Cooperation between somatic mutation and germline-encoded residues enable antibody recognition of HIV-1 envelope glycans

English version

Autoři: Nelson R. Wu ^aff001; Nathan I. Nicely ^aff001; Esther M. Lee ^aff001; Rachel K. Reed ^aff001; Brian E. Watts ^aff001; Fangping Cai ^aff001; William E. Walkowicz ^aff003; Baptiste Aussedat ^aff003; Julia A. Jones ^aff001; Amanda Eaton ^aff002; Ashley M. Trama ^aff001; S. Munir Alam ^aff001; David C. Montefiori ^aff002; Barton F. Haynes ^aff001; Kevin O. Saunders ^aff002
Působiště autorů: Department of Medicine, Duke University Medical Center, Durham, North Carolina, United States of America ^aff001; Duke Human Vaccine Institute, Duke University Medical Center, Durham, North Carolina, United States of America ^aff002; Department of Chemical Biology, Memorial Sloan Kettering Cancer Center, New York, New York, United States of America ^aff003; Department of Surgery, Duke University Medical Center, Durham, North Carolina, United States of America ^aff004; Department of Biomedical Engineering, Duke University Medical Center, Durham, North Carolina, United States of America ^aff005; Department of Immunology, Duke University Medical Center, Durham, North Carolina, United States of America ^aff006; Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina, United States of America ^aff007
Vyšlo v časopise: Cooperation between somatic mutation and germline-encoded residues enable antibody recognition of HIV-1 envelope glycans. PLoS Pathog 15(12): e32767. doi:10.1371/journal.ppat.1008165
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.ppat.1008165

Souhrn

Viral glycoproteins are a primary target for host antibody responses. However, glycans on viral glycoproteins can hinder antibody recognition since they are self glycans derived from the host biosynthesis pathway. During natural HIV-1 infection, neutralizing antibodies are made against glycans on HIV-1 envelope glycoprotein (Env). However, such antibodies are rarely elicited with vaccination. Previously, the vaccine-induced, macaque antibody DH501 was isolated and shown to bind to high mannose glycans on HIV-1 Env. Understanding how DH501 underwent affinity maturation to recognize glycans could inform vaccine induction of HIV-1 glycan antibodies. Here, we show that DH501 Env glycan reactivity is mediated by both germline-encoded residues that contact glycans, and somatic mutations that increase antibody paratope flexibility. Only somatic mutations in the heavy chain were required for glycan reactivity. The paratope conformation was fragile as single mutations within the immunoglobulin fold or complementarity determining regions were sufficient for eliminating antibody function. Taken together, the initial germline V_HDJ_H rearrangement generated contact residues capable of binding glycans, and somatic mutations were required to form a flexible paratope with a cavity conducive to HIV-1 envelope glycan binding. The requirement for the presence of most somatic mutations across the heavy chain variable region provides one explanation for the difficulty in inducing anti-Env glycan antibodies with HIV-1 Env vaccination.

Klíčová slova:

Antibodies – Crystal structure – HIV-1 – Macaque – Mannose – Proline – Somatic mutation – Recombination reactions

Zdroje

1. Rey FA, Lok SM. Common Features of Enveloped Viruses and Implications for Immunogen Design for Next-Generation Vaccines. Cell. 2018;172(6):1319–34. Epub 2018/03/10. doi: 10.1016/j.cell.2018.02.054 29522750.

2. Vigerust DJ, Shepherd VL. Virus glycosylation: role in virulence and immune interactions. Trends Microbiol. 2007;15(5):211–8. Epub 2007/04/03. doi: 10.1016/j.tim.2007.03.003 17398101.

3. Helle F, Vieyres G, Elkrief L, Popescu CI, Wychowski C, Descamps V, et al. Role of N-linked glycans in the functions of hepatitis C virus envelope proteins incorporated into infectious virions. Journal of virology. 2010;84(22):11905–15. Epub 2010/09/17. doi: 10.1128/JVI.01548-10 20844034; PubMed Central PMCID: PMC2977866.

