Abstract
Hepatitis C continues to be a significant public health problem despite advancements in antiviral therapeutics. To eliminate this disease, an effective vaccine against new infections and re-infections is needed. However, to date only one Hepatitis C virus (HCV) envelope protein (E1E2) immunogen, developed by Chiron Inc., has been tested in a Phase I clinical trial (ClinicalTrials.gov identifier NCT00500747). To establish a benchmark for elicitation of broadly neutralizing antibodies (bnAbs) by E1E2, we previously immunized non-human primates (NHPs) with this immunogen and isolated monoclonal nAbs that exhibit neutralization potency comparable to human nAbs. Here we show that NHP nAbs, encoded by germline genes IGHV1-138*01 and IGHV4-NL_5*01 (homologs of human IGHV1-69*10 and IGHV4-59*12, respectively), recognize a relatively conserved E2 region (neutralizing face) proximal to antigenic region 3 (AR3). These NHP AR3-targeting nAbs share highly similar binding modes to human AR3-targeting nAbs, suggesting a similarity in human and NHP immune responses to the same HCV immunogen.
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Data availability
The X-ray coordinates and structure factors have been deposited in the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank under accession codes 9MS9, 9MRZ, 9MNS, 9MNU, 9MNT, 9MNQ, and 9MSC.
References
Falade-Nwulia, O. et al. Oral direct-acting agent therapy for Hepatitis C virus infection: a systematic review. Ann. Intern. Med. 166, 637–648 (2017).
Kanwal, F. et al. Risk of hepatocellular cancer in HCV patients treated with direct-acting antiviral agents. Gastroenterology 153, 996–1005.e1 (2017).
Midgard, H. et al. HCV epidemiology in high-risk groups and the risk of reinfection. J. Hepatol. 65, S33–S45 (2016).
Echeverría, N. et al. In the era of rapid mRNA-based vaccines: Why is there no effective Hepatitis C virus vaccine yet? World J. Hepatol. 13, 1234–1268 (2021).
Torrents de la Peña, A. et al. Structure of the Hepatitis C virus E1E2 glycoprotein complex. Science 378, 263–269 (2022).
Metcalf, M. C. et al. Structure of engineered Hepatitis C virus E1E2 ectodomain in complex with neutralizing antibodies. Nat. Commun. 14, 3980 (2023).
Augestad, E. H. et al. The Hepatitis C virus envelope protein complex is a dimer of heterodimers. Nature 633, 704–709 (2024).
Pileri, P. et al. Binding of Hepatitis C virus to CD81. Science 282, 938–41 (1998).
Evans, M. J. et al. Claudin-1 is a Hepatitis C virus co-receptor required for a late step in entry. Nature. 446, 801–5 (2007).
Scarselli, E. et al. The human scavenger receptor class B type I is a novel candidate receptor for the Hepatitis C virus. EMBO J. 21, 5017–25 (2002).
Ploss, A. et al. Human occludin is a Hepatitis C virus entry factor required for infection of mouse cells. Nature 457, 882–6 (2009).
Lindenbach, B. D. & Rice, C. M. The ins and outs of Hepatitis C virus entry and assembly. Nat. Rev. Microbiol. 11, 688–700 (2013).
Owsianka, A. et al. Monoclonal antibody AP33 defines a broadly neutralizing epitope on the Hepatitis C virus E2 envelope glycoprotein. J. Virol. 79, 11095–11104 (2005).
Law, M. et al. Broadly neutralizing antibodies protect against Hepatitis C virus quasispecies challenge. Nat. Med. 14, 25–7 (2008).
Harman, C. et al. A view of the E2-CD81 interface at the binding site of a neutralizing antibody against Hepatitis C virus. J. Virol. 89, 492–501 (2015).
Kong, L. et al. Hepatitis C virus E2 envelope glycoprotein core structure. Science 342, 1090–4 (2013).
Tzarum, N., Wilson, I. A. & Law, M. The neutralizing face of Hepatitis C virus E2 envelope glycoprotein. Front. Immunol. 9, 1315 (2018).
Kong, L., Jackson, K. N., Wilson, I. A. & Law, M. Capitalizing on knowledge of Hepatitis C virus neutralizing epitopes for rational vaccine design. Curr. Opin. Virol. 11, 148–57 (2015).
Giang, E. et al. Human broadly neutralizing antibodies to the envelope glycoprotein complex of Hepatitis C virus. Proc. Natl. Acad. Sci. USA 109, 6205–6210 (2012).
Weber, T. et al. Analysis of antibodies from HCV elite neutralizers identifies genetic determinants of broad neutralization. Immunity 55, 341–354.e7 (2022).
