Hepatitis A virus (HAV) causes one of the most prevalent infections of man, and remains a worldwide public health problem. The ability to propagate the virus in cultured cells, as well as the recent construction of cDNA clones and the determination of the nucleotide sequence of the viral genome, have made possible numerous new approaches to understanding the biology and pathogenesis of HAV infection and provided insight towards the development of potential subunit vaccines. In this application, we propose experiments designed to identify specific viral sequences that are responsible for the slow and protracted replication cycle of this virus, a characteristic that distinguishes it from all other members of its genus (enterovirus) and family (Picornaviridae). Recombinant viruses will be constructed that are composed of HAV 5' end regulatory sequences and poliovirus coding sequences, or HAV polymerase coding sequences replacing the homologous polio sequences. The efforts of the insertion of these potentially down-regulating HAV sequences into a rapidly-growing, lytic poliovirus will be evaluated by examining plaque size and morphology, temperature sensitivity, viral RNA and protein synthesis, and virus yields. Another uncharacterized HAV gene, the 2A protease coding region, will be analyzed for functions dealing with host cell interaction and virus morphogenesis by expressing genetically engineered proteins containing 2A sequences, both in vivo and in vitro. Finally, we have produced HAV capsid protein (VP1) antigens in both prokaryotic (E. coli) and eukaryotic (baculovirus-infected SF9) cells. We plan to systematically evaluate the ability of different antigenic proteins, expressed from a variety of different vectors and purified by different procedures, to induce neutralizing antibodies and/or to prime the immune system for a neutralizing anamnestic response. We hope that these studies will identify a recombinant protein that will be effective as a candidate immunogen for a subunit vaccine.
Showing the most recent 10 out of 20 publications
