Persistent infection with hepatitis C virus (HCV), a recently identified positive-strand RNA virus, is commonly associated with chronic hepatitis and may be an important factor in the development of primary hepatocellular carcinoma. Effective control of HCV infections will require development of a safe and effective vaccine, but this is dependent upon gaining a better understanding of the antigenic structure of the HCV particle. In order to map important antigenic determinants of HCV, a cytoplasmic T7 expression system will be utilized to express the putative core (C) and envelope (El and E2) proteins of HCV in mammalian cell lines (BSC/T7-6 and OS-T7 cells). Because this transient expression system involves cytoplasmic transcription and translation of viral RNA in mammalian cells, the expression, transport, and processing (especially glycosylation) of these HCV proteins should closely mimic events during natural infection with HCV. Expression of HCV proteins will be monitored by indirect immunofluorescence, flow cytometry, and in immunoblots of cell lysates using antibodies raised in guinea pigs to synthetic peptides representing regions of these three proteins. Whole cells expressing HCV proteins will be used to immunize Balb/c mice for generation of HCV-specific monoclonal antibodies (mAbs). Hybridoma screening will be accomplished using flow cytometry to detect antibodies binding specifically to cells expressing HCV proteins. The specificity of these mAbs will be determined for: (a) BSC/T7-6 cells expressing individual HCV proteins (C, El, or E2), and (b) BSC/T7-6 cells expressing HCV proteins which have defined deletions due to mutagenesis of the cDNA. These mAbs will also be tested for their ability to recognize denatured HCV proteins. Based on these results, mAbs may also be subjected to PEPSCAN analysis, as PEPSCAN has the ability to identify important linear determinants which are not dependent upon glycosylation or other post-translational modifications. To assess whether anti-El and anti-E2 mAbs react with native HCV particles, they will be tested for the ability to capture HCV particles onto a solid-phase support in a modified antigen-capture/polymerase chain reaction (AC/PCR) assay. Competitive-inhibition assays will be developed to determine what proportion of a group of HCV-infected patients have antibodies capable of competing with selected mAbs for binding to El and E2 expressed in T7-6 cells.
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