570. Genetic Vaccination Against Yersinia pestis Using Recombinant Adenovirus Vectors Expressing the V Antigen
Authors: Maria Chiuchiolo, Barry Shea, Julie L. Boyer, Neil R. Hackett, Ronald G. Crystal
Journal: Molecular Therapy (2004)

Aerosolization of Yersinia pestis in a bioterrorism event would cause the pneumonic form of plague, a virulent, rapidly fatal disease that develops within a few days of exposure. At present, no Y. pestis vaccines are available in the USA. In a bioterror-induced outbreak, an effective vaccine must have a rapid onset of immunity with a single dose to prevent secondary cases. The V antigen of Y. pestis is a component of the type III secretory system that injects virulence factors into host cells; it is known that protective immune responses are elicited against V antigen when administered as purified protein, but multiple immunizations are required for efficacy. In the context that replication-defective adenovirus (Ad)-based gene transfer vectors encoding pathogen-specific antigens can stimulate strong immune responses without a requirement for repeat administration, we examined the immune responses generated against V antigen by immunization with an Ad vaccine vector encoding V antigen with mammalian preferred codons. Two vectors were constructed, AdV expressing the native V antigen sequence and AdsecV expressing a V gene fused to the human Igκ signal sequence for extracellular secretion. At 24 hr post-infection of A549 cells with AdsecV, V antigen could be detected in the culture media by Western analysis and in intracellular organelles by indirect immunofluorescence. We tested the hypothesis that anti-V immunity could be obtained by administration of these vectors to mice and that the secreted version would facilitate antigen uptake by antigen presenting cells and therefore be more efficient at inducing a humoral immune response. To compare anti-V antigen antibody titers evoked by each vaccine, BALB/c mice were immunized with a single intramuscular administration of either vector at a dose of 108 and 109 particles units (pu). At 2 wk post-immunization, the anti-V antigen IgG reciprocal titers in serum from AdV-immunized mice (n=5/group) were 280±60 and 3500±900 for the 108 pu and 109 pu vaccine doses, respectively. In contrast, the anti-V antigen IgG reciprocal titers from AdsecV-immunized mouse sera were 3000±400 (108 pu) and 9700±3200 (109 pu). By 4 wk post-immunization, anti-V antigen IgG reciprocal titers were comparable in mice immunized with either AdsecV [2886±410 (108 pu), 3800±420 (109 pu)] or AdV [1600±300 (108 pu), 1800±420 (109 pu)]. The two vectors were also analyzed for stimulation of TH1 (IFN-γ) responses by ELISPOT. Ten days after intramuscular immunization with 109 pu AdV or AdsecV, CD4+T cells were purified and were stimulated for 36 hr with syngeneic dendritic cells pulsed with purified recombinant V antigen. Antigen-specific IFN-γ producing CD4+cells were observed for AdsecV-immunized mice (176±16 IFNγ+/105 CD4+T cells) and for AdV mice (95±37 IFNγ+ cells/105 CD4 cells). In conclusion, both vaccines can elicit robust humoral as well as cellular immune responses, but secreted V antigen stimulates a more rapid humoral response relative to the cell-associated protein.