Broadly Neutralizing Antibodies Targeting New Sites of Vulnerability in Hepatitis C Virus E1E2

Abstract

Increasing evidence indicates that broadly neutralizing antibodies (bNAbs) play an important role in immune-mediated control of hepatitis C virus (HCV) infection, but the relative contribution of neutralizing antibodies targeting antigenic sites across the HCV envelope (E1 and E2) proteins is unclear. Here, we isolated thirteen E1E2-specific monoclonal antibodies (MAbs) from B cells of a single HCV-infected individual who cleared one genotype 1a infection and then became persistently infected with a second genotype 1a strain. These MAbs bound six distinct discontinuous antigenic sites on the E1 protein, the E2 protein, or the E1E2 heterodimer. Three antigenic sites, designated AS108, AS112 (an N-terminal E1 site), and AS146, were distinct from previously described antigenic regions (ARs) 1 to 5 and E1 sites. Antibodies targeting four sites (AR3, AR4-5, AS108, and AS146) were broadly neutralizing. These MAbs also displayed distinct patterns of relative neutralizing potency (i.e., neutralization profiles) across a panel of diverse HCV strains, which led to complementary neutralizing breadth when they were tested in combination. Overall, this study demonstrates that HCV bNAb epitopes are not restricted to previously described antigenic sites, expanding the number of sites that could be targeted for vaccine development.IMPORTANCE Worldwide, more than 70 million people are infected with hepatitis C virus (HCV), which is a leading cause of hepatocellular carcinoma and liver transplantation. Despite the development of potent direct acting antivirals (DAAs) for HCV treatment, a vaccine is urgently needed due to the high cost of treatment and the possibility of reinfection after cure. Induction of multiple broadly neutralizing antibodies (bNAbs) that target distinct epitopes on the HCV envelope proteins is one approach to vaccine development. However, antigenic sites targeted by bNAbs in individuals with spontaneous control of HCV have not been fully defined. In this study, we characterize 13 monoclonal antibodies (MAbs) from a single person who cleared an HCV infection without treatment, and we identify 3 new sites targeted by neutralizing antibodies. The sites targeted by these MAbs could inform HCV vaccine development.

Keywords: Flaviviridae; broadly neutralizing antibodies; epitope; hepatitis C virus; hepatitis C virus clearance; humoral immunity; neutralizing antibodies.

FIG 1

Subject C18 spontaneously cleared one HCV infection and then became persistently infected with a second HCV strain. The limit of detection (LOD) of the viral load assay (50 international units [IU]/ml) is indicated by a dashed line. Values below the LOD are set at 25 IU/ml and marked with open circles. The arrow at 1,483 days postinfection indicates the time point at which MAbs were isolated from circulating B cells.

FIG 2

C18 MAbs targeted six distinct antigenic sites. Hierarchical clustering of 13 C18 MAbs (in bold type) and 13 reference MAbs based on Ward’s minimal variance analysis of MAb binding to a library of strain H77 E1E2 proteins with single alanine mutations spanning the full E1E2 sequence, depicted as an unrooted tree. Bootstrap values greater than 80 are shown. MAbs that clustered together with bootstrap values of >90 were highlighted in the same color and given a label indicating a previously described (AR1, AR2, AR3, and AR4-5) or novel (AS108, AS112, and AS146) antigenic site.

FIG 3

Conformational E1E2 binding. Binding of C18 MAbs to native and denatured E1E2 proteins. HEPC85 and HEPC3 bind to linear and conformational epitopes, respectively, and are shown as controls (31).

FIG 4

Mapping of probable binding residues of each MAb onto the E2 ectodomain crystal structure. One C18 MAb representative of each antigenic cluster in Fig. 2 is shown, with the reference MAb or MAbs with which is shares the most probable binding residues, namely, (A) HEPC153, (B) HEPC111, (C) HEPC167, (D) HEPC108, (E) HEPC146, and (F) HEPC112. For each MAb, up to 15 probable E2-binding residues are shown, marked with colored spheres on the HCV E2 structure (Flyak et al., PDB 6MEI) (34). Probable E1-binding residues are shown in tabular form. These 15 E1E2 binding residues were also subdivided into tertiles of greatest effect (magenta), middle effect (orange), and least effect (yellow) of mutation to alanine on MAb binding. E2 structural components were highlighted in PyMOL, including the AS412 site (purple), front layer (cyan), VR2 (brown), CD81 binding loop (blue), beta sandwich (red), VR3 and post-VR3 (gray), and back layer (green). Dashed line indicates the stalk region of E2 not present in the ectodomain structure. MAb names are color-coded according to hierarchical clustering in Fig. 2.

