This study provides support for the contention that the majority of antibodies capable of inhibiting the interaction of HCV E2 with CD81 are developed to conformational epitopes conserved across genotypes 1 and 2. By producing HMAbs, our approach directly analyzes the human immune response to HCV. We chose an individual with a 2-year history of normal alanine aminotransferase values as the donor for antigen-specific B cells since an asymptomatic infection was more likely to reflect an effective humoral immune response containing HCV-related disease. This individual also had a high NOB titer. In the study by Rosa et al., (35
), 60% of 34 HCV-infected individuals exhibited essentially no NOB activity and the remainder exhibited minimal inhibition of E2 binding to CD81. Thus, the antibody response of the B-cell donor used in the production of the HMAbs described herein is exceptional and cannot be assumed to be representative of the antibody response observed in the majority of HCV-infected individuals. We are currently using the HCV HMAbs described in this report to determine the prevalence and titers of similar antibodies in other HCV-infected individuals.
The B-cell donor was infected with genotype 1b. In an effort to obtain cross-reactive antibodies, we used HCV 1a E2 proteins to identify HMAbs to HCV E2. All of the HMAbs also reacted with E2 from a heterologous HCV 1b isolate, Q1b, that was 80% homologous with the HCV 1a isolate used in the selection of HMAbs. We do not, unfortunately, have sequence information on the HCV 1b isolate from the B-cell donor. It is therefore not possible to comment on the relatedness of Q1b to the homologous 1b isolate. All of the isolated hybridomas secreted antibodies with IgG1 heavy chains, which corroborates recent data indicating that the antibody response to HCV E2 is dominated by IgG1 antibodies (4
). Nine of the HMAbs recognized conformational epitopes sensitive to denaturation of full-length HCV E2. Future studies with E2 deletion mutants or E2 fragments will be required to determine if the epitopes recognized by the HMAbs can be encompassed in discrete domains of E2. Four HMAbs, CBH-4D, -4B, -4G, and -17, did not inhibit the formation of HCV E2-CD81 complexes. One of these antibodies, CBH-4G, reacted with HCV E2-CD81 complexes of genotypes 1a, 1b, 2a, and 2b, confirming that E2 proteins of genotype 2 are capable of binding to CD81-LEL. The broad reactivity of HMAb CBH-4G with E2-CD81 complexes makes it a useful reagent for quantifying titers of antibody capable of inhibiting binding of E2 with CD81 in HCV sera.
Six of the HMAbs recognizing conformational epitopes, CBH-2, -5, -7, -8C, -8E, and -11, inhibited the binding of HCV E2 proteins with CD81-LEL and can be referred to as inhibitory or NOB positive. However, all six antibodies were able to bind to preformed E2–CD81-LEL complexes with some of the same E2 proteins that they efficiently inhibited from binding CD81. Five of the six inhibitory antibodies were reactive with preformed complexes of 2a or 2b E2 proteins with CD81-LEL and not 1a or 1b E2 complexes with CD81. This implies that the tertiary structure of genotype 2 E2 differs from that of genotype 1 E2. However, the ability of an inhibitory antibody to bind to CD81-E2 complexes did not strictly correlate with genotype (i.e., HMAb CBH-7 bound complexes of 1a E2 with CD81-LEL and HMAb CBH-8C did not bind complexes of 2b E2 with CD81-LEL). Thus, the amino acid sequences that underlie the reactivity of inhibitory HMAbs with CD81-E2 complexes may be subject to mutation in vivo. This raises the possibility that sequences in variable regions of E2 affect the reactivity of inhibitory HMAbs with CD81-E2 complexes, even though the actual epitopes recognized by the HMAbs appear to be highly conserved. We also note that the differences in reactivity of the six inhibitory HMAbs with CD81-E2 complexes suggest that the antibodies recognize multiple distinct epitopes. Competition and mutagenesis studies should clarify the total number of unique epitopes recognized by the inhibitory HMAbs.
Previous studies have suggested that the binding site for CD81 in E2 is dependent on E2 conformation (12
). The reactivity of the six inhibitory antibodies with preformed E2-CD82 complexes indicates that the epitopes recognized by these antibodies do not directly overlap the CD81 binding site. If the binding sites of the HMAbs and CD81 overlapped, steric hindrance should have prevented simultaneous binding of E2 with the HMAb and CD81. A second possibility is that the epitopes recognized by the inhibitory HMAbs are located nearby to the CD81 binding site, so that a complex of antibody and E2 has a reduced affinity for CD81 due to antibody-mediated steric hindrance. However, the very similar binding patterns of the six inhibitory HMAbs and the noninhibitory HMAb CBH-4G with complexes of genotype 2 E2 and CD81 argue that the epitopes are equivalently accessible. The most likely explanation is that the affinity of the HMAbs for E2 was higher than the affinity of CD81-LEL for E2. Once an antibody-E2 complex was formed, the inhibitory antibodies stabilized E2 protein in a conformation that had a low affinity for CD81. If a complex of E2 and CD81 was already formed, accessibility of the antibody to its epitope may have been affected by the altered conformation of the E2 in the CD81 complex. Alternatively, the epitopes recognized by the NOB-positive antibodies may always be exposed in a CD81-E2 complex, but subsequent binding of the antibody to this epitope may have a variable ability to dissociate E2 from CD81 (i.e., binding of CBH-5 to a complex of 1a E2 and CD81 results in dissociation of 1a E2 from CD81, and binding of CBH-5 to a complex of 2a E2 and CD81 is not sufficient to dissociate 2a E2 from CD81). In the first case, the E2 protein remains associated with CD81; in the second case, the E2 protein would not be present. We are currently exploring the use of biotinylated CBH-4G and other epitope tags to attempt to differentiate between these two possibilities.
