Display of heterologous antigens on the cell surface is considered a useful technique for vaccine delivery by recombinant lactobacilli. In this study, two recombinant Lactobacillus acidophilus derivatives displaying Salmonella flagellin (FliC) were constructed using different anchor motifs. In one instance, the FliC protein was fused to the C-terminal region of a cell envelope proteinase (PrtP) and was bound to the cell wall by electrostatic bonds. In the other case, the same antigen was conjugated to the anchor region of mucus binding protein (Mub) and was covalently associated with the cell wall by an LPXTG motif. These two recombinant L. acidophilus cell surface displays resulted in dissimilar maturation and cytokine production by human myeloid dendritic cells. The surface-associated antigen was highly sensitive to simulated gastric and small intestinal juices. By supplementation with bicarbonate buffer and soybean trypsin inhibitor, the cell surface antigen was protected from proteolytic enzymes during gastric challenge in vitro. The protective reagents also increased the viability of the L. acidophilus cells upon challenge with simulated digestive juices. These results demonstrate the importance of protecting cells and their surface-associated antigens during oral immunization.

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...

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 recombinantLactobacillusfor oral delivery of oral vaccines against HIV-1 and to evaluate their immunogenicity. A recombinantLactobacillus 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) fromL. acidophiluswith or without coexpression ofSalmonella 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 withL. acidophilusdisplaying both Gag and FliC resulted in an increase of Gag-specific IgA-secreting cells. These results suggested that the Gag-displayingL. acidophiluselicited specific immune responses and the coexistence of FliC conferred an adjuvant effect on local IgA production.

A recombinant Lactobacillus casei expressing a flagellar antigen from Salmonella enterica serovar Enteritidis was constructed and evaluated as a mucosal vaccine. Intragastric immunization of the recombinant strain conferred protective immunity against Salmonella infection in mice. This immunization did not result in antigen-specific antibody in either feces or sera but induced the release of IFN-gamma on restimulation of primed lymphocytes ex vivo. The results suggested that the protective efficacy provided by flagellin-expressing L. casei is mainly attributable to cell-mediated immune responses. In addition, an adjuvant-type effect of the antigen delivery system with L. casei was also observed.