HCV infections are on the rise in the United States, reflecting increasing rates of opioid addiction (Zibbell et al., 2018). An HCV vaccine is urgently needed to control the epidemic, but vaccine development is challenging due to the enormous genetic diversity of the HCV envelope proteins (Yusim et al., 2010). The HCV genome encodes two structural proteins, E1 and E2, that associate to form a noncovalent heterodimer, E1E2 (Freedman et al., 2016). Potent bNAbs isolated from HCV-infected individuals predominantly target conserved epitopes in the front layer of the E2 glycoprotein. The majority of bNAbs that bind to the front layer are derived from VH1-69 genes (Tzarum et al., 2019), which are also associated with bNAbs that target conserved epitopes on influenza virus and HIV-1 envelope glycoproteins (Chen et al., 2019).

We recently described crystal structures of two VH1-69 bNAbs, HEPC3 and HEPC74, isolated from individuals who spontaneously cleared HCV infection (Flyak et al., 2018). Both bNAbs utilized a disulfide motif in their CDRH3 regions to recognize a conserved epitope in the front layer of E2. While the HEPC3 and HEPC74 CDRH3 loops adopted a straight ß-hairpin conformation, the VH1-69-encoded AR3A and AR3C bNAbs that were isolated from an individual with a chronic HCV infection included bent CDRH3 loops that contained an analogous disulfide motif (Kong et al., 2013). Since the two bNAbs with straight CDRH3s were isolated from individuals who spontaneously cleared HCV infection and the two bNAbs with bent CDRH3s were isolated from a single chronically-infected individual, we wondered if some individuals are naturally predisposed to make antibodies with straight or bent CDRH3s and/or whether the straight CDRH3 conformation was related to the ability to clear HCV infection. Among bNAbs isolated from a chronically-infected individual (Law et al., 2008), we found AR3X, a VH1-69-encoded antibody that included a CDRH3 with a disulfide motif and an unusually long 14-amino acid-long insertion in CDRH2 (Figure 1A). AR3X provided an opportunity to explore the structural plasticity of VH1-69-derived anti-HCV bNAbs with a disulfide-containing CDRH3 and to determine the impact of a long CDRH2 insertion on the recognition of the conserved epitope in E2 front layer.

Figure 1

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The most likely scenario resulting in the insertion into the CDRH2 of AR3X involves a duplication event, as the CDRH2 insertion has 69% identity with the N-terminal sequence preceding the CDRH2 (Figure 1B). Similar to other front layer-specific bNAbs with the CDRH3 disulfide motif (Figure 1E), the cysteines in the AR3X CDRH3 region are encoded by the human D gene segment 15 (IGHD2-15) (Figure 1C). The C-terminal portion of the AR3X CDRH3 is likely encoded by human J-gene segment 3*02 (J3*02). Not including the 14-amino acid insertion in CDRH2, AR3X shares 91% nucleotide identity with the V_H1-69 gene segment and includes 17 somatic mutations (Figure 1D). To investigate the importance of the CDRH2 insertion and the effects of somatic mutations on AR3X binding and neutralization, we generated a panel of AR3X variants: AR3X ΔINS (AR3X without the CDRH2 insertion), AR3Xrua (germline precursor of AR3X, which lacks the CDRH2 insertion and somatic mutations), and AR3Xrua + INS (germline precursor of AR3X with the CDRH2 insertion) (Figure 1D).

We evaluated the binding of AR3X and AR3X variants to a panel of E2 ectodomain (E2ecto) proteins representing the E2 envelopes from 19 HCV genotype 1 strains. We also tested the binding of AR3X and AR3X variants to E2ecto proteins from genotypes 2, 3, 4, 5, and 6 strains. AR3X recognized all 19 E2 envelopes from genotype 1 including the 1a116 strain, which was not recognized by other front layer-specific bNAbs that include the CDRH3 disulfide motif (Figure 2A, Figure 2—figure supplement 1; Flyak et al., 2018). AR3X also recognized E2 envelopes from genotypes 2, 3, 4, 5, and 6 (Figure 2A). In contrast to mature AR3X, the AR3X ΔINS protein that lacks the CDRH2 insertion bound only 4 of the 25 variants, indicating that the CDRH2 insertion mediates the breath of binding. While AR3Xrua failed to bind any E2ecto proteins, AR3Xrua + INS recognized 1 of the 25 variants, further highlighting the importance of the CDRH2 insertion in initial recognition of the E2 antigen by naïve B cells. The fact that AR3Xrua + INS only bound to one HCV strain, whereas mature AR3X recognized all strains, indicated that somatic mutations, in addition to the CDRH2 insertion, are required for breath of binding and optimal E2 recognition. Consistent with our previous studies in which the strain 1a157 E2ecto envelope was recognized by HEPC3, HEPC74, AR3C and their germline precursors (Flyak et al., 2018), AR3X and two AR3X variants (AR3X ΔINS, AR3Xrua + INS) also bound to 1a157, suggesting that immunogens based on the genotype 1 1a157 ectodomain sequence could be used to stimulate the development of potent front layer-specific bNAbs (Figure 2A, Figure 2—figure supplement 1).