4. Falkowska E, Kajumo F, Garcia E, Reinus J, Dragic T. Hepatitis C virus envelope glycoprotein E2 glycans modulate entry, CD81 binding, and neutralization. Journal of virology. 2007;81(15):8072–9. Epub 2007/05/18. doi: 10.1128/JVI.00459-07 17507469; PubMed Central PMCID: PMC1951298.

5. Moldt B, Rakasz EG, Schultz N, Chan-Hui PY, Swiderek K, Weisgrau KL, et al. Highly potent HIV-specific antibody neutralization in vitro translates into effective protection against mucosal SHIV challenge in vivo. Proceedings of the National Academy of Sciences of the United States of America. 2012;109(46):18921–5. Epub 2012/10/27. doi: 10.1073/pnas.1214785109 23100539; PubMed Central PMCID: PMC3503218.

6. Hessell AJ, Rakasz EG, Poignard P, Hangartner L, Landucci G, Forthal DN, et al. Broadly neutralizing human anti-HIV antibody 2G12 is effective in protection against mucosal SHIV challenge even at low serum neutralizing titers. PLoS pathogens. 2009;5(5):e1000433. Epub 2009/05/14. doi: 10.1371/journal.ppat.1000433 19436712; PubMed Central PMCID: PMC2674935.

7. Lopez-Ribot JL, Casanova M, Murgui A, Martinez JP. Antibody response to Candida albicans cell wall antigens. FEMS Immunol Med Microbiol. 2004;41(3):187–96. Epub 2004/06/16. doi: 10.1016/j.femsim.2004.03.012 15196567.

8. Wei X, Decker JM, Wang S, Hui H, Kappes JC, Wu X, et al. Antibody neutralization and escape by HIV-1. Nature. 2003;422(6929):307–12. Epub 2003/03/21. doi: 10.1038/nature01470 12646921.

9. Seymour RM, Allan MJ, Pomiankowski A, Gustafsson K. Evolution of the human ABO polymorphism by two complementary selective pressures. Proc Biol Sci. 2004;271(1543):1065–72. Epub 2004/08/06. doi: 10.1098/rspb.2004.2674 15293861; PubMed Central PMCID: PMC1691687.

10. Scanlan CN, Offer J, Zitzmann N, Dwek RA. Exploiting the defensive sugars of HIV-1 for drug and vaccine design. Nature. 2007;446(7139):1038–45. Epub 2007/04/27. doi: 10.1038/nature05818 17460665.

11. Leonard CK, Spellman MW, Riddle L, Harris RJ, Thomas JN, Gregory TJ. 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. 1990;265(18):10373–82. Epub 1990/06/25. 2355006.

12. Astronomo RD, Burton DR. Carbohydrate vaccines: developing sweet solutions to sticky situations? Nat Rev Drug Discov. 2010;9(4):308–24. Epub 2010/04/02. doi: 10.1038/nrd3012 20357803; PubMed Central PMCID: PMC3878310.

13. Go EP, Irungu J, Zhang Y, Dalpathado DS, Liao HX, Sutherland LL, et al. Glycosylation site-specific analysis of HIV envelope proteins (JR-FL and CON-S) reveals major differences in glycosylation site occupancy, glycoform profiles, and antigenic epitopes' accessibility. J Proteome Res. 2008;7(4):1660–74. Epub 2008/03/12. doi: 10.1021/pr7006957 18330979; PubMed Central PMCID: PMC3658474.

14. Mizuochi T, Matthews TJ, Kato M, Hamako J, Titani K, Solomon J, 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. 1990;265(15):8519–24. Epub 1990/05/25. 2341393.

15. Go EP, Chang Q, Liao HX, Sutherland LL, Alam SM, Haynes BF, et al. Glycosylation site-specific analysis of clade C HIV-1 envelope proteins. J Proteome Res. 2009;8(9):4231–42. Epub 2009/07/21. doi: 10.1021/pr9002728 19610667; PubMed Central PMCID: PMC2756219.

16. Go EP, Herschhorn A, Gu C, Castillo-Menendez L, Zhang S, Mao Y, et al. Comparative Analysis of the Glycosylation Profiles of Membrane-Anchored HIV-1 Envelope Glycoprotein Trimers and Soluble gp140. Journal of virology. 2015;89(16):8245–57. Epub 2015/05/29. doi: 10.1128/JVI.00628-15 26018173; PubMed Central PMCID: PMC4524223.