Tzarum, N. et al. An alternate conformation of HCV E2 neutralizing face as an additional vaccine target. Sci. Adv. 6, eabb5642 (2020).
Kumar, A. et al. Structural insights into Hepatitis C virus receptor binding and entry. Nature 598, 521–525 (2021).
Kumar, A. et al. Regions of Hepatitis C virus E2 required for membrane association. Nat. Commun. 14, 433 (2023).
Tzarum, N. et al. Genetic and structural insights into broad neutralization of Hepatitis C virus by human VH1-69 antibodies. Sci. Adv. 5, eaav1882 (2019).
Chen, F., Tzarum, N., Wilson, I. A. & Law, M. VH1-69 antiviral broadly neutralizing antibodies: genetics, structures, and relevance to rational vaccine design. Curr. Opin. Virol. 34, 149–159 (2019).
Flyak, A. I. et al. HCV broadly neutralizing antibodies use a CDRH3 disulfide motif to recognize an E2 glycoprotein site that can be targeted for vaccine design. Cell Host Microbe 24, 703–716.e3 (2018).
Ogega, C. O. et al. Convergent evolution and targeting of diverse E2 epitopes by human broadly neutralizing antibodies are associated with HCV clearance. Immunity 57, 890–903.e6 (2024).
Wu, N. C. & Wilson, I. A. Influenza hemagglutinin structures and antibody recognition. Cold Spring Harb. Perspect. Med. 10, a038778 (2020).
Corti, D. & Lanzavecchia, A. Broadly neutralizing antiviral antibodies. Annu. Rev. Immunol. 31, 705–42 (2013).
Barnes, E., Cooke, G. S., Lauer, G. M. & Chung, R. T. Implementation of a controlled human infection model for evaluation of HCV vaccine candidates. Hepatology. 77, 1757–1772 (2023).
Liang, T. J., Feld, J. J., Cox, A. L. & Rice, C. M. Controlled human infection model - fast track to HCV vaccine? N. Engl. J. Med. 385, 1235–1240 (2021).
Gibbs, R. A. et al. Evolutionary and biomedical insights from the rhesus macaque genome. Science 316, 222–34 (2007).
Lemaitre, J. et al. Non-human primate models of human respiratory infections. Mol. Immunol. 135, 147–164 (2021).
Yu, G. Y. et al. Cynomolgus macaque (Macaca fascicularis) immunoglobulin heavy chain locus description. Immunogenetics 68, 417–428 (2016).
Antinori, S. et al. Non-human primate and human malaria: past, present and future. J. Travel Med. 28, taab036 (2021).
Roark, R. S. et al. Recapitulation of HIV-1 Env-antibody coevolution in macaques leading to neutralization breadth. Science 371, eabd2638 (2021).
He, W. T. et al. Broadly neutralizing antibodies to SARS-related viruses can be readily induced in rhesus macaques. Sci. Transl. Med. 14, eabl9605 (2022).
Vázquez Bernat, N. et al. Rhesus and cynomolgus macaque immunoglobulin heavy-chain genotyping yields comprehensive databases of germline VDJ alleles. Immunity 54, 355–366.e4 (2021).
Lefranc, M. P. IMGT, the international immunogenetics database. Nucleic Acids Res. 31, 307–10 (2003).
Ramesh, A. et al. Structure and diversity of the rhesus macaque immunoglobulin loci through multiple de novo genome assemblies. Front. Immunol. 8, 1407 (2017).
Kaduk, M., Corcoran, M. & Karlsson Hedestam, G. B. Addressing IGHV gene structural diversity enhances immunoglobulin repertoire analysis: Lessons from rhesus macaque. Front. Immunol. 13, 818440 (2022).
Corcoran, M. M. et al. Production of individualized V gene databases reveals high levels of immunoglobulin genetic diversity. Nat. Commun. 7, 13642 (2016).
Guo, Y., Waltari, E., Lu, H., Sheng, Z. & Wu, X. Novel rhesus macaque immunoglobulin germline genes identified by three sequencing approaches. Front. Immunol. 15, 1506348 (2024).
Chen, F. et al. Antibody responses to immunization with HCV envelope glycoproteins as a baseline for B-cell-based vaccine development. Gastroenterology. 158, 1058–1071.e6 (2020).
Chen, F. et al. Functional convergence of a germline-encoded neutralizing antibody response in rhesus macaques immunized with HCV envelope glycoproteins. Immunity 54, 781–796.e4 (2021).
