HCV REPLICATION AND IN VITRO NEUTRALIZATION ASSAY

Hepatitis C virus (HCV) infections represent a worldwide health problem,
with approximately 0.5 to 1.5% of most populations testing positive for
antibodies to HCV. An estimated 50% of infected individuals will become
chronic carriers of HCV and will be at increased risk for the development
of cirrhosis and hepatocellular carcinoma and will also act as a source
of infection for others. Transmission of HCV occurs most readily through
contact with contaminated blood as in blood transfusions and IV drug
abuse; however, the true epidemiology of this agent is only now emerging
through the use of diagnostic assays. Other routes of transmission
include sexual contact, intrafamilial contact, and transmission from
mother to child at birth. The development of a vaccine for this virus
must be considered a high priority. Currently, the only available method
of testing vaccine candidates is the immunization and challenge of
chimpanzees, a very costly and time consuming approach. The development
of an in vitro assay for evaluating the presence of neutralizing
antibodies would greatly facilitate vaccine development. This proposal
will employ a recently developed primary hepatocyte tissue culture system
for the in vitro propagation of HCV to address these needs. The specific
aims of this proposal are: 1) to optimize methods for detecting HCV
polypeptides using a human hepatoma cell line transfected with
recombinant expression vectors encoding various domains of the HCV
polyprotein; 2) to produce antibodies to the capsid and envelope proteins
of HCV expressed in the insect cell/baculovirus system; 3) to define the
authentic HCV proteins expressed in HCV-infected primary hepatocytes
using immunoprecipitation and immunoblot methodology; 4) to develop a
quantitative assay for measuring in vitro infections of primary
hepatocytes with HCV and to use this assay to develop an in vitro
neutralization assay for HCV; 5) to examine panels of human and
chimpanzee sera for the presence of neutralizing antibodies and to
evaluate antisera produced against the envelope proteins of HCV for
neutralizing activity; and 6) to create immortalized human and chimpanzee
hepatocyte cell lines permissive for HCV infections.