17. Behrens AJ, Vasiljevic S, Pritchard LK, Harvey DJ, Andev RS, Krumm SA, et al. Composition and Antigenic Effects of Individual Glycan Sites of a Trimeric HIV-1 Envelope Glycoprotein. Cell reports. 2016;14(11):2695–706. Epub 2016/03/15. doi: 10.1016/j.celrep.2016.02.058 26972002; PubMed Central PMCID: PMC4805854.

18. Doria-Rose NA, Altae-Tran HR, Roark RS, Schmidt SD, Sutton MS, Louder MK, et al. Mapping Polyclonal HIV-1 Antibody Responses via Next-Generation Neutralization Fingerprinting. PLoS pathogens. 2017;13(1):e1006148. Epub 2017/01/05. doi: 10.1371/journal.ppat.1006148 28052137; PubMed Central PMCID: PMC5241146.

19. Walker LM, Simek MD, Priddy F, Gach JS, Wagner D, Zwick MB, et al. A limited number of antibody specificities mediate broad and potent serum neutralization in selected HIV-1 infected individuals. PLoS pathogens. 2010;6(8):e1001028. doi: 10.1371/journal.ppat.1001028 20700449; PubMed Central PMCID: PMC2916884.

20. Walker LM, Huber M, Doores KJ, Falkowska E, Pejchal R, Julien JP, et al. Broad neutralization coverage of HIV by multiple highly potent antibodies. Nature. 2011;477(7365):466–70. Epub 2011/08/19. doi: 10.1038/nature10373 21849977; PubMed Central PMCID: PMC3393110.

21. Walker LM, Phogat SK, Chan-Hui PY, Wagner D, Phung P, Goss JL, et al. Broad and potent neutralizing antibodies from an African donor reveal a new HIV-1 vaccine target. Science. 2009;326(5950):285–9. Epub 2009/09/05. doi: 10.1126/science.1178746 19729618; PubMed Central PMCID: PMC3335270.

22. Bonsignori M, Kreider EF, Fera D, Meyerhoff RR, Bradley T, Wiehe K, et al. Staged induction of HIV-1 glycan-dependent broadly neutralizing antibodies. Sci Transl Med. 2017;9(381). Epub 2017/03/17. doi: 10.1126/scitranslmed.aai7514 28298420; PubMed Central PMCID: PMC5562350.

23. Bonsignori M, Montefiori DC, Wu X, Chen X, Hwang KK, Tsao CY, et al. Two distinct broadly neutralizing antibody specificities of different clonal lineages in a single HIV-1-infected donor: implications for vaccine design. J Virol. 2012;86(8):4688–92. Epub 2012/02/04. doi: 10.1128/JVI.07163-11 22301150; PubMed Central PMCID: PMC3318651.

24. Kong L, Lee JH, Doores KJ, Murin CD, Julien JP, McBride R, et al. Supersite of immune vulnerability on the glycosylated face of HIV-1 envelope glycoprotein gp120. Nature structural & molecular biology. 2013;20(7):796–803. Epub 2013/05/28. doi: 10.1038/nsmb.2594 23708606; PubMed Central PMCID: PMC3823233.

25. Barnes CO, Gristick HB, Freund NT, Escolano A, Lyubimov AY, Hartweger H, et al. Structural characterization of a highly-potent V3-glycan broadly neutralizing antibody bound to natively-glycosylated HIV-1 envelope. Nature communications. 2018;9(1):1251. Epub 2018/03/30. doi: 10.1038/s41467-018-03632-y 29593217; PubMed Central PMCID: PMC5871869.

26. Kong L, Torrents de la Pena A, Deller MC, Garces F, Sliepen K, Hua Y, et al. Complete epitopes for vaccine design derived from a crystal structure of the broadly neutralizing antibodies PGT128 and 8ANC195 in complex with an HIV-1 Env trimer. Acta crystallographica Section D, Biological crystallography. 2015;71(Pt 10):2099–108. doi: 10.1107/S1399004715013917 26457433; PubMed Central PMCID: PMC4601371.

