Our laboratory and others have isolated broadly neutralizing HMAbs to overlapping epitopes of the E2 protein, which we designated domain B (21
). The breadth of their protective potential is exemplified by one antibody in particular that completely neutralized the diversity of HCV quasispecies in an infectious inoculum in a human liver-mouse chimeric model (27
). The successful isolation of these antibodies suggests that domain B is a highly immunogenic region on HCV E2 that contains a cluster of overlapping epitopes that are able to induce potent neutralizing antibodies in patients infected with HCV. The immunogenicity of this region is supported by a study showing that an HCVpp mutant with an alanine substitution at N532A exhibited greater sensitivity to neutralizing sera obtained from individuals infected with HCV genotype 1a, 1b, 2b, 3, 4, or 5 virus (10
). The findings revealed that these sera contain domain B-like neutralizing antibodies directed at residues near N532, in close proximity to residues contributing to domain B epitopes. The elimination of this N-glycosylation site allowed more efficient binding by these domain B-like antibodies to their epitopes surrounding N532 and more efficient virus neutralization (16
). Taken together, these results indicate that the epitopes comprising domain B are frequent targets of the humoral immune response in HCV-infected patients. Two questions are of concern from a vaccine perspective: which of the domain B epitopes are prone to accumulating mutations under immune pressure, leading to virus escape from neutralization, as observed with the antibody response to HVR1, and which domain B epitopes remain relatively invariant to accommodate the interactions of E2 with CD81 required for viral viability? We implemented in this study an approach that allowed escape variants to be amplified for detection by propagating infectious 2a HCVcc under increasing concentrations of a neutralizing antibody and by repeated passaging of the surviving virus to achieve a virus titer of 104
FFU/ml at each antibody concentration. This led to the isolation of escape mutants bearing mutations in a functional region on HCV E2 that has been associated with binding to CD81.
The studies reported here revealed three patterns contributing to the ability of HCV to escape from virus neutralization by domain B antibodies. For CBH-2, two sets of escape mutants containing mutations at D431G and A439E, respectively, were isolated. The induction of the escape mutant at D431G under the selective pressure of CBH-2 mimics the previously observed naturally occurring variant at this site, D431E, which was not neutralized by this antibody (22
). Thus, the design of the in vitro
escape selection method appears to recapitulate the evolution of viral antigenic determinants under immune pressure in humans. Furthermore, the similarity of the replication rate of infectious CBH-2 escape HCVcc mutants to that of wt HCVcc suggests that escape from CBH-2-like antibodies is similar to the observed rapid escape from the antibody response to HVR1 (40
) and does not compromise viral fitness. This pattern of virus escape partially explains the persistent viremia in the context of broadly neutralizing antibodies in the sera of patients with chronic HCV infection.
In the escape studies with HMAb HC-11, wt HCVcc was completely eliminated, and no escape mutants were generated under a starting antibody concentration of 100 μg/ml. By modifying the approach to growing the virus at increasing antibody concentrations starting at a low IC50 level of 0.05 μg/ml, an escape mutant with a single substitution at L438F was observed when the antibody concentration reached 10 μg/ml. When the virus was maintained at a steady concentration of 10 μg/ml, wt HCVcc disappeared, and a second mutant bearing a double substitution, L438F plus N434D, appeared. The L438F plus N434D mutant probably evolved from the L438F single-substitution mutant rather than from wt HCVcc (C). When the mixture of variants composed of 20% wt HCVcc, 70% L438F, and 10% L438F plus N434D was exposed to 100 μg/ml HC-11, wt HCVcc disappeared in a single passage and a second double-substitution mutant, L438F plus T435A, appeared. The pattern of rapid elimination of wt HCVcc with the appearance of the L438F plus T435A mutant again suggested that this variant evolved from the L438F mutant. Surprisingly, the appearance of a second mutation at 434 or 435 was not a compensatory change leading to a virus with greater fitness than the single L438F mutation. In fact, the opposite occurred, and the viral fitness of the L438F plus N434D and L438F plus T435A mutants was substantially less than that of the L438F mutant. Although the fine epitope-mapping studies by alanine substitution indicated that neither residue 434 or 435 is part of the HC-11 epitope (as defined by >80% decrease in binding associated with alanine substitution), a possible explanation is that these 2 residues, in proximity to 438, provide some component (albeit minor) to the HC-11 epitope. This is perhaps more likely with residue T435, as HC-11 bound less than 50% to an alanine substitution mutant at T435A compared to wt HCVpp (A). If that is the case, a more “complete” escape is achieved with the L435F plus T435A double mutation under the increased immune pressure associated with a higher concentration of HC-11 at 100 μg/ml. If T435A does not