Induction of broadly neutralizing antibodies using a secreted form of the hepatitis C virus E1E2 heterodimer as a vaccine candidate

Abstract

SignificanceHepatitis C virus chronically infects approximately 1% of the world's population, making an effective vaccine for hepatitis C virus a major unmet public health need. The membrane-associated E1E2 envelope glycoprotein has been used in clinical studies as a vaccine candidate. However, limited neutralization breadth and difficulty in producing large amounts of homogeneous membrane-associated E1E2 have hampered efforts to develop an E1E2-based vaccine. Our previous work described the design and biochemical validation of a native-like soluble secreted form of E1E2 (sE1E2). Here, we describe the immunogenic characterization of the sE1E2 complex. sE1E2 elicited broadly neutralizing antibodies in immunized mice, with increased neutralization breadth relative to the membrane-associated E1E2, thereby validating this platform as a promising model system for vaccine development.

Keywords: E1E2 envelope glycoproteins; hepatitis C virus; secreted; vaccine.

Fig. 1.

Construction and characterization of mbE1E2, sE1E2.LZ, and sE2. (A) Schematic diagram of full-length mbE1E2, sE1E2 with c-Fos/c-Jun and furin cleavage sites (red), and sE2. Signal sequences (tPA and IgK) and 6× His tags are shown. (B) SDS/PAGE analysis of purified mbE1E2, sE1E2.LZ, and sE2 under reducing conditions. (C) Western blot detection of the purified proteins under reducing and nonreducing conditions using anti-E2 mAb (HCV1) and anti-E1 mAb (H-111) as probes. D, dimer; M, monomer; T, trimer.

Fig. 2.

Immunogenicity assessment of antibody inducted in immunized mice at day 56 by ELISA. (A) Mouse immunization schedule. (B) Anti-mbE1E2 titer. (C) Anti-sE1E2.LZ titer. (D) Anti-sE2 titer. (E) Binding to E1 peptides, E2 peptides, and c-Fos/c-Jun. The numbers presented in D represent the average of duplicate experiments. (F) Anti-mbE1E2 titers at different days postimmunization. In B–D, sera from individual mice immunized with the indicated antigen are analyzed. In E and F, pooled sera from the indicated groups are analyzed. Endpoint titers were calculated by curve fitting in GraphPad Prism software with endpoint OD defined as four times the highest absorbance value of day 0 sera. In D, peptide binding endpoint titers were calculated by curve fitting in GraphPad Prism software with endpoint OD defined as seven times the highest absorbance value of day 0. P values were calculated using Kruskal–Wallis analysis of variance with Dunn’s multiple comparison test, and significant P values are shown (*P < 0.05). B represents previously published data from our team (66), shown here for comparison.

Fig. 3.

Competition ELISA using day 56 pooled serum with paired domain specific antibodies of E2 at domain B (AR3A and HEPC74), domain D (HC84.26 and HC84.1), domain E (HCV1 and HC33.1), and anti-E1E2 antibodies (AR4A and AR5A). Serum competition with HCV E1-specific antibodies (H-111 and IGH526) and nonneutralizing antibodies (CBH-4B and CBH-4G) was also analyzed. The value shown is the percentage inhibition relative to the antibody in the absence of serum.

Fig. 4.

Competition ELISA of individual mouse Day 56 serum at 1:60 dilutions with anti-E2 antibodies: domain B AR3A (A), domain D HC84.26 (B), domain E HCV1 (C); anti-E1E2 antibodies: AR4A (D), AR5A (E); and anti-E1 antibody H-111 (F). P values were calculated using Kruskal–Wallis ANOVA with Dunn’s multiple comparison test and significant P values are shown (**P < 0.01).

Fig. 5.

Kinetics of HCVpp neutralization. (A) Neutralization titers (ID₅₀s) against homologous isolate (H77C, GT1a). (B) The same shown against heterologous isolate (J6, GT2a). Serial dilutions of pooled sera from the indicated days were used, and titers were calculated as serum dilution levels reached at 50% neutralization (ID₅₀) by curve fitting in GraphPad Prism software.

Fig. 6.

Breadth of neutralization against all HCV genotypes with HCVpp. (A–I) Individual immunized mice sera were assessed for neutralization activities at day 56 and day 0 against seven genotypes of HCVpp. Neutralization titers were calculated as serum dilution levels reached at 50% neutralization (ID₅₀) by curve fitting in Graphpad Prism software. Serum dilutions were performed as two-fold dilutions starting at 1:64 for HCVpp neutralization. ID₅₀ values are plotted on a log₁₀ scale on the y axis. P values were calculated using Kruskal–Wallis analysis of variance with Dunn’s multiple comparison test, and significant P values are shown (*P < 0.05). The H77C neutralization data (A) were previously published by our team (66) and are shown here for comparison.

Fig. 7.

Breadth of neutralization against all HCV genotypes with HCVcc. Day 56 immunized mice pooled sera were analyzed for neutralization using chimeric HCVcc with H77C (GT1a), J4 (GT1b), Con1 (GT1b), J8 (GT2b), S52 (GT3a), ED43 (GT4a), SA13 (GT5a), HK (GT6a), QC69 (GT7a). Percent neutralization was calculated using RLU normalized to RLU of supernatant cultured without HCVcc nor serum (100%) and RLU of supernatant cultured with HCVcc without serum (0%). ID₅₀ neutralization was calculated from the sigmoid curve. Dotted line indicates highest concentration of serum.

Fig. 8.

Heat map ID₅₀ showing heterologous neutralization for three immunized groups. HCVpp neutralization (Left) and HCVcc neutralization (Right). Each row corresponds to an HCV genotype represented as HCVpp or HCVcc, and cell colors represent mean group ID₅₀ values from Fig. 6, or pooled serum ID₅₀ values from Fig. 7.