A major problem in hepatitis C virus (HCV) immunotherapy or vaccine design is the extreme variability of the virus. We identified human monoclonal antibodies (mAbs) that neutralize genetically diverse HCV isolates and protect against heterologous HCV quasispecies challenge in a human liver-chimeric mouse model. The results provide evidence that broadly neutralizing antibodies to HCV protect against heterologous viral infection and suggest that a prophylactic vaccine against HCV may be achievable.

Hepatitis C virus (HCV) remains a significant threat to the general health of the world's population, and there is a pressing need for the development of new treatments and preventative vaccines. Here, we describe the generation of retrovirus-based pseudoparticles (HCVpp) incorporating a panel of full-length E1E2 clones representative of the major genotypes 1 through 6, and their application to assess the reactivity and neutralizing capability of antisera and monoclonal antibodies raised against portions of the HCV E2 envelope protein.Rabbit antisera raised against either the first hypervariable region or ectodomain of E2 showed limited and strain specific neutralization. By contrast, the monoclonal antibody (MAb) AP33 demonstrated potent neutralization of infectivity against HCVpp carrying E1E2 representative of all genotypes tested. The concentration of AP33 required to achieve 50% inhibition of infection by HCVpp of diverse genotypes ranged from 0.6 to 32 g/ml. The epitope recognized by MAb AP33 is linear and highly conserved across different genotypes of HCV. Thus, identification of a broadly neutralizing antibody that recognizes a linear epitope is likely to be of significant benefit to future vaccine and therapeutic antibody development.Hepatitis C virus (HCV), a positive-strand RNA virus belonging to the Flaviviridae family, is the major cause of non-A, non-B viral hepatitis. HCV has infected approximately 200 million people worldwide and current estimates suggest that as many as 3 million individuals are newly infected each year (4). Approximately 80% of those infected fail to clear the virus; a chronic infection ensues, frequently leading to severe chronic liver disease, cirrhosis, and hepatocellular carcinoma (2, 41). Current treatments for chronic infection are ineffective for approximately 50% of patients, and there is a pressing need to develop preventative and therapeutic vaccines.Due to the error-prone nature of the RNA-dependent RNA polymerase and the high replicative rate in vivo (30, 46), HCV exhibits a high degree of genetic variability. Crucially, this propensity for genetic change allows the virus to respond to and overcome a variety of selective pressures, including host immunity and antiviral therapy (18,26,37,44,53). HCV can be classified into six genetically distinct genotypes and further subdivided into at least 70 subtypes, which differ by approximately 30% and 15% at the nucleotide level, respectively (59, 61). A significant challenge for the development of vaccines will lie in identifying protective epitopes that are conserved in the majority of viral genotypes and subtypes. This problem is compounded by the fact that the envelope proteins, the natural targets for the neutralizing response, are two of the most variable proteins (10).The envelope proteins E1 and E2 are responsible for cell binding and entry (5,8,16,51,57). They are N-linked glycosylated (23,31,43,62) transmembrane proteins with a Nterminal ectodomain and a C-terminal hydrophobic membrane anchor (12,21,22). In vitro ex...

Because of the lack of a robust cell culture system, relatively little is known about the molecular details of the cell entry mechanism for hepatitis C virus (HCV). Recently, we described infectious HCV pseudo-particles (HCVpp) that were generated by incorporating unmodified HCV E1E2 glycoproteins into the membrane of retroviral core particles. These initial studies, performed with E1E2 glycoproteins of genotype 1, noted that HCVpp closely mimic the cell entry and neutralization properties of parental HCV. Because sequence variations in E1 and E2 may account for differences in tropism, replication properties, neutralization, and response to treatment in patients infected with different genotypes, we investigated the functional properties of HCV envelope glycoproteins from different genotypes/subtypes. Our studies indicate that hepatocytes were preferential targets of infection in vitro, although HCV replication in extrahepatic sites has been reported in vivo. Receptor competition assays using antibodies against the CD81 ectodomain as well as ectopic expression of CD81 in CD81-deficient HepG2 cells indicated that CD81 is used by all the different genotypes/subtypes analyzed to enter the cells. However, by silencing RNA (siRNA) interference assays, our results show that the level of Scavenger Receptor Class-B Type-I (SR-BI) needed for efficient infection varies between genotypes and subtypes. Finally, sera from chronic HCV carriers were found to exhibit broadly reactive activities that inhibited HCVpp cell entry, but failed to neutralize all the different genotypes. In conclusion, we characterize common steps in the cell entry pathways of the major HCV genotypes that should provide clues for the development of cell entry inhibitors and vaccines. (HEPATOLOGY 2005;41:265-274.) H epatitis C virus (HCV) is an RNA-enveloped virus belonging to the Flaviviridae family. HCV exhibits a high degree of genetic heterogeneity. The propensity for genetic change is associated primarily with the error-prone nature of its RNA-dependent RNA polymerase together with the high HCV replicative rate in vivo. 1,2 This results in infected individuals harboring a diverse population of viral variants known as a quasispecies, which evolve in response to a variety of selective pressures. 3 Although HCVspecific immunity develops after primary infection, it frequently fails to eliminate the virus. 4-6 HCV has infected approximately 170 million people worldwide, and approximately 80% of those infected will develop chronic infection. The outcome of chronic infection varies widely between individuals, but large proportions of patients develop serious liver diseases such as cirrhosis and hepatocellular carcinoma. 7 Current therapies are inadequate, and development of appropriate therapeutic and prophylactic vaccines remains a significant challenge.HCV can be classified into six genetically distinct genotypes and further subdivided into at least 70 subtypes, which differ by approximately 30% and 15% at the nu-