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We have evaluated an oral vaccine based on an Salmonella enteric serovar typhi (S. typhi) Ty2 derivative TSB7 harboring deletion mutations in ssaV (SPI-2) and aroC together with a chromosomally integrated copy of eltB encoding the B subunit of enterotoxigenic Escherichia coli heat labile toxin (LT-B) in volunteers. Two oral doses of 10(8) or 10(9)CFU were administered to two groups of volunteers and both doses were well tolerated, with no vaccinemia, and only transient stool shedding. Immune responses to LT-B and S. typhi lipopolysaccharide were demonstrated in 67 and 97% of subjects, respectively, without evidence of anti-carrier immunity preventing boosting of LT-B responses in many cases. Further development of this salmonella-based (spi-VEC) system for oral delivery of heterologous antigens appears warranted.

A previously described attenuated TnphoA mutant (BRD441) of Salmonella enterica serovar Typhimurium C5 (I. Miller, D. Maskell, C. Hormaeche, K. Johnson, D. Pickard, and G. Dougan, Infect. Immun. 57:2758-2763, 1989) was characterized, and the transposon was shown to be inserted in surA, a gene which encodes a peptidylprolyl-cis, trans-isomerase. A defined surA deletion mutation was introduced into S. enterica serovar Typhimurium C5 and the mutant strain, named S. enterica serovar Typhimurium BRD1115, was extensively characterized both in vitro and in vivo. S. enterica serovar Typhimurium BRD1115 was found to be defective in the ability to adhere to and invade eukaryotic cells. Furthermore, S. enterica serovar Typhimurium BRD1115 was attenuated by at least 3 log units when administered orally or intravenously to BALB/c mice. Complementation of the mutation with a plasmid carrying the intact surA gene almost completely restored the virulence of BRD1115. In addition, S. enterica serovar Typhimurium BRD1115 demonstrated potential as a vaccine candidate, since mice immunized with BRD1115 were protected against subsequent challenge with S. enterica serovar Typhimurium C5. S. enterica serovar Typhimurium BRD1115 also showed potential as a vehicle for the effective delivery of heterologous antigens, such as the nontoxic, protective fragment C domain of tetanus toxin, to the murine immune system.

Salmonella enterica serovar Typhi strain CVD 908-htrA is a live attenuated strain which may be useful as an improved oral typhoid vaccine and as a vector for cloned genes of other pathogens. We conducted a phase 2 trial in which 80 healthy adults received one of two dosage levels of CVD 908-htrA in a double-blind, placebo-controlled, crossover study. There were no differences in the rates of side effects among volunteers who received high-dose vaccine (4.5 x 10(8) CFU), lower-dose vaccine (5 x 10(7) CFU), or placebo in the 21 days after vaccination, although recipients of high-dose vaccine (8%) had more frequent diarrhea than placebo recipients (0%) in the first 7 days. Seventy-seven percent and 46% of recipients of high- and lower-dose vaccines, respectively, briefly excreted vaccine organisms in their stools. All blood cultures were negative. Antibody-secreting cells producing antilipopolysaccharide (LPS) immunoglobulin A (IgA) were detected in 100 and 92% of recipients of high- and lower-dose vaccines, respectively. Almost half the volunteers developed serum anti-LPS IgG. Lymphocyte proliferation and gamma interferon production against serovar Typhi antigens occurred in a significant proportion of vaccinees. This phase 2 study supports the further development of CVD 908-htrA as a single-dose vaccine against typhoid fever and as a possible live vector for oral delivery of other vaccine antigens.

