Stamataki, Z., S. Coates, M. J. Evans, M. Wininger, K. Crawford, C. Dong, Y. L. Fong, D. Chien, S. Abrignani, P. Balfe, C. M. Rice, J. A. McKeating, and M. Houghton. Vaccine 25:7773-84. 2007.
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Efforts to develop HCV vaccines have been compromised by the technical difficulties in measuring functional or protective antibody responses. However, the recent development of HCVpp and HCVcc systems has allowed these studies to proceed. This study demonstrates that immunization of rodents with recombinant HCV E1E2/p7 or E2 can elicit polyclonal antibody responses capable of neutralizing HCVpp and HCVcc bearing diverse gps.
Neutralizing antibody responses elicited during infection generally confer protection from infection. Hepatitis C virus (HCV) encodes two glycoproteins E1 and E2 that are essential for virus entry and are the major target for neutralizing antibodies. To assess whether both glycoproteins are required for the generation of a neutralizing antibody response, rodents were immunized with a series of glycoproteins comprising full length and truncated versions. Guinea pigs immunized with HCV-1 genotype 1a E1E2p7, E1E2 or E2 generated high titer anti-glycoprotein antibody responses that neutralized the infectivity of HCVpp and HCVcc expressing gps of the same genotype as the immunizing antigen. Less potent neutralization of viruses bearing the genotype 2 strain J6 gps was observed. In contrast, immunized mice demonstrated reduced anti-gp antibody responses, consistent with their minimal neutralizing activity. Immunization with E2 alone was sufficient to induce a high titer response that neutralized HCV pseudoparticles (HCVpp) bearing diverse glycoproteins and cell culture grown HCV (HCVcc). The neutralization titer was reduced 3-fold by the presence of lipoproteins in human sera. Cross-competition of the guinea pig anti-E1E2 immune sera with a panel of epitope mapped anti-E2 monoclonal antibodies for binding E2 identified a series of epitopes within the N-terminal domain that may be immunogenic in the immunized rodents. These data demonstrate that recombinant E2 and E1E2 can induce polyclonal antibody responses with cross-reactive neutralizing activity, supporting the future development of prophylactic and therapeutic vaccines.
Recombinant HCV-1 envelope gps elicit antibody responses that neutralize HCVpp infectivity. (A) 13 groups of Swiss outbred female mice (10 per group) and (B) 10 groups of Hartley outbred female guinea pigs (10 or 9 per group) were immunized with various preparations of HCV-1 E1E2p7, E1E2 and E2 plus adjuvant listed in the table below. Intramuscular immunizations were performed at 0, 30 and 90 days and the rodents bled two weeks after the last immunization. Immune sera at a final dilution of 1:100 were pre-incubated with HCVpp-H77 for 1h at 37oC and allowed to infect Huh-7.5 cells for 6h. Infection was terminated at 72h and infectivity determined (luciferase activity). Neutralization values are determined from quadruplicate infections and the mean value for each group shown.
List of the antigens used for Immunization and for detection of serum reactivity by EIA: HCV strain HCV-1 (genotype 1a) E2715, E2661, E1E2746, and E1E2p7809 were expressed in CHO cells and extracted from intracellular lysates or extracellular supernatants. (the subscript annotations refer to the final amino acid of the expressed proteins, where the first amino acid of E1 is at position 191 and E2 at 384 within the HCV polyprotein - see the Los Alamos hepatitis C sequence database for details.