FIG 5

C18 MAbs formed five groups based on E1E2 competition binding. Percent inhibition of binding of 2 μg/ml or 0.4 μg/ml of biotinylated MAbs to E1E2 proteins in an ELISA in the presence of blocking MAbs at a concentration of 20 μg/ml. Squares at the intersection between blocking and biotinylated MAbs are color coded by the degree of inhibition. Groups of MAbs that were mutually inhibitory (i.e., binding of each MAb inhibited each other MAb in the group and was inhibited by each other MAb in the group) are marked with thick black boxes. Values are averages of three independent experiments, each performed in duplicate. MAb names are color coded according to hierarchical clustering in Fig. 2 Data not available is marked “na.”

FIG 6

MAbs targeting four antigenic sites were broadly neutralizing. Neutralizing breadth of the C18 MAbs was measured using a diverse panel of genotype 1a and 1b HCV pseudoparticles (HCVpps) and genotype 2–6 HCVccs. Percent neutralization of each strain by each MAb at a 50-μg/ml concentration is shown, with HCVpp or HCVcc on the y axis and MAbs arranged from greatest to least neutralizing breadth on the x axis. HCVpp values are averages of two independent experiments, each performed in duplicate. HCVcc values are from a single experiment performed in duplicate. MAb names are color coded according to hierarchical clustering in Fig. 2.

FIG 7

Polymorphisms outside MAb binding sites influence bNAb resistance. Amino acids present at probable binding residues of HEPC111 (A), HEPC153 (B), and HEPC151-1 (C) across all strains in the HCVpp panel. Homology to the strain H77 sequence is indicated with a dot. Percent neutralization of each HCVpp strain by 50 μg/ml of each MAb is shown. Values are averages of two independent experiments, each performed in duplicate. For each MAb, full neutralization curves were also measured in duplicate with 5-fold serial dilutions of MAb starting at 100 μg/ml for one sensitive and two more resistant HCVpp strains. Error bars indicate standard deviations. MAb names are color coded according to hierarchical clustering in Fig. 2.

FIG 8

C18 MAbs formed three functional groups based on neutralization profiles. Neutralization of 18 HCVpps by 50 μg/ml of each MAb was measured in duplicate, generating a neutralization profile for each MAb (i.e., a ranking of HCVpp strains by relative neutralization sensitivity). Pearson’s correlations were measured between these C18 MAb neutralization profiles and neutralization profiles of reference MAbs. (A) Neutralization testing of reference MAb HEPC74 was performed in parallel with testing of C18 MAbs. Correlation was measured between the neutralization profile of this repeat test of HEPC74 (HEPC74-test) and the previously published HEPC74 neutralization profile (HEPC74-reference, left) or an unrelated MAb (AR5A-reference, right). Each point indicates a single HCVpp, with rank of neutralization sensitivity to one MAb on the x axis and another MAb on the y axis. (B) Pearson r values of pairwise correlations between neutralization profiles of each C18 MAb (y axis) and each reference MAb (x axis). Only r values that are statistically significant (P < 0.05) are shown, and darker green color indicates a stronger positive correlation. The highest r value for each C18 MAb is boxed and in bold type. C18 MAbs clustered into three functional groups, namely, AR3-like, AR1-like, and AR5-like.

FIG 9

C18 MAbs displayed complementary neutralizing breadth. C18 MAbs were tested for neutralization of the HCVpp and HCVcc panels individually at 50 μg/ml or in two different 3-MAb combinations at 50-μg/ml total antibody concentration (i.e., 16.7 μg/ml of each MAb), namely, (A) HEPC146, HEPC153, and HEPC151-1; and (B) HEPC111, HEPC153, and HEPC158. Lines connect neutralization of each HCV strain by each individual MAb and the 3-MAb combination. Blue lines indicate strains not neutralized >50% by the MAb combination. MAb names are color coded according to hierarchical clustering in Fig. 2. Percent neutralization values are the average of duplicate measurements.