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Structural basis for diverse N-glycan recognition by HIV-1–neutralizing V1–V2–directed antibody PG16

Abstract

HIV-1 uses a diverse N-linked-glycan shield to evade recognition by antibody. Select human antibodies, such as the clonally related PG9 and PG16, recognize glycopeptide epitopes in the HIV-1 V1–V2 region and penetrate this shield, but their ability to accommodate diverse glycans is unclear. Here we report the structure of antibody PG16 bound to a scaffolded V1–V2, showing an epitope comprising both high mannose–type and complex-type N-linked glycans. We combined structure, NMR and mutagenesis analyses to characterize glycan recognition by PG9 and PG16. Three PG16-specific residues, arginine, serine and histidine (RSH), were critical for binding sialic acid on complex-type glycans, and introduction of these residues into PG9 produced a chimeric antibody with enhanced HIV-1 neutralization. Although HIV-1–glycan diversity facilitates evasion, antibody somatic diversity can overcome this and can provide clues to guide the design of modified antibodies with enhanced neutralization.

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Figure 1: Cocrystal structure of human antibody PG16 and scaffolded-V1–V2 of HIV-1 gp120 with a high mannose–type N-linked glycan at residue 160gp120 and a hybrid-type N-linked glycan at residue 173gp120.
Figure 2: PG16 and PG9 interactions with V1–V2 domain of HIV-1 gp120.
Figure 3: PG16 interactions with hybrid-type N-linked glycan at residue 156gp120 or 173gp120.
Figure 4: PG16 binds complex-type glycans more tightly than Man5GlcNAc2, whereas PG9 prefers Man5GlcNAc2.
Figure 5: Design and glycan recognition of PG9-16-RSH antibody.
Figure 6: Neutralization activity of PG9, PG16 and PG9-16-RSH.
Figure 7: Effect of sialic acid removal by neuraminidase treatment on PG9, PG16 and PG9-16-RSH antibody-mediated neutralization of HIV-1.
Figure 8: Glycan recognition, binding affinities, and detailed interactions of PG16, PG9 and PG9-16-RSH.

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References

  1. Pantophlet, R. & Burton, D.R. GP120: target for neutralizing HIV-1 antibodies. Annu. Rev. Immunol. 24, 739–769 (2006).

    CAS  Article  PubMed  Google Scholar 

  2. Wyatt, R. & Sodroski, J. The HIV-1 envelope glycoproteins: fusogens, antigens, and immunogens. Science 280, 1884–1888 (1998).

    CAS  Article  PubMed  Google Scholar 

  3. Binley, J.M. et al. Analysis of the interaction of antibodies with a conserved enzymatically deglycosylated core of the HIV type 1 envelope glycoprotein 120. AIDS Res. Hum. Retroviruses 14, 191–198 (1998).

    CAS  Article  PubMed  Google Scholar 

  4. Walker, L.M. et al. A limited number of antibody specificities mediate broad and potent serum neutralization in selected HIV-1 infected individuals. PLoS Pathog. 6, e1001028 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  5. Walker, L.M. et al. Broad neutralization coverage of HIV by multiple highly potent antibodies. Nature 477, 466–470 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  6. Kwong, P.D. et al. Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody. Nature 393, 648–659 (1998).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  7. Wyatt, R. et al. The antigenic structure of the HIV gp120 envelope glycoprotein. Nature 393, 705–711 (1998).

