Inactivated genotype 1a, 2a and 3a HCV vaccine candidates induced broadly neutralising antibodies in mice

Abstract

Objective: A prophylactic vaccine is needed to control the HCV epidemic, with genotypes 1-3 causing >80% of worldwide infections. Vaccine development is hampered by HCV heterogeneity, viral escape including protection of conserved neutralising epitopes and suboptimal efficacy of HCV cell culture systems. We developed cell culture-based inactivated genotype 1-3 HCV vaccine candidates to present natively folded envelope proteins to elicit neutralising antibodies.

Design: High-yield genotype 1a, 2a and 3a HCV were developed by serial passage of TNcc, J6cc and DBN3acc in Huh7.5 cells and engineering of acquired mutations detected by next-generation sequencing. Neutralising epitope exposure was determined in cell-based neutralisation assays using human monoclonal antibodies AR3A and AR4A, and polyclonal antibody C211. BALB/c mice were immunised with processed and inactivated genotype 1a, 2a or 3a viruses using AddaVax, a homologue of the licenced adjuvant MF-59. Purified mouse and patient serum IgG were assayed for neutralisation capacity; mouse IgG and immune-sera were assayed for E1/E2 binding.

Results: Compared with the original viruses, high-yield viruses had up to ~1000 fold increased infectivity titres (peak titres: 6-7 log10 focus-forming units (FFU)/mL) and up to ~2470 fold increased exposure of conserved neutralising epitopes. Vaccine-induced IgG broadly neutralised genotype 1-6 HCV (EC50: 30-193 µg/mL; mean 71 µg/mL), compared favourably with IgG from chronically infected patients, and bound genotype 1-3 E1/E2; immune-sera endpoint titres reached up to 32 000.

Conclusion: High-yield genotype 1-3 HCV could be developed as basis for inactivated vaccine candidates inducing broadly neutralising antibodies in mice supporting further preclinical development.

Keywords: HCV; chronic viral hepatitis; hepatitis C; immune response; molecular biology.

Figure 1

In vitro serial passage resulted in high-yield polyclonal genotype 1a, 2a and 3a HCV. Huh7.5 cells were inoculated with cell culture supernatant containing the specified viruses derived from the previous passage culture at the peak of infection, as determined by immunostaining. Every 2–3 days, passage cultures were split, and % HCV antigen positive cells and HCV infectivity titres were determined by immunostaining and infectivity titration, respectively. Peak supernatant HCV infectivity titres determined for the specified passages are means of three replicates with standard deviations (SD). For TNcc, the later passage line was inoculated with passage 10 virus from an initial passage line. Blue frames, passage subjected to NGS. Green frame, passage used for production of genotype 2a seed stock. Red frame, passage subjected to NGS and used for production of genotype 1a seed stock. The genotype 3a seed stock was based on the later developed DBNcc-HI recombinant. FFU, focus-forming unit; NGS, next-generation sequencing.

Figure 2

Nucleotide changes in serially passaged genotype 1a, 2a and 3a HCV. For TNcc-PP-38.1, J6cc-PP-35 and DBNcc-PP-16, coding and silent changes with allele frequency >10% determined by NGS of the complete ORF are shown. Coding changes with frequency of ≥80% are specified above bars. Genome positions relate to TNcc, J6cc and DBN3acc(GenBank accession numbers JX993348, JQ745650 and KX280714, respectively). For genome positions relating to the H77 (AF009606) reference genome and encoded amino acid changes, see online supplemental tables 4–6. NGS, next-generation sequencing; ORF, open reading frame.

Figure 3

Engineered high-yield genotype 1a, 2a and 3a HCV recombinants had increased viral fitness in cell culture. (A) Specified recombinants were transfected into Huh7.5 cells using the same amount of HCV RNA in vitro transcripts for recombinants directly compared in each graph. (B) In first passage kinetic experiments, cells were infected at multiplicity of infection of 0.002 with original and HI-viruses using supernatants derived from the transfection experiment when peak infectivity titres were observed; polyclonal passage viruses were included for comparison. (A and B) Every 2–3 days, passage cultures were split, and % HCV antigen positive cells and HCV infectivity titres were determined by immunostaining and infectivity titration, respectively. HCV infectivity titres are means of three replicates with SD. FFU, focus-forming unit; MOI, multiplicity of infection.

