Patent 6,923,957

Diseases caused by Salmonella bacteria range from a mild, self-limiting diarrhea to serious gastrointestinal and septicemic disease in humans and animals. Salmonella is a gram-negative, rod-shaped, motile bacterium (nonmotile exceptions include S. gallinarum and S. pullorum) that is non-spore forming. Environmental sources of the organism include water, soil, insects, factory surfaces, kitchen surfaces, animal feces, raw meats, raw poultry, and raw seafoods.

Salmonella infection is a widespread occurrence in animals, especially in poultry and swine, and is one of the most economically damaging of the enteric and septicemic diseases that affect food producing animals. Although many serotypes of Salmonella have been isolated from animals, S. chloeraesuis and S. typhimurium are the two most frequently isolated serotypes associated with clinical salmonellosis in pigs. In swine, S. typhimurium typically causes an enteric disease, while S. choleraesuis (which is host-adapted to swine) is often the etiologic agent of a fatal septicemic disease with little involvement of the intestinal tract. S. dublin and S. typhimurium are common causes of infection in cattle; of these, S. dublin is host adapted to cattle and is often the etiologic agent of a fatal septicemia disease. Other serotypes such as S. gallinarum and S. pullorum are important etiologic agents of salmonellosis in avian and other species. Although these serotypes primarily infect animals, S. dublin and S. chloeraesuis also often cause human disease.

Various Salmonella species have been isolated from the outside of egg shells, including S. enteritidis which may even be present inside the egg yolk. It has been suggested that the presence of the organism in the yolk is due to transmission from the infected layer hen prior to shell deposition. Foods other than eggs have also caused outbreaks of S. enteritidis disease in humans.

S. typhi and S. paratyphi A, B, and C produce typhoid and typhoid-like fever in humans. Although the initial infection with salmonella typically occurs through the gastrointestinal tract, typhoid fever is a systemic disease that spreads throughout the host and can infect multiple organ sites. The fatality rate of typhoid fever can be as high as 10% (compared to less than 1% for most forms of salmonellosis). S. dublin has a 15% mortality rate when the organism causes septicemia in the elderly, and S. enteritidis has an approximately 3.6% mortality rate in hospital/nursing home outbreaks, with the elderly being particularly affected.

Numerous attempts have been made to protect humans and animals by immunization with a variety of vaccines. Many of the vaccines provide only poor to moderate protection and require large doses to be completely efficacious. Previously used vaccines against salmonellae and other infectious agents have generally fallen into four categories: (i) specific components from the etiologic agent, including cell fractions or lysates, intact antigens, fragments thereof, or synthetic analogs of naturally occurring antigens or epitopes (often referred to as subunit vaccines); (ii) antiidiotypic antibodies; (iii) the whole killed etiologic agent (often referred to as killed vaccines); or (iv) an avirulent (attenuated) derivative of the etiologic agent used as a live vaccine.

Reports in the literature have shown that attenuated live vaccines are more efficacious than killed vaccines or subunit vaccines for inducing protective immunity. Despite this, high doses of live vaccines are often required for efficacy and few live-attenuated Salmonella vaccines are commercially available. Ideally, an effective attenuated live vaccine retains the ability to infect the host without causing serious disease and is also capable of stimulating humoral (antibody-based) immunity and cell-mediated immunity sufficient to provide resistance to any future infection by virulent bacteria.

