Immune responses in BALB/c mice following immunization with aromatic compound or purine-dependent Salmonella typhimurium strains.

Two near isogenic strains of Salmonella typhimurium HWSH, stably mutated in either the aroA gene affecting the biosynthesis of aromatic compounds, or the purA gene affecting the biosynthesis of purines, were administered intravenously as live attenuated vaccines to BALB/c mice. HWSH aroA-immunized mice were well protected against intravenous (i.v.) challenge with wild-type virulent HWSH for at least 10 weeks, whereas HWSH purA-immunized mice were unprotected. Furthermore, HWSH aroA-immunized mice could also control a heterologous challenge with virulent Listeria monocytogenes at 7 and 14 days post-immunization, whereas mice receiving a similar dose of HWSH purA could not. Increasing the i.v. dose of HWSH purA compared to HWSH aroA induced some resistance to L. monocytogenes. Induction of early anti-S. typhimurium resistance by HWSH aroA immunization appeared slightly later than the anti-L. monocytogenes resistance. Mice immunized with either vaccine were able to mount S. typhimurium-specific T-cell proliferative responses and produced anti-S. typhimurium humoral antibodies in their serum. The antibody titre was greater in those mice immunized with the aroA mutant.     

Adoptive transfer of immunity to oral challenge with virulent salmonellae in innately susceptible BALB/c mice requires both immune serum and T cells.

The mechanisms of immunity to salmonellae conferred by immunization with live vaccines were studied by adoptive transfer using the mouse-virulent strain Salmonella typhimurium C5 and innately susceptible BALB/c (ltys) mice. This organism cannot establish a sublethal infection in naive BALB/c mice. Animals immunized 2 to 3 months earlier with the S. typhimurium SL3261 aroA live vaccine were used as donors of serum, spleen cells, and mesenteric lymph node cells for naive recipients which were challenged orally with the virulent C5 strain. Simultaneous transfer of both immune serum and immune cells was necessary for protection. Simultaneously depleting the donors of CD4+ and CD8+ T cells by administration of antisera in vivo prior to cell harvesting showed that T cells were necessary for protection. The results demonstrate that both antibody and T cells are required for recall of immunity to oral challenge with virulent salmonellae in innately susceptible mice and suggest that the ability to elicit opsonizing antibody in addition to cell-mediated immunity is important for optimal protection induced by salmonella vaccines.     

Immunity conferred by Aro- Salmonella live vaccines

The specificity of protection conferred by Aro- salmonellae was studied in BALB/c mice challenged 3 months after intravenous (i.v.) vaccination, more than 1 month after the vaccine had been cleared. Oral challenge showed better protection than i.v. challenge. Salmonella typhimurium aroA SL3261 conferred very good protection against wild-type S. typhimurium C5 (over 10,000 x LD50). Cross protection experiments were performed using S. typhimurium, S. enteritidis and S. dublin for vaccination and challenge, including variants of S. typhimurium and S. enteritidis of similar virulence differing in the main LPS antigen (O-4 or O-9). Salmonella typhimurium aroA conferred solid protection against S. typhimurium (O-4), but no protection against wild-type S. enteritidis (O-9). However challenge with LPS variant strains showed that although protection was generally better to strains of the homologous LPS type, specificity of protection was determined more by the parent strain background (S. typhimurium or S. enteritidis) of the challenge than by O-factors 4 or 9, suggesting that other antigens are involved. The nature of the protective antigen(s) in this model is unclear, but it does not appear to be the main O-specific antigen. A S. enteritidis Se795 aroA vaccine gave good protection against wild-type S. enteritidis Se795 2 weeks after vaccination, but much less at 3 months (approximately 10-200 x LD50), although the persistence of the S. enteritidis aroA vaccine in the liver and spleen was similar to that of the S. typhimurium vaccine, and the wild-type Se795 challenge strain was of similar virulence to S. typhimurium C5. A S. dublin aroA vaccine conferred similar protection against wild-type S. dublin (approximately 300 x LD50).     

