A highly immunogenic vaccine platform against encapsulated pathogens using chimeric probiotic Escherichia coli membrane vesicles.

Vaccines against infectious diseases should elicit potent and long-lasting immunity, ideally even in those with age-related decline in immune response. Here we report a rational polysaccharide vaccine platform using probiotic Escherichia coli-derived membrane vesicles (MVs). First, we constructed a probiotic E. coli clone harboring the genetic locus responsible for biogenesis of serotype 14 pneumococcal capsular polysaccharides (CPS14) as a model antigen. CPS14 was found to be polymerized and mainly localized on the outer membrane of the E. coli cells. The glycine-induced MVs displayed the exogenous CPS14 at high density on the outermost surface, on which the CPS14 moiety was covalently tethered to a lipid A-core oligosaccharide anchor. In in vivo immunization experiments, CPS14+MVs, but not a mixture of free CPS14 and empty MVs, strongly elicited IgG class-switch recombination with a Th1/Th2-balanced IgG subclass distribution without any adjuvant. In addition, CPS14+MVs were structurally stable with heat treatment and immunization with the heat-treated MVs-elicited CPS14-specific antibody responses in mouse serum to levels comparable to those of non-treated CPS14+MVs. Notably, the immunogenicity of CPS14+MVs was significantly stronger than those of two currently licensed vaccines against pneumococci. The CPS14+MV-elicited humoral immune responses persisted for 1 year in both blood and lung. Furthermore, the CPS14+MV vaccine was widely efficacious in mice of different ages. Even in aged mice, vaccination resulted in robust production of CPS14-specific IgG that bound to the pneumococcal cell surface. Taken together, the present probiotic E. coli MVs-based vaccine platform offers a promising, generalizable solution against encapsulated pathogens.

Effects of pH on the Properties of Membrane Vesicles Including Glucosyltransferase in Streptococcus mutans.

Streptococcus mutans releases membrane vesicles (MVs) and induces MV-dependent biofilm formation. Glucosyltransferases (Gtfs) are bound to MVs and contribute to the adhesion and glucans-dependent biofilm formation of early adherent bacteria on the tooth surface. The biofilm formation of S. mutans may be controlled depending on whether the initial pH tends to be acidic or alkaline. In this study, the characteristics and effects of MVs extracted from various conditions {(initial pH 6.0 and 8.0 media prepared with lactic acid (LA) and acetic acid (AA), and with NaOH (NO), respectively)} on the biofilm formation of S. mutans and early adherent bacteria were investigated. The quantitative changes in glucans between primary pH 6.0 and 8.0 conditions were observed, associated with different activities affecting MV-dependent biofilm formation. The decreased amount of Gtfs on MVs under the initial pH 6.0 conditions strongly guided low levels of MV-dependent biofilm formation. However, in the initial pH 6.0 and 8.0 solutions prepared with AA and NO, the MVs in the biofilm appeared to be formed by the expression of glucans and/or extracellular DNA. These results suggest that the environmental pH conditions established by acid and alkaline factors determine the differences in the local pathogenic activities of biofilm development in the oral cavity.

Roles of membrane vesicles from Streptococcus mutans for the induction of antibodies to glucosyltransferase in mucosal immunity.

Glucosyltransferase (Gtf) B and GtfC from Streptococcus mutans are key enzymes for the development of biofilm-associated diseases such as dental caries. Gtfs are involved in membrane vesicles (MVs) and function in the formation of biofilms by initial colonizers such as Streptococcus mitis and Streptococcus oralis on the tooth surface. Therefore, MVs may be important virulence factors and targets for the prevention of biofilm-associated disease. To clarify how GtfB encoded by gtfB and GtfC encoded by gtfC associate with MVs and whether MVs are effective as a mucosal immunogen to induce the production of antibodies against Gtfs, MVs from S. mutans UA159 wild-type (WT), gtfB-, gtfC- and gtfB-C- were extracted from culture supernatants by ultracentrifugation and observed by scanning electron microscopy. Compared with GtfB, GtfC was mainly contained in MVs and regulated the size and aggregation of MVs, and the biofilm formation of S. mutans. The intranasal immunization of BALB/c mice with MVs plus a TLR3 agonist, poly(I-C), was performed 2 or 3 times for 5 weeks, with an interval of 2 or 3 weeks. MVs from all strains caused anti-MV IgA and IgG antibody production. In quality analysis of these antibodies, the IgA and IgG antibodies produced by immunization with MVs from WT and gtfB- strains reacted with Gtfs in the saliva, nasal wash and serum but those produced by immunization with MVs from gtfC- and gtfB-C- strains did not. S. mutans MVs mainly formed by GtfC are an intriguing immunogen for the production of anti-Gtf antibodies in mucosal immunogenicity.

Effects of Complex DNA and MVs with GTF Extracted from Streptococcus mutans on the Oral Biofilm.

Streptococcus mutans is one of the principal pathogens for the development of dental caries. Oral biofilms formed by S. mutans are constructed of insoluble glucan formation induced by the principal enzymes, GTF-I and GTF-SI, in sucrose-containing conditions. However, as another means of biofilm formation, extracellular DNA (eDNA) and membrane vesicles (MVs) are also contributors. To explore the roles of eDNA and MVs for biofilm formation, short and whole size pure DNAs, two types of sub-purified DNAs and MVs were extracted from S. mutans by beads destruction, treatment of proteinase K, and ultracentrifugation of culture supernatant, and applied into the biofilm formation assay using the S. mutans UA159 gtfBC mutant, which lost GTF-I and GTF-SI, on a human saliva-coated 96 well microtiter plate in sucrose-containing conditions. Sub-purified DNAs after cell lysis by beads destruction for total 90 and 180 s showed a complex form of short-size DNA with various proteins and MVs associated with GTF-I and GTF-SI, and induced significantly higher biofilm formation of the S. mutans UA159.gtfBC mutant than no sample (p < 0.05). Short-size pure DNA without proteins induced biofilm formation but whole-size pure DNA did not. Moreover, the complex form of MV associated with GTFs and short-size DNA showed significantly higher biofilm formation of initial colonizers on the human tooth surface such as Streptococcus mitis than no sample (p < 0.05). The short-size DNAs associated with MVs and GTFs are important contributors to the biofilm formation and may be one of additional targets for the prevention of oral biofilm-associated diseases.