contribute to the HC-11 epitope, the expected outcome is for the L438F single-substitution mutant to persist at 100 μg/ml of HC-11. The contribution by N434 to the binding pocket for HC-11 is less clear. Alanine substitution at this site, N434A, is associated with only a 12% decrease in binding by HC-11. It is also possible that the addition of the N434D mutation to the L438F mutant at 10 μg/ml of HC-11, which leads to the double-substitution mutant, is a random event. Supporting this possibility is the continued presence of both single (L438F) and double (L438F plus N434D) escape mutants with prolonged exposure to 10 μg/ml of HC-11. If N434 contributes to the HC-11 epitope, the L438F plus N434D double-substitution mutant should be a surviving escape mutant. One can speculate that a stepwise pattern occurs in which the virus first accumulates the L438F mutant and then the T435A mutation becomes a mechanism for the virus to escape from prolonged exposure to a broadly neutralizing antibody at increasing concentrations. The implication for vaccine design is a requirement for the immunogen, containing an HC-11-like epitope, to be able to elicit a high-titer antibody response to avoid virus escape. If the antibody response is of low titer, virus escape can occur. Nonetheless, the escape mutations result in a progressive decrease in viral fitness alongside the virus's survival under the selective pressure of HC-11. The L438F single-substitution mutant yielded at least eight times less virus and the L438F plus T435A double-substitution mutant yielded 256 times less virus than wt HCVcc.
Epitope mapping by alanine scanning localized the binding regions of CBH-2 and HC-11 to two regions of HCV E2 encompassing aa 425 to 443 and aa 529 to 535. Both regions have been reported to contain contact residues that form the site of E2 binding to CD81 (9
). In a proposed model of the tertiary organization of HCV E2, based on the 3-domain organization of the class II viral membrane fusion proteins (25
), the residues in the CD81 binding site on HCV E2 lie in domain I and, more precisely, on its exposed C0
β-sheet (see reference 32a
for domain and strand nomenclature). The proposed HCV E2 domain I is organized so that β-strands C0
, as well as E0
, are consecutive in sequence from aa 418 to 444 and aa 526 to 542, respectively. In contrast, there is a long insertion between D0
, which is part of domain II. The locations of contact residues for the CBH-2 and HC-11 epitopes on C0
provide support for this model, in which these antibodies bind to the same tertiary structure that interacts with CD81.
The studies of the interaction between the HC-11 L438F escape mutant and CD81 suggested that the loss of viral fitness associated with this mutation is caused in part by a decreased ability of the escape mutant to interact with CD81. This implies that both discontinuous binding regions of HC-11, aa 425 to 443 and aa 529 to 535, contribute to the structure required for virus binding to CD81. This is consistent with a recent report that two HCV E2 regions encompassing aa 384 to 444 and aa 522 to 541 are required for proper protein folding to form the CD81 binding region (1
), which is also consistent with the proposed E2 model described above. However, the relative contributions to the structure required for CD81 binding appear different for aa 425 to 443 and aa 529 to 535. The CD81 contact residues within aa 529 to 535 (e.g., 529, 530, and 535) remain absolutely invariant to permit the interactions of E2 with CD81 required for virus viability () (30
). However, the residues within aa 425 to 443, depending on location and substitution, may or may not adversely affect viral fitness or distort the E2 tertiary structure required for this interaction with CD81. The HC-11 L438F-related escape mutants indicate that mutations in this region are more variable and that substitution at L438F is more tolerable from the perspective of virus survival, though it significantly reduces efficiency in binding to CD81. Thus, there appear to be two groups of residues forming the CD81 binding structure of E2. The first group, as exemplified by aa 529, 530, and 535, includes primary contact residues, where any amino acid substitution leads to complete abrogation of CD81 binding. The second group of residues, located in aa 425 to 443, has different degrees of modulating effects on virus binding to CD81. Depending on the location of the residue, such as aa 431, 439, or 438, amino acid substitution may have no effect or variable effects on CD81 binding. It is possibly that the L438F substitution leads to some degree of steric hindrance affecting the binding of the primary contact residues located in aa 529 to 535 to CD81. HC-1 differs from the other two antibodies in that the HC-1 epitope does not contain contact residues in the aa 425 to 443 region. It is probable that the failure of HCV to escape from HC-1 is due to a lack of contact residues in aa 425 to 443, a region having a greater ability to mutate without causing a lethal change to the virus. Collectively, these findings reveal a region on E2 that is responsible for virus escape from broadly neutralizing antibodies, highlight the substantial challenges inherent in developing HCV vaccines, and show that an effective vaccine will need to induce antibodies to intrinsically conserved epitopes in order to lessen the probability of virus escape.