Several attenuation strategies have been utilized to render Salmonella avirulent [Cardenas et al., Clin Microbial Rev. 5:328-342 (1992); Chatfield et al., Vaccine 7:495-498 (1989); Curtiss, in Woodrow et al., eds., New Generation Vaccines, Marcel Dekker, Inc., New York, p. 161 (1990); Curtiss et al., in Kohler et al., eds., Vaccines: new concepts and developments. Proceedings of the 10th Int'l Convocation of Immunology, Longman Scientific and Technical, Harlow, Essex, UK, pp. 261-271 (1987); Curtiss et al., in Blankenship et al., eds., Colonization control of human bacterial enteropathogens in poultry, Academic Press, New York, pp. 169-198 (1991)]. These strategies include the use of temperature sensitive mutants [e.g., Germanier et al., Infect Immun. 4:663-673 (1971)], aromatic and auxotrophic mutants (e.g., -aroA, -asd, -cys, or -thy [Galan et al., Gene 94:29-35 (1990); Hoiseth et al., Nature 291:238-239 (1981); Robertsson et al., Infect Immun. 41:742-750 (1983); Smith et al., Am J Vet Res. 45:59-66 (1984); Smith et al., Am J Vet Res. 45:2231-2235 (1984)]), mutants defective in purine or diaminopimelic acid biosynthesis (e.g., Δpur and Δdap [Clarke et al., Can J Vet Res. 51:32-38 (1987); McFarland et al., Microb Pathog. 3:129-141 (1987); O'Callaghan et al., Infect Immun. 56:419-423 (1988)]), strains altered in the utilization or synthesis of carbohydrates (e.g., ΔgalE [Germanier et al., Infect Immun. 4:663-673 (1971); Hone et al., J Infect Dis. 156:167-174 (1987)]), strains altered in the ability to synthesize lipopolysaccharide (e.g., galE, pmi, rfa) or cured of the virulence plasmid, strains with mutations in one or more virulence genes (e.g., invA) and mutants altered in global gene expression (e.g., -cya -crp, ompR or -phoP [Curtiss (1990), supra; Curtiss et al. (1987), supra; Curtiss et al. (1991)], supra).

In addition, random mutagenesis techniques have been used to identify virulence genes expressed during infection in an animal model. For example, using a variety of approaches, random mutagenesis is carried out on bacteria followed by evaluation of the mutants in animal models or tissue culture systems, such as Signature-Tagged Mutagenesis (STM) [see U.S. Pat. No. 5,876,931].

However, published reports have shown that attempts to attenuate Salmonella by these and other methods have led to varying degrees of success and demonstrated differences in both virulence and immunogenicity [Chatfield et al., Vaccine 7:495-498 (1989); Clarke et al., Can J Vet Res. 51:32-38 (1987); Curtiss (1990), supra; Curtiss et al. (1987), supra; Curtiss et al. (1991), supra]. Prior attempts to use attenuation methodologies to provide safe and efficacious live vaccines have encountered a number of problems.

First, an attenuated strain of Salmonella that exhibits partial or complete reduction in virulence may not retain the ability to induce a protective immune response when given as a vaccine. For instance, ΔaroA mutants and galE mutants of S. typhimurium lacking UDP-galactose epimerase activity were found to be immunogenic in mice [Germanier et al., Infect Immun. 4:663-673 (1971), Hohmann et al., Infect Immun. 25:27-33 (1979); Hoiseth et al., Nature, 291:238-239 (1981); Hone et al., J. Infect Dis. 156:167-174 (1987)] whereas Δasd, Δthy, and Δpur mutants of S. typhimurium were not [Curtiss et al. (1987), supra, Nnalue et al., Infect Immun. 55:955-962 (1987)]. All of these strains, nonetheless, were attenuated for mice when given orally or parenterally in doses sufficient to kill mice with the wild-type parent strain. Similarly, ΔaroA, Δasd, Δthy, and Δpur mutants of S. chloeraesuis were avirulent in mice, but only ΔaroA mutants were sufficiently avirulent and none were effective as live vaccines [Nnalue et al., Infect Immun. 54:635-640 (1986); Nnalue et al., Infect Immun. 55:955-962 (1987)].

Second, attenuated strains of S. dublin carrying mutations in phoP, phoP crp, [crp-cdt] cya, crp cya were found to be immunogenic in mice but not cattle [Kennedy et al., Abstracts of the 97th General Meeting of the American Society for Microbiology. B-287:78 (1997)]. Likewise, another strain of S. dublin, SL5631, with a deletion affecting gene aroA was highly protective against lethal challenge to a heterologous challenge strain in mice [Lindberg et al., Infect Immun. 61:1211-1221 (1993)] but not cattle [Smith et al., Am J Vet Res. 54:1249-1255 (1993)].

