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ABSTRACTOral vaccines that elicit a mucosal immune response may be effective against human immunodeficiency virus type 1 (HIV-1) because its transmission occurs mainly at the mucosa. The aim of this study was to construct recombinant Lactobacillusfor oral delivery of oral vaccines against HIV-1 and to evaluate their immunogenicity. A recombinant Lactobacillus acidophilusstrain expressing the HIV-1 Gag on the bacterial cell surface was established by fusion with the signal peptide and anchor motif of a mucus binding protein (Mub) from L. acidophiluswith or without coexpression of Salmonella entericaserovar Typhimurium flagellin (FliC) fused to a different Mub signal peptide and anchor. Using HEK293 cells engineered to express Toll-like receptor 5 (TLR5), the biological activity of FliC on the bacterial cell surfaces was determined. The surface-exposed flagellin retained its TLR5-stimulating activity, suggesting that the recombinant strain with Gag and FliC dual display might provide a different immunopotency than the strain expressing only Gag. The immunological properties of the recombinant strains were assessed by coculture with human myeloid dendritic cells (DCs). The heterologous antigens on the cell surface affected maturation and cytokine responses of DCs. Acquired immune responses were also investigated by intragastric immunization of mice. The enzyme-linked immunosorbent spot assay showed induction of gamma interferon-producing cells at local mucosa after immunization of mice with the Gag-producing strain. Meanwhile, the immunization with L. acidophilusdisplaying both Gag and FliC resulted in an increase of Gag-specific IgA-secreting cells. These results suggested that the Gag-displaying L. acidophiluselicited specific immune responses and the coexistence of FliC conferred an adjuvant effect on local IgA production.

ABSTRACTLactobacillusspecies are commensal bacteria that have long been recognized as probiotic microbes and are generally regarded as safe (GRAS) for human consumption. We have investigated the use of L. gasserias a vaccine vector for oral immunization against mucosal pathogens. Recent research has shown that the immune response to different lactobacilli can vary widely depending on the species or subspecies of Lactobacillusbeing studied. While some lactobacilli seem to induce oral tolerance, others induce an adaptive immune response. This study characterized the systemic and mucosal immune response to wild-type and genetically modified L. gasseri. L. gasseriprimarily activates TLR2/6, with additional activation through the TLR2 homodimer. To expand the Toll-like receptor (TLR) activation profile of L. gasseriand the immunogenicity of the vector, a plasmid containing fliC, the gene encoding bacterial flagellin, was introduced which resulted in the strong activation of TLR5. The treatment of human myeloid dendritic cells with recombinant lactobacilli expressing flagellin triggered phenotypic maturation and the release of proinflammatory cytokines. In contrast, bacterial treatment also resulted in a statistically significant increase in IL-10 production. In vivostudies established that treatment with L. gasseriled to a diversification of B-cell populations in the lamina propria of the murine colon. Furthermore, treatment with genetically modified L. gasseriled to a significant decrease in the percentage of FoxP3+colonic lymphocytes. Taken together, these data clarify the interaction of L. gasseriwith the host immune system and support further investigation of the in vivoimmunogenicity of L. gasseriexpressing both flagellin and candidate vaccine antigens.