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. Author manuscript; available in PMC: 2022 Apr 13.
Published in final edited form as: Immunity. 2021 Mar 5;54(4):781–796.e4. doi: 10.1016/j.immuni.2021.02.013

Functional convergence of a germline-encoded neutralizing antibody response in rhesus macaques immunized with hepatitis C virus envelope glycoproteins

Fang Chen 1,7, Netanel Tzarum 2,7,8, Xiaohe Lin 1, Erick Giang 1, Rodrigo Velázquez-Moctezuma 3, Elias H Augestad 3, Kenna Nagy 1, Linling He 1, Mayda Hernandez 4, Mallorie E Fouch 4, Ariadna Grinyó i Escuer 4, Deborah Chavez 5, Benjamin J Doranz 4, Jannick Prentoe 3, Robyn L Stanfield 2, Robert Lanford 5, Jens Bukh 3, Ian A Wilson 2,6,*, Jiang Zhu 1,2,*, Mansun Law 1,9,*
PMCID: PMC8046733  NIHMSID: NIHMS1676208  PMID: 33675683

SUMMARY

Human IGHV1-69-encoded broadly neutralizing antibodies (bnAbs) that target the hepatitis C virus (HCV) envelope glycoprotein (Env) E2 are important for protection against HCV infection. An IGHV1-69 ortholog gene, VH1.36, is preferentially used for bnAbs isolated from HCV Env-immunized rhesus macaques (RMs). Here we studied the genetic, structural and functional properties of VH1.36-encoded bnAbs generated by vaccination, in comparison to IGHV1-69-encoded bnAbs from HCV patients. Global B cell repertoire analysis confirmed the expansion of VH1.36-derived B cells in immunized animals. Most E2-specific, VH1.36-encoded antibodies cross-neutralized HCV. Crystal structures of two RM bnAbs with E2 revealed that the RM bnAbs engaged conserved E2 epitopes using similar molecular features as human bnAbs but with a different binding mode. Longitudinal analyses of the RM antibody repertoire responses during immunization indicated rapid lineage development of VH1.36-encoded bnAbs with limited somatic hypermutation. Our findings suggest functional convergence of a germline-encoded bnAb response to HCV Env with implications to vaccination in humans.

Graphical Abstract

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eTOC Blurb

The human IGHV1-69 gene is preferentially utilized in broadly neutralizing antibody (bnAb) responses against HCV infection. Chen et al. discovered that HCV Env immunization of macaques elicited analogous VH1.36 bnAbs on the functional and molecular level. Functional convergence of a corresponding germline-encoded bnAb response within primates has implications for HCV rational vaccine design and testing.

Introduction

Elicitation of broadly neutralizing antibodies (bnAbs) that can cross-neutralize diverse viral strains is a major goal for development of a hepatitis C virus (HCV) vaccine (Kinchen et al., 2018). Most bnAbs isolated from HCV-infected patients bind to a conserved antigenic surface known as the E2 neutralizing face (E2 NF), which overlaps the CD81 receptor binding site [reviewed in (Tzarum et al., 2018)]. E2 NF is an excellent target for rational HCV vaccine design and is composed of three overlapping neutralizing sites: antigenic sites AS412, AS434, and antigenic region 3 (AR3, representing a cluster of discontinuous epitopes formed by the E2 front layer and CD81 binding loop). Virus escape studies indicate that bnAbs targeting AR3 have a high barrier to resistance (Velazquez-Moctezuma et al., 2019). AR3-specific bnAb responses have been reported in multiple individuals and are preferentially encoded by IGHV1-69 (Bailey et al., 2017; Colbert et al., 2019; Giang et al., 2012; Keck et al., 2019; Law et al., 2008; Merat et al., 2019; Merat et al., 2016), which is a human immunoglobulin (Ig) heavy chain variable gene (IGHV or VH) frequently used against other viruses, e.g., influenza A virus and HIV-1 [reviewed in (Chen et al., 2019)]. These observations suggest that such antibodies can be reproducibly induced in the general population by a germline-targeting vaccine strategy. Furthermore, several studies demonstrated that IGHV1-69-encoded bnAbs are associated with natural clearance in HCV patients (Bailey et al., 2017; Merat et al., 2019; Merat et al., 2016) and can protect animals from HCV challenge (de Jong et al., 2014; Giang et al., 2012; Law et al., 2008). Therefore, a vaccine-induced antibody response that effectively activates the IGHV1-69 germline gene will likely be protective.

IGHV1-69-encoded HCV bnAbs targeting AR3 share common features for E2 NF recognition: (1) the majority of the interactions are mediated by the heavy chain; (2) an unusually hydrophobic heavy-chain complementarity-determining region 2 (CDRH2) interacts with a hydrophobic patch on E2 NF; and (3) a relatively low level of somatic hypermutation (SHM) is sufficient for antibody maturation and function (Chen et al., 2019; Flyak et al., 2018; Keck et al., 2016; Krey et al., 2013; Tzarum et al., 2020; Tzarum et al., 2019). An intramolecular disulfide within a long CDRH3 loop is found in several IGHV1-69-encoded bnAbs (Flyak et al., 2018; Flyak et al., 2020; Kong et al., 2013; Tzarum et al., 2019) although not ubiquitous (Keck et al., 2019; Law et al., 2008; Tzarum et al., 2020). Notably, E2 NF is structurally flexible (Kong et al., 2016b) and two conformations, ‘A’ and ‘B’, are recognized by different IGHV1-69-encoded bnAbs (Tzarum et al., 2020). Consequently, the IGHV1-69-encoded bnAbs can adapt different modes to recognize this flexible antigenic surface (Figure S1) (Flyak et al., 2018; Kong et al., 2013; Tzarum et al., 2020; Tzarum et al., 2019).

A comprehensive understanding of how HCV bnAbs are generated is essential for HCV vaccine design. Previous advances in next-generation sequencing (NGS) of Ig transcripts with longitudinal sampling have allowed high-resolution characterization of antibody repertoires (i.e., initial VDJ recombination, CDRH3 length and SHM frequency) and their developmental pathways during infection and vaccination (Dai et al., 2015; Gao et al., 2019; Kong et al., 2016a; Umotoy et al., 2019; Wen et al., 2020; Wu et al., 2015). Such information is crucial for B cell lineage (or ontogeny)-based vaccine design but is not yet available for HCV bnAbs.

Rhesus macaques (RMs) provide an attractive preclinical nonhuman primate (NHP) model for evaluation of human vaccine candidates because of their genetic relatedness to humans (Ramesh et al., 2017). The VH genes of RM and human share 92% homology on average (Sundling et al., 2012a). In a previous study, we immunized four RMs with the Chiron recombinant E1E2 complex from a genotype 1a strain HCV-1 and compared the antibody responses to that of healthy volunteers immunized with the same antigen in a phase I clinical trial (Chen et al., 2020). The animals faithfully recapitulated human antibody responses to HCV Env vaccination. Although the immune sera are mostly strain-specific, bnAbs against AR3 isolated from multiple animals are mostly derived from VH1.36, an ortholog of the human IGHV1-69 gene (Chen et al., 2020). To further our understanding on how bnAbs to HCV are elicited by vaccination, we investigated the genetic, structural and evolutionary bases for their elicitation in the RM model. Crystal structures of two VH1.36-encoded bnAbs, RM02-01 and RM11-43, with the E2 core domain indicated that they share common features for E2 NF recognition with human VH1-69-encoded HCV bnAbs. Lineage tracing of these RM bnAbs revealed rapid development with only a few SHMs. A single serine to isoleucine substitution at position 31 (S31I) in CDRH1 of the precursor antibody is sufficient for high affinity binding and broad neutralization. These findings elucidate how VH1.36-encoded bnAbs were generated in HCV Env-immunized RMs by targeting the same E2 antigenic surface. Such functional convergence of a germline-encoded bnAb response in primates has direct implications for use of the RM model in germline-targeting vaccine approaches.