Third, genetically engineered Salmonella strains that contain a mutation in only a single gene may spontaneously mutate and "revert" to the virulent state. The introduction of mutations in two or more genes tends to provide a high level of safety against restoration of pathogenicity by recombination [Tacket et al., Infect Immun. 60:536-541 (1992)]. However, the use of double or multiple gene disruptions is unpredictable in its effect on virulence and immunogenicity; the introduction of multiple mutations may overattenuate a bacteria for a particular host [Linde et al., Vaccine 8:278-282 (1990); Zhang et al., Microb. Pathog., 26(3):121-130 (1999)].

Of interest to the present invention is the identification of pathogenicity islands (PAIs) in Salmonella and other bacteria, which are large, sometimes unstable, chromosomal regions harboring clusters of genes that often define virulence characteristics in enteric bacteria. The DNA base composition of PAIs often differs from those of the bacterial chromosomes in which they are located, indicating that they have probably been acquired by horizontal gene transfer. One Salmonella pathogenicity island containing genes required for epithelial cell invasion has been identified at around 63 centisomes (cs) on the S. typhimurium chromosome, and has been shown to contain genes encoding components of a type III (contact-dependent) secretion system, secreted effector proteins, and associated regulatory proteins [Millis et al., Mol Microbiol 15(4):749-59 (1995)] A second Salmonella PAI of 40 kb is located at 30.7 and has been designated Salmonella pathogenicity island 2 (SP12) [Shea et al., Proc. Nat'l Acad. Sci. USA, 19;93(6):2593-2597 (1996)]. Nucleotide sequence analysis of regions of SPI2 revealed genes encoding a second type III secretion apparatus that has been suggested to be involved at a stage of pathogenesis subsequent to epithelial cell penetration. Mutations in some genes within SPI2 have been shown to result in attenuation of bacterial virulence in mice. See U.S. Pat. No. 5,876,931; Shea et al., Proc. Natl. Acad. Sci. USA, 93:2593-2597 (1996); Ochman et al., Proc Natl Acad Sci USA, 93(15):7800-7804 (1996); Hensel et al., J. Bacteriol., 179(4):1105-1111 (1997); Hensel et al., Molec. Microbiol., 24(1):155-167 (1997); Dunyak et al., poster presented at 97th General Meeting of the American Society for Microbiology (1997), p. 76.

A need continues to exist for more safe and efficacious live attenuated Salmonella vaccines that ideally do not need to be administered at very large doses.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to safe and efficacious vaccines employing one or more strains of attenuated mutant gram-negative bacteria in which one or more genes homologous to genes within Salmonella pathogenicity island 2 (SPI2) have been inactivated, preferably by deletion of about 5% to about 100% of the gene, most preferably by deletion of about 50% or more of the gene. Specifically contemplated are vaccines comprising one or more species of attenuated mutant Salmonella bacteria in which one or more genes, and preferably two or more genes, within SPI2 have been inactivated. In a preferred embodiment, one or more of the ssa genes, particularly ssaT ssaJ, ssaC or ssaM genes, have been inactivated in the mutant bacteria. The invention is based on results of extensive safety and efficacy testing of these vaccines, including vaccines containing more than one serotype of Salmonella, in animal species other than rodents, including cattle and pigs.