    CAS  Article  PubMed  Google Scholar 

  8. Trkola, A. et al. Human monoclonal antibody 2G12 defines a distinctive neutralization epitope on the gp120 glycoprotein of human immunodeficiency virus type 1. J. Virol. 70, 1100–1108 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Sanders, R.W. et al. The mannose-dependent epitope for neutralizing antibody 2G12 on human immunodeficiency virus type 1 glycoprotein gp120. J. Virol. 76, 7293–7305 (2002).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  10. Scanlan, C.N. et al. The broadly neutralizing anti-human immunodeficiency virus type 1 antibody 2G12 recognizes a cluster of α1→2 mannose residues on the outer face of gp120. J. Virol. 76, 7306–7321 (2002).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  11. Binley, J.M. et al. Profiling the specificity of neutralizing antibodies in a large panel of plasmas from patients chronically infected with human immunodeficiency virus type 1 subtypes B and C. J. Virol. 82, 11651–11668 (2008).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  12. Walker, L.M. et al. Rapid development of glycan-specific, broad, and potent anti-HIV-1 gp120 neutralizing antibodies in an R5 SIV/HIV chimeric virus infected macaque. Proc. Natl. Acad. Sci. USA 108, 20125–20129 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  13. Walker, L.M. et al. Broad and potent neutralizing antibodies from an African donor reveal a new HIV-1 vaccine target. Science 326, 285–289 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  14. Calarese, D.A. et al. Dissection of the carbohydrate specificity of the broadly neutralizing anti-HIV-1 antibody 2G12. Proc. Natl. Acad. Sci. USA 102, 13372–13377 (2005).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  15. Calarese, D.A. et al. Antibody domain exchange is an immunological solution to carbohydrate cluster recognition. Science 300, 2065–2071 (2003).

    CAS  Article  PubMed  Google Scholar 

  16. Doores, K.J., Fulton, Z., Huber, M., Wilson, I.A. & Burton, D.R. Antibody 2G12 recognizes di-mannose equivalently in domain- and nondomain-exchanged forms but only binds the HIV-1 glycan shield if domain exchanged. J. Virol. 84, 10690–10699 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  17. Wang, L.X., Ni, J., Singh, S. & Li, H. Binding of high-mannose-type oligosaccharides and synthetic oligomannose clusters to human antibody 2G12: implications for HIV-1 vaccine design. Chem. Biol. 11, 127–134 (2004).

    PubMed  Google Scholar 

  18. McLellan, J.S. et al. Structure of HIV-1 gp120 V1/V2 domain with broadly neutralizing antibody PG9. Nature 480, 336–343 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. Pejchal, R. et al. A potent and broad neutralizing antibody recognizes and penetrates the HIV glycan shield. Science 334, 1097–1103 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  20. Sattentau, Q.J. Vaccinology: a sweet cleft in HIV's armour. Nature 480, 324–325 (2011).

    CAS  Article  PubMed  Google Scholar 

  21. Leonard, C.K. et al. Assignment of intrachain disulfide bonds and characterization of potential glycosylation sites of the type 1 recombinant human immunodeficiency virus envelope glycoprotein (gp120) expressed in Chinese hamster ovary cells. J. Biol. Chem. 265, 10373–10382 (1990).

    CAS  PubMed  Google Scholar 

  22. Mizuochi, T. et al. Carbohydrate structures of the human-immunodeficiency-virus (HIV) recombinant envelope glycoprotein gp120 produced in Chinese-hamster ovary cells. Biochem. J. 254, 599–603 (1988).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  23. Zhu, X., Borchers, C., Bienstock, R.J. & Tomer, K.B. Mass spectrometric characterization of the glycosylation pattern of HIV-gp120 expressed in CHO cells. Biochemistry 39, 11194–11204 (2000).

    CAS  Article  PubMed  Google Scholar 

  24. Bonomelli, C. et al. The glycan shield of HIV is predominantly oligomannose independently of production system or viral clade. PLoS ONE 6, e23521 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  25. Doores, K.J. et al. Envelope glycans of immunodeficiency virions are almost entirely oligomannose antigens. Proc. Natl. Acad. Sci. USA 107, 13800–13805 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  26. Eggink, D. et al. Lack of complex N-glycans on HIV-1 envelope glycoproteins preserves protein conformation and entry function. Virology 401, 236–247 (2010).