Immunity induced by live attenuated Salmonella vaccines

Studies on the degree and specificity of protection conferred by immunization with aroA salmonella live vaccines in BALB/c mice are described. Animals were immunized i.v. and challenged orally 3 months later to ensure that the vaccine had been cleared from the tissues. Vaccination with Salmonella typhimurium aroA SL3261 conferred very good protection against virulent S. typhimurium C5 (over 10,000 x LD50). The specificity of cross protection was studied using S. typhimurium, Salmonella enteritidis and Salmonella dublin for vaccination and challenge, including challenge with variants of S. typhimurium and S. enteritidis of similar virulence which differed in the main LPS (lipopolysaccharide) antigen (0-4 or 0-9). S. typhimurium SL3261 gave very good protection against S. typhimurium C5 (0-4), but no protection against S. enteritidis Se795 (0-9). However, challenge with strains differing in the main 0 antigens showed that, although protection was generally better to strains expressing the same LPS type as the vaccine, specificity of protection was determined more by the background (S. typhimurium or S. enteritidis) of the parent strain used for the challenge than by 0 factors 4 or 9, suggesting that other factors could be involved. The nature of the antigen(s) responsible for protection in this model is unclear, but it would not appear to be the main 0-specific antigen. An S. enteritidis Se795 aroA vaccine was far less effective than S. typhimurium SL3261; it conferred good protection against the homologous wild type at 2 weeks post-vaccination, but far less at three months (approx 10-200 x LD50). This was unexpected, as the persistence of the S. enteritidis vaccine in the liver and spleen was similar to that of S. typhimurium SL3261, and the S. enteritidis and S. typhimurium challenge strains were of similar virulence. An S. dublin aroA vaccine conferred similar protection against wild type S. dublin (approx 300 x LD50).     

Salmonella typhimurium aroA, htrA, and aroD htrA mutants cause progressive infections in athymic (nu/nu) BALB/c mice.

Athymic (nu/nu) BALB/c mice and their euthymic (nu/+) littermates were inoculated intravenously with live attenuated vaccine strains of Salmonella typhimurium. All strains caused progressive infections in the athymic mice but not in their euthymic littermates. Athymic mice given strain SL3261, an aroA derivative of SL1344, in doses between log 4.7 and 5.7 CFU were all severely ill and were killed by weeks 4 to 5. Athymic mice given log 4.7 CFU of a derivative of S. typhimurium C5 carrying a mutation in htrA, encoding a stress protein, were ill and were killed by week 7 in one experiment but survived to week 13 in another. Athymic mice given log 4.6 CFU of a C5 aroD htrA double mutant were ill and were killed at week 7. Athymic mice given SL3261 had high bacterial counts in the reticuloendothelial system at 4 weeks. Athymic mice given SL3261 or C5 htrA made immunoglobulin G3 (IgG3) (and to a lesser extent IgM) antibody to lipopolysaccharide (LPS), whereas euthymic mice made IgM, IgG1, IgG2a, IgG2b, and IgG3 anti-LPS antibodies. The results indicate that both aroA and htrA strains will produce slow, progressively lethal infections in athymic mice, that the htrA strain is more attenuated than the aroA strain as measured by time to death in this model, and that IgG3 anti-LPS antibody alone cannot suppress the progress of infections by very attenuated strains in athymic mice.     

Moderate immunodeficiency does not increase susceptibility to Salmonella typhimurium aroA live vaccines in mice.