According to one aspect of the present invention, vaccine compositions are provided that comprise an immunologically protective amount, of a first attenuated mutant Salmonella bacterium in which one or more ssa genes are inactivated. In one embodiment, the ssa genes are selected from the group consisting of ssaT, ssaJ, ssaC and ssaM. Suitable amounts will vary but may include about 10⁹bacteria or less. In these mutant bacteria, the inactivated gene(s) is/are preferably inactivated by deletion of a portion of the coding region of the gene. Alternatively, inactivation is effected by insertional mutation. Any species of Salmonella bacteria, particularly S. enterica subspecies and subtypes, may be mutated according to the invention, including Salmonella from serogroups A, B, C₁, C₂, D, and E₁. All of the Salmonella serovars belong to two species: S. bongori and S. enterica. The six subspecies of S. enterica are: S. enterica subsp. enterica (I or 1), S. enterica subsp. salamae (II or 2), S. enterica subsp. arizonae (IIIa or 3a), S. enterica subsp. diarizonae (IIIb or 3b), S. enterica subsp. houtenae (IV or 4), S. enterica subsp. indica (VI or 6). Exemplary subspecies include: S. Choleraesuis, S. Typhimurium, S. Typhi, S. Paratyphi, S. Dublin, S. Enteritidis, S. Gallinarum, S. Pullorum, Salmonella Anatum, Salmonella Hadar, Salmonella Hamburg, Salmonella Kentucky, Salmonella Miami, Salmonella Montevideo, Salmonella Ohio, Salmonella Sendai, Salmonella Typhisuis.

Two or more virulence genes may be inactivated in the mutant Salmonella bacteria, of which at least one gene is a gene within SPI2. In one aspect, the gene is an ssa gene. Preferably, two genes selected from the group consisting of ssaT, ssaJ and ssaC have been inactivated. Most preferably, the combination of ssaT and ssaC, or ssaT and ssaJ have been inactivated.

The vaccine composition may further comprise a second attenuated mutant Salmonella bacterium in which one or more ssa genes have been inactivated. In one aspect, the ssa gene is selected from the group consisting of ssaT, ssaJ and ssaC. Preferably, the first and second mutant Salmonella bacteria are of different serotypes. For cattle, vaccines comprising both S. dublin and S. typhimurium are preferred.

The invention also provides methods of immunizing, i.e., conferring protective immunity on, an animal by administering the vaccine compositions of the invention. Signs of protective immunity are described below. The invention further provides methods of reducing transmission of infection by administering vaccines of the invention in amounts effective to reduce amount or duration of bacterial shedding during infection. Animals that are suitable recipients of such vaccines include but are not limited to cattle, sheep, horses, pigs, poultry and other birds, cats, dogs, and humans. Methods of the invention utilize any of the vaccine compositions of the invention, and preferably, the vaccine comprises an effective amount of an attenuated, non-reverting mutant Salmonella bacterium in which one or more genes within the SPI2 region have been inactivated, either by deleting a portion of the gene(s), or, alternatively, by insertional mutation. In one aspect, methods utilize attenuated bacteria wherein an ssa gene is inactivated, and preferably the ssa gene is selected from the group consisting of ssaT, ssaJ, ssaC, and ssaM.

According to another aspect of the invention, the attenuated mutant Salmonella bacterium may further comprise a polynucleotide encoding a non-Salmonella polypeptide. Administration of the mutant bacteria or a vaccine composition comprising the mutant bacteria thus provides a method of delivering an immunogenic polypeptide antigen to an animal.

Numerous additional aspects and advantages of the invention will become apparent to those skilled in the art upon consideration of the following detailed description of the invention which describes presently preferred embodiments thereof.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides vaccines, or immunogenic compositions, comprising one or more species of attenuated mutant Salmonella bacteria in which one or more virulence genes, preferably genes within SPI2, have been deleted. An advantage of the vaccines of the present invention is that the live attenuated mutant bacteria can be administered as vaccines at reasonable doses, via a variety of different routes, and still induce protective immunity in the vaccinated animals. Another advantage is that mutant bacteria containing inactivations in two different genes are non-reverting, or at least are much less likely to revert to a virulent state.

Risk of reversion can be assessed by passaging the bacteria multiple times (e.g., 5 passages) and administering the resulting bacteria to animals. Non-reverting mutants will continue to be attenuated.

