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Three attenuated Salmonella typhi strains have been constructed by introducing deletions in aroC and aroD or deletions in cya and crp into one of two wild-type parent strains, Ty2 or ISP1820. These mutant strains were designated CVD 906 (ISP1820 AaroC AaroD), CVD 908 (Ty2 AaroC AaroD), and X3927 (Ty2 Acya Acrp). Two studies were conducted with 36 healthy adult inpatient volunteers to determine in a double-blind fashion the safety and immunogenicity of approximately 5 x 104 and 5 x 105 CFU of each of these three vaccine candidates given as a single dose. No statistically significant difference in the incidence of reactions among vaccinees was observed. Fever (oral temperature 2 38.2°C) occurred in 2 of 12 volunteers who received CVD 906, in 0 of 12 who received CVD 908, and in 1 of 12 who received x3927. Vaccine bacteremia without symptoms occurred in 1 of 12 vaccinees who received CVD 906, in 0 of 12 who received CVD 908, and in 2 of 12 who received x3927. Overall, 19 (53%) of 36 vaccinees developed immunoglobulin G antibody to S. typhi lipopolysaccharide after vaccination, with no statistically significant differences in the rate of seroconversion among volunteers in the three groups. We conclude that defined mutations in the aromatic biosynthetic pathway and in the cyclic AMP global regulatory system attenuate S. typhi. Mutant strains CVD 906, CVD 908, and X3927 are highly (and approximately equally) immunogenic but possibly differ in their propensity to induce fever. Further studies are needed to document the apparent relative safety of CVD 908 as a typhoid vaccine and as a vaccine carrier of foreign antigens. Live oral attenuated Salmonella typhi vaccines are likely to replace the parenteral killed whole-cell vaccine as safer and possibly more effective vaccines against typhoid fever for use in healthy adults and children (19). Ingestion of attenuated bacteria mimics natural enteric infection and potentially improves the intensity and duration of the immune response compared with parenterally administered vaccines. These strains may also serve as vaccine carriers of cloned protective antigens of other pathogens. A prototype attenuated S. typhi vaccine strain, Ty21a, developed in the early 1970s by chemical mutagenesis before the advent of recombinant DNA technology (7), has been licensed in the United States and used to prevent typhoid fever. Ty2la has also been used as a carrier of foreign antigens, with variable success (1, 26). However, Ty21a requires multiple doses to achieve acceptable immunogenicity and is a difficult background in which to perform genetic manipulations. Therefore, new oral typhoid vaccines have been sought to serve as immunoprophylaxis against typhoid and as a recombinant vaccine carrier.

Currently, there is no long-term effective treatment for unresectable hepatic malignancies. Salmonella sp. are known to naturally track to the liver during active infection. To develop a biological vector for delivery of Interleukin-2 (IL-2) to the liver for anti-tumor purposes, the avirulent and highly immunogenic chi 4550 strain of Salmonella typhimurium was used as a vector for IL-2. The gene for human IL-2 was cloned into plasmid pYA292 (renamed pIL-2) and inserted into the attenuated Salmonella typhimurium and renamed [chi 4550 (pIL-2)]. This transformant was found to produced biologically active IL-2 demonstrated by NK cell activation in a 4 hour chromium release cytotoxicity assay. To determine anti-tumor potential, MCA-38 murine adenocarcinoma cells were injected intrasplenically into C57BL/6 mice to produce hepatic metastases and metastases were subsequently enumerated after 12 days. Statistical significance was determined by ANOVA with Fisher's test for significance. Hepatic metastases enumerated by blinded observers revealed that the mean number of metastases was 106.4 in control mice, 103.7 in mice gavage fed attenuated salmonella without IL-2 [chi 4550(pYA292)], and 44.3 in mice fed the chi 4550(pIL2); (ANOVA: p < 0.01). Culture of livers and spleens in mice administered a single gavage dose of salmonella demonstrated persistent colonization for up to 4 weeks. No observable toxicity was seen to either IL-2 or salmonella. These studies demonstrate that the chi 4550(pIL2) is a novel form of in vivo biotherapy which produces biologically active IL-2 and employs the oral route of administration to stimulate an immune response against malignancy in the liver.

