Fine mapping of murine antibody responses to immunization with a novel soluble form of hepatitis C virus envelope glycoprotein complex

Abstract

The hepatitis C virus (HCV) envelope glycoprotein E1E2 complex is a candidate vaccine antigen. Previous immunization studies of E1E2 have yielded various results on its ability to induce virus-neutralizing antibodies in animal models and humans. The murine model has become a vital tool for HCV research owing to the development of humanized mice susceptible to HCV infection. In this study, we investigated the antibody responses of mice immunized with E1E2 and a novel soluble form of E1E2 (sE1E2) by a DNA prime and protein boost strategy. The results showed that sE1E2 elicited higher antibody titers and a greater breadth of reactivity than the wild-type cell-associated E1E2. However, immune sera elicited by either immunogen were only weakly neutralizing. In order to understand the contrasting results of binding and serum neutralizing activities, epitopes targeted by the polyclonal antibody responses were mapped and monoclonal antibodies (MAbs) were generated. The results showed that the majority of serum antibodies were directed to the E1 region 211 to 250 and the E2 regions 421 to 469, 512 to 539, 568 to 609, and 638 to 651, instead of the well-known immunodominant E2 hypervariable region 1 (HVR1). Unexpectedly, in MAb analysis, ∼ 12% of MAbs isolated were specific to the conserved E2 antigenic site 412 to 423, and 85% of them cross-neutralized multiple HCV isolates. The epitopes recognized by these MAbs are similar but distinct from the previously reported HCV1 and AP33 broadly neutralizing epitopes. In conclusion, E1E2 can prime B cells specific to conserved neutralizing epitopes, but the levels of serum neutralizing antibodies elicited are insufficient for effective virus neutralization. The sE1E2 constructs described in this study can be a useful template for rational antigen engineering.

Importance: Hepatitis C virus infects 2 to 3% of the world's population and is a leading cause of liver failures and the need for liver transplantation. The virus envelope glycoprotein complex E1E2 produced by detergent extraction of cells overexpressing the protein was evaluated in a phase I clinical trial but failed to induce neutralizing antibodies in most subjects. In this study, we designed a novel form of E1E2 which is secreted from cells and is soluble and compared it to wild-type E1E2 by DNA immunization of mice. The results showed that this new E1E2 is more immunogenic than wild-type E1E2. Detailed mapping of the antibody responses revealed that antibodies to the conserved E2 antigenic site 412 to 423 were elicited but the serum concentrations were too low to neutralize the virus effectively. This soluble E1E2 provides a new reagent for studying HCV and for rational vaccine design.

FIG 1

Characterization of sE1E2 constructs. (A) Schematic illustration of 6 sE1E2 constructs. The numbering corresponds to the amino acid positions in the polyprotein of the genotype 1a H77 strain. TMD, transmembrane domain; Strep-Tag II sequence, WSHPQFEK; long linker sequence, GGSSRSSSSGGGGSGGGG; TEV cleavage site sequence, GENLYFQ. (B) Antigenicity of sE1E2. The sE1E2 constructs were expressed in 293T cells by transient transfection in the presence of kifunensine and captured from the cell supernatants onto microwells by MAb AR4A. Captured sE1E2 was probed by MAbs AR1A, AR1B, AR2A, AR3A, AR4A, AR5A, IGH526, and CD81 LEL-Fc. As expected, poor binding was observed for the cell-associated wild-type E1E2 and for detection with MAb AR4A, which was the capture antibody. However, a binding signal was not detected for MAb AR2A, suggesting the absence of this isolate-specific E2 epitope on the engineered sE1E2 complexes. OD₄₅₀, optical density at 450 nm. (C) sE1E2, sE1E2v4, and E1E2 in cell supernatant and cell lysate. The expressed glycoproteins were captured onto ELISA plates precoated with MAb AR4A and detected with anti-E1 MAb IGH526 and anti-E2 MAb AR1B, as described for panel B. (D) Immunoblot analysis of sE1E2 and sE1E2v4. Soluble E1E2 was treated with TEV protease at 30°C for the specified times, and digested samples were resolved in a reducing SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane for immunoblotting. Mouse anti-E1 MAb A4 and human anti-E2 MAb HCV1 primary antibodies, followed by IRDye800CW anti-mouse (green signal) and IRDye700DX anti-human (red signal) secondary antibodies, were used. Codetection of E1 and E2 generated a yellow signal. (E) SDS-PAGE analysis of purified sE1E2v4. Five micrograms of sE1E2v4 proteins was resolved by reducing (lane 2) and nonreducing (lane 3) SDS-PAGE, and bovine serum albumin (BSA; reduced) (lane 1) was used as a control. The relative abundance of the sE1E2v4 oligomeric forms was determined by densitometry using ImageJ software, and it was found that the sE1E2v4 preparation contained 5%, 19%, 19%, and 37% monomers, dimers, trimers, and higher oligomers, respectively. The numbers on the left are molecular masses (in kilodaltons).

FIG 2

(A) Schematic representation of the study design. Mice were immunized with DNA vaccine at biweekly intervals and boosted with purified E2 protein (arrows), and blood samples obtained at the indicated time points were analyzed for humoral responses. (B) Antibody titers of pooled mouse and human sera to E1E2. Sera were serially diluted, and their reactivity against E1E2 lysate was determined by ELISA. The EC₅₀s of the polyclonal antibody responses were calculated from the titration curve, and values are the means from at least 3 independent experiments. *, P < 0.05). (C) Reactivity of immune sera (diluted 1:300) to native and reduced E1E2. The data shown are the mean values from at least 2 experiments.

FIG 3

Reactivity of sera from E1E2- and sE1E2-immunized animals (A) and MAbs (B) to a library of overlapping E1E2 peptides in ELISA. The MAbs not shown did not bind to the peptides in the ELISA and were assumed to bind discontinuous epitopes. All 7 neutralizing MAbs except 23D9 were mapped to the region from amino acids 412 to 423 of the E1E2 glycoprotein. *, MAb 22C9 was also mapped to this region but was nonneutralizing. Peptides were supplied by the NIH AIDS Research and Reference Reagent Program. Data shown are representative of those from 3 independent experiments performed in duplicate.

FIG 4

E2 residues required for MAb binding to the conserved E2 antigenic site 412 to 423. Expressed proteins with the indicated amino acid mutated to alanine were analyzed in an E1E2 capture ELISA. Bound antibodies were detected by peroxidase-conjugated goat anti-mouse IgG secondary antibody. The binding of antibody to each mutant is expressed as a percentage of the binding to H77 wild-type protein. Residual binding of <50% was defined as positive, and the critical residues are denoted with asterisks. Note the clear dependence of the contacting residues L413, N415, G418, and W420 for the binding of MAb HCV1, as revealed in the X-ray structure (55). Data shown are representative of those from 3 independent experiments performed in duplicate.