The examples herein demonstrate that inactivation of two or more genes within SPI2 does not further attenuate (or overattenuate) the bacteria compared to bacteria in which only a single gene has been inactivated. The examples also demonstrate that deletion of genes within SPI2 result in safe, efficacious vaccines as shown by observable reductions in adverse signs and symptoms associated with infection by wild type bacteria. The exemplary vaccines of the present invention have been shown to confer superior protective immunity compared to other vaccines containing live attenuated bacteria, e.g., Salmo Shield®TD (Grand Laboratories, Inc.) and mutant Salmonella bacteria containing Δrfa K or Δcya Δcrp mutations. The examples further demonstrate that a combination vaccine containing two or more strains of attenuated mutant Salmonella of different serotypes is efficacious and that inclusion of the different serotypes does not cause interference with the immune response.

When two or more genes within SPI2 are inactivated, the two genes may be from the same or different SPI2 "regions" (e.g., the export machinery, effector proteins, and sensor/regulator regions). The genes may also be from the same or different functional groups as illustrated in FIG. 4, which include the structural components, ssa (secretion system apparatus, e.g., ssaB, ssaC, ssaD, ssaE, ssaF, ssaG, ssaH, ssaI, ssaJ, ssaK, ssaL, ssaM, ssaN, ssaO, ssaP, ssaQ, ssaR, ssaS, ssaT, and/or ssaU) secreted targets of the type III secretion system, sse (secretion system effector), the two-component sensor regulator system, ssr (secretion system regulator), and the chaperones for the secreted proteins, ssc (secretion system chaperone).

The nucleotide sequence of ssaT from S. dublin is set forth in SEQ ID NO: 1. The nucleotide sequence of ssaT from S. typhimurium is set forth in SEQ ID NO: 2. As used herein, "ssaT" includes SEQ ID NOS: 1, 2 and other Salmonella species equivalents thereof, e.g., full length Salmonella nucleotide sequences that hybridize to the non coding complement of SEQ ID NO: 1 or 2 under stringent conditions, wherein stringent conditions comprise hybridization in 50% formamide with washing at 650° C. (e.g., as described in FIG. 4 of Shea et al., Proc. Nat'l. Acad. Sci. USA, 93:2593-2597 (1996), incorporated herein by reference), and full length Salmonella nucleotide sequences that have 90% sequence identity to SEQ ID NO: 1 or 2. Salmonella species equivalents can be easily identified by those of ordinary skill in the art and also include nucleotide sequences with, e.g. 90%, 95%, 98% and 99% identity to SEQ ID NO: 1 or 2.

The nucleotide sequence of ssaJ from S. dublin is set forth in SEQ ID NO: 3. The nucleotide sequence of ssaJ from S. typhimurium is set forth in NO: 4. As used herein, "ssaJ" includes SEQ ID NOS: 3, 4, and other Salmonella species equivalents thereof, e.g., full length Salmonella nucleotide sequences that hybridize to the non coding complement of SEQ ID NO: 3 or 4 under stringent conditions, and full length Salmonella nucleotide sequences that have 90% sequence identity to SEQ ID NO: 3 or 4. Salmonella species equivalents can be easily identified by those of ordinary skill in the art and also include nucleotide sequences with, e.g., 90%, 95%, 98% and 99% identity to SEQ ID NO: 3 or 4.

The nucleotide sequence of ssaC from S. dublin is set forth in SEQ ID NO: 5. The nucleotide sequence of ssaC from S. typhimurium is set forth in SEQ ID NO: 6. As used herein, "ssaC" includes SEQ ID NOS: 5, 6, and other Salmonella species equivalents thereof, e.g., full length Salmonella nucleotide sequences that hybridize to the non coding complement of SEQ ID NO: 5 or 6 under stringent conditions, and full length Salmonella nucleotide sequences that have 90% sequence identity to SEQ ID NO: 5 or 6. Salmonella species equivalents can be easily identified by those of ordinary skill in the art and also include nucleotide sequences with, e.g., 90%, 95%, 98% and 99% identity to SEQ ID NO: 5 or 6.

