Broadly Neutralizing Antibody Mediated Clearance of Human Hepatitis C Virus Infection

Abstract

The role that broadly neutralizing antibodies (bNAbs) play in natural clearance of human hepatitis C virus (HCV) infection and the underlying mechanisms remain unknown. Here, we investigate the mechanism by which bNAbs, isolated from two humans who spontaneously cleared HCV infection, contribute to HCV control. Using viral gene sequences amplified from longitudinal plasma of the two subjects, we found that these bNAbs, which target the front layer of the HCV envelope protein E2, neutralized most autologous HCV strains. Acquisition of resistance to bNAbs by some autologous strains was accompanied by progressive loss of E2 protein function, and temporally associated with HCV clearance. These data demonstrate that bNAbs can mediate clearance of human HCV infection by neutralizing infecting strains and driving escaped viruses to an unfit state. These immunopathologic events distinguish HCV from HIV-1 and suggest that development of an HCV vaccine may be achievable.

Keywords: HCV clearance; antibody specificity; binding sites; hepatitis C virus; immunologic memory; neutralizing antibodies.

Figure 1.. Longitudinal neutralizing antibody responses of two subjects with HCV clearance and a third with persistence of infection.

(A) HCV RNA was measured at initial infection and then periodically for more than six years. Dashed line indicates limit of detection (LOD) of the HCV RNA assay. (B) Neutralizing breadth of longitudinal plasma samples or bNAbs HEPC3 and HEPC98 (isolated from C117), bNAbs HEPC74 and HEPC43 (isolated from C110), or CBH-2 (control bNAb). Values are the number out of nineteen HCVpp neutralized at least 50% by a 1:50 dilution of plasma, or by 10 μg/mL of mAb, tested in duplicate. (C) Spearman correlations were measured between neutralization profiles (i.e., patterns of relative neutralization potency across a panel of 19 HCV strains) of C117 or C110 plasma samples and autologous or negative control (CBH-2) bNAbs (see also Fig S1). Significant r values are colored with a yellow to green heatmap.

Figure 2.. Maximum likelihood phylogenetic trees of 5 kb hemigenomic HCV nucleotide sequences obtained by single-genome amplification from plasma at longitudinal time points throughout the course of infection.

Sequences are color-coded by duration of infection at the time of sampling. Transmitted/founder (T/F) sequences inferred by phylogeny and date of sampling are indicated. Identical sequences amplified in multiple independent reactions are boxed. Strains selected for further phenotypic analysis are indicated with an asterisk. All distances are drawn to scale, except distance to the outgroup. Nodes with bootstrap values >80 are indicated with black circles. (A) C117, clearance of a genotype 1a infection. (B) C110, clearance of three genotype 1a infections. (C) P29, persistence of a genotype 1a infection.

Figure 3.. Naturally selected substitutions in C117 E2 confer partial resistance to autologous plasma antibodies and autologous bNAbs.

(A) All substitutions arising longitudinally in E2 over the course of C117 infection. Dots indicate homology with the D17–1a53 (T/F) sequence. (B-D) Binding in an ELISA to longitudinal C117 E1E2 proteins (left) or neutralization of HCVpp expressing longitudinal C117 E1E2 proteins (right) by C117 D388 plasma IgG (B), HEPC3 (C), or HEPC98 (D) All assays were performed in duplicate, and values are means +/− SD. (See Fig S5 for additional independent experiments).

Figure 4.. Naturally selected substitutions in C110 E2 confer partial resistance to autologous plasma antibodies and autologous bNAbs.

