wherein the attenuating mutation is a defined deletion/insertion mutation in the Salmonella cya gene.

5. The composition of claim 4, wherein the recombinant Salmonella strain also has an attenuating mutation in the Salmonella crp gene.

6. The composition of claim 5, wherein the APEC strain has a serotype of O1, O2, O35 or O78.

7. The composition of claim 1, wherein the recombinant Salmonella strain also has a recombinant polynucleotide encoding a desired gene product.

8. The composition of claim 7, wherein the desired gene product is an antigen from an avian pathogenic organism.

9. The composition of claim 8, wherein the avian pathogenic organism is an avian pathogenic Escherichia coli (APEC) strain and the antigen is a fimbriae or an iron-regulated outer membrane protein of the APEC strain.

10. A method for stimulating an immune response in a bird against an avian pathogenic gram-negative (AP.sub.G-N) microbe selected from the group consisting of Escherichia coli serotypes O1, O2, O3, O6, O8, O15, O18, O35, O71, O74, or O78; Salmonella group C strains; Salmonella group D strains; and avian pathogenic species from Bordetella, Haemophilus, Pasturella, Klebsiella, Pseudomonas, or Ornithobacterium, the method comprising administering to the bird an effective amount of a composition comprising live cells of a recombinant Salmonella strain expressing an O-antigen of the AP.sub.G-N microbe against which the immune response is directed, the recombinant Salmonella strain having an rfb/rfc gene cluster of the AP.sub.G-N microbe stably integrated into the Salmonella chromosome and having a mutation in the Salmonella rfb gene cluster or in the Salmonella rfc gene which inactivates expression of Salmonella O-antigen, wherein the recombinant Salmonella strain is an attenuated mutant of a virulent Salmonella strain which is capable of colonizing birds.

11. The method of claim 10, wherein the AP.sub.G-N microbe is an avian pathogenic Escherichia coli (APEC) strain.

12. The method of claim 11, wherein the integrated APEC rfb/rfc gene cluster comprises an attenuating mutation in a Salmonella gene selected from the group consisting of pab, pur, aro, asd, dap, nadA, pncB, galE, pmi, fur, rpsL, ompR, htrA, hemA, cdt, cya, crp, phoP, phoQ, rfc, poxA, and galU.

13. The method of claim 12, wherein the attenuating mutation is a defined deletion/insertion mutation in the Salmonella cya gene.

14. The method of claim 13, wherein the recombinant Salmonella strain also has an attenuating mutation in the Salmonella crp gene.

15. The method of claim 14, wherein the APEC strain has a serotype of O1, O2, O35 or O78.

16. The method of claim 10, wherein the recombinant Salmonella strain also has a recombinant polynucleotide encoding a desired gene product.

17. The method of claim 10, wherein the bird is a chicken or a turkey.

18. The method of claim 17, wherein the composition is administered by coarse spray at day-of-hatch.

19. The method of claim 18, further comprising orally administering to the bird a booster amount of the composition.

20. The method of claim 19, wherein the booster amount is administered on day 13, 14, or 15 after day-of-hatch.

21. A composition for stimulating an immune response in birds against at least two avian pathogenic gram-negative (AP.sub.G-N) microbes, the vaccine comprising a mixture of live cells of first and second recombinant Salmonella strains, the first recombinant Salmonella strain having an rbf/rfc gene cluster of a first AP.sub.G-N microbe integrated into the Salmonella chromosome and expressing an O-antigen of the first AP.sub.G-N microbe and the second recombinant Salmonella strain having an rfb/rfc gene cluster of a second AP.sub.G-N microbe integrated into the Salmonella chromosome and expressing an O-antigen of the second AP.sub.G-N microbe, wherein each of the first and second recombinant Salmonella strains has a mutation in the Salmonella rfb gene cluster or in the Salmonella rfc gene which inactivates expression of Salmonella O-antigen, wherein the first AP.sub.G-N microbe and the second AP.sub.G-N microbe are selected from the group consisting of Escherichia coli serotypes O1, O2, O3, O6, O8, O15, O18, O35, O71, O74, or O78; Salmonella group C or group D strains; and avian pathogenic species from Bordetella, Haemophilus, Pasturella, Klebsiella, Pseudomonas, or Ornithobacterium, and wherein each of the first and second recombinant Salmonella strains is an attenuated mutant of a virulent Salmonella strain which is capable of colonizing birds.

22. A composition for stimulating an immune response in birds against strains of at least two avian pathogenic gram-negative (AP.sub.G-N) microbes each of which strains is selected from the group consisting of Escherichia coli serotypes O1, O2, O3, O6, O8, O15, O18, O35, O71, O74, or O78; Salmonella group C or group D strains; and avian pathogenic species from Bordetella, Haemophilus, Pasturella, Klebsiella, Pseudomonas, or Ornithobacterium, the composition comprising live cells of a recombinant Salmonella strain expressing an O-antigen of each of the AP.sub.G-N microbes against which the immune response is directed, the recombinant Salmonella strain having an rfb/rfc gene cluster of each of the AP.sub.G-N microbes stably integrated into the Salmonella chromosome and having a mutation in the Salmonella rfb gene cluster or in the Salmonella rfbc gene which inactivates expression of Salmonella O-antigen, wherein the recombinant Salmonella strain is an attenuated mutant of a virulent Salmonella strain which is capable of colonizing birds.

23. A method of making a composition for stimulating an immune response in a bird against an avian pathogenic gram-negative (AP.sub.G-N) microbe selected from the group consisting of Escherichia coli serotypes O1, O2, O3, O6, O8, O15, O18, O35, O71, O74, or O78; Salmonella group C or group D strains; and avian pathogenic species from Bordetella, Haemophilus, Pasturella, Klebsiella, Pseudomonas, or Ornithobacterium, comprising the steps of:

(a) selecting a Salmonella strain capable of colonizing the bird;

(b) integrating into the Salmonella chromosome an rfb/rfc gene cluster from the AP.sub.G-N microbe against which the immune response is directed;

(c) introducing a mutation into the Salmonella rfb gene cluster and/or into the Salmonella rfc gene; and

(d) isolating recombinant Salmonella bacteria which express O-antigen characteristic of the AP.sub.G-N strain but which do not express Salmonella O-antigen, wherein steps (b) and (c) can be performed in any order.

