L-selectin and P-selectin are novel biomarkers of cervicovaginal inflammation for preclinical mucosal safety assessment of anti-HIV-1 microbicide.

A major obstacle thwarting preclinical development of microbicides is the lack of a validated biomarker of cervicovaginal inflammation. Therefore, the present study aims to identify novel noninvasive soluble markers in a murine model for assessment of microbicide mucosal safety. By performing cytokine antibody array analysis, we identified two adhesion molecules, L-selectin and P-selectin, which significantly increased when mucosal inflammation was triggered by nonoxynol-9 (N9), an anti-HIV-1 microbicide candidate that failed clinical trials, in a refined murine model of agent-induced cervicovaginal inflammation. We found that patterns of detection of L-selectin and P-selectin were obviously different from those of the two previously defined biomarkers of cervicovaginal inflammation, monocyte chemotactic protein 1 (MCP-1) and interleukin 6 (IL-6). The levels of these two soluble selectins correlated better than those of MCP-1 and IL-6 with the duration and severity of mucosal inflammation triggered by N9 and two approved proinflammatory compounds, benzalkonium chloride (BZK) and sodium dodecyl sulfate (SDS), but not by two nonproinflammatory compounds, carboxymethyl celluose (CMC; microbicide excipients) and tenofovir (TFV; microbicide candidate). These data indicated that L-selectin and P-selectin can serve as additional novel cervicovaginal inflammation biomarkers for preclinical mucosal safety evaluation of candidate microbicides for the prevention of infection with HIV and other sexually transmitted pathogens.

Second-generation Flagellin-rPAc Fusion Protein, KFD2-rPAc, Shows High Protective Efficacy against Dental Caries with Low Potential Side Effects.

Dental caries is one of the most common global chronic diseases affecting all ages of the population; thus a vaccine against caries is urgently needed. Our previous studies demonstrated that a fusion protein, KF-rPAc, in which rPAc of S. mutans is directly fused to the C-terminal of E. coli-derived flagellin (KF), could confer high prophylactic and therapeutic efficiency against caries. However, possible side effects, including the high antigenicity of flagellin and possible inflammatory injury induced by flagellin, may restrict its clinical usage. Here, we produced a second-generation flagellin-rPAc fusion protein, KFD2-rPAc, by replacing the main antigenicity region domains D2 and D3 of KF with rPAc. Compared with KF-rPAc, KFD2-rPAc has lower TLR5 agonist efficacy and induces fewer systemic inflammatory responses in mice. After intranasal immunization, KFD2-rPAc induces significantly lower flagellin-specific antibody responses but a comparable level of rPAc-specific antibody responses in mice. More importantly, in rat challenge models, KFD2-rPAc induces a robust rPAc-specific IgA response, and confers efficient prophylactic and therapeutic efficiency against caries as does KF-rPAc, while the flagellin-specific antibody responses are highly reduced. In conclusion, low side effects and high protective efficiency against caries makes the second-generation flagellin-rPAc fusion protein, KFD2-rPAc, a promising vaccine candidate against caries.

Flagellin-rPAc vaccine inhibits biofilm formation but not proliferation of S. mutans.

As the main etiologic bacterium of dental caries, Streptococcus mutans (S. mutans) has been considered as the primary object of vaccine research. We previously constructed a recombinant flagellin-rPAc fusion protein (KF-rPAc) that consists of an alanine-rich region to proline-rich region fragment of PAc (rPAc) from S. mutans and flagellin KF from E.coli K12 strain. Intranasal (i.n) immunization of KF-rPAc could induce high level of rPAc-specific antibody responses and offer robust protection against dental caries. In caries development, biofilm formation was considered as the necessary process involved. As PAc possesses other activities besides affecting adherence of S. mutans to salivary glycoproteins, we wondered whether rPAc-specific antibody responses induced by KF-rPAc could inhibit biofilm formation. Hence, in the present study, a simple and convenient in vitro biofilm model of S. mutans was constructed without saliva pre-coated. Both serum and saliva from KF-rPAc immunized rats significantly inhibited biofilm formation. Moreover, with the presence of serum or saliva, the biofilm formation is negatively correlated with the level of rPAc-specific antibody, and positively correlated with caries scores in rat. Moreover, in immunized mice, the level of rPAc-specific antibody also negatively correlated with the biofilm formation. Unlike ampicillin, serum of KF-rPAc immunized mice only inhibited biofilm formation but not proliferation. All together, we discovered that besides the well known blocking adherence of S. mutans to salivary glycoproteins by rPAc-specific antibody, flagellin-rPAc vaccine could also protects tooth from caries by inhibiting biofilm structure formation in between bacteria.