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To evaluate the neutralization breadth of AR3X variants, we evaluated antibodies in an in vitro neutralization assay using a panel of 19 genotype 1 HCV pseudoparticles (HCVpp) that represents 94% of the amino acid polymorphisms present at >5% frequency in a reference panel of 643 genotype 1 HCV isolates from GenBank (Munshaw et al., 2012). Only mature AR3X exhibited neutralization activity, neutralizing 17 of 19 HCV strains (Figure 2B, Figure 2—figure supplement 2). The neutralization breadth of AR3X (89%) was slightly lower than the breath of AR3C bNAb (100%) (Flyak et al., 2018), which was isolated from the same HCV-infected individual (Law et al., 2008). AR3X variants failed to neutralize HCV isolates, suggesting that both the CDRH2 insertion and somatic mutations are required for the broad neutralization activity of AR3X.

We and others described two classes of VH1-69 bNAbs with a CDRH3 disulfide motif: bNAbs with a straight CDRH3 (HEPC3 and HEPC74) and bNAbs with a kinked CDRH3 (AR3A and AR3C) (Flyak et al., 2018; Kong et al., 2013; Tzarum et al., 2019; Figure 3). To determine to which class AR3X belongs, we determined the crystal structure of AR3X in complex with E2ecto from the 1b09 HCV strain (Figure 4, Figure 4—figure supplement 1). The 2.2 Å AR3X-E2ecto structure demonstrated that, similar to previously-characterized HCV bNAbs that recognize the neutralizing face of E2 (Flyak et al., 2018; Kong et al., 2013; Tzarum et al., 2019), AR3X binds to the conserved epitope in the E2 front layer (Figure 4A). The AR3X CDRH3 loop contains two cysteines that form a disulfide bond, as seen in multiple other E2 front layer-binding bNAbs, and the AR3X CDRH3 adopts the straight conformation we previously described in the HEPC3 and HEPC74 bNAbs that were isolated from an individual who cleared HCV infection (Flyak et al., 2018; Figure 3). By contrast, the CDRH3s of AR3A and AR3C, which were isolated from the same HCV-infected individual as AR3X (Law et al., 2008), are bent (Kong et al., 2013; Tzarum et al., 2019). The tip of the AR3X CDRH3 loop interacts with the same conserved residues in the front layer of E2 as the CDRH3 tips in the other HCV bNAbs (Figure 4e, Figure 4—figure supplement 2).

Figure 3

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Overall, AR3X has a similar binding footprint to the footprints of HEPC3, HEPC74, AR3C, and AR3A, sharing multiple contact residues in the front layer and CD81 receptor-binding loop (Figure 4—figure supplement 2). As also found for these other front layer-specific bNAbs, AR3X’s contacts with E2ecto almost exclusively involved V_H domain residues, burying 1,250 Ų (98% of the total Fab buried surface area; BSA) (Figure 4B), with the CDRH3 accounting for 44.5% (556 Ų) of the total BSA on the V_H domain (Figure 4B, Figure 4—figure supplement 2). However, in contrast to other front layer-specific bNAbs in which the CDRH3 plays a dominant role in the interactions with E2 envelope (Flyak et al., 2018; Kong et al., 2013; Tzarum et al., 2019), the main contributor to the AR3X-E2ecto binding interface was CDRH2, which accounted for 48.2% (602 Ų) of the total BSA of the V_H domain, with the majority of the binding footprint provided by the CDRH2 insertion (45.4% or 567 Ų of total BSA of the V_H domain) (Figure 4C).

We next investigated the frequency of antibodies with 14-residue CDRH2 insertions. While the size of an insertion or deletion in human antibody genes ranges from 3 to 33 nucleotides (Kanyavuz et al., 2019), AR3X has a unusually long 42-nucleotide insertion, which results in a 31-residue CDRH2 (Kabat definition: [Kabat and National Institutes of Health (U.S.). Office of the Director, 1991]). According to the abYsis database (Swindells et al., 2017), a typical human CDRH2 is 17 residues (relative frequency 67%) (Figure 4D), and CDRH2s longer than 20 residues are rare (relative frequency <1%). To our knowledge, AR3X with its 31-residue CDRH2 represents the longest CDRH2 among antibody structures available in the Protein Data Bank (PDB).

