Recombinant H77C gpE1/gpE2 heterodimer elicits superior HCV cross-neutralisation than H77C gpE2 alone

Abstract

Background & aims: An optimal HCV vaccine requires the induction of antibodies that neutralise the infectivity of many heterogenous viral isolates. In this study, we have focused on determining the optimal recombinant envelope glycoprotein component to elicit cross-neutralising antibodies against global HCV genotypes. We compared the immunoreactivity and antigenicity of the HCV genotype 1a strain H77C-derived envelope glycoprotein heterodimer gpE1/gpE2 with that of recombinant gpE2 alone.

Methods: Characterisation of the envelope glycoproteins was accomplished by determining their ability to bind to a panel of broadly cross-neutralising monoclonal antibodies. Immunogenicity was determined by testing the ability of vaccine antisera to neutralise the infectivity in vitro of a panel of pseudotyped HCV particles in which gpE1/gpE2 derived from representative isolates of the major global HCV genotypes were displayed.

Results: gpE1/gpE2 binds to more diverse broadly cross-neutralising antibodies than gpE2 alone and elicits a broader profile of cross-neutralising antibodies in animals, especially against more heterologous, non-1a genotypes. While not all heterologous HCV strains can be potently inhibited in vitro by gpE1/gpE2 antisera derived from a single HCV strain, the breadth of heterologous cross-neutralisation is shown to be substantial.

Conclusions: Our work supports the inclusion of gpE1/gpE2 in an HCV vaccine in order to maximise the cross-neutralisation of heterogenous HCV isolates. Our data also offers future directions in formulating a cocktail of gpE1/gpE2 antigens from a small selection of HCV genotypes to further enhance cross-neutralisation of global HCV strains and hopefully advance the development of a globally effective HCV vaccine.

Impact and implications: An HCV vaccine is urgently required to prevent the high global incidence of HCV infection and disease. Since HCV is a highly heterogeneous virus, it is desirable for a vaccine to elicit antibodies that neutralise the infectivity of most global strains. To this end, we have compared the immunoreactivity and antigenicity of recombinant H77C E1E2 heterodimer with that of H77C E2 alone and show that the former exhibits more cross-neutralising epitopes and demonstrates a broader cross-neutralisation profile in vitro. In addition, our data suggests a way to further broaden cross-neutralisation using a combination of E1E2 antigens derived from a few different HCV clades. Our work is relevant for the development of an effective global HCV vaccine.

Keywords: HCV; antibody; cross-neutralisation; vaccine.

Fig. 1.. Binding of HCV cross-neutralising human MAbs to purified E1E2 and E2 antigens.

Microtiter plates were coated either with purified recombinant E1E2 (circle, light blue) or E2 (diamond, dark blue) and probed with the 2-fold decreasing concentrations of HCV neutralising human mAbs (starting at 0.5 μg/ml to 10 μg/ml). Bound antibodies were detected by an alkaline phosphatase-conjugated anti-human secondary antibody. The mean optical densities measured at 450 nm for each mAb tested in two independent experiments are plotted vs. mAb concentration (μg/ml). E2-specific antibodies are AR3A, HC33.4, HC84.6 and HC-1AM. E1E2-specific antibodies are AR4A, AR5A. E1-specific antibody is H-111. mAbs, monoclonal antibodies.

Fig. 2.. Homologous neutralisation activity against H77C HCVpp by E1E2 and E2-immunised guinea pig antisera.

(A) Antisera from guinea pigs (G1-G8) either immunised with E1E2 (left) or E2 (right) were serially diluted and their abilities to block entry of HCV pseudoparticles pseudotyped with H77C E1E2 were determined as described. (B) IC₅₀ of these antisera were determined using GraphPad Prism software (version 9). The Mean of IC₅₀ between antisera from E1E2-and E2-immunised guinea pigs were compared. Unpaired Student’s t test showed p values >0.05 (non-significant (n.s.)).

Fig. 3.. Comparison of the neutralisation activity between antisera from E1E2-and E2-immunized guinea pigs.

The infectivity of sera from guinea pigs pre- or post-vaccination with E1E2 and E2 was tested against a panel of eight genotype 1a HCVpp (A) and eight non genotype 1a HCVpp (B) in Huh7.5 cells. Pre- and post-vaccinated sera were diluted at a 1:100 ratio. The amount of virus entry was measured by quantifying the HCVpp-encoded luciferase activity 48 h post incubation and the proportion of infectivity was normalized with HCVpp incubated without serum. Results were shown from at least two independent experiments in triplicate. Mean proportion of infectivity is denoted by a solid line. Student’s t tests were performed to compare between pre- and post-vaccination. ****p <0.0001; ***p <0.001; **p <0.01 and *p <0.05. n.s. indicates no significance. (C) The normalized neutralisation activity of post-vaccination antisera was represented in a heat map. Color codes: >75% neutralisation (red), >50% neutralisation (orange), >25% neutralisation (light green), and <25% neutralisation (white). Patterns of neutralisation from individual guinea pigs immunised with either E1E2 (upper panel) or E2 (lower panel) against a panel of 16 HCVpp are shown. Another representation of this figure is shown in Fig. S2 by arranging the HCVpp as tiers of neutralisation resistance as described. (D) The breadth of the cross-neutralisation conferred by E1E2-or E2-induced antisera was compared. The breadth of cross-neutralisation is determined by the number of isolates (out of the 16 HCVpp tested) that were neutralised by >50%. The mean of this number between guinea pigs immunised with E1E2 or E2 was compared by Student’s t tests. *Indicates statistical significance, p <0.05. HCVpp, HCV pseudoparticles.

Fig. 4.. The cross-neutralisation profile of antisera from E1E2-immunised guinea pigs.

The neutralisation activity of sera from guinea pigs pre- or post-vaccination with E1E2 was tested against an expanded panel of heterologous HCVpp in Huh 7.5 cells. Pre- and post-vaccinated sera were diluted at 1:100. The amount of virus entry was measured by quantifying the HCVpp-encoded luciferase activity 48 h post incubation and the proportion of infectivity was normalized with HCVpp incubated without serum. The data was calculated from three independent experiments, each performed with triplicate wells. Means of infectivity % are indicated. Student’s t tests were performed to compare between pre- and post-vaccination. ****p <0.0001; ***p <0.001; **p <0.01 and *p <0.05. n.s. indicates no significance. The normalized neutralisation activities of post-vaccination antisera against 30 HCVpp are represented in a heat map. The same scale was used as indicated in Fig. 3. HCVpp, HCV pseudoparticles.