Native Folding of a Recombinant gpE1/gpE2 Heterodimer Vaccine Antigen from a Precursor Protein Fused with Fc IgG

Abstract

A recombinant strain HCV1 (hepatitis C virus [HCV] genotype 1a) gpE1/gpE2 (E1E2) vaccine candidate was previously shown by our group to protect chimpanzees and generate broad cross-neutralizing antibodies in animals and humans. In addition, recent independent studies have highlighted the importance of conserved neutralizing epitopes in HCV vaccine development that map to antigenic clusters in E2 or the E1E2 heterodimer. E1E2 can be purified using Galanthis nivalis lectin agarose (GNA), but this technique is suboptimal for global production. Our goal was to investigate a high-affinity and scalable method for isolating E1E2. We generated an Fc tag-derived (Fc-d) E1E2 that was selectively captured by protein G Sepharose, with the tag being removed subsequently using PreScission protease. Surprisingly, despite the presence of the large Fc tag, Fc-d E1E2 formed heterodimers similar to those formed by GNA-purified wild-type (WT) E1E2 and exhibited nearly identical binding profiles to HCV monoclonal antibodies that target conserved neutralizing epitopes in E2 (HC33.4, HC84.26, and AR3B) and the E1E2 heterodimer (AR4A and AR5A). Antisera from immunized mice showed that Fc-d E1E2 elicited anti-E2 antibody titers and neutralization of HCV pseudotype viruses similar to those with WT E1E2. Competition enzyme-linked immunosorbent assays (ELISAs) showed that antisera from immunized mice inhibited monoclonal antibody binding to neutralizing epitopes. Antisera from Fc-d E1E2-immunized mice exhibited stronger competition for AR3B and AR5A than the WT, whereas the levels of competition for HC84.26 and AR4A were similar. We anticipate that Fc-d E1E2 will provide a scalable purification and manufacturing process using protein A/G-based chromatography.

Importance: A prophylactic HCV vaccine is still needed to control this global disease despite the availability of direct-acting antivirals. Previously, we demonstrated that a recombinant envelope glycoprotein (E1E2) vaccine (genotype 1a) elicited cross-neutralizing antibodies from human volunteers. A challenge for isolating the E1E2 antigen is the reliance on GNA, which is unsuitable for large scale-up and global vaccine delivery. We have generated a novel Fc domain-tagged E1E2 antigen that forms functional heterodimers similar to those with native E1E2. Affinity purification and removal of the Fc tag from E1E2 resulted in an antigen with a nearly identical profile of cross-neutralizing epitopes. This antigen elicited anti-HCV antibodies that targeted conserved neutralizing epitopes of E1E2. Owing to the high selectivity and cost-effective binding capacity of affinity resins for capture of the Fc-tagged rE1E2, we anticipate that our method will provide a means for large-scale production of this HCV vaccine candidate.

Keywords: envelope glycoproteins; epitopes; hepatitis C virus; neutralizing antibodies; vaccines.

FIG 1

Purification of E1E2 from an Fc-tagged precursor. (A) Schematic representation of wild-type (WT) and Fc-tagged constructs and polypeptide processing. The E1E2 H77c (GenBank accession no. AF009606) polypeptide was expressed under the control of the CMV promoter (P_CMV) and preceded by the signal sequence from tissue plasminogen activator (tPA). For Fc-tagged E1E2, a duplication of the E2 N-terminal amino acids (384 and 385) (ET) was inserted, followed by the human IgG1 Fc tag (hu IgG1 Fc) and a PreScission protease (PP) recognition sequence (LEVLFQGP). Sizes of the polypeptide regions are shown at the top (aa, amino acids) as well as cleavage sites by signal peptidase (SP). (B) Capture of WT or Fc tag-derived (Fc-d) E1E2 from CHO cell extracts was performed with GNA and protein G Sepharose, respectively, and proteins were separated by SDS-PAGE and blotted with anti-E1 (A4) and anti-E2 (H52) monoclonal antibodies (MAbs). (C) Purified E1E2 antigens (with PP-mediated Fc tag removal step for Fc-d) were separated by SDS-PAGE. (Left) Western blot with anti-E1 (A4) and anti-E2 (H52) MAbs (1 μg loaded per lane); (right) Coomassie brilliant blue G250 (2 μg loaded per lane). (D) Wild-type and Fc tag-derived E1E2 antigens (1 μg/lane) were denatured at 95°C for 5 min in Laemmli buffer with (R) or without (NR) 1% β-mercaptoethanol. Samples were separated by SDS-PAGE and blotted with ant-E1 (A4) and anti-E2 (H52) MAbs.