    CAS  Article  PubMed  Google Scholar 

  27. Go, E.P. et al. Characterization of glycosylation profiles of HIV-1 transmitted/founder envelopes by mass spectrometry. J. Virol. 85, 8270–8284 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  28. Pabst, M., Chang, M., Stadlmann, J. & Altmann, F. Glycan profiles of the 27 N-glycosylation sites of the HIV envelope protein CN54gp140. Biol. Chem. 393, 719–730 (2012).

    CAS  Article  PubMed  Google Scholar 

  29. Mizuochi, T. et al. Diversity of oligosaccharide structures on the envelope glycoprotein gp 120 of human immunodeficiency virus 1 from the lymphoblastoid cell line H9. Presence of complex-type oligosaccharides with bisecting N-acetylglucosamine residues. J. Biol. Chem. 265, 8519–8524 (1990).

    CAS  PubMed  Google Scholar 

  30. Kong, L. et al. Expression-system-dependent modulation of HIV-1 envelope glycoprotein antigenicity and immunogenicity. J. Mol. Biol. 403, 131–147 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  31. Binley, J.M. et al. Role of complex carbohydrates in human immunodeficiency virus type 1 infection and resistance to antibody neutralization. J. Virol. 84, 5637–5655 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  32. Ouellet, M. et al. Galectin-1 acts as a soluble host factor that promotes HIV-1 infectivity through stabilization of virus attachment to host cells. J. Immunol. 174, 4120–4126 (2005).

    CAS  Article  PubMed  Google Scholar 

  33. St-Pierre, C. et al. Host-soluble galectin-1 promotes HIV-1 replication through a direct interaction with glycans of viral gp120 and host CD4. J. Virol. 85, 11742–11751 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  34. van Montfort, T. et al. HIV-1 N-glycan composition governs a balance between dendritic cell-mediated viral transmission and antigen presentation. J. Immunol. 187, 4676–4685 (2011).

    CAS  Article  PubMed  Google Scholar 

  35. Reitter, J.N., Means, R.E. & Desrosiers, R.C. A role for carbohydrates in immune evasion in AIDS. Nat. Med. 4, 679–684 (1998).

    CAS  Article  PubMed  Google Scholar 

  36. Wei, X. et al. Antibody neutralization and escape by HIV-1. Nature 422, 307–312 (2003).

    CAS  Article  PubMed  Google Scholar 

  37. Mouquet, H. et al. Complex-type N-glycan recognition by potent broadly neutralizing HIV antibodies. Proc. Natl. Acad. Sci. USA 109, E3268–E3277 (2012).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  38. Bonsignori, M. et al. Analysis of a clonal lineage of HIV-1 envelope V2/V3 conformational epitope-specific broadly neutralizing antibodies and their inferred unmutated common ancestors. J. Virol. 85, 9998–10009 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  39. Doores, K.J. & Burton, D.R. Variable loop glycan dependency of the broad and potent HIV-1-neutralizing antibodies PG9 and PG16. J. Virol. 84, 10510–10521 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  40. Kabat, E.A., Wu, T.T., Perry, H.M., Gottesman, K.S. & Foeller, C. Sequences of Proteins of Immunological Interest (U.S. Department of Health and Human Services, National Institutes of Health, 1991).

  41. Pancera, M. et al. Crystal structure of PG16 and chimeric dissection with somatically related PG9: structure-function analysis of two quaternary-specific antibodies that effectively neutralize HIV-1. J. Virol. 84, 8098–8110 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  42. Otto, V.I., Schurpf, T., Folkers, G. & Cummings, R.D. Sialylated complex-type N-glycans enhance the signaling activity of soluble intercellular adhesion molecule-1 in mouse astrocytes. J. Biol. Chem. 279, 35201–35209 (2004).

