Induction of mosquitocidal activity in mice immunized with Anopheles gambiae midgut cDNA.

Vaccines that induce mosquito-killing (mosquitocidal) activity could substantially reduce the transmission of certain mosquito-borne diseases, especially vaccines against African malaria vectors, such as the mosquito Anopheles gambiae. To generate and characterize antimosquito immunity we immunized groups of mice with two individual A. gambiae midgut cDNAs, Ag-Aper1 (a secreted peritrophic matrix protein) and AgMuc1 (a midgut-bound mucin), and an A. gambiae midgut cDNA library from blood-fed mosquitoes. We observed significantly increased mortality among mosquitoes that fed on either the AgMuc1- or the cDNA library-immunized mice compared to that of controls, but no differences were observed among those fed on Ag-Aper1-immunized mice. Analysis of the humoral and cellular immune responses from mice showed that the induced mosquitocidal effect was associated with immune profiles characterized by elevated tumor necrosis factor alpha and gamma interferon cytokine levels and very low antibody titers. Furthermore, an additional immunization of cDNA library-immunized mice with midgut protein shifted immunity toward a Th2-type immune response, characterized by elevated antibody titers and high interleukin-5 and interleukin-10 cytokine levels; importantly, mosquitoes feeding on these mice exhibited no undue mortality. Finally, when immune sera was ingested by mosquitoes through a membrane feeder, no effect on mosquito mortality was observed, indicating that serum factors alone were not responsible for the mosquitocidal effect. Our results demonstrate that mosquitocidal immunity in mice can be consistently generated by midgut cDNA immunization and suggest this cDNA-induced mosquitocidal immunity is cell mediated.

Partial protection against experimental vaginal candidiasis after mucosal vaccination with heat-killed Candida albicans and the mucosal adjuvant LT(R192G).

The effectiveness of a mucosal vaccine composed of heat-killed Candida albicans (HK-CA) or C. albicans culture filtrate (CaCF) in conjunction with the mucosal adjuvant LT(R192G) against vulvovaginal candidiasis was examined in an estrogen-dependent murine model. Mice vaccinated intranasally with HK-CA + LT(R192G) exhibited a significant but short-lived protection accompanied by a vigorous delayed-type hypersensitivity response as well as high titers of circulating C. albicans-specific antibodies. Surprisingly, the levels of antigen-specific antibodies in the vaginal secretions of protected mice were negligible and no correlates of vaginal-associated Type 1 or Type 2 cytokines were observed. Vaginal priming with C. albicans before vaccination did not alter the protective outcome. Immunization with CaCF + LT(R192G) induced a discrete level of protection when administered intrarectally but not intranasally. These results suggest that mucosal vaccination can afford partial protection against vulvovaginal candidiasis, but the precise immune mechanisms responsible for protection are complex and as yet, not well understood.

Effectiveness of intranasal immunization with HIV-gp160 and an HIV-1 env CTL epitope peptide (E7) in combination with the mucosal adjuvant LT(R192G).

LT(R192G) is a novel mucosal adjuvant that induces protective immunity when co-administered with certain whole inactivated bacteria or viruses or with subunits of relevant virulence determinants from these pathogens. LT(R192G) stimulates antigen-specific humoral and cellular immune responses, both systemically and in mucosal compartments, and is safe and nontoxic at adjuvant effective doses. Intranasal (IN) immunization of mice with LT(R192G) in conjunction with oligomeric HIV-1 gp160 elevates antigen-specific systemic and mucosal IgG and IgA production and Th1- and Th2-type cytokine responses. Isotype characterization of induced IgG reveals that gp160 alone fails to stimulate IgG2a responses in the absence of adjuvant. Both IgG1 and IgG2a are induced by immunization in the presence of LT(R192G). Additionally, intranasal immunization with a 15-amino acid peptide corresponding to an HIV-1 Env CTL determinant and LT(R192G) induces systemic, peptide-specific CTL activity and Th1 and Th2 cytokine responses that are absent when the adjuvant is excluded from the immunizations. These studies show that LT(R192G) quantitatively and qualitatively enhances cellular and humoral HIV-specific immune responses and that this adjuvant may offer significant advantages toward vaccine development against HIV.

The role of cAMP in mucosal adjuvanticity of Escherichia coli heat-labile enterotoxin (LT).