RESULTS

HCV Env immunization elicited a VH1.36-derived B cell response in multiple RMs

To understand fundamental aspects of vaccine-elicited antibody responses in RMs, we examined circulating B cell repertoires of the four animals from the previous HCV Env immunization study (Chen et al., 2020). Before immunization (week 0), the animals displayed a similar profile for VH gene usage, SHM, and CDRH3 length (Figures S2A-S2D). The VH4 family was most prevalent, followed by VH3 and VH1. In healthy human repertoires, VH3 is the predominant gene family, followed by VH4 and VH1 (Briney et al., 2019; DeWitt et al., 2016; Tzarum et al.,2019. Approximately 2-7% of peripheral B cells in the RMs carry B cell receptors (BCR) derived from the VH1.36 gene (Figure 1A), compared to 4~9% of IGHV1-69-derived B cells in healthy human circulating repertoires (Briney et al., 2019; DeWitt et al., 2016; Tzarum et al., 2019).

Figure 1. Dynamics of global B cell VH repertoires in RMs #30734 and #31881 during HCV Env immunization.

Figure 1.

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(A) Schematic overview of HCV Env immunization and longitudinal sampling for RMs #30734 and #31881. The animals were immunized 5 times with recombinant E1E2 and blood samples were collected 1 or 2 weeks after each immunization. The antibody heavy chain sequences of circulating B cells were determined by next-generation sequencing (NGS), and monoclonal antibodies (mAbs) were isolated from single B cells at specified timepoints. (B) Dynamics of the most frequently used VH genes in the B cell repertoires of #30734 and #31881 during the immunization. Data on VH1.36 are highlighted by dotted lines. See also Figures S2E and S2F. (C) The most frequently used RM VH genes and the corresponding human VH genes (Sundling et al., 2012a). (D) Distribution of VH families at week 0 and week 29. (E) Dynamics of VH1.36-derived B cell response and the E1E2-specific antibody secreting cell (ASC) response during the immunization. The data on the ASC response as previously reported (Chen et al., 2020) were included here for comparison. (F and G) Distribution of CDRH3 length (F) and VH somatic hypermutation (SHM; G) at week 0 and week 29. CDRH3 length was based on Kabat numbering, which is 2 amino acids shorter than that in the IMGT definition. SHM is shown at the nucleotide level.

After immunization, three animals (I.D. #30734, #31881 and #31782) developed a prolonged autologous serum nAb response that peaked after the 4th immunization, whereas animal #31859 mounted a weak nAb response after the 3rd and no neutralizing activity after the 4th immunization (Chen et al., 2020). We analyzed the dynamics of global VH repertoire for #30734 and #31881 by longitudinal NGS (Figure 1A and Table S1). During immunization, VH4-derived B cells were still dominant in the RM B cell repertoires (Figures 1B, S2E and S2F). Prominent B cell expansions were observed for VH1.36 and VH4.40 (ortholog of human VH4-39) for #30734, and VH5.7 (ortholog of human VH5-51) and VH1.36 for #31881 (Figures 1B-1D). Similar to their serum nAb responses, the expansion of VH1.36-derived B cells peaked after the 4th immunization for both animals; at week 29, day 4 post boost, their frequency was 17% for #30734 and 5.2% for #31881. However, at week 30, their frequency quickly dropped to 4.4% and 1.8%, respectively. Such a transient response is consistent with the dynamics of antigen-specific antibody secreting cells (ASC) elicited by the immunization (Figure 1E) (Chen et al., 2020), suggesting that the VH1.36-derived B cells were mostly ASC or plasmablasts. We noted a longer CDRH3 loop in the B cells from the week-29 repertoire, compared to the week-0 repertoire, for #30734, but not for #31881 (Figure 1F). No difference in overall SHM was observed for the two animals at week 0 versus week 29 (Figure 1G).

To investigate the antigen-specific B cell repertoire, we analyzed E2-sorted memory B cells of #31782 and #31859 at day 10 after the 4th immunization (Figure S3 and Table S1). At this timepoint, the #31782 serum nAb response reached a peak, while no neutralizing activity was detected in the #31859 serum (Chen et al., 2020). The two animals exhibited distinct B cell repertoire profiles (Figure S3). VH1.36- and VH3.58-derived B cells (contributing to 32% and 19% of total antigen-specific B cells, respectively) were dominant in the memory B cell repertoire of #31782, while the #31859 repertoire was largely comprised of antigen-specific B cells derived from VH5.7 (49%) and VH4.40 (17%). Collectively, these data demonstrated that HCV Env immunization elicited a VH1.36-derived B cell response related to serum neutralization in 3 of 4 RMs (#30374, #31881 and #31782).

RM VH1.36-encoded antibodies effect cross-neutralizing responses to HCV Env immunization

A panel (n=100) of antigen-specific monoclonal antibodies (mAbs) were isolated from single plasmablasts and memory B cells of the week-29 and week-30 repertoires of #30374 and #31881 [(Chen et al., 2020) and this study]. The VH1.36-encoded mAbs accounted for 20% (11/56) and 7% (3/44) of total mAbs for #30734 and #31881, respectively (Figure 2A). About 71% of the VH1.36-encoded mAbs were capable of cross-neutralizing >50% isolates on a panel of 14 viruses from at least five major genotypes in HCV pseudoparticles (HCVpp) and/or cell culture (HCVcc) systems, while 14% had limited breadth and 14% had no neutralizing activity (Figures 2A, 2B and S4). In contrast, antigen-specific antibodies originating from the VH4 and VH3 families were mostly non-neutralizing (Figures 2A and 2B). Among the eight broadest RM bnAbs, seven originated from VH1.36 (Figure S4A) and displayed high binding affinity (0.1-120 nM) and neutralization against diverse viruses spanning genotypes 1 to 6 (Figures 2C and 2D).

Figure 2. Function and specificity of RM VH1.36-encoded bnAbs.

Figure 2.

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(A) VH gene usage of antigen-specific mAbs isolated from RMs #30734 and #31881. Neutralization breadth is defined as the percentage and genotype of viruses neutralized by the mAbs using a panel of 7 HCVpp and 12 HCVcc. Neutralizes >50% of viruses from at least five genotypes, broadly neutralizing; neutralizes 8-50% viruses, limited breadth; neutralizes only 1 virus, strain-specific. See also Figure S4A. (B) Maximum likelihood (ML) tree of heavy chain aa sequences of the RM mAbs. (C) Binding affinity of VH1.36-encoded bnAbs to HCV E2 core (E2c) from genotypes 1-6 by biolayer interferometry (BLI). (D) Neutralization breadth and potency of VH1.36-encoded bnAbs against 10 HCVcc. Human IGHV1-69-encoded bnAbs HEPC74 and HC84.1 were included as controls. Half-maximum inhibitory concentration (IC50) is shown in μg/ml. ND, not determined. (E) Epitope mapping of VH1.36-encoded bnAbs by alanine scanning mutagenesis. Cells shown binding percentage relative to wild type H77. The E2 sequence logo of the 14 viruses (HCV-1, H77, TN, DH1, UKN1B.12.6, J4, J6, J8, S52, DBN, ED43, UKN4.11.1, SA13 and HK6a) used in this study are shown. See also Figure S4B.

RM VH1.36- and human IGHV1-69-encoded bnAbs target overlapping epitopes and share common genetic features

We showed previously that RM VH1.36-encoded bnAbs competed with human AR3-specific mAb AR3A and block CD81 binding to E1E2 (Chen et al., 2020), suggesting that they target overlapping epitopes on E2 NF. Here, alanine scanning mutagenesis demonstrated that residues within HCV E2 front layer (resides 427-430 and 436-445), CD81 binding loop (residues 527 and 529) and back layer (residue 616) were critical for binding of this class of antibodies (Figure 2E).