Salmonellae carrying appropriate mutations in genes of the aromatic biosynthesis pathway are effective as live vaccines in animals, and they are candidate typhoid vaccines for human use. They are also very effective as carriers of recombinant antigens from other pathogens to the immune system, eliciting circulatory, secretory, and cell-mediated immunity to foreign antigens. Their attenuation is believed to be due to their requirement for the metabolites p-aminobenzoic acid and 2,3-dihydroxybenzoate, which are not available in mammalian tissues. Immunosuppression (e.g., acquired immunodeficiency syndrome) is a major contraindication to the use of live vaccines. If the avirulence of Aro mutants is largely due to their auxotrophy, they should not be markedly more invasive in immunosuppressed animals. We report that wild-type Salmonella typhimurium M525 of intermediate virulence was much more invasive in sublethally irradiated BALB/c mice than in normal BALB/c mice, whereas sublethal irradiation had little if any effect on the invasiveness of an S. typhimurium aorA vaccine strain apart from a delay in its clearance from the reticuloendothelial system. xid mutant CBA/N mice carry an X-linked B-cell functional defect which results in immunoglobulin G3 agammaglobulinemia, and they are known to be more susceptible to salmonellae in late stages of the infection. We found that whereas male (CBA/N x BALB/c)F1 mice (immunodefective) were more susceptible to wild-type S. typhimurium C5 than female littermates (immunocompetent), there was no difference in the response to the S. typhimurium aroA vaccine strain. The results indicate that moderate immunosuppression does not markedly enhance susceptibility to S. typhimurium aroA live vaccines.     

Antibody response and protection against challenge in mice vaccinated intraperitoneally with a live aroA O4-O9 hybrid Salmonella dublin strain.

An auxotrophic Salmonella dublin (O9,12) strain, SL5631, with a deletion affecting gene aroA, was made into a partial diploid expressing the rfb (O-antigen-repeat-unit-specifying) gene cluster of Salmonella typhimurium (O4,12). By use of O4- and O9-specific antisera in indirect immunofluorescence assays, the resulting hybrid SL7103 was shown to express both the O4- and O9-antigen epitopes in the same bacterium. Qualitative and quantitative sugar analyses by gas-liquid chromatography on peralditol acetates of phenol-water-extracted lipopolysaccharides showed that the S. dublin and S. typhimurium repeating units (estimated on the basis of their tyvelose and abequose contents, respectively) were present in approximately equimolar amounts. The SL7103 hybrid auxotroph was avirulent when given intraperitoneally to NMRI mice in a dose of 10(8) CFU and elicited a protective immunity against intraperitoneal challenge with either virulent S. dublin (50% lethal dose of ca. 1.5 x 10(4) CFU versus < 1 x 10(1) CFU in nonimmunized mice) or virulent S. typhimurium (50% lethal dose of ca. 1 x 10(5) versus < 1 x 10(1) CFU in nonimmunized mice). Compared with the protection elicited in homologous systems (S. dublin SL5631 against S. dublin and S. typhimurium SL1479 against S. typhimurium), the protective efficacy of the hybrid was reduced approximately 70-fold against S. dublin challenge and 100-fold against S. typhimurium challenge. Vaccination with S. typhimurium SL1479 conferred no protection against S. dublin challenge, and vaccination with S. dublin SL5631 conferred no protection against S. typhimurium challenge. The protection elicited by the hybrid strain SL7103 is supposed to be mainly a consequence of serum antibodies directed against the immunodominant O4 and O9 epitopes.     

Mice vaccinated with a non-virulent, aromatic-dependent mutant of Salmonella choleraesuis die from challenge with its virulent parent but survive challenge with Salmonella typhimurium

BALB/c mice given a live vaccine of an aroA mutant of Salmonella choleraesuis by intraperitoneal (i.p.) injection were not protected against i.p. challenge with its virulent parental strain but were protected against i.p. challenge with either of two virulent strains of Salmonella typhimurium (O [1], 4, [5], 12). Vaccination with a live vaccine of S. typhimurium aroA protected against challenge with S. typhimurium but not with S. choleraesuis. Intraperitoneal administration of either aroA strain evoked high levels of serum antibody against the homologous lipopolysacharide (LPS) as determined by an enzyme immunoassay. Sera from vaccinated mice also reacted with heterologous LPS but at dilutions at least seven-fold lower than homologous endpoint titres. The vaccination schedule employed with either live-vaccine strain conferred an equal degree of resistance to challenge with Listeria monocytogenes. After mixed infection of mice with equal numbers of virulent S. typhimurium and S. choleraesuis by the i.p. route, the former was isolated in numbers at least 50,000 times greater than the latter from the liver and spleen between days 1 and 5. When mice were vaccinated i.p. with S. choleraesuis aroA, L. monocytogenes or P. multocida before mixed infection, neither serotype showed more than a slight predominance in the liver and spleen during the same period. Thus, in relative terms, vaccination with S. choleraesuis aroA or inoculation with unrelated bacteria suppressed the growth of virulent S. typhimurium in mice but allowed virulent S. choleraesuis to multiply. These results clearly show that S. choleraesuis 38(1) can multiply to kill immunised BALB/c mice.     