FIG 2

Binding of HCV cross-neutralizing monoclonal antibodies to purified E1E2 antigens. Wild-type or Fc tag-derived antigens were used to coat ELISA plates in triplicate and probed with increasing concentrations of neutralizing human HCV monoclonal antibodies. Binding of E2 MAbs was detected by anti-human alkaline phosphatase-conjugated secondary antibody and p-nitrophenylphosphate substrate. The optical densities at 405 to 495 nm (normalized to the maximum, 24 μg/ml, of each MAb concentration tested) from two independent experiments are shown plotted versus MAb concentration (log₁₀ micrograms per milliliter). E2-specific antibodies included HC33.4, HC84.26, and AR3B. E1E2-specific antibodies included AR4A and AR5A. The control human anti-HIV IgG1 was B6 (normalized to the maximum dose of AR3B).

FIG 3

Neutralizing antibodies induced by vaccination with wild-type and Fc tag-derived E1E2. (A) Recombinant E2 HCV1 (1a) (amino acids 384 to 661; GenBank accession no. M62321.1) was used to coat ELISA plates in triplicate and probed with postvaccination mouse sera. Binding of E2-specific antibodies in sera from WT and Fc-d E1E2-vaccinated mice (1,000-, 5,000-, and 10,000-fold dilutions) compared to sera from control-vaccinated mice (1,000-fold dilution) were detected by anti-mouse horseradish peroxidase-conjugated secondary antibody and peroxidase substrate. The optical densities at 450 to 507 nm (OD450-507) (means and SEM) from three independent experiments are shown plotted versus serum dilution. (B) Neutralization of HCVpp H77c (1a) entry was performed using 2-fold dilutions (1:25 to 1:1,600) of pre- and postvaccination sera, and the ID₅₀s were determined (shown as the reciprocal value of the dilution to achieve 50% neutralization). The group mean with SEM is shown from a representative of two independent experiments. Vaccinated mouse groups: control (C), buffer plus alum/MPL; WT, GNA agarose-derived E1E2 H77c plus alum/MPL; and Fc-d, Fc tag-derived E1E2 H77c plus alum/MPL. *, P < 0.05 respective to control by one-way ANOVA by Kruskal-Wallis and Dunn's post hoc tests.

FIG 4

Comparison of neutralizing antibodies toward homologous (1a) and heterologous (5a) HCVpp. Neutralization of homologous HCVpp H77 (1a) and heterologous HCVpp SA13 (5a) was performed using pre- and postvaccination sera (1:50), and the group means with SEM were plotted from representatives of two independent experiments. Positive control, anti-CD81 MAb (1 μg/ml). *, P < 0.05 respective to control by one-way ANOVA and Tukey post hoc test.

FIG 5

Mouse antisera compete for the binding of HCV cross-neutralizing MAbs to E1E2. Competition studies with mouse antisera and a panel of cross-neutralizing human HCV antibodies were done. Microtiter wells containing GNA-purified E1E2 H77c were incubated with diluted postvaccination antiserum (1:100) in triplicate for 1 h, followed by incubation with the indicated MAb. Binding of the MAbs was detected with anti-human alkaline phosphatase-conjugated secondary antibodies. The percentages of MAb binding were calculated relative to the amount of MAb bound in the absence of antiserum. Shown are mean values for each group ± the range from two independent experiments. Vaccinated mouse groups: control (C), buffer plus alum/MPL; WT, GNA agarose-derived E1E2 H77c plus alum/MPL; Fc-d, Fc tag-derived E1E2 H77c plus alum/MPL. E2-specific antibodies included HC33.4, HC84.26, and AR3B. E1E2-specific antibodies included AR4A and AR5A. *, P < 0.05 by one-way ANOVA and Tukey's post hoc test. At n = 2, SEM is actually the same as the range.