The RM VH1.36- and human IGHV1-69-encoded bnAbs share sequence similarity particularly in CDRH2 (Figures 3A and 3B). A hallmark feature of human IGHV1-69-encoded bnAbs is the unusually hydrophobic tip of CDRH2 that can interact with conserved epitopes in diverse viral antigens (Chen et al., 2019; Ekiert et al., 2009; Lang et al., 2017; Lingwood et al., 2012; Tzarum et al., 2020; Tzarum et al., 2019). For most RM VH1.36-encoded bnAbs, a germline-encoded hydrophobic motif, I51I52P52aL53 (Kabat numbering), and a somatically mutated X54 were observed at the CDRH2 tip (Figures 3A and 3B). Both human (except for HC84.1, which binds AS434 instead of AR3) and RM antibodies had medium to long CDRH3 of 14-21 amino acids (aa; Figures 3A and 3C), compared to the 9-15 aa for naïve animals (Figure S2C) and healthy human donors (Briney et al., 2019). Some human IGHV1-69-encoded bnAbs (e.g., AR3A, AR3C, HEPC3, HEPC74 and HC11) utilize the DH2-15 or DH2-21 segment and contain a disulfide motif (CxGGxC) that may help stabilize their CDRH3 (Figure 3A) (Flyak et al., 2018; Keck et al., 2019; Kong et al., 2013; Tzarum et al., 2019). However, such a motif was not observed in AR3B, AR3D, HC-1AM, 212.1.1, 212.10 and HC84.1, or in the RM bnAbs here (Figure 3A). The SHM in the VH of RM VH1.36-encoded bnAbs ranged from 4-8% (average 6%) at the nucleotide level (Figures 3A and 3C), whereas human IGHV1-69-encoded bnAbs appear to have a higher SHM (Figure 3C), likely due to continuous stimulation by evolving virus during chronic infection. In summary, the genetic features of RM VH1.36-encoded bnAbs were similar to human IGHV1-69-encoded bnAbs.

Figure 3. Comparison of genetic features of RM VH1.36- and human IGHV1-69-encoded bnAbs.

Figure 3.

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(A) Heavy-chain CDR amino acid sequences, CDRH3 length, and SHM rate of individual antibodies. The germline genes VH1.36 and IGHV1-69*01 are shown as reference. Note that AR3C, AR3A, HC1AM and 212.10 are encoded by IGHV1-69*06, *15, *17 and *04, respectively. Sequence differences between these gene alleles are reported in (Chen et al., 2019). Dots denote matches to the reference sequence. The hydrophobic motif at CDRH2 tip and the disulfide motif (CxGGxC) in CDRH3 are in red and blue, respectively. Kabat numbering is indicated. (B) Amino acid sequence logo of CDRH1 and CDRH2. (C) Comparison of CDRH3 length and SHM level of RM and human antibodies. P values were calculated using two-tailed Mann-Whitney test. ns, not significant. *** P<0.0001.

Crystal structures of RM VH1.36-encoded bnAbs in complex with E2

To further characterize E2 recognition by RM VH1.36-encoded bnAbs, we determined the crystal structure of the Fab fragments of antibodies RM2-01 and RM11-43 (both isolated from #30734) in complex with the E2 core domain (recombinant E2c3 protein) of HCV isolate HK6a (Bukh et al., 2010; Meunier et al., 2005; Tzarum et al., 2019) (Table S2). The structures elucidated that RM2-01 and RM11-43 bound to epitopes that overlap with AR3 (Figure 4A), consistent with the epitope mapping data (Figure 2E). The two antibodies represented two distinct B cell lineages in the panel of RM VH1.36-derived bnAbs (Figure 3A) (Chen et al., 2020). Sequence alignment of the RM2-01 and RM11-43 VH regions (aa 1-113) showed high sequence identity in their CDRH1 and CDRH2 loops (100% and 88%, respectively), but notable difference in their CDRH3 sequence and length (15 versus 19 aa, Figure S5A). Superposition of the two complexes revealed that RM2-01 and RM11-43 bind E2 in a similar overall orientation (Figure S5B). However, major conformational differences in CDRH3 as well as minor variation in CDRH2, were observed between the two complexes (Figures S5B and S5C), while HK6a E2c3 remained largely unchanged (Figure S5D) with a Cα RMSD of 0.38Å on 127 residues. Buried surface area (BSA) analysis indicated that the majority of the E2 epitope surface area was buried by the VH region [620 Å2 by RM2-01 and 790 Å2 by RM11-43 VH, equivalent to 87% of the total BSA (712 and 907 Å2, respectively) for both antibodies; Figures 4B and S5E]. Consistently, as indicated by biolayer interferometry (BLI), the swapping of the wild-type light chains with other unrelated light chains did not compromise their binding affinity for E2 (Figure 4C).

Figure 4. Crystal structures of HK6a E2c3-RM VH1.36-encoded nAb complexes.

Figure 4.

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(A) Crystal structures of HK6a E2c3-RM2-01 (blue) and HK6a E2c3-RM11-43 (pink) complexes. For clarity, only the Fab variable regions are shown. (B) Buried surface area (BSA) by the CDRs of RM2-01 and RM11-43 mAbs on HK6a E2c3. See also Figure S5E. (C) Biolayer interferometry (BLI) binding affinity of RM2-01 and RM11-43 to HK6a E2c, where their light chains were swapped with light chains from an anti-influenza virus antibody or HIV-1 antibody. (D) Schematic overview of the interactions between HK6a E2c and RM2-01 and RM11-43 heavy chains. The CDR sequences were aligned, and E2 interacting residues are highlighted here in red (salt bridge) orange (pi-cation) blue (hydrogen bonds) and green (hydrophobic interactions). (E) Interactions between E2 (gray) and CDRH1-3 of RM2-01 (blue) and RM11-43 (pink). The interacting residues are labelled and shown in stick representation and hydrogen bonds are shown as dashed lines.

Detailed analysis of the interactions between RM2-01 and RM11-43 antibodies to HK6a E2c3 revealed that, for both antibodies, the CDRH1 loops formed hydrophilic charged (salt bridge and/or π-cation) and hydrophobic interactions with the front layer and CD81 binding loop, whereas the CDRH2 loops only engage in side-chain hydrophobic interactions with the front layer (Figures 4B, 4D and 4E). Both CDRH3s formed hydrophobic interactions and main-chain hydrogen bonds with the front and back layers. Due to low sequence similarity and differences in CDRH3 loop length (15 vs. 19 aa) and conformation (Figures S5A and S5C), the CDRH3 of each mAb created a different set of interactions (Figures 4D, 4E and S5F). Interestingly, the CDRH3 loops of both RM2-01 and RM11-43 made direct interactions with the back layer (Y613 and P612, respectively).