Characterization of aromatic- and purine-dependent Salmonella typhimurium: attention, persistence, and ability to induce protective immunity in BALB/c mice.

Stable transposon-generated auxotrophic mutations in aroA, purA, and purE or aroA and purA together were introduced into Salmonella typhimurium strains which were virulent in mice. Strains harboring any of these mutations were attenuated when tested in BALB/c mice. purE strains were less attenuated than aroA or purA strains. Both aroA and purA mutants persisted for several weeks in the livers and spleens of the mice after intravenous infection, although the numbers of viable cells detected at various times after infection differed. aroA strains persisted at a higher level than purA strains and were effective live vaccines given intravenously or orally. purA strains were ineffective as oral vaccines and were poor intravenous vaccines. Strains harboring both aroA and purA mutations together were ineffective vaccines when administered orally or intravenously even though they persisted in the livers and spleens of the mice for long periods after intravenous infection.     

Salmonella typhimurium aroA mutants as carriers of the Escherichia coli heat-labile enterotoxin B subunit to the murine secretory and systemic immune systems

We investigated the ability of Salmonella typhimurium vaccines to deliver heterologous antigens to the systemic and secretory immune systems of the mouse, while retaining their immunogenicity against salmonellosis. S. typhimurium SL3261, an avirulent aroA mutant, or SL3261 carrying plasmid pBRD026, a pBR322 derivative encoding the gene for Escherichia coli LT-B were used to immunize BALB/c mice orally. Both immunizing strains invaded the mononuclear phagocyte system of the mice, grew slowly until approximately day 14 post-infection, and then were rapidly cleared. No salmonellae were detected in livers, spleens, mesenteric lymph nodes or Peyer's patches by day 42. Mice immunized with either strain and challenged orally with the virulent parent strain, SL1344, several weeks after clearing the immunizing organism, were protected against the lethal S. typhimurium infection. Mice infected with SL3261 (pBRD026) developed substantial levels of IgG and IgA anti-LT-B antibodies 14 days post-infection in both serum and gut samples. The sera neutralized the effects of LT in an in vitro Vero cell assay. Thus, aroA mutants of S. typhimurium can deliver a heterologous antigen from a different enteric pathogen to the murine systemic and secretory immune systems without altering their efficacy against salmonellosis.     

Identification of p60 antibodies in human sera and presentation of this listerial antigen on the surface of attenuated salmonellae by the HlyB-HlyD secretion system.

Antibodies directed against the major secreted protein of Listeria monocytogenes, termed p60, were found more frequently than antilisteriolysin antibodies in sera of listeriosis patients. Anti-p60 antibodies were also identified in all tested sera from healthy individuals. To test whether p60 provides protection against L. monocytogenes, we constructed an attenuated Salmonella typhimurium aroA strain which secretes p60 via the Escherichia coli hemolysin secretion pathway. Application of this Salmonella strain to BALB/c mice prior to an L. monocytogenes infection induced p60 antibodies in these mice and led to a significantly reduced number of viable bacteria in the spleen compared with that in control animals which were primed with the S. typhimurium aroA strain alone.     

Murine antibody response to oral infection with live aroA recombinant Salmonella dublin vaccine strains expressing filamentous hemagglutinin antigen from Bordetella pertussis.