The nucleotide sequence of ssaM from S. dublin is set forth in SEQ ID NO: 30. The nucleotide sequence of ssaM from S. typhimurium is set forth in SEQ ID NO: 7. As used herein, "ssaM" includes SEQ ID NOS: 7, 30 and other Salmonella species equivalents thereof, e.g., full length Salmonella nucleotide sequences that hybridize to the non coding complement of SEQ ID NO: 7 or 30 under stringent conditions, and full length Salmonella nucleotide sequences that have 90% sequence identity to SEQ ID NO: 7 or 30. Salmonella species equivalents can be easily identified by those of ordinary skill in the art and also include nucleotide sequences with, e.g., 90%, 95%, 98% and 99% identity to SEQ ID NO: 7 or 30.

The invention also contemplates that equivalent genes (e.g., greater than 80% homology) in other gram negative bacteria can be similarly inactivated to provide efficacious vaccines.

The nucleotide sequences of other genes in SPI2 can be assembled using sequences available on GenBank (e.g., Accession #'s AJ224892, Z95891, U51927, Y09357 and X99944) which cover SPI2.

As used herein, an "inactivated" gene means that the gene has been mutated by insertion, deletion or substitution of nucleotide sequence such that the mutation inhibits or abolishes expression and/or biological activity of the encoded gene product. The mutation may act through affecting transcription or translation of the gene or its mRNA, or the mutation may affect the polypeptide gene product itself in such a way as to render it inactive.

In preferred embodiments, inactivation is carried by deletion of a portion of the coding region of the gene, because a deletion mutation reduces the risk that the mutant will revert to a virulent state. For example, some, most (e.g., half or more) or virtually all of the coding region may be deleted (e.g., about 5% to about 100% of the gene, but preferably about 20% or more of the gene, and most preferably about 50% or more of the gene may be deleted). Alternatively, the mutation may be an insertion or deletion of even a single nucleotide that causes a frame shift in the open reading frame, which in turn may cause premature termination of the encoded polypeptide or expression of an completely inactive polypeptide. Mutations can also be generated through insertion of foreign gene sequences, e.g., the insertion of a gene encoding antibiotic resistance.

Deletion mutants can be constructed using any of a number of techniques well known and routinely practiced in the art. In one example, a strategy using counterselectable markers can be employed which has commonly been utilized to delete genes in many bacteria. For a review, see, for example, Reyrat, et al., Infection and Immunity 66:4011-4017 (1998), incorporated herein by reference. In this technique, a double selection strategy is often employed wherein a plasmid is constructed encoding both a selectable and counterselectable marker, with flanking DNA sequences derived from both sides of the desired deletion. The selectable marker is used to select for bacteria in which the plasmid has integrated into the genome in the appropriate location and manner. The counterselecteable marker is used to select for the very small percentage of bacteria that have spontaneously eliminated the integrated plasmid. A fraction of these bacteria will then contain only the desired deletion with no other foreign DNA present. The key to the use of this technique is the availability of a suitable counterselectable marker.

In another technique, the cre-lox system is used for site specific recombination of DNA. The system consists of 34 base pair lox sequences that are recognized by the bacterial cre recombinase gene. If the lox sites are present in the DNA in an appropriate orientation, DNA flanked by the lox sites will be excised by the cre recombinase, resulting in the deletion of all sequences except for one remaining copy of the lox sequence. Using standard recombination techniques, it is possible to delete the targeted gene of interest in the Salmonella genome and to replace it with a selectable marker (e.g., a gene coding for kanamycin resistance) that is flanked by the lox sites. Transient expression (by electroporation of a suicide plasmid containing the cre gene under control of a promoter that functions in Salmonella of the cre recombinase should result in efficient elimination of the lox flanked marker. This process would result in a mutant containing the desired deletion mutation and one copy of the lox sequences.

