Cloning of Th1 and Th2-Inducing Antigens of Porphyromonas gingivalis

Objective: Porphyromonas gingivalis (Pg) is a consensus pathogen that induces periodontal disease in humans and animal models. Recent evidence suggests that host Th1 pro-inflammatory responses may exacerbate, and Th2 anti-inflammatory responses ameliorate, periodontal destruction. T cell expression cloning strategies have proven successful in identifying microbial antigens that preferentially induce Th1 responses, and that are useful in studies of immunopathogenesis and vaccine development. In the present investigation, we used this approach to identify Pg antigens that may serve as vaccine candidates. Methods: A Pg expression library with 7-8X genome coverage was constructed in E. coli. A total of 500 pools containing 40-50 clones each were ‘fed’ to antigen-presenting cells from IL-10-deficient C57Bl/6 mice, and were used to stimulate Pg-specific T cell lines derived from syngeneic splenocytes from mice previously immunized with viable Pg. T cell proliferation was assessed by 3H-thymidine uptake, and supernatants were assayed for IFNg and IL-10 production by ELISA. Pools of clones exhibiting a positive response were broken down and individual clones were re-assayed as above. Results: In the first round of screening, 9/500 pools stimulated T cells to proliferate and to produce IFNg (Th1 clones). An additional pool induced T cell proliferation but no IFNg production (putative Th2 clone). No IL-10 was detected in any pool. The pool that induced the strongest IFNg response and the pool that only induced proliferation were broken down to a single clone per well and re-screened. One Th1 and one Th2 clone were isolated from the original pools. Sub-cloning and characterization of the corresponding Pg genes are in progress. Conclusion: T cell expression cloning is an efficient approach for the discovery of Pg protein antigens that are involved in Th1 vs. Th2 specification.

This work supported by grant DE-13747 from the N.I.D.C.R.