27. Pejchal R, Doores KJ, Walker LM, Khayat R, Huang PS, Wang SK, et al. A potent and broad neutralizing antibody recognizes and penetrates the HIV glycan shield. Science. 2011;334(6059):1097–103. doi: 10.1126/science.1213256 21998254; PubMed Central PMCID: PMC3280215.

28. Calarese DA, Scanlan CN, Zwick MB, Deechongkit S, Mimura Y, Kunert R, et al. Antibody domain exchange is an immunological solution to carbohydrate cluster recognition. Science. 2003;300(5628):2065–71. Epub 2003/06/28. doi: 10.1126/science.1083182 12829775.

29. Murin CD, Julien JP, Sok D, Stanfield RL, Khayat R, Cupo A, et al. Structure of 2G12 Fab2 in complex with soluble and fully glycosylated HIV-1 Env by negative-stain single-particle electron microscopy. Journal of virology. 2014;88(17):10177–88. Epub 2014/06/27. doi: 10.1128/JVI.01229-14 24965454; PubMed Central PMCID: PMC4136306.

30. Pegu A, Yang ZY, Boyington JC, Wu L, Ko SY, Schmidt SD, et al. Neutralizing antibodies to HIV-1 envelope protect more effectively in vivo than those to the CD4 receptor. Science translational medicine. 2014;6(243):243ra88. Epub 2014/07/06. doi: 10.1126/scitranslmed.3008992 24990883; PubMed Central PMCID: PMC4562469.

31. Gautam R, Nishimura Y, Pegu A, Nason MC, Klein F, Gazumyan A, et al. A single injection of anti-HIV-1 antibodies protects against repeated SHIV challenges. Nature. 2016;533(7601):105–9. Epub 2016/04/28. doi: 10.1038/nature17677 27120156; PubMed Central PMCID: PMC5127204.

32. Campbell CT, Llewellyn SR, Demberg T, Morgan IL, Robert-Guroff M, Gildersleeve JC. High-throughput profiling of anti-glycan humoral responses to SIV vaccination and challenge. PloS one. 2013;8(9):e75302. Epub 2013/10/03. doi: 10.1371/journal.pone.0075302 24086502; PubMed Central PMCID: PMC3781036.

33. Pantophlet R, Trattnig N, Murrell S, Lu N, Chau D, Rempel C, et al. Bacterially derived synthetic mimetics of mammalian oligomannose prime antibody responses that neutralize HIV infectivity. Nature communications. 2017;8(1):1601. Epub 2017/11/19. doi: 10.1038/s41467-017-01640-y 29150603; PubMed Central PMCID: PMC5693931.

34. Luallen RJ, Lin J, Fu H, Cai KK, Agrawal C, Mboudjeka I, et al. An engineered Saccharomyces cerevisiae strain binds the broadly neutralizing human immunodeficiency virus type 1 antibody 2G12 and elicits mannose-specific gp120-binding antibodies. Journal of virology. 2008;82(13):6447–57. Epub 2008/04/25. doi: 10.1128/JVI.00412-08 18434410; PubMed Central PMCID: PMC2447081.

35. Doores KJ, Fulton Z, Hong V, Patel MK, Scanlan CN, Wormald MR, et al. A nonself sugar mimic of the HIV glycan shield shows enhanced antigenicity. Proceedings of the National Academy of Sciences of the United States of America. 2010;107(40):17107–12. Epub 2010/09/21. doi: 10.1073/pnas.1002717107 20852065; PubMed Central PMCID: PMC2951454.

36. Astronomo RD, Lee HK, Scanlan CN, Pantophlet R, Huang CY, Wilson IA, et al. A glycoconjugate antigen based on the recognition motif of a broadly neutralizing human immunodeficiency virus antibody, 2G12, is immunogenic but elicits antibodies unable to bind to the self glycans of gp120. Journal of virology. 2008;82(13):6359–68. Epub 2008/04/25. doi: 10.1128/JVI.00293-08 18434393; PubMed Central PMCID: PMC2447108.