    CAS  Article  PubMed  Google Scholar 

  43. Sánchez -Felipe, L., Villar, E. & Munoz-Barroso, I. α2–3- and α2–6- N-linked sialic acids allow efficient interaction of Newcastle Disease Virus with target cells. Glycoconj. J. 29, 539–549 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  44. Guo, Y. et al. Analysis of hemagglutinin-mediated entry tropism of H5N1 avian influenza. Virol. J. 6, 39 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  45. Durocher, Y. & Butler, M. Expression systems for therapeutic glycoprotein production. Curr. Opin. Biotechnol. 20, 700–707 (2009).

    CAS  Article  PubMed  Google Scholar 

  46. Scheid, J.F. et al. Sequence and structural convergence of broad and potent HIV antibodies that mimic CD4 binding. Science 333, 1633–1637 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  47. Wu, X. et al. Focused evolution of HIV-1 neutralizing antibodies revealed by structures and deep sequencing. Science 333, 1593–1602 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  48. Zhu, J. et al. Somatic populations of PGT135–137 HIV-1-neutralizing antibodies identified by 454 pyrosequencing and bioinformatics. Front. Microbiol. 3, 315 (2012).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  49. Wu, X. et al. Rational design of envelope identifies broadly neutralizing human monoclonal antibodies to HIV-1. Science 329, 856–861 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  50. Heyndrickx, L. et al. International network for comparison of HIV neutralization assays: the NeutNet report II. PLoS ONE 7, e36438 (2012).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  51. Euler, Z. et al. Activity of broadly neutralizing antibodies, including PG9, PG16, and VRC01, against recently transmitted subtype B HIV-1 variants from early and late in the epidemic. J. Virol. 85, 7236–7245 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  52. May, A.P., Robinson, R.C., Vinson, M., Crocker, P.R. & Jones, E.Y. Crystal structure of the N-terminal domain of sialoadhesin in complex with 3′ sialyllactose at 1.85 A resolution. Mol. Cell 1, 719–728 (1998).

    CAS  Article  PubMed  Google Scholar 

  53. Somers, W.S., Tang, J., Shaw, G.D. & Camphausen, R.T. Insights into the molecular basis of leukocyte tethering and rolling revealed by structures of P- and E-selectin bound to SLe(X) and PSGL-1. Cell 103, 467–479 (2000).

    CAS  Article  PubMed  Google Scholar 

  54. Zhuravleva, M.A., Trandem, K. & Sun, P.D. Structural implications of Siglec-5-mediated sialoglycan recognition. J. Mol. Biol. 375, 437–447 (2008).

    CAS  Article  PubMed  Google Scholar 

  55. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

    CAS  Article  PubMed  Google Scholar 

  56. McCoy, A.J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  57. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  PubMed  Google Scholar 

  58. Adams, P.D. et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D Biol. Crystallogr. 58, 1948–1954 (2002).

    Article  PubMed  Google Scholar 

  59. Davis, I.W. et al. MolProbity: all-atom contacts and structure validation for proteins and nucleic acids. Nucleic Acids Res. 35, W375–W383 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

  60. Li, M. et al. Human immunodeficiency virus type 1 env clones from acute and early subtype B infections for standardized assessments of vaccine-elicited neutralizing antibodies. J. Virol. 79, 10108–10125 (2005).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  61. Seaman, M.S. et al. Multiclade human immunodeficiency virus type 1 envelope immunogens elicit broad cellular and humoral immunity in rhesus monkeys. J. Virol. 79, 2956–2963 (2005).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  62. Haynes, B.F. et al. Cardiolipin polyspecific autoreactivity in two broadly neutralizing HIV-1 antibodies. Science 308, 1906–1908 (2005).