In another approach, it is possible to directly replace a desired deleted sequence in the Salmonella genome with a marker gene, such as green fluorescent protein (GFP), β-galactosidase, or luciferase. In this technique, DNA segments flanking a desired deletion are prepared by PCR and cloned into a suicide (non-replicating) vector for Salmonella. An expression cassette, containing a promoter active in Salmonella and the appropriate marker gene, is cloned between the flanking sequences. The plasmid is introduced into wild-type Salmonella. Bacteria that incorporate and express the marker gene (probably at a very low frequency) are isolated and examined for the appropriate recombination event (i.e., replacement of the wild type gene with the marker gene).

In order for a modified strain to be effective in a vaccine formulation, the attenuation must be significant enough to prevent the pathogen from evoking severe clinical symptoms, but also insignificant enough to allow limited replication and growth of the bacteria in the recipient. The recipient is a subject needing protection from a disease caused by a virulent form of Salmonella or other pathogenic microorganisms. The subject to be immunized may be a human or other mammal or animal, for example, farm animals including cows, sheep, pigs, horses, goats and poultry (e.g., chickens, turkeys, ducks and geese) and companion animals such as dogs and cats; exotic and/or zoo animals. Immunization of both rodents and non-rodent animals is contemplated.

An "immunologically protective amount" of the attenuated mutant bacteria is an amount effective to induce an immunogenic response in the recipient that is adequate to prevent or ameliorate signs or symptoms of disease, including adverse health effects or complications thereof, caused by infection with wild type Salmonella bacteria. Either humoral immunity or cell-mediated immunity or both may be induced. The immunogenic response of an animal to a vaccine composition may be evaluated, e.g., indirectly through measurement of antibody titers, lymphocyte proliferation assays, or directly through monitoring signs and symptoms after challenge with wild type strain.

The protective immunity conferred by a vaccine can be evaluated by measuring, e.g., reduction in clinical signs such as mortality, morbidity, temperature number and % of days of diarrhea, milk production or yield, average daily weight gain, physical condition and overall health and performance of the subject.

When a combination of two or more different serotypes of bacteria are administered, it is highly desirable that there be little or no interference among the serotypes such that the host is not prevented from developing a protective immune response to one of the two or more serotypes administered. Interference can arise, e.g., if one strain predominates in the host to the point that it prevents or limits the host from developing a protective immune response to the other strain. Alternatively, one strain may directly inhibit the other strain.

In addition to immunizing the recipient, the vaccines of the invention may also promote growth of the recipient and/or boost the recipient's immunity and/or improve the recipient's overall health status. Components of the vaccines of the invention, or microbial products, may act as immunomodulators that may inhibit or enhance aspects of the immune system. For example, the vaccines of the invention may signal pathways that would recruit cytokines that would have an overall positive benefit to the host.

The vaccines of the present invention also provide veterinary and human community health benefit by reducing the shedding of virulent bacteria by infected animals. Either bacterial load being shed (the amount of bacteria, e.g., CFU/ml feces) or the duration of shedding (e.g., number of % of days shedding is observed) may be reduced, or both. Preferably, shedding load is reduced by about 10% or more compared to unvaccinated animals preferably by 20% or more, and/or shedding duration is reduced by at least 1 day, or more preferably 2 or 3 days, or by 10% or more or 20% or more.

While it is possible for an attenuated bacteria of the invention to be administered alone, one or more of such mutant bacteria are preferably administered in conjunction with suitable pharmaceutically acceptable excipient(s), diluent(s), adjuvant(s) or carrier(s). The carrier(s) must be "acceptable" in the sense of being compatible with the attenuated mutant bacteria of the invention and not deleterious to the subject to be immunized. Typically, the carriers will be water or saline which will be sterile and pyrogen free.

