Our understanding of the humoral immune response to hepatitis C virus (HCV) is limited because the virus can be studied only in humans and chimpanzees and because previously described neutralization assays have not been robust or simple to perform. Nevertheless, epidemiologic and laboratory studies suggested that neutralizing Ab to HCV might be important in preventing infection. We have recently described a neutralization assay based on the neutralization of pseudotyped murine retrovirus constructs bearing HCV envelope glycoproteins on their surface. We have applied the assay to well characterized clinical samples from HCV-infected patients and chimpanzees, confirmed the existence of neutralizing Ab to HCV, and validated most previously reported neutralizations of the virus. We did not find neutralizing anti-HCV in resolving infections but did find relatively high titers (>1:320) of such Ab in chronic infections. Neutralizing Ab was directed not only to epitope(s) in the hypervariable region of the E2 envelope protein but also to one or more epitopes elsewhere in the envelope of the virus. Neutralizing Ab was broadly reactive and could neutralize pseudotype particles bearing the envelope glycoproteins of two different subgenotypes (1a and 1b). The ability to assay neutralizing anti-HCV should permit an assessment of the prospects for successful Ab-mediated passive and active immunoprophylaxis against hepatitis C. H epatitis C virus (HCV) is a small enveloped virus containing single-stranded positive sense RNA. It infects up to 170 million people worldwide and is a major cause of chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Although HCV accounts for only Ϸ12% of acute hepatitis in the United States, its high rate of persistence (70-80%) makes it responsible for almost half of the economic burden of this disease.A better understanding of the pathogenesis of hepatitis C and its control is hampered by certain characteristics of the virus. (i) HCV is genetically and probably serologically heterogeneous.(ii) The only animal model for HCV infection is the chimpanzee. (iii) Although the virus replicates sparingly in some cell lines, the in vitro method developed for detecting neutralizing antibodies (Nt Ab) to HCV in this system is so difficult to perform that it has not been widely used. Consequently, although the two envelope proteins of HCV, E1 and E2, have been expressed individually and as heterodimers, and Ab to them has been measured, there is no confirmation that such Ab accurately reflects the response to the virus or that it is Nt Ab.Previously, we identified Nt Ab to HCV by their ability to prevent replication of the virus in a lymphoid cell line (1, 2) or to prevent hepatitis C in chimpanzees (3, 4). These and a small number of similar studies (5) have provided, for almost a decade, the only direct evidence for Ab-mediated neutralization of HCV. Consequently, although considerable new knowledge has been gained about the cellular immune response to HCV, little is known about the role of the hum...

The lack of a cell culture system to support hepatitis C virus (HCV) replication has hampered studies of this frequent cause of chronic liver disease. However, pseudotyped retroviral particles (pp) bearing the HCV envelope glycoproteins have provided a different approach to HCV studies. We used genotype 1a pp to detect neutralizing antibodies (NtAb) in eight chimpanzees and four humans infected with 1a strains, and developed pp of genotypes 2a, 3a, 4a, 5a, and 6a to study cross-reactivity. NtAb was detected in one of four chimpanzees and none of three humans with acute resolving infection, suggesting that NtAb is not required for HCV clearance. NtAb were detected at high titer in two of four chimpanzees and, in Patient H, all with persistent infection; responses paralleled humoral responses to envelope 1 and 2 proteins and, in some cases, correlate also with antibodies to the hypervariable region 1, previously thought to be the primary site of neutralization. NtAb raised during 1a infections could neutralize HCVpp of genotypes 4a, 5a, and 6a but had only limited reactivity against 2a and 3a. The detection of high-titer NtAb with cross-genotype reactivity has important implications for the development of active and passive immune-prophylaxis strategies against HCV. Finally, we found that HCVpp infectivity was enhanced by human or chimpanzee sera; apolipoprotein C1 alone or as a component of high-density lipoproteins caused this enhancement. Future studies of the in vivo role of apolipoprotein C1 might provide additional insights into the infection process of HCV.neutralizing antibodies ͉ high-density lipoproteins