The goal of this project is the production of a cross-protective flavivirus vaccine, based on the epitope identified by the murine monoclonal antibody 4G2. This epitope appears to be present on the 50-60 Kd envelope (E) glycoprotein of all members of the Flavivirus family. Initially, the production of a murine monoclonal antiidiotype antibody was attempted. The 4G2 antibody, or its Fab fragment, was used as an immunogen in mice, either alone or coupled to BSA or KLH. Fusions were performed using splenocytes from immunized animals, and the resultant hybridomas screened for reactivity against 4G2. Testing of over 700 hybrids resulting from 30 fusions yielded no reactive clones. In a new approach, the 4G2 antibody will be employed to determine the exact cross-protective epitope on E. E will be fragmented by enzyme and/or chemical means, the reactivity of the fragment(s) followed by electrophoresis and western blotting, and reactive fragments sequenced. The 4G2 epitope as displayed on both Japanese Encephalitis Virus (JEV) and West Nile virus (WN) is stable to heating at 100 degree centigrade for up to six minutes. We have determined that a two-minute heat treatment at 100 degree centigrade will inactivate up to 8 logs of viral infectivity; therefore, such a step is used to inactivate the virus so it can be used under non-containment conditions. The dengue virus and WN epitopes are stable to denaturing agents such as mercaptoethanol and dithiothreitol, whereas the JEV epitope is sensitive to these denaturing agents. However, JEV was chosen as the source of E because large quantities of purified virus can be produced, and an animal model for testing the protective efficacy of vaccine candidates is available. The electrophoretic mobility and western blot reactivity of JEV E is unchanged by boiling before electrophoresis, so the heating step is not employed during these studies. Western blot analysis has shown that 4G2 reactivity is retained by a 17-18 Kd tryptic/chymotryptic fragment of E. The E- glycoprotein is quite susceptible to trypsin cleavage, with epitope reactivity lost after exposure to 0.5 mg/ml trypsin for 4 hours at 37 degree centigrade; the optimum digestion time has not yet been determined.