Any adjuvant known in the art may be used in the vaccine composition, including oil-based adjuvants such as Freund's Complete Adjuvant and Freund's Incomplete Adjuvant, mycolate-based adjuvants (e.g., trehalose dimycolate), bacterial lipopolysaccharide (LPS), peptidoglycans (i.e., mureins, mucopeptides, or glycoproteins such as N-Opaca, muramyl dipeptide [MDP], or MDP analogs), proteoglycans (e.g., extracted from Klebsiella pneumoniae), streptococcal preparations (e.g., OK432), Biostim™ (e.g., 01K2), the "Iscoms" of EP 109 942, EP 180 564 and EP 231 039, aluminum hydroxide, saponin, DEAE-dextran, neutral oils (such as miglyol), vegetable oils (such as arachis oil), liposomes, Pluronic® polyols, the Ribi adjuvant system (see, for example GB-A-2 189 141), or interleukins, particularly those that stimulate cell mediated immunity. An alternative adjuvant consisting of extracts of Amycolata, a bacterial genus in the order Actinomycetales, has been described in U.S. Pat. No. 4,877,612. Additionally, proprietary adjuvant mixtures are commercially available. The adjuvant used will depend, in part, on the recipient organism. The amount of adjuvant to administer will depend on the type and size of animal. Optimal dosages may be readily determined by routine methods.

The vaccine compositions optionally may include vaccine-compatible pharmaceutically acceptable (i.e., sterile and non-toxic) liquid, semisolid, or solid diluents that serve as pharmaceutical vehicles, excipients, or media. Any diluent known in the art may be used. Exemplary diluents include, but are not limited to, polyoxyethylene sorbitan monolaurate, magnesium stearate, methyl- and propylhydroxybenzoate, talc, alginates, starches, lactose, sucrose, dextrose, sorbitol, mannitol, gum acacia, calcium phosphate, mineral oil, cocoa butter, and oil of theobroma.

The vaccine compositions can be packaged in forms convenient for delivery. The compositions can be enclosed within a capsule, caplet, sachet, cachet, gelatin, paper, or other container. These delivery forms are preferred when compatible with entry of the immunogenic composition into the recipient organism and, particularly, when the immunogenic composition is being delivered in unit dose form. The dosage units can be packaged, e.g., in tablets, capsules, suppositories or cachets.

The vaccine compositions may be introduced into the subject to be immunized by any conventional method including, e.g., by intravenous, intradermal, intramuscular, intramammary, intraperitoneal, or subcutaneous injection; by oral, transdermal, sublingual, intranasal, anal, or vaginal, delivery. The treatment may consist of a single dose or a plurality of doses over a period of time.

Depending on the route of administration, suitable amounts of the mutant bacteria to be administered include ˜10⁹bacteria or less, provided that an adequate immunogenic response is induced by the vaccinee. Doses of ˜10¹⁰or less or ˜10¹¹or less may be required to achieve the desired response. Doses significantly higher than ˜10¹¹may not be commercially desirable.

Another aspect of the invention involves the construction of attenuated mutant bacteria that additionally comprise a polynucleotide sequence encoding a heterologous polypeptide. For example, for Salmonella, a "heterologous" polypeptide would be a non-Salmonella polypeptide not normally expressed by Salmonella bacteria. Such attenuated mutant bacteria can be used in methods for delivering the heterologous polypeptide or DNA. For example, Salmonella could be engineered to lyse upon entry into the cytoplasm of a eukaryotic host cell without causing significant damage, thereby becoming a vector for the introduction of plasmid DNA into the cell. Suitable heterologous polypeptides include immunogenic antigens from other infectious agents (including gram-negative bacteria, gram-positive bacteria and viruses) that induce a protective immune response in the recipients, and expression of the polypeptide antigen by the mutant bacteria in the vaccine causes the recipient to be immunized against the antigen. Other heterologous polypeptides that can be introduced using the mutant Salmonella include immunomodulatory molecules e.g., cytokines or "performance" proteins such as growth hormone, GRH, and GDF-8.

Claim 1 of 7 Claims

1. A vaccine composition comprising an immunologically protective amount of a first attenuated, non-reverting mutant Salmonella bacterium in which the ssaT and ssaJ genes have been inactivated,

wherein said ssaT gene comprises the nucleic acid sequence of SEQ ID NO: 1 or 2, and wherein said ssaC gene comprises the nucleic acid sequence of SEQ ID NO: 5 or 6.

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