The envelope glycoprotein E2 of hepatitis C virus (HCV) is the target of neutralizing antibodies and is presently being evaluated as an HCV vaccine candidate. HCV binds to human cells through the interaction of E2 with the tetraspanin CD81, a putative viral receptor component. We have analyzed four different E2 proteins from 1a and 1b viral isolates for their ability to bind to recombinant CD81 in vitro and to the native receptor displayed on the surface of Molt-4 cells. A substantial difference in binding efficiency between these E2 variants was observed, with proteins derived from 1b subtypes showing significantly lower binding than the 1a protein. To elucidate the mechanism of E2-CD81 interaction and to identify critical regions responsible for the different binding efficiencies of the E2 variants, several mutants were generated in E2 protein regions predicted by computer modeling to be exposed on the protein surface. Functional analysis of these E2 derivatives revealed that at least two distinct domains are responsible for interaction with CD81. A first segment centered around amino acid residues 613 to 618 is essential for recognition, while a second element including the two hypervariable regions (HVRs) modulates E2 receptor binding. Binding inhibition experiments with anti-HVR monoclonal antibodies confirmed this mapping and supported the hypothesis that a complex interplay between the two HVRs of E2 is responsible for modulating receptor binding, possibly through intramolecular interactions. Finally, E2 proteins from different isolates displayed a profile of binding to human hepatic cells different from that observed on Molt-4 cells or isolated recombinant CD81, indicating that additional factors are involved in viral recognition by target liver cells.

The hypervariable region 1 (HVR1) of the putative envelope protein E2 of hepatitis C virus (HCV) contains a principal neutralization epitope, and anti-HVR1 antibodies have been shown to possess protective activity in ex vivo neutralization experiments. However, the high rate of variability of this antigenic fragment may play a major role in the mechanism of escape from host immune response and might represent a major obstacle to developing an HCV vaccine. Thus, even if direct experimental evidence of the neutralizing potential of anti-HVR1 antibodies by active immunization is still missing, the generation of a vaccine candidate with a cross-reactive potential would be highly desirable. Hepatitis C virus (HCV) is the major etiologic agent of blood transfusion-associated and sporadic non-A non-B hepatitis worldwide, with an estimated prevalence of 0.4% to 2% in the blood donor population. 1 Despite a wide array of humoral and cell-mediated host immune responses, HCV infection leads to chronic disease in about 70% of cases, among which a significant proportion eventually develops cirrhosis and hepatocellular carcinoma. 2 Interferon treatment (also in combination with ribavirin) is the only antiviral therapy available at the moment, but it is effective only in 20% to 40% of patients, 3-5 thus making the development of an HCV vaccine a high priority target.Vaccination of chimpanzees (the only other species susceptible to HCV infection) using recombinant forms of the putative envelope proteins E1 and E2 has been shown to induce protective immunity against low-dose challenge with the homologous virus, whose efficacy seems to correlate with the titer of anti-E2 antibodies. [6][7][8] More recently, HCV was shown to bind to its putative receptor component CD81 via E2. 9 These data point toward the E2 protein as a likely candidate for vaccine development. However, a major concern still remains as to whether the anti-E2 response elicited by one recombinant protein would be effective against heterologous viral inocula. 10 In fact, HCV displays a high rate of mutation during replication and exists in the bloodstream of infected patients as a quasispecies, [11][12][13][14][15] which fluctuates during the course of the disease mainly as a result of immune pressure. [15][16][17][18][19][20][21][22][23] The major target of this immune response is the 27 amino acid-long N-terminal segment of the E2 glycoprotein. This protein fragment is the most variable region of the whole HCV polyprotein 11 and contains a principal neutralization determinant, 24 but anti-hypervariable region 1 (HVR1) antibodies specific for one variant display only a limited ability to block different viral variants. 24 In this scenario, the most difficult task in developing an HCV vaccine would be to find a solution to the issue of viral variability.We have recently described a novel approach to this problem by selecting highly cross-reacting "synthetic variants" of the HCV HVR1 from a vast repertoire of HVR1 surrogates as fusion to the major coat protein of ba...

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Hepatitis C Virus (HCV) is a major cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma, worldwide, and the development of an effective vaccine represents a high priority goal. The Hyper Variable Region 1 (HVR1) of the second Envelope protein (E2) of HCV contains a principal neutralizing determinant, but it is highly variable among different isolates and it is involved in the escape from host immune response. Thus, to be effective, a vaccine should elicit a cross-reacting humoral response against the majority of viral variants. We show that it is possible to achieve a broadly cross-reactive immune response in rabbits by immunization with mimotopes of the HVR1. selected from a specialized phage library using HCV patients' sera. At least some of the cross-reacting anti-mimotope antibodies, elicited in rabbits, recognize discontinuous epitopes in a manner similar to those induced by the virus in infected patients.