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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.

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.