37. Saunders KO, Nicely NI, Wiehe K, Bonsignori M, Meyerhoff RR, Parks R, et al. Vaccine Elicitation of High Mannose-Dependent Neutralizing Antibodies against the V3-Glycan Broadly Neutralizing Epitope in Nonhuman Primates. Cell reports. 2017;18(9):2175–88. doi: 10.1016/j.celrep.2017.02.003 28249163; PubMed Central PMCID: PMC5408352.

38. Scanlan CN, Ritchie GE, Baruah K, Crispin M, Harvey DJ, Singer BB, et al. Inhibition of mammalian glycan biosynthesis produces non-self antigens for a broadly neutralising, HIV-1 specific antibody. J Mol Biol. 2007;372(1):16–22. Epub 2007 Jun 16. doi: 10.1016/j.jmb.2007.06.027 17631311

39. Bonomelli C, Doores KJ, Dunlop DC, Thaney V, Dwek RA, Burton DR, et al. The glycan shield of HIV is predominantly oligomannose independently of production system or viral clade. PloS one. 2011;6(8):e23521. Epub 2011/08/23. doi: 10.1371/journal.pone.0023521 21858152; PubMed Central PMCID: PMC3156772.

40. Doores KJ, Bonomelli C, Harvey DJ, Vasiljevic S, Dwek RA, Burton DR, et al. Envelope glycans of immunodeficiency virions are almost entirely oligomannose antigens. Proceedings of the National Academy of Sciences of the United States of America. 2010;107(31):13800–5. Epub 2010/07/21. doi: 10.1073/pnas.1006498107 20643940; PubMed Central PMCID: PMC2922250.

41. Schrödinger L. The PyMOL Molecular Graphics System 2.0 ed.

42. Scanlan CN, Ritchie GE, Baruah K, Crispin M, Harvey DJ, Singer BB, et al. Inhibition of mammalian glycan biosynthesis produces non-self antigens for a broadly neutralising, HIV-1 specific antibody. J Mol Biol. 2007;372(1):16–22. Epub 2007/07/17. doi: 10.1016/j.jmb.2007.06.027 17631311.

43. Doores KJ, Burton DR. Variable loop glycan dependency of the broad and potent HIV-1-neutralizing antibodies PG9 and PG16. Journal of virology. 2010;84(20):10510–21. doi: 10.1128/JVI.00552-10 20686044; PubMed Central PMCID: PMC2950566.

44. Kepler TB. Reconstructing a B-cell clonal lineage. I. Statistical inference of unobserved ancestors. F1000Res. 2013;2:103. Epub 2014/02/21. doi: 10.12688/f1000research.2-103.v1 24555054; PubMed Central PMCID: PMC3901458.

45. Jones PT, Dear PH, Foote J, Neuberger MS, Winter G. Replacing the complementarity-determining regions in a human antibody with those from a mouse. Nature. 1986;321(6069):522–5. Epub 1986/05/04. doi: 10.1038/321522a0 3713831.

46. Georgiev IS, Rudicell RS, Saunders KO, Shi W, Kirys T, McKee K, et al. Antibodies VRC01 and 10E8 neutralize HIV-1 with high breadth and potency even with Ig-framework regions substantially reverted to germline. J Immunol. 2014;192(3):1100–6. Epub 2014/01/07. doi: 10.4049/jimmunol.1302515 24391217; PubMed Central PMCID: PMC4140862.

47. Jardine JG, Sok D, Julien JP, Briney B, Sarkar A, Liang CH, et al. Minimally Mutated HIV-1 Broadly Neutralizing Antibodies to Guide Reductionist Vaccine Design. PLoS pathogens. 2016;12(8):e1005815. Epub 2016/08/26. doi: 10.1371/journal.ppat.1005815 27560183; PubMed Central PMCID: PMC4999182 following competing interests: WRS is a co-founder and stock holder in Compuvax, Inc. which has programs in non-HIV vaccine design that might benefit indirectly from this research.

48. Klein F, Diskin R, Scheid JF, Gaebler C, Mouquet H, Georgiev IS, et al. Somatic mutations of the immunoglobulin framework are generally required for broad and potent HIV-1 neutralization. Cell. 2013;153(1):126–38. Epub 2013/04/02. doi: 10.1016/j.cell.2013.03.018 23540694; PubMed Central PMCID: PMC3792590.