We have investigated the effect of preexisting immunity to homologous (Salmonella typhimurium) or heterologous (S. dublin) serotypes of Salmonella on the ability of an attenuated S. typhimurium aroA aroD vector (BRD509) to immunize mice against the heterologous antigen fragment C (FrgC). We studied two strains, BRD847 and BRD937, expressing FrgC carried on plasmids that differ only with respect to the promoter controlling FrgC expression, the nirB promoter in the case of BRD847 and the htrA promoter in the case of BRD937. Mice were preimmunized orally with S. typhimurium BRD509, S. dublin aroA aroD (BRD620), or saline. Forty-four days later, they were immunized orally with BRD847 or BRD937. Prior immunity to S. typhimurium severely depressed the serum immunoglobulin G (IgG) and IgA anti-FrgC response in both BRD847- and BRD937-immunized mice. Mice with existing immunity to S. dublin also had lower IgG anti-FrgC geometric mean titers (GMTs) than did mice preimmunized with saline, but this difference was significant only in the case of mice immunized with BRD937. However, in nonimmune mice or in mice preimmunized with S. typhimurium or S. dublin, the anti-FrgC IgG GMTs were always higher in mice in the BRD937 groups than in the equivalent BRD847 groups. This is reflected in the effect of prior immunity on the ability of oral immunization with BRD847 or BRD937 to protect mice from challenge with a lethal dose of tetanus toxin. All of the mice preimmunized with saline and then immunized with BRD847 or BRD937 survived challenge. Only 20% of the animals immunized with BRD847 and 60% of the mice in the BRD937 group survived tetanus toxin challenge if they were preimmunized with BRD509. Preexisting immunity to S. dublin did not affect the ability of BRD937 to immunize mice against tetanus, but it did reduce the efficiency of BRD847: only 60% percent of the mice survived challenge. The intestinal secretory IgA responses to FrgC were very similar in the BRD847 and BRD937 groups. Prior immunity did depress the IgA anti-FrgC titers but only significantly so in the mice preimmunized with BRD509. These results show that preexisting Salmonella immunity, particularly to homologous serotypes, can severely compromise the ability of live Salmonella vectors to deliver heterologous antigens to the mammalian immune system. However, the results also indicate that this may be overcome by the design of more powerful in vivo expression systems.

The properties of two candidate Salmonella typhi-based live oral typhoid vaccine strains, BRD691 (S. typhi Ty2 harboring mutations in aroA and aroC) and BRD1116 (S. typhi Ty2 harboring mutations in aroA, aroC, and htrA), were compared in a number of in vitro and in vivo assays. BRD1116 exhibited an increased susceptibility to oxidative stress compared with BRD691, but both strains were equally resistant to heat shock. Both strains showed a similar ability to invade Caco-2 and HT-29 epithelial cells and U937 macrophage-like cells, but BRD1116 was less efficient at surviving in epithelial cells than BRD691. BRD1116 and BRD691 were equally susceptible to intracellular killing within U937 cells. Similar findings were demonstrated in vivo, with BRD1116 being less able to survive and translocate to secondary sites of infection when inoculated into the lumen of human intestinal xenografts in SCID mice. However, translocation of BRD1116 to spleens and livers in SCID mice occurred as efficiently as that of BRD691 when inoculated intraperitonally. The ability of BRD1116 to increase the secretion of interleukin-8 following infection of HT-29 epithelial cells was comparable to that of BRD691. Therefore, loss of the HtrA protease in S. typhi does not seem to alter its ability to invade epithelial cells or macrophages or to induce proinflammatory cytokines such as IL-8 but significantly reduces intracellular survival in human intestinal epithelial cells in vitro and in vivo.

The genes encoding murine macrophage migration inhibitory factor (MIF), IL-2, IFN-gamma or TNF-alpha were cloned individually into an expression plasmid under the control of the inducible promoter nirB and transfected into the aroA- aroD- deletion mutant strain of Salmonella typhimurium (BRD509). These S. typhimurium derivatives (henceforward called constructs and termed GIDMIF, GIDIL2, GIDIFN and GIDTNF) expressed their respective cytokines in vitro under anaerobic conditions and stably colonized BALB/c mice up to 14 days after oral administration. The highly susceptible BALB/c mice that had received the constructs orally and that had been subsequently infected via the footpad with Leishmania major, developed significantly reduced disease compared with control mice administered the untransfected Salmonella strain (BRD509). Importantly, a combination of GIDMIF, GIDIFN, and GIDTNF administered orally after L. major infection was able to significantly limit lesion development and reduced parasite loads by up to three orders of magnitude. Spleen and lymph node cells of mice administered this combination expressed markedly higher levels of inducible nitric oxide synthase (iNOS) compared with those from mice receiving an equivalent dose of the control strain of Salmonella (BRD509). These data therefore demonstrate the feasibility of therapeutic treatment in an infectious disease model using cytokines delivered by attenuated Salmonella. The protective effect observed correlates with the induction of inducible nitric oxide synthase in vivo.

