Genetic Markers Of Virulence &Adaptation to Hepatitis A

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Abstract

Our objectives are to determine the genetic basis for virulence and adaptation to cell culture of HAV and to use this information to develop a strain of HAV suitable for use as an attenuated vaccine. Determinants of cell culture adaptation and of virus virulence are being identified by studying chimeric viruses encoded by infectious cDNA clones containing genes from a virulent and an attenuated virus. Thirteen different recombinant chimeric viruses were compared for their pathogenicity for tamarins. The ability to cause acute hepatitis depended on the genotypes of two of the seven genes encoding nonstructural proteins. A virus was virulent if these two genes were from the wild-type parent and conversely a virus was attenuated if these two genes were from the attenuated parent. Other viral genes did not have a discernible effect on virulence. Each of the two genes appeared independently to increase serum liver enzyme levels but both genes were required for development of severe histopathology. In general, the pattern of fecal virus shedding suggested severe hepatitis reflected higher levels of viral replication. The time from inoculation to seroconversion was consistently shorter if either gene was from wild-type. If both genes were from the attenuated parent, time to seroconversion was prolonged and mutations selectively appeared in the second gene. A new chimeric virus was engineered by incorporating multiple mutations that had selectively appeared in the second virulence gene during a lengthy infection in a tamarin. This new recombinant virus displayed the high level of replication and the short time to seroconversion characteristic of the virulent virus but did not cause disease, making it a vaccine candidate. It is currently being evaluated in tamarins for possible oral administration. We established a cell line of liver cells which is unique in that it supports the replication of clinical isolates (virulent virus) of HAV. However, virus passaged only four times in these cells became attenuated for tamarins. The basis for this attenuation is being analyzed since these cells appear to apply different selective pressures to virus replication than do other cells we have used. In all cell culture systems studied thus far (except for our unique liver cell line) viral replication requires a specific amino acid change in one of the nonstructural proteins not involved in virulence. This site is being mutagenized and the effect of different amino acid substitutions on replication in vitro and in vivo is being determined. We have isolated chimpanzee monoclonal antibodies that neutralize HAV and are preparing scale-up expression vectors so that we can make enough antibodies to determine if they can be used for passive immunoprophylaxis in humans. We have shown that an ELISA to the 3C proteinase of HAV can be used to identify infection during vaccine trials.