Although the CDH3s of AR3X, AR3A, AR3C, HEPC3, and HEPC74 CDRH3s make similar binding footprints on the E2 surface (Figure 5), the difference in Fab approach angles and the presence of the long insertion in the AR3X CDRH2 result in different footprints on E2 for the V_H1-69–encoded CDRH2 loops of the bNAbs: AR3X CDRH2 contacts the C-terminus of the E2 α1-helix, the portion of the E2 front layer between the α-helix, variable region 2 (residues 446–448), and the back layer of E2 (residues 444, 445) (Figure 4F and Figure 4—figure supplement 2). In contrast, the AR3A and AR3C CDRH2 contacts are reduced to hydrophobic residues in α1-helix (Kong et al., 2013), whereas the HEPC3 and HEPC74 CDRH2s contact the E2 α1-helix and the portion of the E2 front layer between the α1-helix and variable region 2 (residues 446–448) (Flyak et al., 2018).

Figure 5

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A feature of VH1-69-derived antibodies is the presence of two hydrophobic residues at the tip of the CDRH2 loop that facilitate interactions with hydrophobic epitopes. The CDRH2s of AR3A and AR3C contain an Ile/Val-Pro-Met/Leu-Phe motif in which hydrophobic residues interact with the E2 front layer and CD81 binding loop (Chen et al., 2019). The CDRH2s of HEPC3 and HEPC74 are less hydrophobic and contain a Thr/Ser-Pro-Ile-Phe/Ser motif (Chen et al., 2019). In addition to hydrophobic interactions with the E2 front layer, the HEPC3 CDRH2 also makes a single hydrogen bond with E2 (Flyak et al., 2018). By contrast, AR3X is a not a typical VH1-69 antibody in which hydrophobic residues in CDRH2 mediate the binding to hydrophobic residues in E2 (Chen et al., 2019). Instead, the AR3X CDRH2 forms eight hydrogen bonds with the E2 glycoprotein, four of which are mediated by AR3X residue Arg52g (AR3X-E2ecto: Pro52c-His445, Pro52e-Thr444, Arg52g-Ala440, Arg52g-Phe442, Arg52g-Tyr443, Arg52g-Pro612, Asn52n-Tyr443, Trp52i-Tyr613) (Figure 4F, Figure 4—figure supplement 2). Notably, these differences in binding interactions have functional implications, as these mAbs differ in potency of neutralization of individual HCV strains in the HCVpp panel. For example, the AR3X neutralization IC₅₀ for strain 1b21 is ~17 fold lower than the IC₅₀ of HEPC3 (1.2 vs. 20.5 µg/mL). In contrast, the AR3X neutralization IC₅₀ for strain 1a142 is ~9 fold higher than the IC₅₀ of HEPC3 (16.2 vs. 1.9 µg/mL) (Figure 2; Flyak et al., 2018).

A signature feature of the AR3A/AR3C and HEPC3/HEPC74 types of HCV bNAbs is the long CDRH3 that forms multiple main chain–main chain hydrogen bonds with E2 front layer residues (Flyak et al., 2018; Tzarum et al., 2019). Similar to other front layer-specific bNAbs with a CDRH3 disulfide motif, the first cysteine residue of the AR3X CDRH3 (Cys100a) hydrogen bonds with E2 residue Cys429 (Figure 4G, Figure 4—figure supplement 2; Flyak et al., 2018; Kong et al., 2013; Tzarum et al., 2019). Three additional hydrogen bonds (AR3X-E2ecto: Arg99-Asp431, Arg99-Asn430, Asn96-Asp431), as well as a salt bridge between CDRH3 (Arg100b) and a CD81 binding loop residue (Glu531), further stabilize the interaction of AR3X with E2. The AR3X-E2ecto crystal structure also shows contacts between the AR3X CDRH3 and N-glycans attached to E2 residues Asn423 and Asn430 (Figure 4G).