Frey, S. E. et al. Safety and immunogenicity of HCV E1E2 vaccine adjuvanted with MF59 administered to healthy adults. Vaccine 28, 6367–73 (2010).
Ray, R. et al. Characterization of antibodies induced by vaccination with Hepatitis C virus envelope glycoproteins. J. Infect. Dis. 202, 862–6 (2010).
Peres, A. et al. Population-level genomic analysis of immunoglobulin loci variation in rhesus macaques reveals extensive germline diversity. Immunity 59, 213–228.e6 (2026).
Lees, W. D., Saha, S., Yaari, G. & Watson, C. T. Digger: directed annotation of immunoglobulin and T cell receptor V, D, and J gene sequences and assemblies. Bioinformatics. 40, btae144 (2024).
Frumento, N., Flyak, A. I. & Bailey, J. R. Mechanisms of HCV resistance to broadly neutralizing antibodies. Curr. Opin. Virol. 50, 23–29 (2021).
Connolly, M. L. The molecular surface package. J. Mol. Graph. 11, 139–41 (1993).
Wu, T. T. & Kabat, E. A. An analysis of the sequences of the variable regions of Bence Jones proteins and myeloma light chains and their implications for antibody complementarity. J. Exp. Med. 132, 211–50 (1970).
Olson, R. D. et al. Introducing the Bacterial and Viral Bioinformatics Resource Center (BV-BRC): a resource combining PATRIC, IRD and ViPR. Nucleic Acids Res. 51, D678–D689 (2023).
Goffard, A. & Dubuisson, J. Glycosylation of hepatitis C virus envelope proteins. Biochimie 85, 295–301 (2003).
He, L. et al. Proof of concept for rational design of Hepatitis C virus E2 core nanoparticle vaccines. Sci. Adv. 6, eaaz6225 (2020).
Gomez-Escobar, E., Roingeard, P. & Beaumont, E. Current Hepatitis C vaccine candidates based on the induction of neutralizing antibodies. Viruses 15, 1151 (2023).
Burm, R., Collignon, L., Mesalam, A. A. & Meuleman, P. Animal models to study Hepatitis C virus infection. Front. Immunol. 9, 1032 (2018).
Bailey, J. R., Barnes, E. & Cox, A. L. Approaches, progress, and challenges to Hepatitis C vaccine development. Gastroenterology 156, 418–430 (2019).
Ströh, L. J., Nagarathinam, K. & Krey, T. Conformational flexibility in the CD81-binding site of the Hepatitis C virus glycoprotein E2. Front. Immunol. 9, 1396 (2018).
Nabel, G. J. Designing tomorrow’s vaccines. N. Engl. J. Med. 368, 551–60 (2013).
Burton, D. R. & Hangartner, L. Broadly neutralizing antibodies to HIV and their role in vaccine design. Annu. Rev. Immunol. 34, 635–59 (2016).
Burton, D. R. Antibodies, viruses and vaccines. Nat. Rev. Immunol. 2, 706–13 (2002).
Dormitzer, P. R., Ulmer, J. B. & Rappuoli, R. Structure-based antigen design: a strategy for next generation vaccines. Trends Biotechnol. 26, 659–67 (2008).
Schultheiß, C. et al. B cells expressing mutated IGHV1-69-encoded antigen receptors related to virus neutralization show lymphoma-like transcriptomes in patients with chronic HCV infection. Hepatol. Commun. 8, e0503 (2024).
Warren, W. C. et al. Sequence diversity analyses of an improved rhesus macaque genome enhance its biomedical utility. Science 370, eabc6617 (2020).
Cirelli, K. M. et al. Slow delivery immunization enhances HIV neutralizing antibody and germinal center responses via modulation of immunodominance. Cell 177, 1153–1171.e28 (2019).
Cheng, H., Concepcion, G. T., Feng, X., Zhang, H. & Li, H. Haplotype-resolved de novo assembly using phased assembly graphs with hifiasm. Nat. Methods 18, 170–175 (2021).
Kent, W. J. BLAT—the BLAST-like alignment tool. Genome Res. 12, 656–64 (2002).
Madeira, F. et al. Search and sequence analysis tools services from EMBL-EBI in 2022. Nucleic Acids Res. 50, W276–W279 (2022).
Gouet, P., Courcelle, E. & Stuart, D. I. & Métoz, F. ESPript: analysis of multiple sequence alignments in PostScript. Bioinformatics 15, 305–8 (1999).
Abhinandan, K. R. & Martin, A. C. Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains. Mol. Immunol. 45, 3832–9 (2008).
Tiller, T. et al. Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning. J. Immunol. Methods 329, 112–24 (2008).