Structural comparison of E2 recognition by RM VH1.36- versus human IGHV1-69-encoded bnAbs

E2 recognition by human IGHV1-69-encoded bnAbs has been well described (Flyak et al., 2018; Flyak et al., 2020; Kong et al., 2013; Tzarum et al., 2020; Tzarum et al., 2019), revealing common binding features as well as different binding modes based on relative CDR dispositions, angles of approach, and interacting E2 residues (Figure S1). Structure elucidation of HK6a E2c3-RM2-01 and HK6a E2c3-RM11-43 complexes enabled detailed comparison of E2 recognition by RM VH1.36- and human IGHV1-69-encoded bnAbs. Despite variation in their germline sequences, the human and RM bnAbs share a similar hydrophobic CDRH2 tip for antigen interaction that is dominated by the heavy chain (Figure 3A). Structural comparison of RM2-01 and RM11-43 with representative human IGHV1-69-encoded bnAbs (AR3A for AR3A/B/C/D, U1 and HC11 group; 212.1.1 for 212.1.1 and HC1AM group; HEPC74 for HEPC74 and HEPC3 group) (Flyak et al., 2018; Tzarum et al., 2020; Tzarum et al., 2019) illustrated that these two RM bnAbs bound to an antigenic surface overlapping with the epitopes recognized by the human bnAbs (Figures 5A and 5B). However, variation in the angle of Fab approach to E2 resulted in notable differences in the CDRH footprint (Figure S6). CDRH3 of human IGHV1-69-encoded bnAbs interact with an exposed surface formed by the front layer and CD81 binding loop, while their CDRH2 (except for HEPC3/74) engage with a hydrophobic groove formed by the front layer N terminus (aa 421-428), helix α1 C terminus (aa 442-443), and CD81 binding loop tip (W529) (Figure 5B). In contrast, CDRH3 of RM2-01 and RM11-43 interacted with the hydrophobic groove, while CDRH2 only contacted hydrophobic residues of helix α1 (aa 438-443).

Figure 5. Comparison of the E2 complex structures of RM VH1.36-encoded with human IGHV1-69-encoded bnAbs.

Figure 5.

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(A) Comparison of RM2-01 (blue), RM11-43 (violet), AR3A (teal), 212.1 (yellow), and HEPC74 (salmon) epitopes. The E2c structures are shown in surface representation and the interacting residues are colored and labelled. (B) The position of the CDRH2 and CDRH3 in the E2c3-Fab structures. The E2c3 is shown in surface presentation with the front layer and CD81 binding loop as a cartoon and colored by cyan and blue, respectively. The CDRH2 tip (aa 51-56) and CDRH3 are shown in cartoon representation. (C) Superposition of E2 from RM2-01, RM11-43, AR3A, and 212.1.1 complexes indicating similarity of the front layer conformation in RM2-01 and RM11-43 complexes to that of the AR3A complex. (D) Interactions between E2 (gray) and CDRH2 of VH1.36-encoded or representative IGHV1-69-encoded bNAbs. Interacting residues are labelled and shown in stick representation.

CDRH3 of RM2 −01 and RM11-43 also interacted with the α2 helix of the back layer (aa 614-617; Y613 for RM2-01 and P612 for RM11-43) (Figures 4D and 4E) that contains residues critical for CD81 receptor binding (Y613 and W616) (Gopal et al., 2017; Roccasecca et al., 2003). In most E2-bnAb structures, the α2 helix region is shielded by the front layer and only partially exposed, and therefore unable to interact with bnAbs. In our structures of H77 E2c3-Fab 212.1.1 and H77 E2c3-Fab HC1AM, the E2 front layer adopts a different conformation that exposes the helix α2 region (termed ‘B’ conformation), allowing interaction of the antibodies with two back layer residues, Y613 and W616 (Figure 5A) (Tzarum et al., 2020). Superposition of E2 from HK6a E2c3-RM2-01 and HK6a E2c3-RM11-43 on H77 E2c3-AR3A (‘A’ conformation) and H77 E2c3-212.1.1 (‘B’ conformation) indicated that the two RM bnAbs bound to the E2 ‘A’ conformation (Figure 5C), suggesting that the interactions with the back layer α2 helix were a result of the unique angle of approach for RM2-01 and RM11-43.

Analysis of the human bnAb structures indicated that the CDRH2 tip of the AR3A-like bnAbs were positioned vertical to the hydrophobic groove and interacted with both front layer and CD81 binding loop (Figure 5D). For 212.1.1 and HC1AM, as a result of changes in the front layer conformation, CDRH2 bound more deeply into the hydrophobic groove and engaged the front layer, CD81 binding loop, and back layer α2 helix (Figure 5D). In contrast, CDRH2 of RM2-01 and RM11-43 was positioned parallel to the surface of the front layer and used residues upstream to the tip to interact only with the front layer, similar to interactions observed for HEPC3/74 CDRH2 (Figure 5D).

RM VH1.36-encoded bnAbs require only a few SHMs for partial or full function

The heavy chains of RM2-01 and RM11-43 differed from their inferred germline precursors (GLs) by 14 (11.3%) and 7 (5.5%) aa, respectively (Figure 6A). Mutations were observed in framework (FR) 1, CDRH1, CDRH2, FR3 and CDRH3. Only a few (2 for RM2-01 and 4 for RM11-43) appeared to have direct interactions with E2 NF: R30 (FR1) and I31 (CDRH1) were shared by both antibodies, and G54 (CDRH2) and K98 (CDRH3) were in RM11-43 only (Figures 4D and 4E). To examine the minimum requirements for affinity maturation, we compared the inferred heavy chain GLs for RM2-01 and RM11-43, as well as a GL with mature CDRH3 (GM), to the affinity-matured antibodies (MA) (Figure 6A). We also created heavy-chain GM and MA variants by substituting somatic mutations singly or in combination, with a focus on epitope-contacting CDRs and residues at the hydrophobic CDRH2 tip. The heavy chain variants were paired with their corresponding mature light chains and tested for their ability to bind and neutralize diverse HCV isolates.

Figure 6. Effects of SHMs on RM VH1.36-encoded bnAb function.

Figure 6.

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(A) Alignment of heavy-chain aa sequences of RM2-01 and RM11-43 inferred germline precursors (GL), GL with the mature antibody CDRH3 (GM), and mature antibodies (MA). Dots denote matches to the reference sequence. Kabat numbering is indicated. (B and C) ELISA binding of RM2-01 (B) and RM11-43 (C) antibody variants to the autologous HCV-1 (left, serial dilutions starting from 20 μg/ml) and diverse heterologous strains (right, at 20 μg/ml). (D and E) BLI binding affinity (left) and HCVpp neutralization (right) of RM2-01 antibody variants (D) and RM11-43 antibody variants (E) against diverse HCV viral strains. Antibodies were tested at a single concentration of 50 μg/ml in HCVpp. Data shown are the average of at least 3 experiments. NB, no binding. WB, weak binding.

For both antibodies, GL, GM and GM(G30R) variants showed little activity towards the isolates tested (Figures 6B-6E). Instead, high binding for both autologous and heterologous viruses was achieved with an S31I substitution in CDRH1 (Figures 6B-6E). S31I, together with R30G and R30G/G54V/K98R for RM2-01 and RM11-43, respectively, enabled broad binding against heterologous viruses. Consistently, reversion of the S31I or G30R/S31I mutations in the mature antibodies markedly decreased their binding breadth to heterologous viruses. For RM11-43, MA(G54V) slightly increased neutralization of J6, and MA(K98R) largely improved both binding and neutralization against heterologous viruses, whereas MA (R30G/I31S/G54V/K98R) greatly impaired binding and neutralization for all viruses tested. Substitution of individual residues at the CDRH2 tip with alanine (I51A and P52aA for RM2-01, I52A and L53A for RM11-43, and I56A for both antibodies) had a relatively minor effect on antibody function. Together, these results demonstrate that affinity maturation of VH1.36-encoded bnAbs required only a few somatic mutations in VH with a single S31I substitution in CDRH1 being sufficient to confer high-affinity binding to autologous virus. As this mutation was widely present in RM VH1.36-encoded bnAbs (Figure 3A), it may represent a common mechanism of affinity maturation for this antibody class.