49. Zhou T, Georgiev I, Wu X, Yang ZY, Dai K, Finzi A, et al. Structural basis for broad and potent neutralization of HIV-1 by antibody VRC01. Science. 2010;329(5993):811–7. Epub 2010/07/10. doi: 10.1126/science.1192819 20616231; PubMed Central PMCID: PMC2981354.

50. Li SC, Goto NK, Williams KA, Deber CM. Alpha-helical, but not beta-sheet, propensity of proline is determined by peptide environment. Proceedings of the National Academy of Sciences of the United States of America. 1996;93(13):6676–81. Epub 1996/06/25. doi: 10.1073/pnas.93.13.6676 8692877; PubMed Central PMCID: PMC39085.

51. von Heijne G. Proline kinks in transmembrane alpha-helices. J Mol Biol. 1991;218(3):499–503. Epub 1991/04/05. doi: 10.1016/0022-2836(91)90695-3 2016741.

52. Vermeer AW, Norde W. The thermal stability of immunoglobulin: unfolding and aggregation of a multi-domain protein. Biophys J. 2000;78(1):394–404. Epub 2000/01/05. doi: 10.1016/S0006-3495(00)76602-1 10620303; PubMed Central PMCID: PMC1300647.

53. Goto Y, Ichimura N, Hamaguchi K. Effects of ammonium sulfate on the unfolding and refolding of the variable and constant fragments of an immunoglobulin light chain. Biochemistry. 1988;27(5):1670–7. Epub 1988/03/08. doi: 10.1021/bi00405a043 3130099.

54. Lee JH, Andrabi R, Su CY, Yasmeen A, Julien JP, Kong L, et al. A Broadly Neutralizing Antibody Targets the Dynamic HIV Envelope Trimer Apex via a Long, Rigidified, and Anionic beta-Hairpin Structure. Immunity. 2017;46(4):690–702. doi: 10.1016/j.immuni.2017.03.017 28423342; PubMed Central PMCID: PMC5400778.

55. McLellan JS, Pancera M, Carrico C, Gorman J, Julien JP, Khayat R, et al. Structure of HIV-1 gp120 V1/V2 domain with broadly neutralizing antibody PG9. Nature. 2011;480(7377):336–43. doi: 10.1038/nature10696 22113616; PubMed Central PMCID: PMC3406929.

56. Garces F, Sok D, Kong L, McBride R, Kim HJ, Saye-Francisco KF, et al. Structural evolution of glycan recognition by a family of potent HIV antibodies. Cell. 2014;159(1):69–79. Epub 2014/09/27. doi: 10.1016/j.cell.2014.09.009 25259921; PubMed Central PMCID: PMC4278586.

57. Doores KJ, Kong L, Krumm SA, Le KM, Sok D, Laserson U, et al. Two classes of broadly neutralizing antibodies within a single lineage directed to the high-mannose patch of HIV envelope. Journal of virology. 2015;89(2):1105–18. doi: 10.1128/JVI.02905-14 25378488; PubMed Central PMCID: PMC4300629.

58. Mouquet H, Scharf L, Euler Z, Liu Y, Eden C, Scheid JF, et al. Complex-type N-glycan recognition by potent broadly neutralizing HIV antibodies. Proceedings of the National Academy of Sciences of the United States of America. 2012;109(47):E3268–77. doi: 10.1073/pnas.1217207109 23115339; PubMed Central PMCID: PMC3511153.

59. Lavie M, Hanoulle X, Dubuisson J. Glycan Shielding and Modulation of Hepatitis C Virus Neutralizing Antibodies. Frontiers in immunology. 2018;9:910. Epub 2018/05/15. doi: 10.3389/fimmu.2018.00910 29755477; PubMed Central PMCID: PMC5934428.

60. West AP Jr., Diskin R, Nussenzweig MC, Bjorkman PJ. Structural basis for germ-line gene usage of a potent class of antibodies targeting the CD4-binding site of HIV-1 gp120. Proc Natl Acad Sci U S A. 2012;109(30):E2083–90. Epub 2012/06/30. doi: 10.1073/pnas.1208984109 22745174; PubMed Central PMCID: PMC3409792.