Heat-labile enterotoxin (LT) produced by enterotoxigenic Escherichia coli (ETEC) and cholera toxin (CT) produced by Vibrio cholerae have been shown to function as potent mucosal adjuvants. A number of studies have examined the effects of different mutations at either the active site or the protease site of LT and CT and the influence of those mutations on toxicity and adjuvanticity. However, different observations reported by various groups using a variety of animal models with different antigens or different routes of immunization have provided contradictory findings and evoked many questions regarding the underlying mechanisms of mucosal adjuvanticity of LT and CT. In this study, the role of cAMP in mucosal adjuvanticity was examined by comparing three LT active site mutants (S61F, A69G, E112K), a protease site mutant (R192G) and recombinant LT-B for toxicity, cAMP activity and mucosal adjuvanticity using tetanus toxoid (TT) as a model antigen. While all mutants examined showed reduced toxicity, the effects of each mutation on its ability to function as an adjuvant varied. Following intranasal immunization, native LT as well as protease and active site mutants of LT induced serum anti-TT IgG and their responses were virtually indistinguishable from one another. In addition, LT-B was also able to enhance production of serum anti-TT IgG, though at a level significantly lower than that achieved by native LT and mutants. Following oral immunization, the best serum anti-TT IgG responses were obtained with native LT and mutants that retained the ability to induce accumulation of cAMP. Despite the nearly identical serum anti-TT IgG responses following intranasal immunization, there was a strong correlation between the ability to induce accumulation of cAMP in cultured Caco-2 cells and the ability to elicit production of antigen-specific Th1 or Th2 cytokines.

Effectiveness of a vaccine composed of heat-killed Candida albicans and a novel mucosal adjuvant, LT(R192G), against systemic candidiasis.

The incidence of fungal infections caused by the opportunistic yeast Candida albicans has increased significantly in recent years. The ability to vaccinate selected patients against the organism would be advantageous. In this paper we describe a potential anti-C. albicans vaccine consisting of heat-killed C. albicans (HK-CA) in combination with the novel mucosal adjuvant LT(R192G), a genetically detoxified form of the heat-labile toxin of enterotoxigenic Escherichia coli. Groups of male CBA/J mice were immunized intranasally on three occasions at weekly intervals with 2 x 10(7) HK-CA per dose, alone or in conjunction with 10 micrograms of LT(R192G) per dose. Two weeks following the last application of antigen, some animals were challenged intravenously (i.v.) with 10(4), 10(5), or 10(6) viable C. albicans to assess protection as measured by survival and/or culture. Some groups of animals were footpad tested with C. albicans mannan to assess delayed-type hypersensitivity (DTH), and all the animals were bled for antibody assays. In two independent studies, all the animals immunized with HK-CA plus LT(R192G) were able to eradicate 10(4) C. albicans completely, as determined by kidney culture 4 weeks after challenge. Animals immunized with HK-CA only had reduced levels of C. albicans compared to the adjuvant or saline-only control. Greatly enhanced survival was observed when mice immunized with HK-CA plus LT(R192G) were challenged with 10(5) live C. albicans as well. Animals immunized with HK-CA plus LT(R192G) developed a significant DH response, while those given HK-CA alone developed only marginal DH responses. High immunoglobulin G (IgG) levels to cytoplasmic antigens developed in mice immunized with HK-CA plus LT(R192G), but they were found only after i.v. challenge. Addition of adjuvant shifted the antibody isotype production in i.v.-challenged animals to a response dominated by IgG2a. Clearly, intranasal immunization with killed C. albicans in conjunction with LT(R192G) afforded significant levels of protection. This novel approach offers new possibilities for the development of an effective vaccine against candidiasis for use in humans.

New approaches to mucosal immunization.

Every year more than 17 million deaths worldwide are caused by infectious diseases. The great majority of these deaths occur in underdeveloped countries and are attributed to diseases preventable by existing vaccines, or diseases that could potentially be prevented with new vaccines. The fact that most human and veterinary pathogens establish infection in the host by initiating contact at a mucosal surface, provide the rationale for the development of mucosal vaccines. An increasing number of strategies have been proposed to facilitate mucosal immunization. Among the most widely investigated strategies are the use of attenuated microorganisms; the inclusion of immunizing antigens in lipid-based carriers, the genetic creation of transgenic plants and the use of mucosal adjuvants derived from bacterial toxins. This review provides a brief summary of the most recent advances in the field of mucosal immunization with an special emphasis on a promising genetically detoxified mucosal adjuvant, LT(R192G), derived from the heat-labile toxin of enterotoxigenic E. coli. We present evidence regarding the safety, immunogenicity, and efficacy of LT(R192G) for the development of a new generation of mucosal vaccines.