Salmonella aro vaccines are able to confer solid protection against homologous virulent challenge in several animal species. Calves were protected against virulent S. typhimurium challenge following administration of a single oral dose of live BRD562 vaccine. Immune responses elicited by the S. typhimurium aro vaccine strain BRD562 were studied following administration to calves by either the oral or subcutaneous route. Serum antibodies to Salmonella polypeptides, following oral or subcutaneous vaccination, were detected by immunoblotting and the route of inoculation found to affect both the antibody isotype and the antigens detected. Oral, but not subcutaneous, immunisation induced bovine serum IgA antibodies against Salmonella antigens of 30 kDa and 65 kDa and bovine IgG2 antibodies against a 35 kDa antigen. Subcutaneous vaccination triggered responses against antigens of 52 kDa, 54 kDa and 57 kDa which were not detected by immune plasma of animals immunised orally. Antibody responses to LPS were poor in animals inoculated by either route. Subcutaneous vaccination elicited T-cell responses against Salmonella antigens as measured by in vitro peripheral blood cell thymidine incorporation. These studies show that the S. typhimurium vaccine strain BRD562 is capable of inducing both humoral and cellular immune responses. Further studies are necessary to identify the nature of the antigens responsible for protection. Oral or subcutaneous inoculation of BRD562(pTETnir15) failed to induce serum antibodies against the fragment C of tetanus toxin (TetC) but was effective in mice. Oral vaccination with this recombinant vaccine induced mucosal IgA against TetC. This is the first time that Salmonella recombinant vaccines have been shown to successfully elicit antibodies against a guest antigen in cattle after one single oral inoculation.

A single-dose, oral Salmonella typhi vaccine strain has been sought as a carrier or vector of cloned genes encoding protective antigens of other pathogens. Such a hybrid vaccine, administered orally, would stimulate immune responses both at the mucosal surface and in the systemic compartment and would potentially provide protection against multiple pathogens. S. typhi CVD 908 and CVD 906, which harbor deletions in aroC and aroD, were further engineered by deletion in htrA to produce strains CVD 908-htrA and CVD 906-htrA, which are unable to sustain growth and are severely impaired in their ability to survive in host tissues. These strains were fed to humans at doses of 5 x 10(7) to 5 x 10(9) CFU with buffer, and safety and immune responses were assessed. CVD 908-htrA and CVD 906-htrA were well tolerated in volunteers; mild diarrhea in 3 of 36 volunteers and mild fever in 1 volunteer were the only notable adverse responses. The vaccine strains were not detected in blood cultures and only transiently detected in stool. Serum immune responses to S. typhi lipopolysaccharide and H antigens were observed in 75 to 100% of volunteers who received 5 x 10(8) to 5 x 10(9) CFU, and cells secreting S. typhi-specific antibodies were found in all volunteers after ingestion of either strain. Sixty-three percent to 83% of volunteers developed lymphoproliferative responses to S. typhi flagellar and particulate antigens after the higher doses. These studies demonstrate the potential of CVD 908-htrA as a live vector for the delivery of heterologous genes, and a clinical trial of such a construct is planned.

The mechanisms which regulate mucosal IgA responses to orally administered protein vaccines are not yet fully elucidated. We have used two delivery systems, soluble tetanus toxoid (TT) with the mucosal adjuvant cholera toxin (CT) and recombinant Salmonella expressing Tox C, a fragment of TT, to assess the nature of CD4+ T helper (Th) cells and derived cytokines which support mucosal IgA responses in both normal and cytokine knockout (interferon gamma knockout; IFN-gamma-/- and IL-4-/-) mice. Our results provide important new information regarding Th cell and cytokine regulation of mucosal IgA responses. Whereas TT coadministered with CT induces predominant TT-specific Th2-type responses, rSalmonella delivery of Tox C induced dominant Th1-type responses along with synthesis of the Th2-cytokine IL-10. Both vaccine regimen elicited high levels of mucosal S-IgA and IL-6 production by macrophages. Further oral immunization of IFN-gamma-/- and IL-4-/- mice with rSalmonella Tox C also induced macrophage-derived IL-6 and Th2-derived IL-10 as well as S-IgA responses, suggesting that IFN-gamma from Th1-type cells as well as traditional Th2 cells producing IL-4 and IL-5 are not essential for mucosal IgA responses. Rather, induction of second level Th2 cells producing IL-10 together with high levels of IL-6 from other cell sources may be sufficient for mucosal IgA responses in the absence of traditional Th2 cells. These studies were facilitated by the development of a sensitive new luminometry assay which allowed detection of cytokines and cell surface molecules which are below the levels of detection by current solid phase assays.