Two plasmids which express either nearly intact or truncated filamentous hemagglutinin (FHA) from Bordetella pertussis and which are marked with a tetracycline resistance (Tcr) gene were transformed into Salmonella dublin SL1438, an aroA deletion mutant intended for use as an attenuated oral vaccine against salmonellosis. These S. dublin recombinants, when fed to mice, induced serum immunoglobulin, immunoglobulin M (IgM), and sometimes IgA antibody responses to FHA and S. dublin. In addition, IgA antibodies against FHA were found in gut wash fluids. S. dublin carrying pDB2300, a multicopy plasmid encoding truncated FHA protein, induced a better antibody response than did S. dublin carrying pDB2000, a low-copy-number plasmid encoding full-sized FHA. Administration of tetracycline to mice enhanced the stability of recombinant plasmids, and tetracycline-treated mice developed higher anti-FHA titers. Although neither strain examined is suitable for use in a human oral vaccine, these data demonstrated that an immune response against B. pertussis FHA could be induced by oral administration of live attenuated recombinant strains of S. dublin and suggested that development of a live oral attenuated vaccine against pertussis may be possible.     

Evaluation of Salmonella typhimurium strains harbouring defined mutations in htrA and aroA in the murine salmonellosis model.

Derivatives of the mouse-virulent Salmonella typhimurium strain SL1344 were constructed harbouring defined mutations in htrA, aroA or htrA aroA combined. When administered orally or intravenously to BALB/c mice, all the mutants were found to be highly attenuated. All mutants were able to confer significant protection against lethal challenge with SL1344 after a single oral dose of live organisms. SL1344 htrA mutants persisted in livers and spleens at a lower level than SL1344 aroA mutants after intravenous administration. SL1344 htrA aroA mutants persisted at an even lower level and were cleared from the livers and spleens of mice within 21 days of intravenous administration. Thus htrA and htrA aroA mutants can be considered as potential oral vaccines against salmonellosis.     

Effect of a purA mutation on efficacy of Salmonella live-vaccine vectors.

We made delta aroA, delta purA, and delta aroA delta purA derivatives of a strain of Salmonella dublin and isolated a nalidixate-resistant mutant of each construct. An inoculum of each of the nearly isogenic nalidixate-resistant auxotrophs was administered to BALB/c mice by gavage. The ability of each strain to colonize, invade, persist in tissues, and evoke serum and mucosal antibody responses to the lipopolysaccharide of the parent strain was examined. Only the delta aroA strain colonized, invaded, persisted, and (more importantly) evoked sustained significant serum and mucosal antibody responses. Neither the delta purA nor the delta aroA delta purA strain showed any of these abilities. These observations demonstrate that the purA defect, which causes a requirement for adenine, reduces the live-vaccine efficacy of attenuated Salmonella strains and may limit the effectiveness of Salmonella strains as carriers of heterologous antigens. These findings may be important in the selection of attenuated S. typhi strains for use in humans either as antityphoid live vaccines or as vectors for antigens of other pathogens.     

Specific lung mucosal and systemic immune responses after oral immunization of mice with Salmonella typhimurium aroA, Salmonella typhi Ty21a, and invasive Escherichia coli expressing recombinant pertussis toxin S1 subunit.

Pertussis toxin (PT) is considered an essential protective component for incorporation into new generation vaccines against Bordetella pertussis, the causative agent of whooping cough. Traditionally, antipertussis vaccination has employed an intramuscular route. An alternative to this approach is to stimulate mucosal and systemic immune responses by oral immunization with live vaccine carrier strains of Salmonella spp. or Escherichia coli. Recombinant S1 subunit of pertussis toxin was expressed in the attenuated aroA mutant of Salmonella typhimurium, SL3261, in the human typhoid vaccine strain Salmonella typhi Ty21a, and in E. coli CAG629 containing the Shigella flexneri plasmid pWR110, which encodes bacterial invasiveness of epithelial cells. Expression of recombinant PT S1 subunit (rPT-S1) did not affect in vitro invasiveness of the tested strains, which retained the ability to adhere to and invade the embryonic human intestinal cell line HI-407. Following oral immunization of mice with the live vaccine strains expressing rPT-S1, immunoglobulin G (IgG), IgA, and IgM responses were monitored. IgG specific to PT was detected in serum samples of mice, while IgG and IgA specific to PT were detected in lung washes after oral immunization with living Salmonella spp. or E. coli (pWR110) expressing rPT-S1. Utilization of live oral vaccines expressing B. pertussis antigens, which stimulate both a systemic and lung mucosal response, may provide an attractive alternative to purified component vaccines against whooping cough.     