Figure 4

Engineered high-yield genotype 1a, 2a and 3a HCV recombinants showed increased sensitivity to neutralisation by human-derived nAb. Recombinants with in vivo derived genotype(isolate) 1a(TN), 2a(J6) and 3a(DBN) core-NS2 sequences, as well as TNcc-HI, J6cc-HI, DBNcc-HI, the genotype 1a HCV seed stock and DBN3acc with envelope protein substitutions acquired during in vitro passage were subjected to neutralisation with human mAb AR3A and AR4A, and polyclonal antibody C211. Data points are means of three replicates with SD; curves were fitted, and EC50 were calculated with the formula y=100/(1+10^{(log10EC50−X)×hillslope}) using GraphPad prism. Fold increase in neutralisation sensitivity was calculated as [(EC50 of 1a(TN), 2a(J6) or 3a(DBN) virus with in vivo derived envelope protein sequence)/(EC50 of respective virus with in vitro derived envelope protein substitutions)]. Virus stock envelope protein sequences were confirmed by Sanger sequencing. EC50, half maximal effective concentration; gt, genotype; mAb, monoclonal antibody; nAb, neutralising antibody.

Figure 5

Immunisation with inactivated genotype 1a, 2a or 3a HCV elicited antibodies neutralising cell culture infectious HCV of the same genotype. Groups of three mice were immunised with inactivated genotype 1a, 2a and 3a HCV or OVA formulated with adjuvant AddaVax. Purified serum IgG from individual mice was used to neutralise recombinants containing in vivo derived genotype(isolate) 1a(TN), 2a(J6) and 3a(S52) specific core-NS2. Data points are means of three replicates with SD; curves were fitted, and EC50 were calculated with the formula y=100/(1+10^{(log10EC50−X)×hillslope}) using GraphPad prism. Each concentration–response curve specified by unique symbols represents data from one animal. Virus stock envelope protein sequences were confirmed by Sanger sequencing. EC50, half maximal effective concentration; nAb, neutralising antibody; OVA, ovalbumin.

Figure 6

Immunisation with inactivated genotype 1a, 2a or 3a HCV elicited broadly nAb. Purified IgG from mice of each group, immunised with either genotype 1a, 2a or 3a HCV, was pooled using the same amount of IgG from each animal. IgG pools were used to neutralise recombinants containing in vivo derived genotype(isolate) 1a(TN), 1b(J4), 2a(J6), 2b(J8), 3a(S52), 4a(ED43) and 5a(SA13) core-NS2 sequences; 6a(HK6a) contained two vital cell culture adaptive substitutions in E1 and E2. Data points are means of three replicates with SD; curves were fitted, and EC50 were calculated with the formula y=100/(1+10^{(log10EC50−X)×hillslope}) using GraphPad prism. Virus stock envelope protein sequences were confirmed by Sanger sequencing. EC50, half maximal effective concentration; nAb, neutralising antibody.

Figure 7

Vaccine-induced nAb responses compare favourably with those in patients with chronic HCV infection. Purified IgG from patients chronically infected with HCV of genotype 1a, 2a/2b or 3a at the specified concentrations was used to neutralise recombinants containing in vivo derived genotype(isolate) 1a(TN), 2a(J6), 3a(S52) and 5a(SA13) core-NS2 sequences. Genotype 2a versus 2b patients are indicated by grey versus black open circles. Data points obtained in neutralisation assays with the same concentrations of purified mouse IgG shown in figure 6 are replotted for comparison. All data points are means of three replicates. Virus stock envelope protein sequences were confirmed by Sanger sequencing. gt, genotype; nAb, neutralising antibody.

Figure 8

Immunisation with inactivated genotype 1a, 2a or 3a HCV elicited antibodies binding to HCV envelope proteins. Binding capacity of pooled (A) purified serum IgG or (B and C) immune-sera from mice immunised with inactivated genotype 1a, 2a or 3a HCV or OVA to E1/E2 complexes of the specified HI-recombinants was evaluated by ELISA. Values are optical density (OD) reads at 450 nm following subtraction of mean OD of eight negative controls. Data points are means of two replicates with SD. (A) Positive controls: instead of serum IgG, well-characterised primary antibodies were used: AP33 for binding to TNcc-HI and DBNcc-HI E1/E2 and H77.39 for binding to J6cc-HI E1/E2. Negative controls: no IgG or immune-sera were used, and TNcc-HI, J6cc-HI and DBNcc-HI E1/E2 were incubated with secondary antibody only; for negative controls, OD reads were ~0.05. (A and B) In the OVA graphs, data points reflecting binding to J6cc-HI E1/E2 and DBNcc-HI E1/E2 were nudged by 0.04 and 0.08 units in the y direction, respectively. (C) Immune-sera endpoint titres were determined as the highest serum dilution yielding an OD >2 fold mean OD of negative controls. OVA, ovalbumin.