We and others previously described HCV bNAbs that utilize the VH1-69 gene segment and a germline-encoded disulfide motif in CDRH3 to recognize the conserved epitope in E2 front layer (Flyak et al., 2018; Keck et al., 2019; Kong et al., 2013; Tzarum et al., 2019). Here we structurally characterized a front layer-specific HCV bNAb that is encoded by the VH1-69 gene that includes an ultralong insertion in CDRH2 as well as the disulfide motif in CDRH3. We found that AR3X, isolated from the same chronically-infected patient as AR3A or AR3C (Law et al., 2008), surprisingly exhibits the straight CDRH3 conformation found in the HEPC3 or HEPC74 bNAbs isolated from individuals who spontaneously cleared HCV infection (Figure 3). This indicates that a single individual can produce potent HCV-specific bNAbs using the common VH1-69 and D2-15 genes that bind to the conserved region of E2 in at least three different configurations (straight CDRH3 with CDRH2 insertion, straight CDRH3 without CDRH2 insertion, or bent CDRH3 without CDRH2 insertion), highlighting the intrinsic plasticity of the VH1-69–encoded CDRH1 and CDRH2 loops that accommodate different antibody approach angles (Figure 5). It’s likely that the CDRH3s of these bNAbs dictate the preferential mode of engagement of bNAb germline precursors with the conserved epitope in the E2 front layer. Overall, these data demonstrate that B cells using VH1-69 and D2-15 genes can follow multiple pathways of affinity maturation to achieve broad neutralizing activity.

In the four bNAbs that were previously characterized structurally (Flyak et al., 2018; Kong et al., 2013; Tzarum et al., 2019), the first cysteine residue of the CDRH3 hydrogen bonds with E2 residue Cys429 (Figure 4G, Figure 4—figure supplement 2). We hypothesize that after the initial recognition of the front layer by CDRH3, the VH1-69-encoded CDRH1 and CDRH2 further stabilize the interaction while subsequent somatic mutations increase the bNAb affinity and breadth. Other antibodies that utilize a CDRH3 stabilized by a disulfide bond have been also described in the literature (Sui et al., 2009; Thomson et al., 2008; Ying et al., 2015). For example, M336, a potent human antibody that neutralizes severe acute respiratory syndrome coronavirus (Ying et al., 2014), is encoded by the VH1-69 gene segment and includes a germline-encoded disulfide bond in its CDRH3 (Ying et al., 2015).

Nucleotide insertions and deletions play an important role in diversification of the antibody repertoire (de Wildt et al., 1999; Reason and Zhou, 2006; Wilson et al., 1998). Insertions are produced by sequence duplications; while the average size of insertion varies from 3 to 33 nucleotides, the majority of antibodies contain short insertions (Kanyavuz et al., 2019; Wilson et al., 1998). AR3X with its 42-nucleotide insertion in CDRH2 represents an interesting case of an antibody that utilizes an ultralong CDRH2 to bind its epitope (Figure 4). The insertion was required for recognition of E2 glycoproteins across multiple HCV strains, as evidenced by the poor binding activity of AR3X variants lacking the CDRH2 insertion (Figure 2). While several neutralizing antibodies with insertions have been described (Kepler et al., 2014; Krause et al., 2011), AR3X is unique for its exceptionally long CDRH2 insertion, which makes extensive contacts with E2, but does not change the preconfigured mode of AR3X interaction with E2 based on its straight CDRH3 containing a disulfide motif. Thus the conserved epitope in the HCV E2 front layer, which is recognized by multiple human bNAbs containing a disulfide motif in their CDRH3s (Figure 5), remains a promising target for lineage-based immunogen design.

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Author details

1. Andrew I Flyak

   Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, United States

   Contribution

   Conceptualization, Data curation, Software, Formal analysis, Supervision, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing

   Competing interests

   AIF and JRB are inventors of International Patent Application, Serial no. PCT/US2019/029315, pertaining to some of the antibodies presented in this article.

   "This ORCID iD identifies the author of this article:" 0000-0002-8722-479X

2. Stormy E Ruiz

   1. Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, United States
   2. Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, United States

   Contribution

   Formal analysis, Investigation, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-0892-9626

3. Jordan Salas

   Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, United States

   Contribution

   Data curation, Formal analysis, Investigation, Writing - review and editing

   Competing interests

   No competing interests declared

4. Semi Rho

   Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, United States

   Contribution

   Data curation, Formal analysis, Investigation, Writing - review and editing

   Competing interests

   No competing interests declared

5. Justin R Bailey

   Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, United States

   Contribution

   Supervision, Funding acquisition, Investigation, Writing - review and editing

   Competing interests

   AIF and JRB are inventors of International Patent Application, Serial no. PCT/US2019/029315, pertaining to some of the antibodies presented in this article.

6. Pamela J Bjorkman

   Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, United States

   Contribution

   Conceptualization, Supervision, Funding acquisition, Project administration, Writing - review and editing

   For correspondence

   bjorkman@caltech.edu

   Competing interests

   Reviewing editor, eLife

   "This ORCID iD identifies the author of this article:" 0000-0002-2277-3990

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