Replication of HAV is being studied in order to find ways to improve the in vitro replication of the virus. As the virus presently grows slowly and to relatively low titer, vaccine production is inefficient. Several different constructs using the infectious cDNA of HAV as the backbone have been made. These include placing the highly active encephalomyocarditis virus (EMCV) internal ribosomal entry site (IRES) into the 5' non-coding region of the HAV genome. This resulted in a viable virus but without enhanced growth characteristics. Constructs with the poliovirus Ires are now underway. Several genes have been cloned into the HAV genome along with the second IRES in order to have the HAV genome serve as a bicistronic message. The poliovirus 2A protease was inserted behind the EMCV IRES but it was not infectious. We are now inserting the polio 2A as a fusion protein with the HAV. Immunonologic studies on hepatitis A virus have concentrated on T-cel immunity and experimental vaccinology using the chimpanzee model. Chimpanzees were immunized with the expression product of a recombinant vaccinia virus containing the entire open reading frame of the HAV genome. A dose response experiment was performed and it was clear that this vaccine was highly immunogenic and protective. With the higher doses of vaccine th animals seroconverted quickly after the first dose. Chimpanzee T-cell lines specific for hepatitis A have been established and are continuing to be evaluated. A cytotoxic T-cell clone has been established and several T-cell lines specific for hepatitis A have been selected. Both cytotoxic and helper activity are being studied and the epitopes to which these cells react are being evaluated.
