The Combinations Chitosan-Pam3CSK4 and Chitosan-Monophosphoryl Lipid A: Promising Immune-Enhancing Adjuvants for Anticaries Vaccine PAc.

We previously demonstrated that recombinant protein PAc could be administered as an anticaries vaccine. However, the relatively weak immunogenicity of PAc limits its application. In the present study, we investigated the effect of two adjuvant combinations of chitosan plus Pam3CSK4 (chitosan-Pam3CSK4) and of chitosan plus monophosphoryl lipid A (chitosan-MPL) in the immune responses to the PAc protein in vivo and in vitro PAc-chitosan-Pam3CSK4 or PAc-chitosan-MPL promoted significantly higher PAc-specific antibody titers in serum and saliva, inhibited Streptococcus mutans colonization onto the tooth surfaces, and endowed better protection effect with significantly less caries activities than PAc alone. Chitosan-Pam3CSK4 and chitosan-MPL showed no statistically significant differences. In conclusion, our study demonstrated that the chitosan-Pam3CSK4 and chitosan-MPL combinations are promising for anticaries vaccine development.

Identification of FimH derivatives as adjuvant vaccinated with PAc that enhance protection against Streptcoccus mutans colonization.

FimH is the adhesin of type I fimbriae expressed on Escherichia coli that can mediate specific adherence to host cells. High binding mutations in FimH are related to the adaptive evolution of bacteria. However, additional roles that these allelic variations may play remain elusive. To investigate novel biological functions of the mutations in FimH, we introduced four different variants of FimH by incorporating single amino acid substitutions at specific sites, namely A25P, G73R, A106, and T158P, respectively. In this study, adjuvant potential of FimH variants was evaluated by investigating their ability to trigger innate immune response to DC2.4 and adaptive immunity to improve immunological characteristics. The data revealed that purified A106 and T158P up-regulated the expression of co-stimulatory molecules critically involved in DC2.4 activation by interaction with TLR4, whereas A25P and G73R did not induce the phenotypic maturation of DC2.4. Besides, the culture of DC2.4 with A106 and T158P enhanced the release of cytokines and protein phagocytosis. When formulated with PAc, T158P elicited more robust PAc-specific IgG and IgA antibody responses compared to PBS, PAc and PAc+K12 groups and inhibited bacteria colonization. Collectively, the results confirmed that the T158P mutation located around the inter-domain interface of the protein induced a specific enhancement effect on adjuvant characteristics.

Activity of the Chimeric Lysin ClyR against Common Gram-Positive Oral Microbes and Its Anticaries Efficacy in Rat Models.

Dental caries is a common disease caused by oral bacteria. Streptococcus mutans and Streptococcus sobrinus are the primary cariogenic microbes that often survive as biofilms on teeth. In this study, we evaluated the activity of ClyR, a well-known chimeric lysin with extended streptococcal host range, against common Gram-positive oral microbes and its anticaries efficacy in rat models. ClyR demonstrated high lytic activity against S. mutans MT8148 and S. sobrinus ATCC6715, with minor activity against Streptococcus sanguinis, Streptococcus oralis, and Streptococcus salivarius, which are considered as harmless commensal oral bacteria. Confocal laser scanning microscopy showed that the number of viable cells in 72-h aged S. mutans and S. sobrinus biofilms are significantly (p < 0.05) decreased after treatment with 50 µg/mL ClyR for 5 min. Furthermore, continuous administration of ClyR for 40 days (5 µg/day) significantly (p < 0.05) reduced the severity of caries in rat models infected with a single or a mixed bacteria of S. mutans and S. sobrinus. Therefore, ClyR could be a promising agent or additive for the prevention and treatment of dental caries.

Antibiofilm Activities of a Novel Chimeolysin against Streptococcus mutans under Physiological and Cariogenic Conditions.

Streptococcus mutans often survives as a biofilm on the tooth surface and contributes to the development of dental caries. We investigated the efficacy of ClyR, an engineered chimeolysin, against S. mutans biofilms under physiological and cariogenic conditions. Susceptibility tests showed that ClyR was active against all clinical S. mutans isolates tested as well as S. mutans biofilms that displayed resistance to penicillin. The S. mutans biofilms that formed on hydroxyapatite discs under physiological sugar conditions and cariogenic conditions were reduced ∼2 logs and 3 logs after treatment with 100 µg/ml ClyR, respectively. In comparison, only a 1-log reduction was observed in the chlorhexidine gluconate (ChX)-treated group, and no killing effect was observed in the NaF-treated group. A mouse dental colonization model showed that repeated use of ClyR for 3 weeks (5 µg/day) reduced the number of colonized S. mutans cells in the dental plaques significantly (P < 0.05) and had no harmful effects on the mice. Furthermore, toxicity was not noted at concentrations exceeding those used for the in vitro and in vivo studies, and ClyR-specific antibodies could not be detected in mouse saliva after repeated use of ClyR in the oral cavity. Our data collectively demonstrate that ClyR is active against S. mutans biofilms both in vitro and in vivo, thus representing a preventative or therapeutic agent for use against dental caries.