Akinobu Kajikawa

Assessment of Lactobacillus gasseri as a candidate oral vaccine vector

ABSTRACT 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.

Dissimilar Properties of Two Recombinant Lactobacillus acidophilus Strains Displaying Salmonella FliC with Different Anchoring Motifs

ABSTRACT 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.

Innate and acquired immune responses induced by recombinant Lactobacillus casei displaying flagellin-fusion antigen on the cell-surface

Bacterial flagellins are known as antigens that induce innate immune responses through TLR5 and boost immune responses in combination with other antigens. The aim of the present study was to determine the immunological properties of recombinant Lactobacillus casei producing flagellin and flagellin-fusion antigens in vitro and in vivo. Recombinant lactobacilli expressing Salmonella FliC and FliC fused to truncated SipC on the cell-surface were constructed. Fusion and non-fusion flagellin associated with L. casei retained the ability to induce IL-8 production by Caco-2 cells. Immunization of mice with these recombinant strains induced antigen-specific antibodies and cytokine production. The results showed that the outside epitope of the heterologous antigen was recognized more easily by the immune system than the inside epitope. The immune responses elicited by the Lactobacillus-associated antigens were mainly Th1 while that by the soluble antigen was Th2, although some of the responses were mixed.

Adjuvant Effects for Oral Immunization Provided by Recombinant Lactobacillus casei Secreting Biologically Active Murine Interleukin-1β

ABSTRACT Vaccine delivery systems using lactic acid bacteria are under development, but their efficiency is insufficient. Autologous cytokines, such as interleukin-1β (IL-1β), are potential adjuvants for mucosal vaccines and can be provided by recombinant lactic acid bacteria. The aim of this study was the construction and evaluation of recombinant Lactobacillus casei producing IL-1β as an adjuvant delivery agent. The recombinant strain was constructed using an expression/secretion vector plasmid, including a mature IL-1β gene from mouse. The biological activity of the cytokine was confirmed by IL-8 production from Caco-2 cells. In response to the recombinant L. casei secreting IL-1β, expression of IL-6 was detected in vivo using a ligated-intestinal-loop assay. The release of IL-6 from Peyers patch cells was also detected in vitro. Intragastric immunization with heat-killed Salmonella enterica serovar Enteritidis (SE) in combination with IL-1β-secreting lactobacilli resulted in relatively high SE-specific antibody production. In this study, it was demonstrated that recombinant L. casei secreting bioactive murine IL-1β provided adjuvant effects for intragastric immunization.

Mucosal Immunogenicity of Genetically Modified Lactobacillus acidophilus Expressing an HIV-1 Epitope within the Surface Layer Protein

Surface layer proteins of probiotic lactobacilli are theoretically efficient epitope-displaying scaffolds for oral vaccine delivery due to their high expression levels and surface localization. In this study, we constructed genetically modified Lactobacillus acidophilus strains expressing the membrane proximal external region (MPER) from human immunodeficiency virus type 1 (HIV-1) within the context of the major S-layer protein, SlpA. Intragastric immunization of mice with the recombinants induced MPER-specific and S-layer protein-specific antibodies in serum and mucosal secretions. Moreover, analysis of systemic SlpA-specific cytokines revealed that the responses appeared to be Th1 and Th17 dominant. These findings demonstrated the potential use of the Lactobacillus S-layer protein for development of oral vaccines targeting specific peptides.

Establishment and Evaluation of an in vitro M Cell Model using C2BBe1 Cells and Raji Cells

In vitro M cell models, consisting of co-cultures of Caco-2 cells and lymphoid cells, were developed and examined to observe bacterial transport. However, under our experimental conditions, the differentiation of Caco-2 cells into M cell-like cells could not be induced efficiently. To obtain a functionally stable M cell model based on human cells, C2BBe1 cells were screened and co-cultured with human Raji cells. In our co-cultures, increased sialyl Lewis A antigen expression and decreased Ulex europeaus agglutinin 1 binding were observed. Regarding the functional properties of the model, microsphere and lactic acid bacteria transport across the C2BBe1 co-cultures were increased compared with the levels seen in monocultures. The C2BBe1 monolayers that were co-cultured with Raji cells exhibited some M cell features; therefore, we consider our M cell model to be useful for investigating the interactions of bacteria with M cells.