RM VH1.36-encoded bnAbs achieve breadth through rapid lineage development

Following identification of key residues for antigen recognition by the RM VH1.36-encoded bnAbs, we traced their developmental pathways to gain further insight into their ontogeny. The heavy-chain repertoires of #30734 and #31881 at 9 time points (Figure 1A) were interrogated using 10 single-cell sorted VH1.36-encoded bnAbs as references. Two-dimensional identity-divergence analysis and CDRH3-based lineage tracing revealed similar patterns of heavy-chain expansion for all 10 bnAbs (Figures 7A and S7). In all cases, the first notable expansion was observed after the 3rd immunization (week 13). After the 4th immunization, the response peaked at week 29, followed by a quick contraction at week 30, which was consistent with the circulating VH1.36-derived B cell expansion. Analysis of the repertoire NGS data from the preimmunization and early timepoints (weeks 0-12) revealed highly similar heavy chains (>90% nucleotide sequence identity) in the naïve repertoire for 6 of the 10 VH1.36-encoded bnAbs (Figure S7) and rapid accumulation of SHMs in CDRH1 and CDRH2 for all analyzed antibodies (Figure 7B).

Figure 7. Longitudinal sequence analysis of the RM VH1.36-encoded bnAb lineages.

Figure 7.

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(A) Identity-divergence analysis of the heavy chains of RM9-37-, RM2-01- and RM11-43-like antibodies in circulating B cell repertoires. Color coding denotes sequence density. See also Figure S7. (B) CDRH1 and CDRH2 aa sequence logo of the VH1.36-encoded bnAb-like heavy chains identified from the NGS-derived RM B cell repertoires at early time points during the immunization. (C) CDR sequence alignment of the RM2-01-, RM11-48- and RM9-37-like heavy chains identified from indicated time points. Dots denote matches to the reference sequence. Epitope-interacting residues in the crystal structures are colored in blue for the RM2-01 and RM11-43 somatic variants. (D and E) ELISA binding (D), BLI binding and HCVpp neutralization (E) of synthesized antibodies containing NGS-derived heavy chains and RM2-01, RM11-43 or RM9-37 light chains. ND, not determined.

We then traced antibody lineages for RM2-01/RM9-93, RM11-43 and RM9-37 (the most broad and potent VH1.36-encoded bnAbs) in #30734. We found that RM2-01 and RM9-93 were members of the same clonal lineage based on V and J gene usage, CDR3 length, and CDR3 sequence identity (Chen et al., 2020). B cell precursors of the RM2-01/RM9-93 and RM9-37 lineages were observed in the week 0 repertoire, while the RM11-43 lineage was first detected at week 1 (Figure 7A). These sequences also exhibited SHMs at key contact residues for RM2-01 and RM11-43, e.g. aa 30 in FR1, aa 31 in CDRH1, aa 54-56 in CDRH2 tip, and aa 97-100 in CDRH3 (Figure 7C).

To validate our findings from the repertoire analysis, selected heavy chains of the RM2-01, RM11-43 and RM9-37 sub-lineages from week 0 and early timepoints were paired with their respective light chains for functional evaluation. The reconstituted antibodies exhibited high binding and neutralization for E1E2 derived from multiple HCV isolates similar to the corresponding VH1.36-encoded bnAbs (Figures 7D and 7E). These data indicated that the naïve RM repertoire contained BCRs that encoded high-affinity, HCV-reactive, VH1.36 antibodies, from which a rapid response could be mounted on HCV Env immunization. These activated B cells continued to evolve and finally converged to the affinity-matured bnAbs upon vaccine boosts.

DISCUSSION

“Germline-endowed” antibody responses are observed, not only for HCV, but also for other viral pathogens, e.g. influenza virus (Joyce et al., 2016; Pappas et al., 2014; Wheatley et al., 2015), HIV-1 (Wu et al., 203266105811; Zhou et al., 2015), rotavirus (Di Niro et al., 2010; Weitkamp et al., 2005), and SARS-CoV-2 (Robbiani et al., 2020; Yuan et al. 2020). These antibodies are biased for VH gene usage, and it may be feasible to activate these germline pathways reproducibly to generate the desired antibody response. As these VH genes are unique to humans and are not expressed in standard animal models like rodents, designed immunogens have been only investigated in transgenic mice, e.g., for elicitation of VH1-2-encoded VRC01 class bnAbs against HIV-1 (Huang et al., 2020; Tian et al., 2016) and IGHV1-69-encoded bnAbs against influenza virus (Sangesland et al., 2019).

Human antibody responses against AR3 within HCV E2 NF are characterized by their exceptional neutralization breadth, conserved epitope targeting, low SHM, and restricted IGHV1-69 gene usage (Chen et al., 2019). This genetic bias underscores an interesting relationship between HCV and the bnAb response that is highly relevant for rational design of a germline-targeting HCV vaccine. Some germline-reverted precursors of human IGHV1-69-encoded bnAbs bind HCV E2 with high affinity and neutralization breadth is acquired by affinity maturation (Flyak et al., 2018; Tzarum et al., 2019). These attributes, along with limited SHM, suggest the potential to target IGHV1-69-derived precursor B cells for efficient elicitation of bnAbs by vaccination. In our previous study (Chen et al., 2020), AR3-specific antibody responses can be readily elicited by HCV Env immunization in both humans and RMs, albeit at low titer. Intriguingly, the RM AR3-targeting bnAbs exhibit biased usage of VH1.36, which is 94.8% identical to human IGHV1-69. Of note, the IgBlast database (Ye et al., 2013) was used here for RM Ig gene identification and genetic assignment. The incomplete reference sequences of RM Ig alleles may affect identification of authentic SHM but will not change B cell lineage assignment as they are defined by CDRH3 sequence identity. With advances in NGS of the BCR loci of RMs, genes/alleles have been identified and several alleles may be associated with the VH1.36 gene (Cirelli et al., 2019; Ramesh et al., 2017; Vigdorovich et al., 2016). Due to the lack of availability of B cells from the human subjects, we were not able to directly compare the human and RM antibody responses. Nonetheless, comparison of bnAbs from immunized RMs with AR3-targeting bnAbs isolated from HCV-infected patients revealed functional convergence of a germline-encoded bnAb response to HCV Env in humans and RMs. The RM and human antibodies shared common features such as broad neutralizing activity, heavy-chain-mediated binding, hydrophobic motif in CDRH2, and rapid affinity maturation with limited SHM.

To counteract structural flexibility and variation in E2 NF in different HCV viruses, human IGHV1-69-encoded bnAbs employ different modes of recognition (Figure S1). The crystal structure of an E2 core with a IGHV1-69-encoded bnAb, AR3C, marked an important step towards atomic-level understanding of HCV neutralization by human bnAbs and established a critical role for the hydrophobic CDRH2 tip and a long CDRH3 in recognition of E2 NF (Kong et al., 2013). Previous structures of E2 with ten different IGHV1-69-encoded bnAbs that target AR3 (Flyak et al., 2018; Flyak et al., 2020; Tzarum et al., 2020; Tzarum et al., 2019) extended our understanding of HCV recognition by this class of bnAbs. These structures suggested that IGHV1-69-encoded bnAbs can be clustered into sub-groups based on their genetic and structural characteristics. For instance, the CDRH3 of AR3A, AR3C, HC11, HEPC3, and HEPC74 acquire a β-hairpin conformation and contain a germline-encoded disulfide that stabilizes CDRH3. For AR3B and AR3D, CDRH3 adopts a similar conformation yet lacks the disulfide. Another feature to differentiate IGHV1-69-encoded bnAbs is the angle of approach and CDR footprint on E2 NF (Figure 5). Our previous study (Tzarum et al., 2020) indicates that IGHV1-69-encoded bnAbs can also be characterized based on the binding conformation of the front layer region within E2 NF (‘A’ or ‘B’ conformation, Figure 5C). Taken together, structural analysis suggests that plasticity of the E2 NF can accommodate interactions with different IGHV1-69-encoded bnAbs that vary in CDR sequence and CDRH3 length. In this study, we extended this structural understanding to RM VH1.36-encoded bnAbs. Both RM2-01 and RM11-43 share overall similar characteristics with human IGHV1-69-encoded bnAbs (hydrophobic CDRH2 for interaction with a hydrophobic groove in E2 NF and heavy-chain dominant antigen interaction). Despite differences in CDRH3 length, the two RM bnAbs created their own subgroup, characterized by different CDRs footprints; CDRH2 interacted only with the front layer, but the bAbs could also interact with back-layer residues when E2 was in the ‘A’ conformation. This mode of E2 binding underscored how versatile the IGHV1-69 family and orthologs were in recognition of diverse antigenic surfaces including the flexible E2 NF. Further investigation is warranted on whether this binding mode can also be used by human IGHV1-69-encoded bnAbs.