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Salmonella typhimurium strains with deletion (delta) of the adenylate cyclase (cya) and cyclic AMP receptor protein (crp) genes are avirulent for mice and induce a high level of protective immunity to oral challenge with up to 10,000 times what would be a lethal dose of wild-type virulent S. typhimurium cells. This immunity begins as early as seven days after immunization and lasts for at least four months. S. typhimurium delta cya delta crp mutants stably maintain plasmids and give high-level expression of cloned gene products; in this they appear superior to other avirulent S. typhimurium strains. S. typhimurium delta cya delta crp strains with a delta asd mutation (abolishing production of aspartate beta-semialdehyde dehydrogenase), have an obligate requirement for diaminopimelic acid (DAP). This strain can be used in conjunction with plasmid vectors lacking antibiotic resistance markers but having the wild-type asd+ gene from Streptococcus mutans to complement the delta asd chromosomal mutation. The Asd+ plasmid vector can be used to express a diversity of colonization and virulence antigens from other pathogens. In the delta cya delta crp delta asd S. typhimurium vaccine strain, the plasmid is completely stable in the absence of any exogenous selective pressure either in vitro or in vivo.

Salmonella typhi organisms which express genes encoding protective antigens of other pathogens have been developed for use as experimental oral vaccines. A ⌬asd S. typhi strain attenuated by deletions in cya, crp, and cdt which contains hepatitis B core (HBc) and pre-S genes encoded on an Asd ؉ pBR-based plasmid vector was constructed. Healthy adult volunteers ingested a single dose of 5 ؋ 10 5 to 5 ؋ 10 8 CFU of strain 4073 (⌬cya ⌬crp ⌬cdt S. typhi Ty2), 6 ؋ 10 7 or 1 ؋ 10 9 CFU of strain 4632(pYA3149), a further derivative of 4073 deleted in asd and containing the Asd ؉ vector without the HBc-pre-S fusion, or 3 ؋ 10 7 or 7 ؋ 10 8 CFU of strain 4632(pYA3167), a derivative containing the vector with the HBc-pre-S fusion. 4073 was generally well tolerated by 22 volunteers. No volunteer had fever or positive blood cultures; 4 of 22 volunteers shed vaccine organisms in the stool in the first 48 h only. Two of 18 volunteers who received one of the plasmid-containing derivatives of 4073 developed low-grade fevers on day 10 or 12 after ingestion. One of these volunteers had positive blood cultures on days 7 and 8. Seven of these 18 volunteers had vaccine organisms detected in their stools in the first 48 h only. Most volunteers developed S. typhi-specific serum responses and developed S. typhi-specific antibody-secreting cells. However, no volunteer developed serum antibody to hepatitis pre-S or pre-S-specific antibody-secreting cells. Although the parent strain 4073 was well tolerated, induced immunoglobulin G seroconversion to S. typhi lipopolysaccharide in 80 to 100% of vaccinees and stimulated specific IgA-secreting lymphocytes in 80 to 100% of vaccinees given a single oral dose of 2 ؋ 10 7 and 5 ؋ 10 8 CFU, 4073 derivatives containing the Asd ؉ vector with and without sequences encoding the HBc-pre-S fusion caused occasional febrile reactions at high doses and did not stimulate detectable immune responses to hepatitis B antigens.

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An attenuated strain of Salmonella typhi ⌬cya ⌬(crp-cdt) ⌬asd expressing a gene encoding a hepatitis B virus core-pre-S protein was tested in female adult volunteers for its ability to elicit a systemic and a mucosal immune response. Specifically, our purpose was to evaluate the potential of such a vaccine strain to induce specific secretory immunoglobulin A (sIgA) at genital and rectal surfaces. Oral and rectal routes of immunization were compared: oral immunization induced seroconversion against the bacterial lipopolysaccharide (LPS) in six out of seven volunteers, while after rectal immunization only one out of six volunteers seroconverted against LPS. To our disappointment, the latter volunteer was also the only one who seroconverted against the carried antigen (pre-S1), demonstrating the poor ability of this live vaccine to induce an immune response against the carried antigen. Anti-LPS sIgA was found in both the vaginal and cervical secretions of a volunteer who presented a strong seroconversion after oral immunization (16-fold increase in anti-LPS IgG). Smaller amounts of anti-LPS sIgA were found in the rectal secretions of one orally and one rectally immunized volunteer and in the saliva of three orally and one rectally immunized woman. Our data show for the first time that it is possible to induce specific sIgA in the genital and rectal tracts of women by using an S. typhi vaccine strain.