24. The method of claim 23, wherein the selected Salmonella strain is an attenuated mutant of a virulent Salmonella strain.

25. The method of claim 24, wherein the selected Salmonella strain is a virulent Salmonella strain and the method further comprises introducing into the virulent Salmonella strain an attenuating mutation in a Salmonella gene selected from the group consisting of pab, pur, aro, asd, dap, nadA, pncB, galE, pmi, fur, rpsL, ompR, htrA, hemA, cdt, cya, crp, phoP, phoQ, rfc, poxA, and galU, and then isolating mutants having attenuated virulence as compared to the virulent Salmonella strain.

26. The composition of claim 1, wherein the mutation which inactivates expression of Salmonella O-antigen is a mutation in the Salmonella rfb gene cluster.

27. The method of claim 11, wherein the mutation which inactivates expression of Salmonella O-antigen is a mutation in the Salmonella rfb gene cluster.

28. the method of claim 25, wherein step (c) comprises introducing a mutation into the Salmonella rfb gene cluster.

29. The composition of claim 26, wherein the recombinant Salmonella strain also has a recombinant polynucleotide encoding a desired gene product.

30. The composition of claim 1, wherein the recombinant Salmonella strain is selected from the group consisting of Salmonella typhimurium, Salmonella enteriditis, Salmonella heidelberg, Salmonella gallinorum, Salmonella hadar, Salmonella agona, Salmonella kentucky and Salmonella infantis.
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PATENT DESCRIPTION BACKGROUND OF THE INVENTION

This invention relates generally to vaccines for poultry and other birds and, more particularly, to vaccines for protecting poultry and other birds against infection by avian pathogenic gram-negative bacteria.

DESCRIPTION OF THE RELATED ART

Avian pathogenic E. coli (APEC) strains cause a number of related diseases in poultry and other birds, including air sacculitis, cellulitis, colibacillosis, coligranuloma, colisepticemia, Hjarre's disease, omphalitis, peritonitis, salpingitis, synovitis (Gross, W. B. in Diseases of Poultry, Calnek et al., eds., Iowa State University Press, p. 138-144, 1991; Messier et al., Avian Diseases 37:839-844, 1993). These diseases and other diseases caused by gram-negative avian pathogens can lead to increased rates of feed conversion carcass condemnation, or death of the animal, resulting in millions of dollars lost to the poultry industry each year. Norton, R. A. Broiler Industry, February 1998, pp. 28-32.

Contamination of poultry products by Salmonella is a significant source of Salmonella infection in humans, which causes gastroenteritis, and thus is a major public health concern. Oral administration of live cells from S. typhimurium strains having attenuating deletions in the cya and crp genes has been shown to provide excellent protection against wild-type Salmonella challenge in chickens (Hassan et al., Res. Microbiol. 141:839-850, 1990; Hassan et al., Infect. Immun. 62:5519-5527, 1994) and systems for stable expression of heterologous antigens in such strains have been developed (Hone et al., Microb. Pathog. 5:407-418, 1988; Strugnell, et al., Gene 88:57-63, 1990; Galan et al., Gene 94:29-35; 1990; Nakayama et al., Bio/Technology 6:693-697, 1995).

APEC strains are represented primarily by only a few serotypes, O1, O2, O35 and O78 (Cloud et al., Avian Dis. 29:1084-1093, 1985, Glantz et al., Avian Dis. 6:322-328, 1962; Gross, supra), while Salmonella serotypes most prevalent in poultry are in the B, C and D groups. O-serotypes of gram-negative bacteria such as E. coli and Salmonella are determined at the molecular level by the so-called O-antigen structure, also termed O-specific chain or O-polysaccharide (O-PS), which generally is comprised of varying lengths of polymerized identical sugar units anchored in the bacterial outer membrane as the outermost component of lipopolysaccharide (LPS) molecules (Helander et al., in Molecular Biology and Biotechnology: A Comprehensive Desk Reference, R. A. Meyers, ed., VCH Publishers, Inc., 1995).

Specific antibody against LPS O-antigen has been shown to be protective in mammalian models of extraintestinal E. coli infections in humans (Cryz et al., Vaccine 13;449-453, 1995; Pluschke et al., Infect. Immun. 49:365-370, 1985) and LPS O-antigen has been recognized as a protective antigen for other gram negative pathogens (Ding et al., J. Med. Microb. 31:95-102, 1990; Michetti et al., Infect. Immun. 60:1786-1792, 1992; Robbins et al., Clin. Infect Dis. 15:346-361, 1992). In addition, several research groups have reported using attenuated Salmonella and Salmonella-E. coli hybrids as vaccine delivery vehicles for O-antigens of several human pathogens, including Shigella sonnei, Vibrio cholerae, and Pseudomonas aeruginosa. (13lack et al., J. Infect. Dis. 155:1260-1265, 1987; Formal et al., Infect. Immun. 34:746-750, 1981; Pier et al., Infect. Immun. 63:2818-2825, 1995); (Morona et al., U.S. Pat. No. 5,110,588). However, until the work described herein, immunization of poultry with live, attenuated Salmonella expressing an APEC O-antigen had not been reported.

LPS O-antigen made by E. coli and Salmonella bacteria is comprised of lipid A, an R-core oligosaccharide, and the O-specific polysaccharide (O-PS), which are covalently linked in that order. Sugiyama et al., J. Bacteriol. 173:55-58, 1991. In S. typhimurium, synthesis of the R-core moiety is directed by the rfa locus and certain housekeeping genes such as galE, galU, and pgi, while O-PS synthesis is directed by the rfb gene cluster, which encodes enzymes involved in biosynthesis of the monomer sugar unit, and the rfc gene, which encodes the O-antigen polymerase responsible for the polymerization of the sugar unit into a high molecular weight polysaccharide chain. Sugiyama et al., supra.

