A Hepatitis C Virus Envelope Polymorphism Confers Resistance to Neutralization by Polyclonal Sera and Broadly Neutralizing Monoclonal Antibodies

Abstract

Hepatitis C virus (HCV) infection is a global health problem, with millions of chronically infected individuals at risk for cirrhosis and hepatocellular carcinoma. HCV vaccine development is vital in the effort toward disease control and eradication, an undertaking aided by an increased understanding of the mechanisms of resistance to broadly neutralizing antibodies (bNAbs). In this study, we identified HCV codons that vary deep in a phylogenetic tree of HCV sequences and showed that a polymorphism at one of these positions renders Bole1a, a computationally derived, ancestral genotype 1a HCV strain, resistant to neutralization by both polyclonal-HCV-infected plasma and multiple broadly neutralizing monoclonal antibodies with unique binding epitopes. This bNAb resistance mutation reduces replicative fitness, which may explain the persistence of both neutralization-sensitive and neutralization-resistant variants in circulating viral strains. This work identifies an important determinant of bNAb resistance in an ancestral, representative HCV genome, which may inform HCV vaccine development.

Importance: Worldwide, more than 170 million people are infected with hepatitis C virus (HCV), the leading cause of hepatocellular carcinoma and liver transplantation in the United States. Despite recent significant advances in HCV treatment, a vaccine is needed. Control of the HCV pandemic with drug treatment alone is likely to fail due to limited access to treatment, reinfections in high-risk individuals, and the potential for resistance to direct-acting antivirals (DAAs). Broadly neutralizing antibodies (bNAbs) block infection by diverse HCV variants and therefore serve as a useful guide for vaccine development, but our understanding of resistance to bNAbs is incomplete. In this report, we identify a viral polymorphism conferring resistance to neutralization by both polyclonal plasma and broadly neutralizing monoclonal antibodies, which may inform HCV vaccine development.

FIG 1

Codons 242, 424, and 570 show evidence of deep phylogenetic variation, and evolution at the 424 position is constrained. (A) Using a reference alignment of 390 genotype 1a HCV genomes and Bayesian phylogenetics, the identity of the most likely ancestral amino acid at each position across the HCV genome was calculated, generating the Bole1a genome (39). The histogram depicts the distribution of posterior probabilities with which a single most likely ancestral amino acid could be predicted at each of 3,012 codons. The posterior probabilities of leucine (L) at 242, arginine (R) at 424, and aspartic acid (D) at 570 are indicated. (B) Frequency of each observed amino acid at positions 242, 424, and 570 in a reference alignment of 390 genotype 1a sequences. Letters are standard IUPAC amino acid abbreviations.

FIG 2

Mutation of arginine (R) 424 to serine (S) confers resistance to neutralization by plasma. A panel of 13 Bole1a E1E2 constructs encoding different combinations of amino acids at codons 242, 424, and 570 was generated using site-directed mutagenesis. Each Bole1a E1E2 variant construct was used to generate HCVpp. HCVpp were tested for neutralization by the use of 5 to 10 HCV-infected plasma samples in duplicate, and reported values represent the results of an average of 1 to 4 independent experiments. HCVpp with the same amino acid polymorphism at 242, 424, or 570 were grouped for analysis. Horizontal lines indicate median percent neutralization. Significance was tested using one-way ANOVA with a Tukey test for multiple comparisons (****, P < 0.0001; ns, not significant). Samples with percent neutralization values of <0 are not shown.

FIG 3

Mutation of arginine (R) 424 to serine (S) confers resistance to neutralization by plasma. (A) Bole1a_R424 and Bole1a_S424 HCVpp were tested for neutralization by the use of an additional panel of 19 HCV-infected plasma samples in duplicate. Each point represents the mean of two replicate values. Each line indicates neutralization by a unique plasma sample. P values were calculated using a paired two-tailed Student's t test. (B) Bole1a_R424 and Bole1a_S424 E1E2 sequences were cloned into full-length replication-competent HCV (HCVcc) and used to produce infectious supernatants. Viral supernatants were incubated with a 1:100 dilution of HCV-infected plasma for 1 h and then used to infect Huh7.5.1 cells in triplicate. Values shown are means of results from replicate wells. Each line indicates neutralization by a unique plasma sample. P values were calculated using a paired two-tailed Student's t test. (C) E1E2 variants 1a53, 1a79, and H77 were mutated via site-directed mutagenesis to produce 1a53_S424, 1a79_S424, and H77_R424. Wild-type and mutant E1E2 HCVpp were made from these constructs and were then tested in duplicate for neutralization sensitivity using the panel of 19 HCV-patient plasma samples. Each point represents the mean of two replicate values, and each line indicates neutralization by a unique plasma sample. P values were calculated using a paired two-tailed Student's t test. Percentage neutralization values of <0 are not shown. (D) Bole1a_R424 and Bole1a_S424 HCVpp were purified by ultracentrifugation through a sucrose cushion and then analyzed by Western blotting. Blots were probed with human anti-E2 (HC33.1.53) (25) and mouse anti-HIV1 p24.