(A) E2 polymorphisms where the D178-A31 (Infxn 2 T/F) sequence differs from both D0-A4 (Infxn 1) and Bole1a (an inferred ancestral reference sequence), and substitutions arising over the course of Infxn 2. Homology to the D0-A4 sequence is indicated with a dot. (B-D) Neutralization of HCVpp expressing longitudinal C110 E1E2 strains from Infxn 1 and 2 by D436 C110 plasma (B), HEPC43 (C), or HEPC74 (D). (E) E2 polymorphisms where the D1260-B3 (Infxn 3) sequence differs from both D0-A4 (Infxn 1) and Bole1a, and substitutions arising over the course of Infxn 3. Homology to the D0-A4 sequence is indicated with a dot. (F-H) Neutralization of HCVpp expressing longitudinal C110 E1E2 strains from Infxns 1 and 3 by D1875 C110 plasma (F), HEPC43 (G), or HEPC74 (H). (I) Substitutions arising in longitudinal P29 E1E2 clones. (J) Neutralization of HCVpp expressing longitudinal P29 E1E2 strains by autologous D416 plasma. All assays were performed in duplicate, and values are means +/− SD. (See Fig S5 for additional independent experiments.)

Figure 5.. Naturally selected HEPC3 and HEPC74/43 resistance substitutions lead to progressive loss of viral fitness.

Relative entry into hepatoma cells of HCVpp expressing: (A) Longitudinal natural C117 E1E2 strains (B) C117 D17 E1E2 before and after introduction by site directed mutagenesis (SDM) of the substitutions that arose outside of HVR1. Substitutions present in each SDM are indicated, with homology to the D17 sequence indicated with a dot. (C) Longitudinal natural C110 E1E2 strains (D) C110 D0 E1E2 before and after introduction by SDM of polymorphisms present in the front layer and VR2 of Infxn 2 viruses. (E) Longitudinal natural P29 E1E2 strains. HCVpp entry values were normalized to the entry of the chronologically earliest strain from each subject. Each symbol represents an independent experiment. Horizontal lines indicate means. (F-G) Infectivity of serial dilutions of chimeric replication competent cell culture virus (HCVcc) expressing longitudinal C117 (F) or C110 (G) E1E2 strains, measured as focus forming units/mL (FFU/mL) in Huh7.5.1 cells 48 hours after inoculation. Values are normalized for input HCV viral copy number. (See Fig S6 for second independent experiment). All assays were performed in duplicate, and values are means +/− SD.

Figure 6.. HEPC3 and HEPC74/43 resistance substitutions reduced E2 binding affinity for CD81 and SR-B1.

Binding of serial dilutions of soluble E2 (sE2) protein to CD81 or SR-B1 expressed on the surface of Chinese hamster ovary (CHO) cells, measured as median fluorescent intensity (MFI) of cells after incubation with concentration-normalized, His-tagged sE2 and a fluorescently labelled anti-His antibody. MFI values were normalized to binding of the chronologically earliest strain from each subject. (A-B) Longitudinal naturally occurring sE2 strains from C117. (C-D) C117 D17–1a53 sE2 or site-directed mutants D17-SDM3 (N434D, F465Y, D610H) or D17-SDM5 (N434D, H444Y, F465Y, P498S, D533N, D610H). (E-F) Longitudinal naturally occurring sE2 strains from C110. (G-H) C110 D0-A4 sE2 or site-directed mutant D0-SDM (L438I, F442I, N445H, R461L, A466D, A475T).

Figure 7.. BNAb resistance/loss of fitness substitutions selected in C117 and C110 E2 overlap with HEPC3 and HEPC74 binding epitopes, as well as the CD81 binding site.

Binding and resistance substitution positions are indicated with colored spheres superimposed on the HEPC3-E2ecto_1b09 structure. E2 structure is gray. (A) Main chain atoms of critical binding residues and/or contact residues of HEPC3 (yellow spheres) and C117 resistance/loss of fitness substitutions (magenta spheres). (B) Main chain atoms of critical binding residues and/or contact residues of HEPC74 (orange spheres) and C110 resistance/loss of fitness substitutions (blue spheres). (C) Main chain atoms of critical binding residues of CD81LEL (cyan spheres) and resistance/loss of fitness substitutions of C117 (magenta) or C110 (blue). Resistance/loss of fitness substitutions are indicated that fall at contact or critical binding residues (**) or within two amino acids of a contact or critical binding residue (*).