Lieu, R. et al. Rapid and robust antibody Fab fragment crystallization utilizing edge-to-edge beta-sheet packing. PLoS ONE. 15, e0232311 (2020).
Bailey, J. R., Urbanowicz, R. A., Ball, J. K., Law, M. & Foung, S. K. H. Standardized method for the study of antibody neutralization of HCV pseudoparticles (HCVpp). Methods Mol. Biol. 1911, 441–450 (2019).
Chen, F. et al. The conserved bridging domain on HCV E1E2 glycoprotein complex is targeted by neutralizing antibodies from diverse lineages. Preprint at bioRxiv https://doi.org/10.1101/2025.11.05.686883 (2025).
Bartosch, B., Dubuisson, J. & Cosset, F. L. Infectious Hepatitis C virus pseudo-particles containing functional E1-E2 envelope protein complexes. J. Exp. Med. 197, 633–42 (2003).
Lavillette, D. et al. Characterization of host-range and cell entry properties of the major genotypes and subtypes of Hepatitis C virus. Hepatology 41, 265–74 (2005).
Meunier, J. C. et al. Evidence for cross-genotype neutralization of Hepatitis C virus pseudo-particles and enhancement of infectivity by apolipoprotein C1. Proc. Natl. Acad. Sci. USA 102, 4560–5 (2005).
Li, Y. P., Ramirez, S., Mikkelsen, L. & Bukh, J. Efficient infectious cell culture systems of the Hepatitis C virus (HCV) prototype strains HCV-1 and H77. J. Virol. 89, 811–23 (2015).
Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–26 (1997).
McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).
Schritt, D. et al. Repertoire builder: high-throughput structural modeling of B and T cell receptors. Mol. Syst. Des. Eng. 4, 761–768 (2019).
Adams, P. D. et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D Biol. Crystallogr. 58, 1948–54 (2002).
Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–32 (2004).
Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010).
Krissinel, E. & Henrick, K. Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372, 774–97 (2007).
Acknowledgements
The authors thank Henry Tien for automated robotic crystal screening at The Scripps Research Institute and Xiaoping Dai for help with in-house data collection. This work was funded in part by NIH AI168251 (M.L., I.A.W., and R.L.S.) and AI168917 (M.L.). X-ray diffraction data were collected at the Advanced Photon Source (APS) beamline 23ID-B (GM/CA CAT) and the Stanford Synchrotron Radiation Lightsource (SSRL) beamline 12-2. GM/CA CAT is funded in whole or in part with federal funds from the National Cancer Institute (Y1-CO-1020) and the National Institute of General Medical Sciences (NIGMS) (Y1-GM-1104). Use of the APS was supported by the US Department of Energy (DOE), Basic Energy Sciences, Office of Science, under Contract DE-AC02-06CH11357. The SSRL is a Directorate of SLAC National Accelerator Laboratory, and an Office of Science User Facility operated for the US DOE of Science by Stanford University. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research, and by the NIH, NIGMS (including P41GM103393), and the National Center for Research Resources (NCRR) (P41RR001209). The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of NIAID, NIGMS, NCRR, or NIH. This investigation used resources that were supported by the Southwest National Primate Research Center grant P51 OD011133 from the Office of Research Infrastructure Programs, National Institutes of Health. Research reported in this publication was supported by the Office of the Director, National Institutes of Health under Award Numbers S10OD028732 and S10OD032443.
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Conceptualization: Y.T.K.N., F.C., R.L.S., M.L., I.A.W. Methodology: Y.T.K.N., F.C., R.L.S., M.L., I.A.W. Investigation: Y.T.K.N., F.C., E.G., S.S., N.T., L.A.U., R.L.S., C.C. Visualization: Y.T.K.N., F.C., R.L.S., C.W. Supervision: R.L.S., C.T.W., M.L., I.A.W. Writing—original draft: Y.T.K.N., F.C., R.L.S., S.S., C.T.W., M.L., I.A.W. Writing—review and editing: Y.T.K.N., F.C., R.L.S., S.S., C.C., C.T.W., M.L., I.A.W.
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C.T.W. is co-founder/CSO of Clareo Biosciences, Inc. The other authors declare no competing interests.
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Nguyen, Y.T.K., Chen, F., Giang, E. et al. Structural and genetic signatures of two classes of HCV E2 neutralizing face antibodies from non-human primates immunized with a recombinant E1E2. npj Vaccines (2026). https://doi.org/10.1038/s41541-026-01449-1
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DOI: https://doi.org/10.1038/s41541-026-01449-1