One group investigating the genes required for synthesis of LPS O-antigen in E. coli O9 introduced a plasmid containing the rfb locus from E. coli O9 into S. typhimurium wild-type and mutant strains with defects in the rfb, rfc, or rfe loci and reported that the wild-type strain containing the plasmid expressed LPS specific for both E. coli O9 and S. typhimurium on the cell surface, while the rfc mutant was expressed only O9-specific LPS. E. coli O-antigen was also synthesized in the S. typhimurium rfb mutant but not in the rfe mutant. Sugiyama et al., supra. This group concluded that gene products of the S. typhimurium rfa and E. coli O9 rfb loci can cooperate to synthesize E. coli O9-antigen on the R-core of S. typhimurium. However, this group did not report whether any of these recombinant S. typhimurium constructs could grow within an animal host or generate a protective host immune response against wild-type E. coli O9 or S. typhimurium. Accordingly, a need exists for a bivalent vaccine to control Salmonella and E. coli infection in poultry. Such a vaccine would simultaneously benefit the public health and reduce the costs of poultry production.

SUMMARY OF THE INVENTION

In one embodiment the present invention is directed to a vaccine that protects birds against infection by an avian pathogenic gram-negative (AP.sub.G-N) microbe. The vaccine comprises live cells of a recombinant Salmonella strain expressing O-antigen of the AP.sub.G-N microbe due to integration into the Salmonella chromosome of the rfb gene cluster and the rfc gene of the AP.sub.G-N (hereinafter used interchangeably with AP.sub.G-N rfb/rfc gene cluster). The recombinant Salmonella strain, which is an attenuated mutant of a virulent Salmonella strain, does not express O-antigen of the virulent Salmonella strain due to a mutation in the Salmonella rfb gene cluster and/or in the Salmonella rfc gene. In a preferred embodiment, the AP.sub.G-N microbe is an APEC strain and the AP.sub.G-N rfb/rfc gene cluster is an APEC rfb/rfc gene cluster.

This recombinant Salmonella strain has other features that make it particularly useful as a vaccine in poultry and other birds. First, the vaccine can be formulated for oral administration and oral vaccines are known to stimulate the gut associated lymphoid tissue (GALT), including mucosal, humoral and cellular immune responses. Oral, live vaccines also cost less to produce and are easier to administer in the field than injectable vaccines. Second, the lack of expression of LPS O-antigen specific for the carrier strain avoids any interference that the carrier LPS O-antigen might have on expression of the AP.sub.G-N O-antigen or its recognition by the vaccine recipient's immune system. Third, the recombinant Salmonella strain can protect against both AP.sub.G-N microbes and the parental Salmonella strain because it expresses other cell-surface antigens of the parental Salmonella strain. In addition, for vaccines expressing O-antigen from an APEC strain, use of Salmonella rather than E. coli as the carrier bacteria should provide a more vigorous immune response against the APEC O-antigen because while S. enterica subspecies persist in the spleen and bursa of Fabricius, E. coli does not effectively invade these lymphoid tissues or is quickly killed even if occasionally successful in entering them.

In some embodiments, the recombinant Salmonella strain used in the vaccine also contains a recombinant polynucleotide encoding a desired gene product. A preferred gene product is an antigen from an avian pathogenic gram-positive (AP.sub.G-P) microbe or from a eukaryotic avian pathogen.

In another embodiment, the invention provides a multivalent vaccine for immunizing birds against at least two avian pathogenic gram-negative (AP.sub.G-N) microbes which comprises live cells of a recombinant Salmonella strain expressing an O-antigen of each of the AP.sub.G-N microbes, the recombinant Salmonella strain having an rfb/rfc gene cluster of each of the AP.sub.G-N microbes integrated into the Salmonella chromosome and having a mutation in the Salmonella rfb gene cluster or in the Salmonella rfc gene which inactivates expression of Salmonella O-antigen, wherein the recombinant Salmonella strain is an attenuated mutant of a virulent Salmonella strain. In a preferred embodiment, one or both of the AP.sub.G-N microbes is an APEC strain.

In yet another embodiment, the invention provides a multivalent vaccine for immunizing a bird against at least two AP.sub.G-N microbes which comprises a mixture of live cells of first and second recombinant Salmonella strains, the first recombinant Salmonella strain having an rfb/rfc gene cluster of a first AP.sub.G-N microbe integrated into the Salmonella chromosome and expressing O-antigen of the first AP.sub.G-N microbe and the second recombinant Salmonella strain having an rfb/rfc gene cluster of a second AP.sub.G-N microbe integrated into the Salmonella chromosome and expressing O-antigen of the second AP.sub.G-N microbe, wherein each of the first and second recombinant Salmonella strains has a mutation in the Salmonella rfb gene cluster or in the Salmonella rfc gene which inactivates expression of Salmonella O-antigen, and wherein each of the first and second recombinant Salmonella strains is an attenuated mutant of a virulent Salmonella strain. In a preferred embodiment, one or both of the recombinant Salmonella strains in the multivalent vaccine express O-antigen of an APEC strain.

The present invention in other embodiments is directed to methods for immunizing birds against infection by AP.sub.G-N microbes. These methods include a method for immunizing a bird against an AP.sub.G-N microbe which comprises administering to the bird an immunologically effective amount of a vaccine comprising live cells of a recombinant Salmonella strain expressing O-antigen of the AP.sub.G-N microbe, the recombinant Salmonella strain having an rfb/rfc gene cluster of the AP.sub.G-N microbe stably integrated into the Salmonella chromosome and having a mutation in the Salmonella rfb gene cluster or in the Salmonella rfc gene which inactivates expression of Salmonella O-antigen, wherein the recombinant Salmonella strain is an attenuated mutant of a virulent Salmonella strain. The methods also include a method for simultaneously immunizing a bird against more than one AP.sub.G-N microbes which comprises administering to the bird an immunologically effective amount of a multivalent vaccine as described above. Also included is a method for simultaneously immunizing a bird against an AP.sub.G-N microbe and the carrier Salmonella species which comprises administering to the bird an immunologically effective amount of any of the vaccines as described above.

