Graduation Year

2005

Document Type

Dissertation

Degree

Ph.D.

Degree Granting Department

Biology

Major Professor

My Lien Dao, Ph.D.

Committee Member

Daniel V. Lim, Ph.D.

Committee Member

Kenneth E. Ugen, Ph.D.

Committee Member

Valerie J. Harwood, Ph.D.

Keywords

Mutans streptococcus, Prime-boost, Saliva, Secretory IgA, WapA

Abstract

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 wallassociated 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 antiAgA 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 pcDNAwapA 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.

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