1992;66:3225C3229. to measure neutralization by PF6-AM autologous sera, antibodies neutralizing earlier sequence variants were detected at earlier time points than antibodies neutralizing later variants, indicating clearance and development of viral variants in response to pressure from neutralizing antibodies. To demonstrate the effects of amino acid substitution on neutralization, site-directed mutagenesis of a pseudoparticle envelope sequence revealed amino acid substitutions in hypervariable region 1 that were responsible for a dramatic decrease in neutralization sensitivity over time. Additionally, high-titer neutralizing antibodies peaked at the time of viral clearance in all spontaneous resolvers, while chronically evolving subjects displayed low-titer or absent neutralizing antibodies throughout early acute contamination. Conclusions These findings show that during acute hepatitis C computer virus contamination in vivo, virus-specific neutralizing antibodies drive sequence evolution and, in some individuals, play a role in determining the outcome of infection. Introduction The World Health Organization estimates that 170 million persons are infected with the hepatitis C computer virus (HCV) worldwide, of which four million are in the United States.1,2 While ~30% of acute HCV infections are spontaneously resolved, the majority progress to chronic contamination. Persistent viremia can lead to complications such as PF6-AM cirrhosis and hepatocellular carcinoma, making HCV a major cause of liver disease worldwide. 3,4 The HCV genome encodes a mutation prone polymerase, resulting in the presence of the computer virus as a quasispecies, defined as a collection of genetically related but unique viral variants. 5 The capacity of the computer virus to mutate continually most likely contributes to the establishment of chronicity, as variants that escape immune responses have a survival advantage. Little is known regarding the role of HCV-specific neutralizing antibodies (nAb) in modulating HCV pathogenesis or driving viral sequence development. Establishment of HCV glycoprotein-bearing retroviral pseudoparticles (pp) has only recently allowed for detailed studies of the nAb response,6,7 the majority of which used HCVpp expressing heterologous envelope sequences, usually the reference strain H77.8C13 The considerable heterogeneity of HCV, particularly within the envelope genes, may distort results from heterologous assays, resulting in an under-representation of nAb responses. To date, only a small number of neutralization PF6-AM studies have used HCVpp expressing autologous, or person-specific, envelope sequences: two in the setting of single-source HCV outbreaks,14,15 and four studies of the nAb response in individual H, a well-studied individual from whom the H77 reference strain originated.11,13,16,17 During the acute phase, antibodies to heterologous HCV envelope proteins have been shown to appear later and at lower titers compared to antibodies directed against non-structural proteins, suggesting that nAbs may play only a minor role in spontaneous resolution.10,13 However, the quick evolution and greater variability of the envelope genes compared to the rest of the HCV genome suggests that the circulating viral quasispecies is modulated by ongoing humoral immune pressure. It is possible that the use of autologous HCVpp is necessary to detect strain-specific antibodies appearing during acute contamination. In support of this hypothesis, autologous HCVpp studies reported correlations between nAb responses in acute HCV with KIAA0288 both control of viremia14 and spontaneous resolution,15 associations not reported with heterologous antigen-based assays. To assess the impact of immune pressure exerted by HCV nAb responses on viral sequence evolution, we measured neutralization of subject-specific HCVpp in an autologous setting. Our results provide strong evidence that HCV nAb responses in acute contamination have a direct impact on viral sequence evolution and that spontaneous resolution of the computer virus may be associated with the magnitude of the nAb response. Materials and Methods Participants Blood samples were obtained from consenting HCV-infected adults participating in a prospective study of young intravenous drug users as previously explained.18 At each visit, participants were provided counseling to reduce the risks of drug use. Blood was drawn for isolation of serum, plasma, and PBMC in.