Protective immunity elicited by a divalent DNA vaccine encoding both the L7/L12 and Omp16 genes of Brucella abortus in BALB/c mice

This study was designed to evaluate the immunogenicity and the protective efficacy of a divalent fusion DNA vaccine encoding both the Brucella abortus L7/L12 protein (ribosomal protein) and Omp16 protein (outer membrane lipoprotein), designated pcDNA3.1-L7/L12-Omp16. Intramuscular injection of this divalent DNA vaccine into BALB/c mice elicited markedly both humoral and cellular immune responses. The specific antibodies exhibited a dominance of immunoglobulin G2a (IgG2a) over IgG1. In addition, the dual-gene DNA vaccine elicited a strong T-cell proliferative response and induced a large amount of gamma interferon-producing T cells upon restimulation in vitro with recombinant fusion protein L7/L12-Omp16, suggesting the induction of a typical T-helper-1-dominated immune response in vivo. This divalent DNA vaccine could also induce a significant level of protection against challenge with the virulent strain B. abortus 544 in BALB/c mice. Furthermore, the protection level induced by the divalent DNA vaccine was significantly higher than that induced by the univalent DNA vaccines pcDNA3.1-L7/L12 or pcDNA3.1-Omp16. Taken together, the results of this study verify for the first time that the Omp16 gene can be a candidate target for a DNA vaccine against brucellosis. Additionally, a divalent genetic vaccine based on the L7/L12 and Omp16 genes can elicit a stronger cellular immune response and better immunoprotection than the relevant univalent vaccines can.     

Immunization of mice against Plasmodium vinckei with a combination of attenuated Salmonella typhimurium and malarial antigen.

Infection with the blood stage of the malaria parasite Plasmodium vinckei is uniformly lethal in mice. We found that immunization of BALB/c mice with a combination of killed P. vinckei antigens and an attenuated (aroA) Salmonella typhimurium strain induces high levels of protection against challenge with live P. vinckei. This is especially significant because, in our previous studies, immunization of mice with killed P. vinckei antigens and adjuvants such as Bordetella pertussis, complete Freund adjuvant, and saponin failed to induce protective immunity. Immunization with attenuated S. typhimurium alone did not provide any nonspecific immunity. In vivo depletion of CD4+ T cells in the mice immunized with attenuated S. typhimurium and P. vinckei antigens caused the loss of their immunity. Expression of this immunity required the presence of a spleen. These results support our previous hypothesis that a blood stage malaria vaccine may need both induction of CD4+ T cells specific for the parasite and modification of the spleen with a vaccine vehicle. Therefore, attenuated Salmonella strains such as the one used in this study, when expressing recombinant malarial antigens, might fulfill this requirement.     

A DNA vaccine encoding Cu,Zn superoxide dismutase of Brucella abortus induces protective immunity in BALB/c mice

