Validation of a novel vaccine delivery system for Chlamydia trachomatis using recombinant gas vesicles derived from Halobacterium salinarium

Chlamydia trachomatis is the leading cause of bacterial sexually transmitted disease worldwide and while antibiotic treatment is effective in eliminating the pathogen, up to 70% of all infections are asymptomatic. Despite sustained efforts over the past 2 decades, an effective chlamydial vaccine remains elusive, due in large part to the lack of an effective delivery system. We explored the use of gas vesicles derived from Halobacterium salinarium as a potential display and delivery vehicle for chlamydial antigens of vaccine interest. Various size gene fragments coding for the major outer membrane protein (MOMP), outer membrane complex B (OmcB), polymorphic outer membrane protein D (PompD) and polymorphic outer membrane protein B (Pomp B) were integrated into and expressed as part of the gas vesicle protein C (gvpC) on the surface of these stable structures. The gas vesicle purifications are free of any viable Halobacterium cells and constitute a pure preparation of gas vesicles. The presence of the recombinant proteins was confirmed by Western blots probed using anti-gvpC and anti-Chlamydia antibodies as well as sera from Chlamydia trachomatis and Chlamydia pneumoniae-positive patients. Tissue culture evaluation revealed stability and a time-dependent degradation of recombinant gas vesicles (r-Gv) in human and animal cell lines. In vitro assessment using human foreskin fibroblasts (HFF) confirmed Toll-like receptor (TLR) 4 and 5 engagement by wild type and r-Gv, leading to MyD88 activation, TNF-&agr;, IL-6 and IL-12 production. Successful construction of a polymorphic outer membrane protein E/F (Pomp E/F) fusion linked to a chloramphenicol resistance cassette was successful displayed on the surface of gas vesicles. The Pomp E/F fusion was detected by anti- Chlamydia antibodies and resulted in a shift in the GvpC band detected via Western blot. The data suggest that r-GV could be an effective, naturally adjuvanting, time-release antigen delivery system for immunologically relevant Chlamydia vaccine antigens, which are readily recognized by human immune sera.