In still another embodiment, the invention provides a method of making a vaccine for immunizing a bird against an AP.sub.G-N microbe strain. The method comprises the steps of selecting a Salmonella strain capable of colonizing the bird, integrating into the Salmonella chromosome an rjb/rfc gene cluster from the AP.sub.G-N microbe, introducing a mutation into the Salmonella rfb gene cluster and/or into the Salmonella rfc gene, and isolating recombinant Salmonella bacteria which express O-antigen characteristic of the AP.sub.G-N microbe but which do not express Salmonella O-antigen. In one embodiment the selected Salmonella strain is an attenuated mutant of a virulent Salmonella strain. In another embodiment, the selected Salmonella strain is a virulent Salmonella strain and the method further comprises the step of introducing into the virulent Salmonella strain an attenuating mutation into a Salmonella virulence gene and isolating mutants having attenuated virulence as compared to the virulent Salmonella strain.

Among the several advantages achieved by the present invention, therefore, is the provision of live, recombinant Salmonella vaccines capable of protecting birds and particularly poultry against infection by APEC strains and other AP.sub.G-N microbes, and methods for making such vaccines, the provision of multivalent vaccines useful for simultaneously protecting birds against infection by two or more AP.sub.G-N microbes, the provision of methods for immunizing birds against APEC strains and other AP.sub.G-N microbes, and the provision of a method for immunizing birds against both APEC and Salmonella bacteria.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a genetic map of the suicide cosmid pMEG219 showing the following features: apir-dependent origin of replication (R6K ori); the tetracycline resistance genes from Tn10 (tetR, tetA); a mobilization fragment from plasmid RK2 (mob), the kanamycin resistance gene (Kan) and double cos sites (cos) from pCOS2EMBL; and the S. typhimurium .DELTA.cya-27 allele; with the location of NotI and PvuII restriction sites described in the text indicated;

FIGS. 2(A & B) shows digitized images of western blots of LPS isolated from the indicated bacteria strain probed with (FIG. 2A) anti-S. typhimurium group B LPS antibody or (FIG. 2B) anti-O78 LPS antibody;

FIGS. 3(A & B) shows digitized images of western blots of LPS isolated from the indicated bacteria strain probed with (FIG. 3A) anti-S. typhimurium group B LPS antibody or (FIG. 3B) anti-O 78 LPS antibody, with a molecular weight marker included in the gel for the blot shown in FIG. 3A;

FIG. 4 is a genetic map of pMEG287 showing the location of genes discussed in the text; and

FIG. 5 is a genetic map of pMEG055 showing the location of genes discussed in the text.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery that Salmonella enterica subspecies, in particular S. typhimurium, can be engineered to express O antigen of AP.sub.G-N microbes, in particular of O78 and O1 APEC strains, in an immunogenic form on the cell surface without such expression interfering with in vivo growth or colonization of the poultry GALT by the Salmonella carrier. In addition, the inventor herein has discovered that oral administration of such recombinant Salmonella strains to chickens results in a significant reduction in the ability of virulent APEC strains to infect and cause disease in the vaccinated chickens.

Thus, one embodiment of the invention is a vaccine for immunization of birds against an avian pathogenic gram-negative microbe (AP.sub.G-N) comprising live cells of a recombinant Salmonella strain expressing O-antigen of the AP.sub.G-N strain. The AP.sub.G-N O-antigen is expressed in an immunogenic form, meaning that the AP.sub.G-N O-antigen moiety is part of a complete LPS O-antigen molecule in which the AP.sub.G-N O-antigen is attached to an LPS core moiety and, upon administration to a bird, is capable of generating antibodies that react with LPS O-antigen of the wild-type AP.sub.G-N microbe. Typically, the LPS core moiety is synthesized by the recombinant Salmonella carrier, but in some embodiments the LPS core may be from the same AP.sub.G-N microbe as the O-antigen or from a different AP.sub.G-N microbe.

It is contemplated that the vaccine can be used to immunize all types of birds, including chickens, turkeys, ducks, geese, pheasants and other domesticated birds categorized as poultry, as well as non-domesticated birds such as wild turkeys and exotic species such as parrots, parakeets, etc., against any AP.sub.G-N microbe now known or subsequently determined to be pathogenic in birds. Such AP.sub.G-N microbes include but are not limited to: (1) APEC strains such as E. coli serotypes O1, O2, O3, O6, O8, O15, O18, O35, O71, O74, O78, O87, O88, O95, O103 and O109; (2) avian pathogenic Salmonella strains, e.g., group C and group D strains; and (3) species of the following genera: Campylobacter, Bacteroides, Bordetella, Haemophilus, Pasteurella, Francisella, Actinobacillus, Klebisella, Moraxella, Pseudomonas, Proteus, and Ornithobacterium. Preferably, the recombinant Salmonella strain expresses O-antigen of an APEC strain. More preferably, the APEC O-antigen is characteristic of the O1, O2, O35 or O78 serotypes and most preferably the O-antigen is O1 LPS, O2 LPS or O78 LPS.

The recombinant Salmonella strain used in the vaccine is prepared by integrating the rfb/rfc gene cluster from a desired AP.sub.G-N microbe into the chromosome of a suitable Salmonella strain. Salmonella strains for use as the carrier bacteria may be derived from any Salmonella species that is capable of colonizing birds, preferably poultry. Such species include but are not limited to S. typhimurium, S. enteriditis, S. gallinarum, S. pullorum, S. arizona, S. heidelberg, S. anatum, S. hadar, S. agana, S. montevideo, S. kentucky, S. infantis, S. schwarzengrund, S. saintpaul, S. brandenburg, S. instanbul, S. cubana, S. bredeney, S. braenderup, S. livingstone, S. berta, S. california, S. senfenberg, and S. mbandaka. As used herein, "colonizing" means the Salmonella species is able to attach to, invade and persist in one or more of the following tissues in the vaccinated bird: lung, spleen, liver, bursa of Fabricius, and ceca. Preferably, the recombinant Salmonella strain is derived from S. typhimurium, S. gallinarum, S. pullorum, or S. enteriditis. Most preferably, the AP.sub.G-N rfb/rfc gene cluster is inserted into a strain of S. typhimurium.