With longitudinal NGS, we further characterized the BCR repertoires and VH1.36-encoded bnAb lineage development in the immunized RMs. The vaccine-primed B cell repertoires were predominantly enriched in B cells originated from the VH4 and VH3 families (Figures S2E and S2F), but the VH3- and VH4-derived virus-specific antibodies were mostly non-neutralizing or neutralizing with limited breadth (Figures 2A and 2B). In comparison, the VH1.36-derived B cells were less prevalent and underwent a rapid and transient expansion (Figures 1B, 1D and 1E). This finding may explain that, despite the induction of broad and potent VH1.36-encoded bnAbs (Figure 2), their serum levels were still too low to achieve broad neutralization (Chen et al., 2020).

Similar to human IGHV1-69-encoded bnAbs targeting the influenza HA stem (Lingwood et al., 2012; Pappas et al., 2014), the RM HCV VH1.36-encoded bnAbs achieved neutralization breadth through rapid lineage development with limited SHM. A S31I substitution in CDRH1 of RM2-01 and RM11-43 appeared to be sufficient to confer high affinity to autologous virus. Broader reactivity against heterologous viruses could be achieved with a few additional somatic mutations, for example, G30R in FR1, V54G in CDRH2, and R98K in CDRH3 (Figure 6). In addition, B cell precursors of several VH1.36-encoded bnAb lineages were identified in the naïve repertoires. Key somatic mutations for affinity maturation and neutralization appeared to be present prior to immunization or accumulate shortly after immunization. Such B cell precursors or intermediates are highly desirable for bnAb development. However, the quick contraction of the VH1.36-derived bnAb response during this set of immunizations remains a significant concern. As vaccine-induced immunity relies on development of long-term antibody titers or serological memory, future HCV vaccine design should focus on how to improve breadth, potency and durability of the bnAb responses. A possible approach is to display an optimized, NF-bearing E2 core multivalently on nanoparticles to achieve such bnAb responses (He et al., 2020). Such virus-like particle (VLP)-inspired vaccine solutions may warrant further investigation in the RM model.

With the failure in the clinical trial of an experimental HCV vaccine (NCT01436357) focusing on eliciting T cell responses (Cox et al., 2019), there is increasing interest in developing HCV vaccine antigens to elicit bnAbs. Hitherto, only one vaccine trial has been performed to elicit nAbs and we showed previously that RMs immunized with the same antigen produces an antibody response highly reminiscent of the human vaccination (Chen et al., 2020). The RM antibody responses target a similar set of antigenic sites with most antibodies directed to non-neutralizing or isolate-specific epitopes. Importantly, RM VH1.36-encoded bnAbs with similar features to human IGHV1-69-encoded bnAbs were isolated here, indicating that the RM model is useful for testing HCV vaccine antigens, particularly those based on ontogeny-targeting strategies, to elicit bnAbs. RMs, like humans, are “endowed” with a specific germline gene in their antibody repertoire that can readily target the conserved E2 NF. Detailed comparison of RM VH1.36-encoded and IGHV1-69-encoded bnAbs, and analysis of bnAb lineage development in the context of RM B cell repertoires during immunization, have therefore greatly expanded our understanding of RM as a preclinical model for HCV vaccine development.

Limitations of the Study

We determined crystal structures for only two RM VH1.36-encoded bnAbs, RM2-01 and RM11-43. Both antibodies were isolated from the same donor animal and engaged E2 using a binding mode that differs from that observed for human IGHV1-69-encoded HCV bnAbs characterized so far. Further investigation is warranted as to whether this binding mode is also used by human antibodies, or whether human binding modes are also applicable to RM antibodies. Moreover, overall classification of RM Ig germline genes is at present incomplete and gene assignment can vary between databases. The lack of standardized nomenclature of RM antibody genes and incomplete reference sequences of RM Ig alleles may affect identification of authentic SHM.

STAR★METHODS

RESOURCE AVAILABILITY

Lead contact

Further information and requests for resources and reagent should be directed to and will be fulfilled by the lead contact, Mansun Law, DPhil (mlaw@scripps.edu).

Materials availability

Antibodies generated in this study are available from the Lead Contact with a completed Materials Transfer Agreement.

Data and code availability

All data and code to understand and assess the conclusions of this research are available in the main text, the Supplementary Materials, and the PDB (accession codes: 7JTF and 7JTG). Additional data related to this paper may be requested from the authors.

EXPERIMENTAL MODELS AND SUBJECT DETAILS

Rhesus macaque PBMC

PBMC samples from rhesus macaques (#30734, #31881, #31782 and #31859) were collected from a previous HCV Env immunization study (Chen et al., 2020). All procedures and experiments in RMs were performed in accordance with protocols reviewed and approved by the Institutional Animal Care and Use Committees (IACUC) of TBRI and The Scripps Research Institute (TSRI).

METHOD DETAILS

Production of monoclonal antibodies and antibody variants

Monoclonal antibodies (mAbs) were generated from single plasmablasts (CD3−CD20− CD80+HLA-DR+) and E2-specific memory B cells (CD3−CD20+CD27+IgG+ E2+) of animals #30734 and #31881 as previously described (Chen et al., 2020). The Ig heavy-chain and light-chain variable genes from single plasmablasts and E2-specific memory B cells were amplified (Sundling et al., 2012b) and cloned into human Igγ1, Igκ and Igλ expression vectors (Tiller et al., 2008). The heavy-chain variants and NGS identified heavy chains were produced by GeneArt gene synthesis (Thermo Fisher Scientific) or Q5 site-directed mutagenesis kit (NEB). Plasmids containing paired antibody heavy- and light-chain genes were co-transfected (1:1 ratio) into ExpiCHO cells using ExpiFectamine CHO Transfection Kit (Thermo Fisher Scientific). Antibody-containing supernatants were harvested 14 days after transfection, filtered through 0.22 μm filters and purified over Protein A-Sepharose 4 Fast Flow (GE healthcare) column per manufacturer’s instruction.

Library preparation for NGS

The dynamics of global B cell repertoires of animals #30734 and #31881 were analyzed by longitudinal NGS of the full-length antibody variable domains of total B cells from PMBCs collected at indicated time points (Figure 1A). For antigen-specific B cell repertoire analysis, E2-specific memory B cells were bulk sorted from PBMCs of animals #31782 and #31859 collected at day 10 after the 4th immunization. A 5′-rapid amplification of cDNA ends (RACE) protocol previously reported for unbiased sequencing of RM B cell repertoires (Dai et al., 2015; Wen et al., 2020) was applied. Briefly, total RNA was extracted from 1~5 million PBMCs into 30 μl of water with RNeasy Mini Kits (Qiagen). 5′-RACE was performed with SMARTer RACE cDNA Amplification Kit (Clontech). The cDNA was purified and eluted in 20 μl of elution buffer (NucleoSpin PCR Clean-up Kit, Clontech). The Ig PCRs were set up with Platinum Taq High- Fidelity DNA Polymerase (Life Technologies) in a total volume of 50 μl with 5 μl of cDNA as template, 1 μl of 5′-RACE primer, and 1 μl of 10 μM reverse primer. To facilitate deep sequencing on the Ion GeneStudio S5 system, the forward primers contained a P1 adaptor, while the reverse primer contained an A adaptor and an Ion XpressTM barcode (Life Technologies) to differentiate the libraries from various time points. A total of 25 cycles of PCRs were performed and the PCR products (~620 bp) were gel purified (Qiagen).

