Neutralization resistance of hepatitis C virus can be overcome by recombinant human monoclonal antibodies

Abstract

Immunotherapy and vaccine development for hepatitis C virus (HCV) will depend on broadly reactive neutralizing antibodies (NAbs). However, studies in infectious strain JFH1-based culture systems expressing patient-derived Core-NS2 proteins have suggested neutralization resistance for specific HCV strains, in particular, of genotype 2. To further examine this phenomenon, we developed a panel of HCV genotype 2 recombinants for testing of sensitivity to neutralization by chronic-phase patient sera and lead human monoclonal antibodies (HMAbs). The novel Core-NS2 recombinants, with patient-derived genotype 2a (strain T9), 2b (strains DH8 and DH10), and 2c (strain S83) consensus sequences, were viable in Huh7.5 hepatoma cells without requirement for adaptive mutations, reaching HCV infectivity titers of 3.9-4.5 log10 focus-forming units per milliliter. In in vitro neutralization assays, we demonstrated that the novel genotype 2 viruses as well as prototype strains J6/JFH1(2a) and J8/JFH1(2b), all with authentic envelope proteins, were resistant to neutralization by genotype 2a, 2b, 2c, 2j, 2i, and 2q patient sera. However, these patient sera had high titers of HCV-specific NAbs, because they efficiently reduced the infectivity of J6(2a) and J8(2b) with deleted hypervariable region 1. The genotype 2a, 2b, and 2c viruses, found resistant to polyclonal patient sera neutralization, were efficiently neutralized by two lead HMAbs (AR4A and HC84.26).

Conclusion: Using novel 2a, 2b, and 2c cell-culture systems, expressing authentic envelope proteins, we demonstrated resistance of HCV to patient-derived polyclonal high-titer NAbs. However, the same genotype 2 culture viruses were all sensitive to HMAbs recognizing conformational epitopes, indicating that neutralization resistance of HCV can be overcome by applying recombinant antibodies. These findings have important implications for HCV immunotherapy and vaccine development.

Fig. 1

Phylogenetic analysis of the Core-NS2 sequence of isolates used for intergenotypic JFH1-based cell-culture viruses. Genotype 1-7 isolates used for HCV Core-NS2 recombinants were previously described^–. Novel genotype 2a, 2b, and 2c isolates used in this study are indicated with diamonds. Alignment was made using ClustalW in MEGA software. The phylogenetic tree was generated using the Jukes Cantor model and Neighbor-joining algorithm. The percentages (≥80%) of 1000 replicates in which the associated sequences clustered together in the bootstrap test are shown; 80% was considered significant. The unit is the number of nucleotide substitutions per site.

Fig. 2

HCV infectivity titers in cell cultures after transfection with the novel genotype 2 Core-NS2 recombinants. Huh7.5 cells were transfected with RNA transcripts from T9/JFH1(2a), DH8/JFH1(2b), DH10/JFH1(2b), and S83/JFH1(2c) recombinants, and the previously developed J6/JFH1(2a) and J8/JFH1(2b) recombinants. The HCV infectivity titers are shown in bars for each recombinant virus at indicated time points. Data from different recombinants are from different experiments. J6/JFH1 was included as a common positive control; data from one representative experiment are shown. Error bars indicate standard errors of the mean (SEM) from at least triplicate determinations. The lower limit of detection in the experiments was 10^2.3 FFU/mL.

Fig. 3

Phylogenetic analysis of the HCV Core-E1 sequence of 28 genotype 2 patient samples. The genetic relatedness of the Core-E1 sequences (nts 868-1288) is shown. The corresponding Core-E1 sequences previously published for genotype 2a, 2b, 2c, 2i, 2j, and 2q isolates are included and marked with a blue box. The genotype 2 isolates used for development of novel recombinants, T9(2a), DH8(2b), DH10(2b), and S83(2c), are marked with a red box and the isolates used in previously developed recombinants, J6(2a) and J8(2b), are marked with a star. The sera selected for neutralization studies are marked with triangles. H77 is included as an out-group. The alignment was made using ClustalW in MEGA software and manually rearranged to obtain a codon-based nucleotide alignment. The phylogenetic tree was generated using the Jukes Cantor model and Neighbor-joining algorithm. The percentages (≥80%) of 1000 replicates in which the associated clustered together in the bootstrap test are shown. The unit is the number of nucleotide substitutions per site.

Fig. 4

Genotype 2 Core-NS2 HVR1-deleted HCV recombinant viruses tested against sera from 19 patients chronically infected with HCV genotype 2. Sera were used in neutralization assays in 5-fold dilution series (1:200 to 1:625,000). The graphs show the doses-response curves of genotype 2 Core-NS2 HVR1-deleted recombinant viruses J6/JFH1_ΔHVR1 (2a) and J8/JFH1_ΔHVR1 (2b) against 19 serum samples from patients with chronic HCV genotype 2; curves were fitted as described in Materials and Methods. The reciprocal serum dilution with a 50% reduction in FFU (IC₅₀-values) is indicated with two significant digits. Graph A-C show the result against J6/JFH1_ΔHVR1, while graphs D-F show the results against J8/JFH1_ΔHVR1. The genotype 2 subtype of the patient derived viruses is listed above the graphs and divided accordingly. The IC₅₀-values were determined by nonlinear regression (Graph-Pad Prism Software) as described in Materials and Methods. Error bars indicate standard errors of the mean (SEM) from three determinations.

Fig. 5

Genotype 2 Core-NS2 HCV recombinant viruses tested against two human monoclonal antibodies, AR4A and HC84.26. The dose-response neutralization of the genotype 2 Core-NS2 recombinant viruses J6/JFH1(2a), T9/JFH1(2a), J8/JFH1(2b), DH8/JFH1(2b), DH10/JFH1(2b), and S83/JFH1(2c) using HMAb AR4A (A) and HC84.26 (B) was determined in FFU reduction assays; curves were fitted as described in Materials and Methods. Each recombinant was tested against the two HMAbs at concentrations ranging from 0.008 to 25 μg/mL. The concentration with a 50% reduction in FFU (IC50-values) is indicated. An isotype-matched control was included for both HMAbs at 25 μg/mL and tested against all recombinant viruses (shown as color coded open symbols). Error bars indicate standard errors of the mean (SEM) from four determinations.