Two of the inhibitory antibodies, CBH-2 and CBH-5, were able to prevent the binding of intact HCV virions to CD81. Two other inhibitory antibodies, CBH-7 and CBH-11, did not inhibit the interaction of HCV virions with CD81. The failure of CBH-11 to inhibit binding of HCV virions to CD81 may reflect the poor reactivity of CBH-11 with HCV some 1a isolates (such as isolate Q1a). HMAbs CBH-2 and CBH-5 did not bind preformed 1a E2–CD81-LEL complexes and inhibited binding of virions to CD81. CBH-7 bound to preformed 1a E2–CD81-LEL complexes and did not inhibit binding of virions to CD81. Therefore, failure to bind to preformed complexes of E2 and CD81 may be a better predictor of the ability of an antibody to prevent binding of HCV virions to CD81 in vivo than is inhibition of formation of the CD81-E2 complex. One implication of this proposal would be that of the inhibitory HMAbs, CBH-2 has the greatest potential to effectively inhibit binding of multiple isolates of HCV to CD81 in vivo. The other inhibitory HMAbs would have only limited ability to inhibit binding of HCV 2a/2b virions to CD81. However, testing of CBH-2 and other inhibitory antibodies with HCV virions and recombinant E2 proteins generated from the same sera would be required to confirm this.
The observation that a fraction of the E2 antibodies isolated from this HCV PCR-positive B-cell donor could inhibit the interaction of E2 with CD81 raises an important question. If the B-cell donor had a high titer of potentially neutralizing antibodies, why did this individual continue to exhibit plasma viremia? The most obvious explanation is that CD81 is not the primary receptor for HCV. Antibodies recognizing different epitopes that interfere with the binding of HCV to the putative primary receptor would be the antibodies with neutralization activity. The donor may have had a relatively low titer of this type of antibody. If one assumes that CD81 is involved in HCV infectivity, a second explanation is that antibodies that can inhibit the binding of E2 to CD81 will neutralize the infectivity of the majority of HCV virions but have little effect on cells that are already infected. Studies of the infectivity of HCV innocula in chimpanzee have demonstrated that antibody-coated virions exhibit markedly reduced infectivity compared to free virions (17
). Other studies with HCV sera have found that the ability of virions from sera to attach and enter target cells is critically dependent on whether the virions are free or coated with antibodies (21
). In addition, studies of E2 expression in mammalian systems indicate that little or no envelope protein is expressed on the cell surface (8
). Thus, antibodies would have limited opportunity to bind to infected cells. Clearance of cells that are HCV infected would therefore depend on the action of cytotoxic T lymphocytes, which may or may not be effective (reviewed in reference 33
). Assuming that CD81 is a receptor or coreceptor for HCV, individuals with a strong NOB-positive antibody response to E2 may be at a steady state in which minimal de novo infection of susceptible cells occurs while the existing infected cells persist and continue to induce liver damage. Studies in which HCV antisera with high and low NOB activity are assessed for infectivity in naive chimpanzees will be required to more firmly establish a correlation between inhibition of E2-CD81 binding and true virus neutralization.
Overall, multiple HMAbs that recognized conserved epitopes and could inhibit the interaction of HCV E2 with CD81 were obtained from an HCV-infected individual with a high-titer immune response to E2. The antibodies that recognize these epitopes will be useful as reagents to better define the structure of HCV E2. The antibodies will also be very useful reagents in assessing the importance of the interaction between HCV E2 and CD81 in HCV infection. More importantly, if CD81 is confirmed to be a receptor or coreceptor for HCV, the antibodies that inhibited binding of HCV virions to human CD81, CBH-2, and CBH-5 have the potential to mediate virus neutralization. The absence of a true in vitro model for virus neutralization, however, will require that the fundamental proof be obtained by the ability of selected HMAbs to prevent or modify HCV infection in appropriate animal models. If successful, broadly reactive neutralizing antibodies will likely have therapeutic utility. Analogous to the success achieved with hepatitis B immunoglobulin in liver transplantation (7
), one possible application is to suppress HCV infection in liver transplant recipients with broadly reactive neutralizing HMAbs.