This study was conducted to evaluate the immunogenicity and protective efficacy of a DNA vaccine encoding Brucella abortus Cu,Zn superoxide dismutase (SOD). Intramuscular injection of plasmid DNA carrying the SOD gene (pcDNA-SOD) into BALB/c mice elicited both humoral and cellular immune responses. Animals injected with pcDNA-SOD developed SOD-specific antibodies which exhibited a dominance of immunoglobulin G2a (IgG2a) over IgG1. In addition, the DNA vaccine elicited a T-cell-proliferative response and also induced the production of gamma interferon, but not interleukin-10 (IL-10) or IL-4, upon restimulation with either recombinant SOD or crude Brucella protein, suggesting the induction of a typical T-helper-1-dominated immune response in mice. The pcDNA-SOD (but not the control vector) induced a strong, significant level of protection in BALB/c mice against challenge with B. abortus virulent strain 2308; the level of protection was similar to the one induced by B. abortus vaccine strain RB51. Altogether, these data suggest that pcDNA-SOD is a good candidate for use in future studies of vaccination against brucellosis.     

Test of the virulence and live-vaccine efficacy of auxotrophic and galE derivatives of Salmonella choleraesuis.

Aromatic compound-dependent (aro) derivatives of three mouse-virulent strains of Salmonella choleraesuis (Salmonella cholerae-suis) were constructed and shown to be nonvirulent for mice (intraperitoneal [i.p.] 50% lethal dose [LD50], greater than 5 X 10(6) CFU). A pur derivative, and a thy derivative, each of a different virulent parent, remained moderately virulent (i.p. LD50S for BALB/c mice, ca. 10(5) and 5 X 10(4) CFU, respectively). Tested as live vaccines i.p., the aro strains were ineffective in salmonella-susceptible BALB/c and C57BL/6 mice but were somewhat effective in salmonella-resistant CBA/J mice and in outbred CD-1 mice. The pur and thy strains were effective as live vaccines in BALB/c mice when given in sublethal doses. Two previously isolated nonvirulent galE derivatives of S. choleraesuis (i.p. LD50 in BALB/c mice, greater than 10(6) CFU) were also ineffective as live vaccines in BALB/c and C57BL/6 mice. The main antigenic difference between S. choleraesuis (O-6,7) and S. typhimurium (O-4,12) is in O-antigen character, thought to largely determine the specificity of protection in salmonellosis. Paired, nearly isogenic O-6,7 and O-4,12 derivatives were constructed from an aro S. typhimurium strain of proven efficacy as a live vaccine. Used as live vaccines, the O-4,12 member protected BALB/c mice against challenge with virulent S. typhimurium, whereas the O-6,7 member did not protect against virulent S. choleraesuis. However, BALB/c mice vaccinated with the O-6,7 member and mice vaccinated with an aro S. choleraesuis strain were protected against challenge with a moderately virulent (LD50, 5 X 10(4) CFU) O-6,7 derivative of an S. typhimurium strain.     

Protection against Leishmania major infection in genetically susceptible BALB/c mice by gp63 delivered orally in attenuated Salmonella typhimurium (AroA- AroD-).

The gene encoding the Leishmania major (L. major) promastigote surface glycoprotein, gp63, was introduced into the Salmonella typhimurium (S. typhimurium) aroA- aroD- live oral vaccine strain BRD509 and expressed under the control of a constitutive tac promoter in plasmid pKK233-2. This construct (GID101) expressed gp63 in vitro and was used to immunize highly susceptible BALB/c mice by the oral route. The plasmid was relatively stably inherited by bacteria growing or persisting in the mesenteric lymph nodes of immunized mice. Mice immunized with GID101 developed significant resistance against a challenge infection with L. major compared to controls immunized with BRD509 alone. Spleen and lymph node cells from immunized mice developed a strong in vitro proliferative T-cell response to killed or live L. major. The activated T cells secreted interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) which was abrogated by treatment with anti-CD4 but not with anti-CD8 antibody. The cells did not produce detectable levels of interleukin-4 (IL-4). The immunized mice also produced significant amounts of leishmanial specific IgG2a antibody but did not develop delayed-type hypersensitivity (DTH) to live parasites. No IgG1 antibody was detected. These data therefore demonstrate that gp63 gene delivered orally by a vaccine strain of S. typhimurium can preferentially induce the development of Th-1 subset of CD4+ T cells and protective immunity in the highly susceptible BALB/c mice.