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, ϳ12% 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. IMPORTANCEHepatitis 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. H epatitis C virus (HCV) is a leading cause of liver cirrhosis and hepatocellular carcinoma in developed countries, with an estimated 170 million people being infected worldwide (1, 2). Of particular concern in the United States is the increasing number of cases in the 15-to 24-year-old age group, while the national rate of symptomat...

Structural flexibility at a major conserved antibody target on hepatitis C virus E2 antigen

“…From the present study, the CD81bs front layer resembles a highly flexible loop despite being held by two disulfide bonds and despite high sequence conservation, which is generally not associated with flexible loops in viral proteins. High flexibility is consistent with the observation that soluble E2 and E1E2 can elicit antibodies that can bind the CD81bs with high affinity but, nonetheless, cannot neutralize the virus (27)(28)(29)(30)(31). Some of these non-NAbs or weak NAbs could be similar to mAb 8, which recognizes a conformation likely to be poorly accessible on the virus (Fig.…”

“…Third, antibody responses to the CD81bs are frequently detected in sera from subjects immunized with the candidate vaccine immunogens: soluble E2 or E1E2. However, these serum antibodies are often weak or nonneutralizing despite high anti-E1E2 antibody titers (14,(27)(28)(29)(30). Indeed, mAbs have been isolated to the different components of the CD81bs, but only a few (mentioned above) cross-neutralize the highly diverse HCV genotypes (30)(31)(32)(33).…”

“…However, these serum antibodies are often weak or nonneutralizing despite high anti-E1E2 antibody titers (14,(27)(28)(29)(30). Indeed, mAbs have been isolated to the different components of the CD81bs, but only a few (mentioned above) cross-neutralize the highly diverse HCV genotypes (30)(31)(32)(33). In a panel of 37 mAbs isolated from mice immunized with E1E2, 20 (54%) and four (11%) bind to the E2 front layer and CD81bl, respectively, but none neutralize HCV (30).…”

“…Indeed, mAbs have been isolated to the different components of the CD81bs, but only a few (mentioned above) cross-neutralize the highly diverse HCV genotypes (30)(31)(32)(33). In a panel of 37 mAbs isolated from mice immunized with E1E2, 20 (54%) and four (11%) bind to the E2 front layer and CD81bl, respectively, but none neutralize HCV (30). The results indicate that the E2 front layer and CD81bl are highly immunogenic but likely contain structural features that act as decoys for the immune system, skewing the response to nonneutralizing epitopes during immunization.…”

Altered Glycosylation Patterns Increase Immunogenicity of a Subunit Hepatitis C Virus Vaccine, Inducing Neutralizing Antibodies Which Confer Protection in Mice

“…The resulting purified adenoviral bands were isolated using an 18.5-gauge needle and twice dialyzed against 4% sucrose. Adenovirus concentrations were measured at 10 12 times the dilution factor times the OD at 260 nm (27,28,(38)(39)(40). It was reported that N-glycans associated with HCV E2 could modulate receptor-binding affinity, as well as neutralizing-epitope recognition (41).…”