| Streptococcus mutans is the main causative agent of dental caries, which is a widespread infectious disease. A number of surface molecules are involved in the pathogenicity of this organism, including adherence and aggregation factors. The wall-associated protein A (WapA) of Streptococcus mutans GS-5 was previously demonstrated to be a sucrose-dependent adherence and aggregation factor, and is a larger precursor to extracellular antigen A (AgA), a candidate antigen for a dental caries vaccine.; The full-length wapA gene and a C-terminal truncated version agA encoding the AgA were cloned into the mammalian expression vector pcDNA 3.1/V5/His-TOPO. The above constructs were mixed with a cationic lipid and used to transfect Chinese hamster ovary (CHO) cells. Transient expression of the wapA and agA genes was observed at 24 h post-transfection, as shown by Western immunoblot analysis. In CHO, cells WapA containing the membrane and wall-spanning region was found in apoptotic bodies, whereas the soluble AgA, which lacked the hydrophobic region, was found in extracellular medium.; A higher salivary IgA level was observed in mice immunized with the pcDNA- wapA vaccine as compared to those immunized with the pcDNA- agA vaccine. Furthermore, the anti-WapA antibody inhibited S. mutans sucrose-dependent adherence, suggesting potential protection of the tooth against S. mutans colonization, while anti-AgA had no significant effect. Indeed, prediction and analysis of protein epitopes showed that WapA contains highly promiscuous MHC-II binding motifs that are absent from AgA. Immunodot assay confirmed that WapA bound biotin-labeled dextran, whereas AgA did not. These data indicated that full-length wall-associated WapA is a better candidate vaccine antigen than the soluble AgA.; In co-immunization studies pcDNA-ctb was preferable to pcDNA-il-5 as genetic adjuvant. A comparable secondary response was obtained by priming with either pcDNA-wapA or WapA followed by a WapA boost, thus demonstrating the pcDNA-wapA as a valid contender primary vaccine. The successful utilization of the caries DNA-based vaccine protocol would represent a highly significant new approach to this important worldwide health problem. |