Attenuated Salmonella typhi vaccine strain CVD 908, which harbors deletion mutations in aroC and aroD, has been shown to be well-tolerated and highly immunogenic, eliciting impressive serum antibody, mucosal IgA and cell-mediated immune responses. A further derivative prepared by introducing a deletion in htrA (which encodes a heat-shock protein that also has activity as a serine protease in CVD 908 (Chatfield et al., unpublished data) resulted in CVD 908-htrA. In phase 1 clinical trials, CVD 908-htrA appears very attractive as a live oral vaccine candidate. Both CVD 908 and CVD 908-htrA are useful as live vector vaccines to deliver foreign antigens to the immune system. Conditions that enhance the expression and immunogenicity of foreign antigens carried by CVD 908 and CVD 908-htrA are being investigated.

In this study, we constructed strain KR21 (chi 4550 delta cya delta crp delta asd/pYA292asd(+)-toxC+) and compared it with BRD847 (aroA aroD/pnirB-toxC) for the ability to induce humoral and cellular immunity after a single oral or intravenous immunization in 3- to 4-week-old BALB/c mice. ToxC-specific serum immunoglobulin G (IgG) was detectable in animals orally immunized with either BRD847 or KR21. However, after intravenous immunization, IgG was detected only in BRD847-immunized animals. Measurement of immunoglobin types IgG1 and IgG2a suggests that a Th1 cellular response is prominent after immunizations with either system. ToxC-specific IgA was detected in fecal and vaginal samples of animals immunized orally and intravenously with BRD847, while those immunized with KR21 failed to show fecal or vaginal IgA responses. Delayed-type hypersensitivity was used as a measure of induction of T-cell responses in vivo. Mice immunized either orally or intravenously with BRD847 showed significant ear swelling responses after ToxC injections, while KR21-immunized animals failed to show a cellular response. These data indicate that the aroA aroD/pnirB system holds greater potential for inducing global immunity after a single dose when directly compared with the balanced lethal system (delta cya delta crp delta asd/pYA292asd+).

Attenuated Salmonella strains are currently being evaluated as live vectors for the delivery of heterologous antigens to the mammalian mucosal and systemic immune systems. An approach to improving the stability of heterologous antigen expression during vaccination is to drive expression of the foreign protein from promoters, e.g. nirB, that become activated when Salmonella enter the host. Salmonella strains were constructed that harboured similar multicopy plasmids encoding the lacZ gene. In each strain, lacZ expression was driven from either the nirB, htrA or groE promoters. Expression of LacZ increased in all vaccine strains as they were shifted from conditions of low to high temperature. In addition, expression of lacZ driven from the htrA and nirB promoters significantly increased when the Salmonella entered eukaryotic cells, including macrophages. Expression of lacZ from the groE promoter was significantly elevated in macrophages but not in cells derived from epithelia. These promoters may be useful for optimising heterologous antigen expression within immune cells of the host.

Genetically defined, live attenuated Salmonella vaccines are proving useful both as oral vaccines against salmonellosis and for the development of multivalent vaccines based on the expression of heterologous antigens in such strains. Several candidate attenuated S. typhi strains are at present being evaluated as new single dose oral typhoid vaccines in human volunteers. The emergence of such a vaccine will facilitate the development of multivalent vaccines for humans. Many antigens from different infectious organisms have been expressed in attenuated Salmonella. A focus of this work has been on developing vaccines against parasitic diseases. This review will summarize the efforts that have been made in this area.

Safe, live attenuated Salmonella strains can be produced by introducing defined non-reverting mutations into the chromosome. Such rationally attenuated strains have proved to be excellent oral vaccines in several animal species and can therefore be considered as candidate vaccines against invasive salmonellosis in both animals and man. A panel of attenuating lesions is now available from which it is possible to tailor the level of attenuation and hence produce strains with different immunogenic properties. Because of the spectrum of immune responses produced by such Salmonella vaccine strains they have been utilised extensively as vectors for delivering heterologous antigens to the mammalian immune system. We have focussed on the development of a single dose oral tetanus vaccine based on attenuated Salmonella strains expressing a non-toxic, immunogenic protein derived from tetanus toxin (fragment C). Several different expression systems have been used for fragment C and candidate vaccine strains have been constructed that are capable of protecting orally immunised mice against a lethal challenge with tetanus toxin. An oral tetanus vaccine may help to reduce the mortality rate from tetanus in the developing world by overcoming the problems associated with the implementation of vaccine programmes using the current parenteral vaccine.