Characterization of flagellins isolated from a highly motile strain of Lactobacillus agilis

BackgroundMost lactic acid bacteria are non-motile but some of them are flagellated and exhibit motility. So far, motile lactobacilli have rarely been studied, and characteristics of their flagellins are poorly understood. In this study, a highly motile strain of Lactobacillus agilis was recruited for transcriptional analysis and characterization of its flagellins.ResultsUnlike another motile lactic acid bacteria of intestinal isolate, Lactobacillus ruminis, flagellar filaments of the L. agilis strain probably consist of two homologous but distinct flagellins. Glycosylation of the flagellar filaments and their resistance to heat, acid and SDS were also observed. The immunological activity of the flagellins was evaluated through the stimulation of Caco-2 cells. The results show that TLR5-stimulating activity of the protein is attenuated, likely due to an incomplete TLR5-recognition site.ConclusionsThe flagella filaments of L. agilis BKN88 consist of two homologous glycosylated flagellins, which likely have an incomplete TLR5-recognition site. The characteristics of the flagellin are presumably a consequence of adaptation as a commensal microbe in the gastrointestinal tract.

Development of Recombinant Vaccines in Lactobacilli for Elimination of Salmonella

Many Lactobacillus and Lactococcus strains are generally regarded as safe for consumption because they are utilized for food fermentation or inhabit the intestinal mucosa as commensals. Recently, vaccine delivery systems using lactic acid bacteria (LAB) have been under development. Our research group has been investigating the development of oral mucosal vaccines against Salmonella enterica serovar Enteritidis (SE) using Lactobacillus casei IGM393 as an antigen delivery vehicle. Recombinant lactobacilli expressing SE antigens, FliC, SipC, and OmpC, have been constructed and orally administered to mice. Antigen specific immune responses and protective immunity were elicited after the immunization. For adjuvant-delivery, IL-1β-secreting L. casei was also engineered and its effects evaluated in vitro and in vivo. This article reviews a novel approach to the elimination of Salmonella via the development of a vaccine in lactobacilli.

Introduction of bifunctional alcohol/acetaldehyde dehydrogenase gene (adhE) in Fructobacillus fructosus settled its fructophilic characteristics

Fructophilic lactic acid bacteria (FLAB) are unique in the sense that they prefer D-fructose over D-glucose as main carbon source. If D-glucose is metabolised, electron acceptors are required and significant levels of acetate are produced. These bacteria are found in environments rich in D-fructose, such as flowers, fruits and the gastrointestinal tract of insects feeding on fructose-rich diets. Fructobacillus spp. are representatives of this unique group, and their fructophilic characteristics are well conserved. In this study, the bifunctional alcohol/acetaldehyde dehydrogenase gene (adhE) from Leuconostoc mesenteroides NRIC 1541T was cloned into a plasmid and transferred to Fructobacillus fructosus NRIC 1058T. Differences in biochemical characteristics between the parental strain (NRIC 1058T) and the transformants were compared. Strain 1-11, transformed with the adhE gene, did not show any fructophilic characteristics, and the strain grew well on D-glucose without external electron acceptors. Accumulation of acetic acid, which was originally seen in the parental strain, was replaced with ethanol in the transformed strain. Furthermore, in silico analyses revealed that strain NRIC 1058T lacked the sugar transporters/permeases and enzymes required for conversion of metabolic intermediates. This may be the reason for poor carbohydrate metabolic properties recorded for FLAB.

Nod2 is required for antigen-specific humoral responses against antigens orally delivered using a recombinant Lactobacillus vaccine platform

Safe and efficacious orally-delivered mucosal vaccine platforms are desperately needed to combat the plethora of mucosally transmitted pathogens. Lactobacillus spp. have emerged as attractive candidates to meet this need and are known to activate the host innate immune response in a species- and strain-specific manner. For selected bacterial isolates and mutants, we investigated the role of key innate immune pathways required for induction of innate and subsequent adaptive immune responses. Co-culture of murine macrophages with L. gasseri (strain NCK1785), L. acidophilus (strain NCFM), or NCFM-derived mutants—NCK2025 and NCK2031—elicited an M2b-like phenotype associated with TH2 skewing and immune regulatory function. For NCFM, this M2b phenotype was dependent on expression of lipoteichoic acid and S layer proteins. Through the use of macrophage genetic knockouts, we identified Toll-like receptor 2 (TLR2), the cytosolic nucleotide-binding oligomerization domain containing 2 (NOD2) receptor, and the inflammasome-associated caspase-1 as contributors to macrophage activation, with NOD2 cooperating with caspase-1 to induce inflammasome derived interleukin (IL)-1β in a pyroptosis-independent fashion. Finally, utilizing an NCFM-based mucosal vaccine platform with surface expression of human immunodeficiency virus type 1 (HIV-1) Gag or membrane proximal external region (MPER), we demonstrated that NOD2 signaling is required for antigen-specific mucosal and systemic humoral responses. We show that lactobacilli differentially utilize innate immune pathways and highlight NOD2 as a key mediator of macrophage function and antigen-specific humoral responses to a Lactobacillus acidophilus mucosal vaccine platform.