NGS analysis

NGS was performed on the Ion S5 GeneStudio system. Briefly, heavy-chain libraries from different time points were quantitated using Qubit® 2.0 Fluorometer with Qubit® dsDNA HS Assay Kit, and then mixed at an equal ratio for sequencing. Template preparation and (Ion 530) chip loading were performed on Ion Chef using the Ion 520/530 Ext Kit, followed by sequencing on the Ion S5 system with default settings. An improved version of the Antibodyomics pipeline (Wen et al., 2020) was adapted for RM library and used to process the raw data and determine distributions for germline gene usage, somatic hypermutation, germline divergence, and CDRH3 loop length. The two-dimensional (2D) identity-divergence plots were constructed to visualize specific antibody lineages in the context of antibody repertoire at each time point. CDR3 identity of 95% was used as the cutoff for identifying sequences evolutionarily related to a reference antibody (shown as magenta dots on the 2D plots). A consensus or a manually selected sequence was used for antibody synthesis and functional characterization.

Expression and purification of the soluble E2 and Fabs for crystallization

The E2 constructs were expressed and proteins were purified as previously described (Tzarum et al., 2019). The heavy-chain and light-chain sequences of RM2-01 and RM11-43 Fab fragments were generated and expressed in ExpiCHO cells. The mAbs were purified on a protein G affinity column followed by size exclusion chromatography using a Superdex-200 column (GE Healthcare) in 50 mM NaCl, 20 mM Tris-HCl (pH 7.2) buffer.

Crystallization and structural determination of HK6a E2c3-Fab complexes

The E2-Fab complexes were formed by overnight incubation of purified E2 and Fabs in a molar ratio of 1:1.25 (E2:Fab) at room temperature followed by size exclusion chromatography (Superdex-200) to remove unbound Fabs using 20 mM Tris and 50 mM NaCl (pH 7.2) buffer. Crystallization experiments were performed using the vapor diffusion sitting drop method at 20 °C. Crystals of HK6a E2c3-RM2-01 and HK6a E2c3-RM11-43 diffracted to 3.35 Å and 2.60 Å, respectively (Table S2). Crystals of the E2c3 HK6a-RM2-01 complex were obtained using a reservoir solution of 30% PEG 400, 0.1M Cd chloride, 0.1M sodium acetate, pH 4.6, and crystals of E2c3 HK6a-RM11-43 complex from 20% (w/v) PEG 3350, 0.2M sodium sulfate, pH 6.6. Prior to data collection, E2c3 HK6a-RM11-43 crystals were cryoprotected with 10% ethylene glycol (no additional cryoprotectant was required for E2c3 HK6a-RM2-01 crystals) and flash cooled in liquid nitrogen. Diffraction data sets were collected at the Advanced Photon Source (APS, Table S2). Data were integrated and scaled using HKL2000 (Otwinowski and Minor, 1997). The E2c3-Fab structures were solved by molecular replacement using Phaser (McCoy et al., 2005) with E2c3 HK6a-AR3A (PDB entry 6BKB) as the search model. Structure refinement was carried out in Phenix (Adams et al., 2002) and model building with COOT (Emsley and Cowtan, 2004). Final refinement statistics are summarized in Table S2.

KD determination

KD values were determined by bio-layer interferometry (BLI) using an Octet RED instrument (ForteBio, Inc.). IgGs at ~10 μg/ml in a kinetics buffer (1 × PBS, 0.01% BSA and 0.002% Tween 20, pH 7.4) were immobilized onto protein G-coated biosensors. A two-fold concentration gradient of E2, starting at 10μM, 20μM or 40μM depending on the binding affinity, was used in a titration series of five. The kon and koff values of E2 protein for each IgG were measured in real time to determine KD values. All binding data were collected at 30 °C.

HCV neutralization assays

HCV pseudoparticles (HCVpp) were generated by co-transfection of 293T cells with pNL4-3.lucR−E− and the corresponding expression plasmids encoding the E1E2 from isolate HCV-1, H77, UKN1B 12.6, J6, S52, UKN4.11.1 or SA13 (Bartosch et al., 2003; Lavillette et al., 2005; Li et al., 2015; Meunier et al., 2005) at a 4:1 ratio by polyethylenimine (PEI, Polysciences). HCVpp neutralization was carried out on Huh 7 cells with mAbs (50 μg/ml) as previously described (Bailey et al., 2019). Virus infectivity was detected with Bright-Glo luciferase assay system (Promega), and percent neutralization was calculated as the virus infectivity inhibited at the antibody concentrations divided by the infectivity without antibody after background subtraction. The background infectivity of the pseudotype virus was defined by infecting cells with virus made only with pNL4-3.lucR-E-. Pseudoparticles displaying the vesicular stomatitis virus envelope glycoprotein G (VSVpp) were used as a control for nonspecific neutralizing activity.

For HCV cell culture (HCVcc) neutralization, HCV isolates H77, H77ΔHVR1, TN, DH1, J4, J6, J8, S52, DBN, ED43, SA13 and HK6a (Carlsen et al., 2014; Gottwein et al., 2009; Jensen et al., 2008; Li et al., 2015; Pedersen et al., 2013; Prentoe et al., 2011; Scheel et al., 2011) were propagated in Huh 7.5 cells. Neutralization was performed as previously described (Prentoe and Bukh, 2019). Briefly, viruses were preincubated with mAb (at 50 μg/ml or a dilution series) for 1 h at 37°C and then added to Huh7.5 cells plated at 7 × 103 in poly-D-lysine-coated 96-well plates the day before. After 4 h incubation with the virus/antibody mix, the cells were washed and incubated in fresh medium for a total infection time of 48 h. Cells were then fixed and stained with 9E10 antibody. The data were normalized with 8 replicates of virus only and analyzed using GraphPad Prism.

ELISA

Methods for measuring anti-E1E2 antibody titers have been described elsewhere (Major and Law, 2019). Briefly, Costar High Binding Half-Area 96-well plates (Corning) were coated overnight at 4 °C with 5 μg/ml of G. nivalis lectin (GNL, Vector Laboratories). After blocking with 4% nonfat milk (Bio-Rad) in PBS + 0.05% Tween-20 (PBS-T), plates were incubated with batch-diluted cell lysates from 293T cells expressing E1E2 at room temperature for 1 h. Serial dilutions of antibodies were then added and incubated at room temperature for at least 1 h. Horseradish peroxidase (HRP)-conjugated goat anti-human IgG Fc antibody (1:2,000, Jackson ImmunoResearch) was used for detection.

Epitope mapping by alanine scanning mutagenesis

High-throughput scanning mutagenesis using an HCV E1E2 alanine scanning library (genotype 1a, strain H77; reference sequence NC_004102) was performed as described previously (Gopal et al., 2017). Binding of the mAb to an individual alanine mutant was expressed as percentage of the mAb binding signal obtained with wild type E1E2.

Bioinformatics

Antibody sequences were submitted to IgBLAST (https://www.ncbi.nlm.nih.gov/igblast/) and ImMunoGeneTics information system (IMGT, http://www.imgt.org/) for gene identification and genetic assignment. Multiple-sequence alignments were performed using MegAlign Pro program in Lasergene 17 (DNAstar). Maximum-likelihood phylogenetic tree was constructed using MEGA X software.

