Sandra M. Kelly

Avirulent Salmonella typhimurium Δcya Δcrp oral vaccine strains expressing a streptococcal colonization and virulence antigen

Salmonella typhimurium SR-11 strains lacking adenylate cyclase and the cyclic AMP receptor protein (CRP) due to deletion (Δ) mutations in the cya and crp genes, respectively, are avirulent for mice and induce high level protective immunity against subsequent challenge with wild-type virulent S. typhimurium SR-11 cells. The avirulence of these Δcya Δcrp mutants has been enhanced by elimination of the 100 kb virulence plasmid pStSR100 without impairing immunogenicity. The present report confirms the avirulence and immunogenicity of these mutant strains, demonstrates that immunization of both four- and eight-week-old mice has no adverse effect on weight gain, and that immunity lasts at least ninety days following initial immunization. Avirulent S. typhimurium strains have been endowed with the ability to produce several streptococcal colonization and virulence antigens for the purpose of constructing recombinant bivalent oral vaccine strains. Important antigenic determinants of the Streptococcus sobrinus surface protein antigen A (SpaA), presumed to be a critical colonization antigen of S. sobrinus , are expressed at high level by the Δcya Δcrp S. typhimurium strains. The recombinant vaccine strains are stable in vitro and in animals (for a period of at least eight days) where they localize to the gut-associated lymphoid tissue (GALT).

Live oral avirulent Salmonella vaccines

Infection of animals and humans with Salmonella is a consequence of oral consumption of food or fluids contaminated with Salmonella. Once in the intestine, Salmonella usually attach to, invade, and proliferate in enterocytes or the cells of the gut associated lymphoid tissue (GALT). The latter route of infection can lead to disease or to an asymptomatic carrier state or stimulate the induction of mucosal, systemic and cellular immune responses. Infection of animals with virulent invasive Salmonella can result in suppression of the immune responses which in turn can facilitate the establishment of a carrier state. It is possible to attenuate Salmonella by introducing mutations that (i) confer auxotrophy, (ii) interfere with sugar metabolism and LPS biosynthesis or (iii) affect some global means of regulating genes needed for the full display of virulence. Oral immunization of animals such as mice and chickens with avirulent Salmonella strains usually is not associated with suppression but rather with stimulation of mucosal, systemic and cellular immune responses. Vaccination by injection of killed vaccines or bacterins does not lead to the induction of either mucosal or cellular immune responses, and humoral immunity may be relatively short lived. Thus, killed vaccines are inferior to orally administered live avirulent Salmonella vaccines which induce a long-lasting protective immunity. In this manuscript we discuss desirable attributes of a safe, efficacious live attenuated Salmonella vaccine, describe attenuated Salmonella mutants so far isolated and their properties and present information on the evaluation of a live attenuated Salmonella oral vaccine for poultry.(ABSTRACT TRUNCATED AT 250 WORDS)

Stabilization of recombinant avirulent vaccine strains in vivo

Salmonella strains attenuated by various mutational alterations and expressing heterologous colonization and virulence antigens specified by cloned genes have begun to be widely used as vaccines for oral immunization to induce protective immunity against the pathogens supplying the genes for the colonization or virulence antigens. Problems associated with plasmid instability and/or poor expression of cloned gene products have frequently been encountered and regulatory agencies are now banning use of antibiotic resistance markers in live attenuated vaccine strains. We have therefore developed a balanced lethal host-vector system in which the chromosome of the attenuated vaccine strain contains a deletion mutation that impose a requirement for diaminopimelic acid (DAP), an essential constituent of the rigid layer of the cell wall of all Gram-negative and some Gram-positive microbes. The plasmid cloning vector contains the wild-type allele for this gene allowing the recombinant avirulent Salmonella to be independent of DAP. Since DAP is not prevalent in nature, especially in the animal host, essentially 100% of the surviving avirulent Salmonella recovered from an immunized animal host still contain the recombinant plasmid and express the foreign colonization or virulence antigen. Occasional loss of the plasmid renders the avirulent Salmonella DAP-requiring, which quickly results in DAPless death with lysis of the bacterium to release its antigenic contents, an occurrence which might further enhance the immune response to the foreign colonization or virulence antigen. We describe below strains of bacteria, recombinant vectors and the methods to make use of this system in a diversity of situations for development of live recombinant avirulent vaccines as well as for other potential applications.

Immunology and Immunopathology of the Intestines: Recombinant Avirulent Salmonella Vaccine Strains with Stable Maintenance and High Level Expression of Cloned Genes in Vivo

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.

Antitumor mechanisms of attenuated Salmonella typhimurium containing the gene for human interleukin-2: A novel antitumor agent?☆☆☆

Currently, there is no long-term effective treatment for unresectable hepatic malignancies. Salmonella species 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 antitumor purposes, the thi 4550 attenuated strain of Salmonella typhimurium was used as a vector for IL-2. The gene for human IL-2 was cloned into plasmid pYA292 and inserted into the attenuated S typhimurium and renamed (thi 4550(pIL-2)]. MCA-38 murine adenocarcinoma cells were injected intrasplenically into C57BL/6 mice to produce hepatic metastases that were subsequently enumerated after 12 days. We previously have demonstrated that the thi 4550(pIL-2) produces biologically active IL-2 and that a single gavage feeding of 10(7) thi 4550(pIL-2) significantly reduced the number of hepatic metastases when compared with animals fed salmonella lacking the IL-2 gene or nontreated controls. The aims of the current studies were to determine which host effector cell populations were responsible for the antitumor effect seen with thi 4550(pIL-2) by depletion of natural killer (NK), cytotoxic T lymphocytes (CD8+), T helper (CD4+) cells, and Kupffer cells. Multiple experiments were conducted for each host effector cell population depleted. We found a consistent reduction in the mean number of hepatic metastases in animals fed thi 4550(pIL-2) (55.6 metastases; n = 54) when compared with controls (162.3 metastases; n = 53) (P < .0001). Depletion of NK cells and CD8+ T cells significantly inhibited the antitumor effect of thi 4550(pIL-2) (analysis of variance [ANOVA], P < .01). Elimination of CD4+ T cells and Kupffer cells had no significant impact on the antitumor effect of thi 4550(pIL-2) (ANOVA, P value was not significant). Salmonella IL-2 may represent a novel form of in vivo biotherapy for unresectable hepatic malignancies that employs the oral route of administration. Furthermore, both NK cells or CD8+ cells are required for the antitumor effect seen while CD4+ T cells and Kupffer cells do not appear to be as essential.