Antigenic analysis of Punta Toro virus and identification of protective determinants with monoclonal antibodies

Hybridomas producing monoclonal antibodies to the three major structural proteins of Punta Toro virus (PTV) were established by fusion of spleen cells with Sp2/0-Ag-14 mouse plasmacytoma cells. Thirty-six independently derived monoclonal antibodies were evaluated in neutralization, hemagglutination inhibition, and ELISA assays and the isotype, antigen specificities, and cross-reactivities were determined. These antibodies were also assessed for their ability to provide protection in a murine model. Both G1- and G2-specific antibodies were obtained which neutralized virus infectivity in vitro and inhibited hemagglutination, whereas nucleocapsid-specific antibodies exhibited neither activity. All of the anti-G1 antibodies were PTV-specific, whereas anti-G2 and anti-nucleocapsid antibodies exhibited varying patterns of cross-reactivity with heterologous phleboviruses. All of the G1-reactive monoclonal antibodies, which bound to epitopes in two distinct topological sites as determined by competitive binding assays, provided efficient protection to both immunocompetent and immunosuppressed mice. In contrast, of the 23 G2-reactive antibodies, only 8 were able to protect immunocompetent mice and only one was able to protect immunosuppressed animals. The degree of protection achieved in vivo did not correlate directly with the neutralization titers determined in vitro.