We have described a new class of live attenuated salmonella vaccines harbouring lesions in htrA, a stress protein gene previously. The virulence and invasiveness of Salmonella htrA mutants was investigated in three models of increased susceptibility to Salmonella infection. These included BALB/c mice, either given sublethal whole body irradiation (350 R) or administered rabbit anti-TNF alpha antiserum, and (CBA/NfemaleXBALB/cmale)F1 male mice which express the xid sex-linked B cell defect of CBA/N mice and are more susceptible to salmonellae than female littermates. Salmonella typhimurium htrA mutants derived from virulent strains, C5046 (C5 htrA::TnphoA) and BRD726 (SL1344 delta htrA) were not more invasive in immunosuppressed mice than in normal controls in the three mouse models of defective immunity. The results indicate that susceptibility to S. typhimurium htrA vaccines derived from virulent parents is not enhanced by conditions of impaired resistance to infection.

An attenuated derivative of the fully calf virulent Salmonella typhimurium strain ST4/74 was constructed by introducing stable mutations into genes aroA and aroD rendering the strain dependent for growth on aromatic compounds. The strain (BRD562) was used to vaccinate orally ten Friesian calves 7 days after birth with approximately 10(10) live organisms. The only effects of vaccination in the calves was a transient faecal excretion of the organism, mild transient diarrhoea and mild pyrexia in one animal. Two of these animals were tested for the presence of BRD562 at necropsy 56 days after birth but none were shown to be infected. Eight of the vaccinated calves were subjected to an oral challenge infection with ST4/74 28 days after vaccination and seven out of the eight animals survived. The only effect of challenge in the surviving calves was mild pyrexia and transient excretion of ST4/74 in their faeces; only one of the seven animals was found to be infected at necropsy 28 days after challenge. The eighth challenged calf scoured severely and was killed in extremis 7 days after challenge. Four unvaccinated control calves infected orally with ST4/74 28 days after birth all scoured severely and were euthanased for humane reasons 5-7 days later. This study suggests that vaccination with an attenuated strain of S. typhimurium harbouring mutations in two aro genes is a safe and effective way of protecting young calves against experimental S. typhimurium infection.

Because of their invasive properties, Salmonella species are uniquely suited for use as live oral vaccines, and mutants attenuated in general metabolic functions show promise as vaccine strains. We have identified a Salmonella choleraesuis transposon mutant which is deficient in abilities to attach to, invade or penetrate through animal cells. This mutant is avirulent in mice, yet is able to persist within the liver, spleen and gut-associated lymphoid tissue for periods which may be adequate for induction of secretory, humoral and cell-mediated immunity. This and other strains attenuated in factors specifically required for Salmonella pathogenesis may have potential as vaccine strains.

Derivatives of the mouse-virulent Salmonella typhimurium strain SL1344 were constructed harboring stable mutations in aroC alone or in aroC and aroA together. Fifty percent lethal doses after intravenous inoculation of the mutants into BALB/c mice were determined, and the mutants were as highly attenuated as were SL1344 aroA derivatives. All aro-dependent derivatives persisted in vivo at similar levels and for similar intervals in the livers and spleens of BALB/c mice infected intravenously. Mice vaccinated orally with 10(10) live organisms of the different derivatives survived and were well protected against oral challenge with virulent S. typhimurium. A Salmonella typhi Ty2 derivative, designated WBL2000, was constructed that harbors well-defined, stable mutations in both aroA and aroC.

Salmonella species can be rationally attenuated by introducing non-reverting defined mutations into the genome to produce live vaccine strains. Several genes have been identified which when mutated, will attenuate Salmonellae. In particular, salmonella strains harbouring mutations in genes involved in the pre-chorismate biosynthetic pathway make excellent oral vaccines evoking strong humoral, local and cellular immune responses in the host. Because of the spectrum of immune responses induced by live vaccine strains they have the potential to be used for delivery of heterologous antigens to the mammalian immune system. A number of antigens from other bacteria, viruses and parasites have been expressed in live salmonella vaccine strains. Such hybrid strains have the potential to be used as multivalent vaccines against a number of infectious diseases.

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 BA LB/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-γ (IFN-γ) 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. [ABSTRACT FROM AUTHOR]

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 BA LB/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-γ (IFN-γ) 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. [ABSTRACT FROM AUTHOR]