As used herein, an AP.sub.G-N rfb/rfc gene cluster contains all the genes necessary for synthesis of O-antigen characteristic of that AP.sub.G-N microbe, including all rfb genes whose products are required for biosynthesis of the monomer sugar unit, as well as the rfc gene, which encodes the O-antigen polymerase needed for polymerization of the sugar units. The terms rfb genes and ifc gene are intended to mean those genes of any AP.sub.G-N microbe identified as encoding gene products having the same function in O-antigen biosynthesis as the products of an E. coli rjb/rfc gene cluster. In most E. coli and Salmonella serotypes, the rfc gene is tightly linked to the rfb gene cluster, which in E. coli is about 10 kb in length and located near 45 minutes on the E. coli genetic map. However, in some E. coli strains, as in S. typhimurium, the rfc gene is not linked to the rfb gene cluster and can be found a significant distance away. For example, in S. typhimurium, the rfb gene cluster, which is about 21 kb, extends from 44.9-45.3 centisomes on the genetic map of S. typhimurium while the rfc gene is located at 35.7 centisomes.

A functionally complete AP.sub.G-N rfb/rfc gene cluster can readily be cloned using routine cosmid cloning methodology, as previously described for cloning bacteria rfb/rfc gene clusters. See, for example, Viret et al., EP 0 564 689 B1; Neal et al., FEMS Microbiol. Lett. 82:345-352, 1991; Haraguchi et al., Microb. Pathog. 6:123-132, 1989; Microb. Pathog. 10:351-361, 1991; Heuzenroeder et al., Mol. Microbiol. 3:295-302, 1989; Bastin et al., Mol. Microbiol. 5:2223-2231, 1991; and Valvano et al., Infect. Immun. 57:937-943, 1989. General cosmid cloning techniques are also described in detail in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, 1989. In brief, a cosmid library of chromosomal DNA from the desired AP.sub.G-N microbe is prepared in the E. coli K-12 strain, which does not produce O-antigen due to mutations in the rfb region. The cosmid library is plated and isolated colonies are screened for expression of the AP.sub.G-N O-antigen using antisera specific for the O-antigen of the desired AP.sub.G-N strain. The AP.sub.G-N chromosomal DNA fragment can be isolated from positive cosmid clones and further characterized using standard techniques, such as restriction mapping and subcloning, to determine the minimal amount of AP.sub.G-N DNA required to direct synthesis of AP.sub.G-N O-antigen. This minimal amount is herein referred to as the rfb/rfc gene cluster. For those AP.sub.G-N microbes where the rfc gene is located a considerable distance from the rfb gene cluster, a single AP.sub.G-N chromosomal fragment containing a functional rfb/rfc gene cluster may not be represented in the cosmid library. In such cases, additional routine cloning and screening experiments may need to be performed to clone the rfc gene and construct a recombinant fragment containing both the rfb genes and the rfc gene. The rfb/rfc gene cluster is then transfected as a cosmid or plasmid into the selected Salmonella strain and the LPS produced by the resulting recombinant is analyzed by methods known in the art and those briefly described below.

It has been reported that the Salmonella LPS core moiety can not accept heterologous O-antigen of some bacteria, resulting in unbound O-antigen, which is probably less immunogenic than complete LPS molecules carrying the heterologous O-antigen covalently attached to the core moiety of the carrier bacteria (EP 0 564 689 B1; Morona et al., U.S. Pat. No. 5,110,588). Recombinant strains expressing complete LPS O-antigen can be distinguished from those expressing unbound O-antigen by SDS-PAGE analysis of LPS extracted from late exponential or stationary cultures using a rapid small-scale mini-prep method (Hitchcock and Brown, J. Bacteriol. 154:269-277, 1983). The separated LPS molecules can be detected in the gel by silver staining or by immunoblot analysis using an antibody specific for the O-antigen. Complete LPS O-antigen (also referred to herein as smooth, or S-type LPS) will produce a ladder of high molecular weight bands detectable by either silver staining or immunoblot analysis, whereas unbound O-antigen does not produce a ladder and will be detected as a smear by silver staining or probing with antibody. In such cases, the Salmonella rfa locus, which directs synthesis of the LPS core, can be swapped with an rfa locus of another bacteria, such as E. coli K-12 or the AP.sub.G-N microbe, to derive a combination of rfa and AP.sub.G-N rfb/rfc gene clusters that will direct synthesis of AP.sub.G-N O-antigen in an immunogenic form.

To assure stable expression of the heterologous antigen and eliminate the need for use of antibiotics to maintain an extrachromosomal element in the Salmonella strain, the AP.sub.G-N rfb/rfc gene cluster is integrated into the Salmonella chromosome. This can be accomplished by any known methodology, preferably by a defined deletion/insertion mutation into a virulence gene to produce an attenuated mutant as described below. Alternatively, AP.sub.G-N rfb/rfc gene cluster can be integrated in a manner that does not attenuate the recombinant Salmonella strains.

The recombinant Salmonella strain also contains a mutation in the Salmonella rfc gene which inactivates expression of the Salmonella O-antigen polymerase. Salmonella mutants defective in the O-antigen polymerase produce LPS molecules termed semirough (SR LPS), which have at most one O-antigenic sugar unit attached to any given core unit instead of the usual 30-40 sugar units seen in Rfc.sup.+ strains. These SR LPS molecules are less likely to interfere with the ability of the much longer APEC LPS O-antigen to stimulate a protective immune response. The recombinant Salmonella strain can be prepared from a strain already containing an rfc mutation or the rfc mutation can be introduced at the same time as, or after, integration of the APEC rfb/rfc gene clusters into the Salmonella chromosome.