QUANTIFICATION AND STATISTICAL ANALYSIS

Graphpad Prism v9 was used for all statistical analyses. The significance of differences in CDRH3 length and SHM between RM and human antibodies was calculated using unpaired, two-tailed Mann-Whitney U tests. Data were considered statistically significant at *P < 0.05, **P < 0.001, ***P < 0.0001.

Supplementary Material

2

KEY RESOURCES TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER
Antibodies
PE/Cy7 anti-human CD3 (clone SP34-2) BD Biosciences Cat#557749
PerCP/Cy5.5 anti-human CD20 (clone 2H7) BioLegend Cat#302326; RRID: AB_893283
APC anti-human CD20 (clone 2H7) BioLegend Cat#302310; RRID: AB_314258
PE/Cy5 anti-human CD80 (clone L307.4) BD Biosciences Cat#559370
BV421 anti-human HLA-DR (clone L243) BioLegend Cat#307636; RRID: AB_2561831
PE anti-human CD27 (clone O323) BioLegend Cat#302808; RRID: AB_314300
FITC anti-human IgG (clone G18-145) BD Biosciences Cat#555786
HRP-conjugated goat anti-human IgG Fc Jackson ImmunoResearch Cat# 109-035-008; RRID: AB_2337579
AR3A Giang et al., 2020 N/A
HEPC74 Bailey et al., 2017 N/A
HC84.1 Krey et al., 2013 N/A
212.1.1 Keck et al., 2019 N/A
RM2-01 Chen et al., 2020 N/A
RM11-43 Chen et al., 2020 N/A
Bacterial and Virus Strains
JFH1-based HCV recombinant with Core-NS2 of H77 Li et al., 2015; Carlsen et al., 2014 N/A
JFH1-based HCV recombinant with Core-NS2 of H77ΔHVR1 Prentoe et al., 2011 N/A
JFH1-based HCV recombinant with Core-NS2 of TN Carlsen et al., 2014 N/A
JFH1-based HCV recombinant with Core-NS2 of DH1 Carlsen et al., 2014 N/A
JFH1-based HCV recombinant with Core-NS2 of J4 Carlsen et al., 2014 N/A
JFH1-based HCV recombinant with Core-NS2 of J6 Carlsen et al., 2014 N/A
JFH1-based HCV recombinant with Core-NS2 of J8 Carlsen et al., 2014 N/A
JFH1-based HCV recombinant with Core-NS2 of S52 Carlsen et al., 2014 N/A
JFH1-based HCV recombinant with Core-NS2 of DBN Carlsen et al., 2014 N/A
JFH1-based HCV recombinant with Core-NS2 of ED43 Prentoe et al., 2011 N/A
JFH1-based HCV recombinant with Core-NS2 of SA13 Jensen et al., 2008 N/A
JFH1-based HCV recombinant with Core-NS2 of HK6a Prentoe et al., 2011 N/A
Biological Samples
PBMC samples from rhesus macaques Chen et al., 2020 N/A
Chemicals, Peptides, and Recombinant Proteins
HCV-1 E2ΔTM Chen et al., 2020 N/A
HK6a E2c3 Tzarum et al., 2019 N/A
Critical Commercial Assays
Alexa Fluor™ 647 Protein Labeling Kit Thermo Fisher Scientific Cat#A20173
ExpiFectamine CHO Transfection Kit Thermo Fisher Scientific Cat#A29127
Deposited Data
HK6a E2c3-RM2-01 structure This paper PDB: 7JTF
HK6a E2c3-RM11-43 structure This paper PDB: 7JTG
Experimental Models: Cell Lines
ExpiCHO-S cells Thermo Fisher Scientific Cat#A29127; RRID: CVCL_5J31
Huh 7 cells APATH N/A
Huh 7.5 cells APATH N/A
HEK293T cells ATCC Cat# CRL-3216; RRID:CVCL_0063
Oligonucleotides
Primers for mAb cloning Sundling et al., 2012b N/A
Primers for NGS library Dai et al., 2015 N/A
Recombinant DNA
Human Igγ1 expression vector Tiller et al., 2008 N/A
Human Igκ expression vector Tiller et al., 2008 N/A
Human Igλ expression vector Tiller et al., 2008 N/A
HCV-1 E1E2 expression vector Li et al., 2015 N/A
H77 E1E2 expression vector Meunier et al., 2005 N/A
UKN1B 12.6 E1E2 expression vector Lavillette et al., 2005 N/A
J6 E1E2 expression vector Meunier et al., 2005 N/A
S52 E1E2 expression vector Meunier et al., 2005 N/A
UKN4.11.1 E1E2 expression vector Lavillette et al., 2005 N/A
SA13 E1E2 expression vector Meunier et al., 2005 N/A
HK6a E1E2 expression vector Meunier et al., 2005 N/A
 
Software and Algorithms
Prism 9 GraphPad https://www.graphpad.com/scientific-software/prism/; RRID:SCR_002798
Lasergene 17 DNAstar https://www.dnastar.com/software/lasergene/; RRID:SCR_000291
MEGA X MEGA https://www.megasoftware.net/; RRID:SCR_000667
IgBLAST NCBI https://www.ncbi.nlm.nih.gov/projects/igblast/; RRID:SCR_002873
abYsis abYsis http://www.abysis.org/abysis/; RRID:SCR_000756
IMGT/V-QUEST IMGT http://www.imgt.org/; RRID: SCR_012780
PyMOL Schrodinger LLC https://www.schrodinger.com/pymol; RRID: SCR_000305
Open in a new tab

Highlights.

  • HCV Env immunization elicited a VH1.36-encoded bnAb response in rhesus macaques

  • Macaque VH1.36- and human IGHV1-69-encoded bnAbs share common features

  • Macaque bnAbs adopt different binding modes to recognize a flexible antigenic surface

  • bnAbs mature functionally through rapid lineage development with few SHM

ACKNOWLEDGEMENTS

This work was funded in part by NIH Grants AI079031 (to M.L.), AI123861 (to M.L., J.Z. and R.L.), AI123365 (to M.L.) and AI106005 (to M.L. and I.A.W.), Independent Research Fund Denmark (DFF-4004-00598 and DFF-8020-00391B to J.B.), Novo Nordisk Foundation (NNF17OC0029372 and NNF19OC0054518 to J.B.), Candys Foundation (to J.P., E.A. and J.B.) and Lundbeck Foundation (R303-2018-3396 to R.V-M and R324-2019-1375 to J.P.). Integral Molecular, Inc. is supported by U.S. Department of Health and Human Services (HHS) contract (HHSN272201400058C to B.J.D). We thank E. Davidson for coordinating epitope mapping, X. Dai and M. Elsliger for crystallographic and computational support, and H. Tien for automated crystal screening. X-ray data sets were collected at APS beamline 23ID-B (GM/CA CAT). GM/CA@APS has been funded in whole or in part with Federal funds from the National Cancer Institute (ACB-12002) and the National Institute of General Medical Sciences (AGM-12006). This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. This investigation used resources that were supported by Southwest National Primate Research Center (SNPRC) grant P51 OD011133 and U42OD010442 from the Office of Research Infrastructure Programs, NIH. This is manuscript #30055 from The Scripps Research Institute.

Footnotes

DECLARATION OF INTERESTS

The authors declare no conflict of interest.

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Associated Data

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Supplementary Materials

2
NIHMS1676208-supplement-2.pdf (2.1MB, pdf)

Data Availability Statement

All data and code to understand and assess the conclusions of this research are available in the main text, the Supplementary Materials, and the PDB (accession codes: 7JTF and 7JTG). Additional data related to this paper may be requested from the authors.

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