FIG 4

Mutation of arginine (R) 424 to serine (S) confers resistance to binding of plasma antibodies. (A) Lysates of cells transfected with Bole1a_R424 and Bole1_S424 E1E2 expression constructs were used in enzyme-linked immunosorbent assays (ELISAs) to assess E1E2 binding by IgG in the same panel of 19 HCV-infected plasma samples. Each of the 19 plasma samples was assayed in duplicate, and antibody binding was detected using HRP-conjugated anti-human IgG secondary antibody. Each point represents the mean of two replicate values, and each line indicates binding by IgG from a unique plasma sample. P values were calculated using a paired two-tailed Student's t test. OD₄₅₀, optical density at 450 nm. (B) Lysates of E1E2-transfected cells used for ELISA measurements were analyzed by Western blotting in three dilutions to confirm that equal quantities of E1E2 were present. Blots were probed using anti-E2 antibody HC33.1.53 (25).

FIG 5

Mutation of arginine 424 to serine confers resistance to neutralization by a diverse array of broadly neutralizing monoclonal antibodies. (A) A panel of six broadly neutralizing monoclonal human antibodies (CBH-5, HC84.22, HC84.26, AR3A, AR4A, and HC33.4) was selected to assess relative levels of neutralization sensitivity of Bole1a_R424 and Bole1a_S424 variants. The E2 core structure published by Kong and colleagues (20) (Protein Data Bank accession no. 4MWF) is shown with colors modified. The front layer is cyan; the CD81-binding loop is blue; the central β-sandwich is red. The 424 position is indicated with an arrow. Known critical binding residues of AR3A and HC84.22 are indicated with purple and yellow spheres, respectively. Critical binding residues of HC33.4 and AR4A are not present in this structure, but approximate binding positions are indicated along with their known critical binding residues. CBH-5 and HC84.26 (not shown) share multiple binding residues with AR3A and HC84.22, respectively. (B) Bole1a_R424 and Bole1a_S424 E1E2 sequences were cloned into full-length replication-competent HCV (HCVcc) and used to produce infectious virus. Titers of viral supernatants were determined and used to infect target cells in duplicate in neutralization assays with serial dilutions of six different broadly neutralizing MAbs or control IgG. Values shown are means, and error bars indicate standard deviations of the results of comparisons between replicates. (C) IC₅₀s calculated from the curves presented in panel B. For curves with only the highest antibody concentration producing more than 50% neutralization, the IC₅₀ is reported as ∼50 μg/ml; for curves with maximum neutralization of less than 50%, the IC₅₀ is reported as >50 μg/ml. (D) Bole1a_R424 and Bole1a_S424 HCVpp were tested for sensitivity to neutralization by a panel of six broadly neutralizing MAbs. Each HCVpp was incubated with 10 μg/ml MAb for 1 h prior to incubation with Hep3B target cells. Percent neutralization was calculated by comparing infection of HVCpp incubated with MAb to infection of HCVpp incubated with nonspecific IgG. Each construct was tested in duplicate. Values shown are means, and error bars indicate standard deviations. P values were calculated using a paired two-tailed Student's t test. Percentage neutralization values of <0 are not shown. (E) HCVpp 1a53, 1a53_S424, 1a79, 1a79_S424, H77, and H77_R424 were tested for neutralization sensitivity as described for Bole1a HCVpp in the panel D legend.

FIG 6

Mutation of arginine 424 to serine confers resistance to binding of broadly neutralizing monoclonal antibodies. (A) Binding of MAbs AR3A, CBH-5, HC84.22, and AR4A to Bole1a_R424 and Bole1a_S424 E1E2 lysates was measured by ELISA. MAbs were assayed in duplicate with 2.5-fold serial dilutions, starting at 10 μg/ml, and binding was detected using HRP-conjugated anti-human IgG secondary antibody. Values shown represent the means of the results determined with two replicates, and error bars indicate standard deviations. P values were determined by two-way ANOVA (****, P < 0.0001). (B) To compare the binding results seen under native and denatured E1E2 conditions, Bole1a_R424 and Bole1a_S424 E1E2 lysates were diluted either in a denaturing buffer containing sodium dodecyl sulfate (SDS) or in phosphate-buffered saline (PBS). The denatured lysates were also boiled for 5 min and then cooled on ice. Both native and denatured lysates were then added to G. nivalis lectin-coated plates, and binding of serial dilutions of MAb HC33.4 was quantitated in duplicate. Data shown represent the means of the results of two independent experiments, and error bars indicate standard deviations. (C) Binding of MAbs HC84.22, HC33.4, CBH-5, and AR4A to 1a53, 1a53_S424, 1a79, 1a79_S424, H77, and H77_R424 E1E2 lysates was measured by ELISA as described in the panel A legend. P values were determined by two-way ANOVA (*, P < 0.05; ****, P < 0.0001).

FIG 7

Bole1a_S424 carries a fitness cost. Bole1a_R424 and Bole1a_S424 E1E2 sequences were cloned into full-length replication-competent HCV (HCVcc). RNA was in vitro synthesized and transfected into Huh7.5.1 cells, and culture supernatants were collected. Transfection supernatants were subsequently used to infect Huh7.5.1 cells to expand viral stocks. Huh7.5.1 cells were infected in duplicate with serial dilutions of Bole1a_R424 HCVcc and Bole1a_S424 HCVcc supernatants. After 48 h, infection was quantified as the number of spot-forming units per milliliter of supernatant. Viral RNA was extracted from the same infection supernatants, and the viral RNA load was quantitated using real-time PCR and an international unit (IU) viral load standard. Specific infectivity is expressed as the number of spot-forming units per IU. Values shown represent means of the results of two independent experiments, and error bars indicate ranges.