Salmonella rfc mutants can be created using routine techniques known in the art. For example, random transposon insertions can be made in the chromosome of the desired Salmonella strain (see, e.g., Curtiss, U.S. Pat. No. 5,672,345) and mutants having a Rfc phenotype (i.e., expression of SR LPS) isolated (Collins et al., J. Bacteriol.:173:2521-2529, 1991). Alternatively, a deletion mutation can be introduced into the Salmonella rfc gene using recombinant DNA techniques such as those described in the Examples herein. In brief, the rfc gene of S. typhimurium has been cloned and sequenced and is believed to be conserved among Salmonella strains of serogroups A, B and D1 (Collins et al., supra). Based on this information the ifc gene from a Salmonella strain belonging to one of these serogroups can be cloned and used to construct a suicide plasmid containing a deletion in the cloned rfc gene. Introduction of this plasmid into an rfc.sup.+ Salmonella strain will lead to homologous recombination between the chromosomal rfc gene and the .DELTA.rfc plasmid. The transformed cells are then cultured on media lacking selection for the plasmid and the isolates screened for the Rfc phenotype.

Salmonella mutants can be tested for an Rfc phenotype by SDS-PAGE analysis of LPS isolated from the bacteria and detection of a high proportion of SR LPS with an O-sugar unit specific for the Salmonella strain. Another procedure that can be used to screen Salmonella mutants for the Rfc phenotype is by a bacteriophage sensitivity assay, using a phage specific for s-type LPS. For example, S. typhimurium and other Salmonella group B strains having an Rfc.sup.+ phenotype (i.e., express s-type LPS) are lysed by P22 whereas Rfc mutants of these strains are resistant to P22-mediated lysis as P22 does not recognize the SR LPS expressed by these mutant strains.

It is also contemplated that a recombinant Salmonella strain expressing AP.sub.G-N O-antigen from more than one AP.sub.G-N microbe can be constructed using techniques similar to those described herein for preparing strains expressing a single AP.sub.G-N O-antigen.

Because the vaccine comprises live cells of the recombinant Salmonella strain, it is possible that live, recombinant Salmonella bacteria could be transmitted to human consumers of poultry food products such as eggs and meat. In addition, S. enterica species can cause disease in very young birds. Thus, an important feature of the invention is that the recombinant Salmonella strain is an attenuated mutant of a virulent strain of an S. enterica species. As used herein, an attenuated mutant means that the recombinant Salmonella strain has the ability to colonize and replicate in a vaccinated bird but which is substantially incapable of causing the disease symptoms associated with infection of the particular avian species being treated or infection of humans by its virulent counterpart. By the term "substantially incapable of causing disease symptoms" is meant the attenuated mutant either produces no disease symptoms or produces less severe and/or a fewer number of such symptoms. However, an attenuated mutant strain is not necessarily incapable of causing some effect on normal physiological function in the vaccine recipient or a human and may be a pathogen in avian species other than the intended vaccine recipient as well as in other nonhuman hosts.

The recombinant Salmonella strain can be derived from naturally occurring avirulent mutants of virulent strains of S. enterica species, or by introducing attenuating mutations into wild-type, virulent strains using well-known techniques. Attenuating mutations can be in biosynthetic genes, regulatory genes and/or genes involved in virulence. (See Doggett and Brown, in Mucosal Vaccines, Kiyono et al., eds., Academic Press, San Diego, 1996, pp. 105-118). Examples of genes whose mutation will lead to attenuation include, but are not limited to a mutation in a pab gene, a pur gene, an aro gene, asd, a dap gene, nadA, pncB, galE, pmi, fur, rpsL, ompR, htrA, hemA, cdt, cya, crp, phoP, phoQ, rfc, poxA, galU and combinations thereof. Mutations can be insertions, partial or complete deletions or the like so long as expression of the gene is diminished and virulence is decreased. The skilled artisan will readily appreciate that any suitable gene mutation can be used in the present invention so long as the mutation of that gene renders the microorganism attenuated. Preferably, the carrier microbes of the invention have at least two mutations, each of which act to attenuate the microbe and which, in combination, significantly increase the probability that the microbe will not revert to wild-type virulence.

Methods are known in the art that can be used to generate mutations to produce the attenuated recombinant Salmonella strain of the present invention. For example, the transposon, Tn10, can be used to produce chromosomal deletions in a wide variety of bacteria, including Salmonella (Kleckner et al., J. Mol. Biol. 116:125-159, 1977; EPO Pub. No. 315,682; U.S. Pat. No. 5,387,744).

Recently, new methods have become available for producing specific deletions in genes. These methods involve initially selecting a gene in which the deletion is to be generated. In one approach the gene can be selected from a genomic library obtained commercially or constructed using methods well known in the art (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., 1989, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.). Clones containing the gene are isolated from the genomic library by complementation of a strain which contains a mutation in the same gene. Alternatively, when the DNA sequence of the gene is known, selected primers for the polymerase chain reaction method (PCR) can amplify the gene, often with some flanking sequence, from a sample of bacteria or from purified genomic DNA and the PCR product can be inserted into a cloning vector.

A specific deletion in the selected gene can be generated by either of two general methods. The first method generates a mutation in a gene isolated from a population of clones contained in a genomic DNA library using restriction enzymes and the second method generates the mutation in a gene of known sequence using PCR. Using the first method, the position of the gene on a vector is identified using transposon tagging and a restriction map of the recombinant DNA in the vector is generated. Information derived from the transposon tagging allows all or a portion of a gene to be excised from the vector using the known restriction enzyme sites. The second method, which is based upon PCR methodology, can be used when the DNA sequence of the gene is known. According to this method, divergent PCR primers amplify the upstream and downstream regions flanking a specified segment of DNA to be deleted from the gene and generate a PCR product consisting of the cloning vector and upstream and downstream flanking nucleotide sequences (Innes et al. Eds., PCR Protocols, 1990, Academic Press, New York). In a variation of this method, PCR products are produced representing portions of the gene or flanking sequence, which are then joined together in a cloning vector.

