Protective immunity against Streptococcus mutans infection: Studies on nirB promoter controlled expression of cloned S. mutans adhesin in an attenuated Salmonella strain and its immunoenhancing role on host immunity

Attenuated Salmonella enterica serovar Typhimurium has been used for targeted delivery of recombinant antigens to the gut-associated lymphoid tissues. One potential problem associated with this vaccine approach is the likelihood of in vivo instability of the plasmid constructs caused by constitutive hyperexpression of the heterologous immunogen. The aim of this study was to generate and characterize an expression system encoding the saliva-binding region (SBR) of Streptococcus mutans AgI/II adhesin, either alone or linked with the mucosal adjuvant cholera toxin A2/B subunits (CTA2/B), under the control of the inducible nirB promoter. This promoter is activated in an anaerobic environment and within macrophages, which are the primary antigen-presenting cells involved in phagocytosis and processing of Salmonella. The gene encoding the chimeric SBR-CTA2/B was subcloned into a plasmid, which contained the nirB promoter. The resulting plasmid, pSBR-CTA2/BnirB, was introduced into serovar Typhimurium by electroporation. A similar procedure was followed to clone the gene encoding SBR for expression in serovar Typhimurium under nirB control. Subsequent protein analyses confirmed that these clones expressed SBR or SBR-CTA2/B only under anaerobic growth conditions. To determine the ability of these clones to colonize host tissues, the vaccine strains were administered either by the intragastric (i.g.) or intranasal (i.n.) route to mice. High numbers of the recombinant strains persisted in Peyer's patches (PP), spleen, and nasal tissues for at least 21 days following challenge. This study provides quantitative evidence for the colonization of Salmonella strains expressing a recombinant protein under the control of the inducible nirB promoter in PP or nasal tissues following a single i.g. or i.n. administration of the bacteria, respectively. To evaluate the effectiveness of these vaccine strains in inducing a protective immune response against S. mutans infection, BALB/c mice were immunized by either the i.n. or i.g. route with a single initial immunization and a booster immunization at week 17 after the initial immunization. Anti-SBR antibodies were detected by enzyme-linked immunosorbent assay (ELISA) in serum and saliva of the experimental animals by week 3 after immunization. The serum immunoglobulin G (IgG) subclass profiles were indicative of T helper type 1 responses against both the vector and SBR antigen. To determine the effectiveness of these responses on protection against S. mutans infection, mice were challenged after the second immunization with a virulent strain of S. mutans. Mice immunized with Salmonella clones expressing SBR or SBR-CTA2/B demonstrated a significant reduction in the number of S. mutans present in plaque compared to the control groups. These results provide evidence for the effectiveness of the Salmonella vector expressing the SBR antigen in inducing mucosal and systemic immune responses to SBR. Furthermore, the induction of a salivary anti-SBR response corresponded with protection against S. mutans colonization of tooth surfaces.