    CAS  Article  PubMed  Google Scholar 

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Acknowledgements

We thank J. Stuckey for assistance with figures and members of the Structural Biology Section and Structural Bioinformatics Core, Vaccine Research Center for discussions or comments on the manuscript; A. Kumar for sharing the ELISA binding protocol with biotinylated lectins; P. Azadi and S. Archer-Hartmann, University of Georgia, for performing glycan analyses and glycopeptide mapping; J. Baalwa (Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama, USA), D. Ellenberger (International Laboratory Branch, Division of Global HIV/AIDS, Center for Global Health, Centers for Disease Control and Prevention, Atlanta, Georgia, USA), D. Gabuzda (Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, Massachusetts, USA), F. Gao (Duke Human Vaccine Institute, Duke University Medical Center, Durham, North Carolina, USA), B. Hahn (Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama, USA), K. Hong (State Key Laboratory for Infectious Disease Control and Prevention, National Center for AIDS/STD Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China), J. Kim (US Military HIV Research Program, Henry M. Jackson Foundation, Bethesda, Maryland, USA), F. McCutchan (US Military HIV Research Program, Henry M. Jackson Foundation, Bethesda, Maryland, USA), D. Montefiori (Department of Surgery, Duke University, Durham, North Carolina, USA), L. Morris, (Centre for HIV and STIs, National Institute for Communicable Diseases, Johannesburg, South Africa), J. Overbaugh (Program for Appropriate Technology in Health, Seattle, Washington, USA), E. Sanders-Buell (US Military HIV Research Program, Henry M. Jackson Foundation, Bethesda, Maryland, USA), G. Shaw (Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama, USA), R. Swanstrom (University of North Carilina Center for AIDS Research, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA), M. Thomson (University of Birmingham, Birmingham, UK), S. Tovanabutra (Department of Retrovirology, Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand), C. Williamson (Institute of Infectious Diseases and Molecular Medicine, Division of Medical Virology, University of Cape Town and National Health Laboratory Services, Cape Town, South Africa) and L. Zhang (Department of Public Health, Center for Disease Control and Prevention in Jiangxi Province, Nanchang, China) contributing the HIV-1 Envelope plasmids used in our neutralization panel. Support for this work was provided by the US National Institutes of Health Intramural Research Programs (Vaccine Research Center, National Institute of Allergy and Infectious Diseases, and the National Institute of Diabetes and Digestive and Kidney Diseases), by grants from the International AIDS Vaccine Initiative's Neutralizing Antibody Consortium and by the Center for HIV AIDS Vaccine Immunology Grant AI 5U19 AI 067854-06 from the US National Institutes of Health (J.E., K.E.L., J.B., S.M.A. and B.F.H.), by US National Institutes of Health grant 1R21 AI101035 (M.N.A. and L-X.W.) and by the US National Institutes of Health (NIH/NCRR)-funded grant P41 RR018502-01 to the Complex Carbohydrate Research Center. Use of sector 22 (Southeast Region Collaborative Access team) at the Advanced Photon Source was supported by the US Department of Energy, Basic Energy Sciences, Office of Science under contract number W-31-109-Eng-38.

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M.P., S.S.-u.-H., N.A.D.-R., J.S.M., B.F.H., G.J.N., J.R.M., C.A.B. and P.D.K. designed research and analyzed the data; M.P., S.S.-u.-H., N.A.D.-R., R.T.B., K.D., M.K.L., S.L., R.P.S., Y.Y., B.Z., R.P., J.E., K.E.L., J.B. and S.M.A. performed research; D.R.B. and W.C.K. contributed PG9 and PG16 antibodies; M.N.A. and L.-X.W. contributed N-glycans; M.P., S.S.-u.-H., N.A.D.-R., C.A.B. and P.D.K. wrote the paper, with all principal investigators providing comments or revisions.

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Correspondence to Carole A Bewley or Peter D Kwong.

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Pancera, M., Shahzad-ul-Hussan, S., Doria-Rose, N. et al. Structural basis for diverse N-glycan recognition by HIV-1–neutralizing V1–V2–directed antibody PG16. Nat Struct Mol Biol 20, 804–813 (2013). https://doi.org/10.1038/nsmb.2600

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