The DNA containing the mutant gene can be introduced into the bacterial host by transformation using chemical means or electroporation, by recombinant phage infection, or by conjugation. In preferred embodiments, the mutant gene is introduced into the chromosomes of the bacteria which can be accomplished using any of a number of methods well known in the art such as, for example, methods using temperature-sensitive replicons (Hamilton et al., J. Bacteriol. 171:4617-4622, 1989), linear transformation of recBC mutants (Jasin et al., J. Bacteriol. 159:783-786, 1984), or host restricted replicons known as suicide vectors (Miller et al., J. Bacteriol. 170:2575-2583, 1988). The particular method used is coupled with an appropriate counter selection method such as, for example, fusaric acid resistance or sucrose resistance followed by subsequent screening for clones containing the mutant allele based upon phenotypic characteristics or by using PCR, nucleic acid hybridization, or an immunological method.

The recombinant Salmonella strain used in the present invention can also be used to deliver a desired gene product to the vaccinated bird. The term "gene product" as used herein refers to any biological product or products produced as a result of the biochemical reactions that occur under the control of a gene. The gene product can be, for example, an RNA molecule, a peptide, a protein, or a product produced under the control of an enzyme or other molecule that is the initial product of the gene, i.e., a metabolic product. For example, a gene can first control the synthesis of an RNA molecule which is translated by the action of ribosomes into an enzyme which controls the formation of glycans in the environment external to the original cell in which the gene was found. The RNA molecule, the enzyme, and the glycan are all gene products as the term is used here. Examples of desired gene products include but are not limited to antigens, various host cell proteins, pharmacologically active products, toxins, and apoptosis-modulating agents.

An antigen or immunogen is intended to mean a molecule containing one or more epitopes that can stimulate a host immune system to make a secretory, humoral and/or cellular immune response specific to that antigen. An epitope can be a site on an antigen to which an antibody specific to that site binds. For protein antigens, an epitope could comprise 3 amino acids in a spatial conformation which is unique to the epitope; generally, an epitope will consist of at least 6 consecutive amino acids, or more usually, at least 10-12 consecutive amino acids of the antigen. The term "epitope" is intended to be interchangeable with the term "antigenic determinant" as used herein. The term "epitope" is also intended to include T-helper cell epitopes in which an antigenic determinant is recognized by T-helper cells through association with major histocompatibility complex class II molecules. In addition, the term epitope includes any antigen, epitope or antigenic determinant which is recognized by cytotoxic T cells when presented by a MHC class I molecule on the surface of an antigen presenting cell. A cytotoxic T cell epitope can comprise an amino acid sequence of between about 6 to about 11 consecutive amino acids, and preferably comprises a sequence of 8 or 9 consecutive amino acids.

If the desired gene product is an antigen from a bacterial, fungal, parasitic or viral disease agent, the recombinant Salmonella strain can be used to vaccinate birds against diseases caused by such agents at the same time as vaccinating against an AP.sub.G-N microbe. For example, the recombinant Salmonella strain could be used to deliver an antigen from an avian pathogenic microbe that does not express O-antigen such as gram-positive (AP.sub.G-P) bacteria. Such microbes include but are not limited to species of Mycoplasma, Listeria, Borrelia, Chlamydia, Clostridia, Corynebacteria, Coxiella, Eysipelothrix, Flavobacteria, Staphylococcus, and Streptococcus. Examples of fungal and parasitic avian pathogens known to infect poultry are species of Amoebotaenia, Aproctella, Ascaridia, Aspergillus, Candida, Capillaria, Cryptosporidium, Cyathostroma, Dispharynx, Eimeria, Fimbriaria, Gongylonemia, Heterakis, Histomonas, Oxyspirura, Plasmondium, Raillietina, Strongyloides, Subulura, Syngamus, Tetrameres, and Trichostrongylus. Viruses known to infect poultry include adenoviruses (e.g., hemorrhagic enteritis virus), astroviruses, coronaviruses (e.g., Infectious bronchitis virus), paramyxoviruses (e.g., Newcastle disease virus), picornaviruses (e.g., avian encephalomyelitis virus), pox viruses, retroviruses (e.g., avian leukosis/sarcoma viruses), reoviruses, and rotaviruses. Preferred gene products for use as antigens are polysaccharides and proteins, including glycoproteins and lipoproteins. Antigen-encoding genes from these prokaryotic and eukaryotic organisms can be cloned and expressed in the recombinant Salmonella strain using standard techniques.

In other embodiments, the desired gene product directs the expression of a gamete-specific antigen which is capable of eliciting an immune response that confers an antifertility effect upon the immunized animal (See, U.S. Pat. No. 5,656,488).

As used herein, vaccine means an agent used to stimulate the immune system of an animal so that protection is provided against an antigen not recognized as a self-antigen by the immune system. Immunization refers to the process of inducing a continuing high level of antibody and/or cellular immune response in which T-lymphocytes can either kill the pathogen and/or activate other cells (e.g., phagocytes) to do so in the immunized animal, which is directed against a pathogen or antigen to which the animal has been previously exposed. In this application the phrase "immune system" is intended to refer to the anatomical features and mechanisms by which an avian species produces antibodies against an antigenic material which invades the cells of the vertebrate or the extra-cellular fluid of the individual and is also intended to include cellular immune responses.

In the case of antibody production, the antibody so produced can belong to any of the immunological classes, such as immunoglobulins, A, D, E, G or M. Of particular interest are vaccines which stimulate production of immunoglobulin A (IgA) since this is the principle immunoglobulin produced by the secretory system of avian species, although vaccines of the invention are not limited to those which stimulate IgA production. For example, vaccines of the nature described herein are likely to produce a broad range of other immune responses in addition to IgA formation, for example cellular and humoral immunity. Immune responses to antigens are well studied and widely reported.

