Lactobacillus species 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. gasseri as 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 Lactobacillus being 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. gasseri primarily activates TLR2/6, with additional activation through the TLR2 homodimer. To expand the Toll-like receptor (TLR) activation profile of L. gasseri and 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 vivo studies established that treatment with L. gasseri led to a diversification of B-cell populations in the lamina propria of the murine colon. Furthermore, treatment with genetically modified L. gasseri led to a significant decrease in the percentage of FoxP3 ؉ colonic lymphocytes. Taken together, these data clarify the interaction of L. gasseri with the host immune system and support further investigation of the in vivo immunogenicity of L. gasseri expressing both flagellin and candidate vaccine antigens.Several species of lactobacilli have been investigated as antigen delivery vehicles against a spectrum of infectious agents, including Helicobacter pylori, tetanus, enterotoxigenic Escherichia coli, severe acute respiratory syndrome (SARS)-associated coronavirus, rotavirus, Brucella abortis, and human papillomavirus (reviewed in reference 34). Despite the growing evidence that lactobacilli are useful as vaccine vectors (21), it is increasingly clear that different species and even subspecies of lactobacilli interact distinctly with the host immune system (1, 32). Several studies have analyzed the abilities of various Lactobacillus species to induce dendritic cell (DC) maturation and cytokine production, showing a range of outcomes that appear to favor tolerance on one end of the spectrum and immune activation on the other (7, 22). There also are clear differences in the ability of lactobacilli species to survive the hostile environment of the upper gastrointestinal (GI) tract and colonize the lower GI tract. Functional genomic studies have greatly improved our understanding of some of these characteristics, but much less is known about the molecular and genetic basis of host immunomodulation by Lactobacillus species. One strategy to develop lactobacilli as va...

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