The avirulent microbes of this invention can also be used as vectors for synthesis of other proteins, including immunoregulatory molecules made by avian species and pharmacologically active products that might stimulate or suppress various physiological functions (i.e., growth rate, fat or protein content, etc.).

The desired gene product is encoded by a recombinant polynucleotide. The term "recombinant polynucleotide" is defined herein to refer to the result of laboratory manipulations which results in the introduction into the Salmonella strain of a promoter operably linked to a gene from various endogenous and/or exogenous sources. The promoter is one that is functional in the Salmonella strain to produce expression of the gene. The gene can be of chromosomal, plasmid, or viral origin. A gene as used herein is any biological unit of heredity capable of producing a desired gene product. It is not, however, necessary that the gene be a complete gene as is present in the parent organism and capable of producing or regulating the production of a macromolecule such as for example, a functioning polypeptide. The recombinant polynucleotide may, thus, encode all or part of an antigenic product. A gene can also refer to a polynucleotide having a sequence mutated from the naturally-occurring sequence found in the parent organism. The recombinant polynucleotide can also refer to a long section of DNA coding for several gene products, one or all of which can be antigenic or part of a biosynthetic pathway that leads to the desired gene product. For example, such a long section of DNA could encode 5 to 15 proteins necessary for the synthesis of fimbrial antigens (fimbriae), which mediate adhesion of pathogens to host cells (Baumler et al., supra). The induction of an immune response against fimbriae can provide protection against the pathogen. It is to be understood that the term gene as used herein further includes DNA molecules lacking introns such as, for example, is the case for cDNA molecules, so long as the DNA sequence encodes the desired gene product. The recombinant polynucleotide encoding the desired gene product may include DNA sequences other than the promoter such as termination sequences and other regulators of prokaryotic gene expression.

The recombinant polynucleotide encoding a desired gene product can be transferred into the Salmonella strain in the form of a plasmid, phage or cosmid vector by various means such as conjugation, electroporation, or transformation (uptake of naked DNA from the external environment, which can be artificially induced by the presence of various chemical agents, such as calcium ions). Other methods such as transduction are also suitable, wherein the recombinant DNA in the form of a transducing phage or cosmid vector is packaged within a phage. Once the recombinant polynucleotide is in the carrier Salmonella, it may continue to exist as a separate autonomous replicon or it may insert into the Salmonella chromosome and be reproduced along with the chromosome during cell division.

Preferably, the recombinant polynucleotide is incorporated into a "balanced-lethal" system which selects for microorganisms containing and capable of expressing the desired gene product by linking the survival of the microorganism to the continued presence of the recombinant polynucleotide. "Balanced-lethal" mutants of this type are characterized by a lack of a functioning native chromosomal gene encoding an enzyme which is essential for cell survival, preferably an enzyme which catalyzes a step in the biosynthesis of diaminopimelic acid (DAP) and even more preferably a gene encoding beta aspartate semialdehyde dehydrogenase (Asd). DAP pathway enzymes and Asd are required for cell wall synthesis. "Balanced-lethal" mutants also contain a recombinant gene which can serve to complement the non-functioning chromosomal gene and this complementing recombinant gene is structurally linked to the recombinant polynucleotide encoding the desired gene product. Loss of the complementing recombinant gene causes the cells to die by lysis when the cells are in an environment where DAP is lacking. This strategy is especially useful since DAP is not synthesized by eukaryotes and, therefore, is not present in infected avian tissues. Methods of preparing these types of "balanced lethal" microbes are disclosed in U.S. Pat. No. 5,672,345.

Administration of the vaccine to a bird may be by any known or standard technique, including mucosal or intramuscular injection. Preferred administration methods include oral ingestion or broncho-nasal-ocular spraying. These methods allow the recombinant Salmonella to easily reach the gut-associated lymphoid tissue (GALT) or bronchus-associated lymphoid tissue (BALT) and induce antibody formation and cell mediated immunity. A particularly preferred administration method is to vaccinate newborn birds, e.g., on the day of hatch, by coarse spray.

After growth and harvesting of the recombinant Salmonella strain, the bacterial cells may be lyophilized, particularly if they are to be mixed in foodstuffs. The vaccine comprised of the recombinant Salmonella bacteria may be prepared using any excipient that is pharmaceutically acceptable for the type of bird being immunized. For example, if the vaccine is to be administered in solid form, the cells may be coated with and/or encapsulated in a material that is non-toxic to the inoculated bird and compatible with the bacteria. Solid carriers such as talc or sucrose may also be used. If the administration is to be in liquid form, the cells may be suspended in a suitable liquid carrier, including for example, skim milk, normal saline and/or other non-toxic salts at or near physiological concentrations, and other suitable liquid carriers known to those of skill in the art. Where desirable, adjuvants may also be added to enhance the antigenicity. When the vaccine is intended for administration as a spray, the recombinant Salmonella cells may be suspended in a suitable buffer such as BSG (buffered saline with gelatin, Curtiss III, R., J. Bacteriol. 89:28-40, 1965).

The dosage required will vary depending on the quantity and antigenicity of the APEC LPS O-antigen expressed by the recombinant Salmonella strain as well as the type, size and age of bird to be vaccinated. For example, a lower dosage may be required for vaccinating newborn birds than the dosage suitable for vaccinating older birds. Routine experimentation will easily establish the required amount. Generally, the dosage will be in concentrations ranging from 10.sup.5 to 10.sup.9 live cells per bird. A preferred dosage for spray vaccination of newborn chickens with vaccines comprising live recombinant S. typhimurium cells is about 10.sup.6 to 10.sup.8 live cells/bird and a particularly preferred dosage is about 5.times.10.sup.7 live cells/bird.

PATENT EXAMPLES available on request

PATENT PHOTOCOPY available on request

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