61. Corti D, Suguitan AL Jr., Pinna D, Silacci C, Fernandez-Rodriguez BM, Vanzetta F, et al. Heterosubtypic neutralizing antibodies are produced by individuals immunized with a seasonal influenza vaccine. J Clin Invest. 2010;120(5):1663–73. Epub 2010/04/15. doi: 10.1172/JCI41902 20389023; PubMed Central PMCID: PMC2860935.

62. Wrammert J, Koutsonanos D, Li GM, Edupuganti S, Sui J, Morrissey M, et al. Broadly cross-reactive antibodies dominate the human B cell response against 2009 pandemic H1N1 influenza virus infection. J Exp Med. 2011;208(1):181–93. Epub 2011/01/12. doi: 10.1084/jem.20101352 21220454; PubMed Central PMCID: PMC3023136.

63. Lingwood D, McTamney PM, Yassine HM, Whittle JR, Guo X, Boyington JC, et al. Structural and genetic basis for development of broadly neutralizing influenza antibodies. Nature. 2012;489(7417):566–70. Epub 2012/08/31. doi: 10.1038/nature11371 22932267.

64. Bassing CH, Swat W, Alt FW. The mechanism and regulation of chromosomal V(D)J recombination. Cell. 2002;109 Suppl:S45–55. Epub 2002/05/02. doi: 10.1016/s0092-8674(02)00675-x 11983152.

65. Weigert M, Perry R, Kelley D, Hunkapiller T, Schilling J, Hood L. The joining of V and J gene segments creates antibody diversity. Nature. 1980;283(5746):497–9. Epub 1980/01/31. doi: 10.1038/283497a0 6766210

66. Brack C, Hirama M, Lenhard-Schuller R, Tonegawa S. A complete immunoglobulin gene is created by somatic recombination. Cell. 1978;15(1):1–14. Epub 1978/09/01. doi: 10.1016/0092-8674(78)90078-8 100225

67. Sakano H, Kurosawa Y, Weigert M, Tonegawa S. Identification and nucleotide sequence of a diversity DNA segment (D) of immunoglobulin heavy-chain genes. Nature. 1981;290(5807):562–5. Epub 1981/04/16. doi: 10.1038/290562a0 6783961.

68. Early P, Huang H, Davis M, Calame K, Hood L. An immunoglobulin heavy chain variable region gene is generated from three segments of DNA: VH, D and JH. Cell. 1980;19(4):981–92. Epub 1980/04/01. doi: 10.1016/0092-8674(80)90089-6 6769593.

69. Soto C, Bombardi RG, Branchizio A, Kose N, Matta P, Sevy AM, et al. High frequency of shared clonotypes in human B cell receptor repertoires. Nature. 2019;566(7744):398–402. Epub 2019/02/15. doi: 10.1038/s41586-019-0934-8 30760926.

70. Willis JR, Finn JA, Briney B, Sapparapu G, Singh V, King H, et al. Long antibody HCDR3s from HIV-naive donors presented on a PG9 neutralizing antibody background mediate HIV neutralization. Proceedings of the National Academy of Sciences of the United States of America. 2016;113(16):4446–51. doi: 10.1073/pnas.1518405113 27044078; PubMed Central PMCID: PMC4843476.

71. Briney B, Inderbitzin A, Joyce C, Burton DR. Commonality despite exceptional diversity in the baseline human antibody repertoire. Nature. 2019;566(7744):393–7. Epub 2019/01/22. doi: 10.1038/s41586-019-0879-y 30664748; PubMed Central PMCID: PMC6411386.

72. Amzel LM, Poljak RJ. Three-dimensional structure of immunoglobulins. Annu Rev Biochem. 1979;48:961–97. Epub 1979/01/01. doi: 10.1146/annurev.bi.48.070179.004525 89832.

73. Porter RR. Structural studies of immunoglobulins. Science. 1973;180(4087):713–6. Epub 1973/05/18. doi: 10.1126/science.180.4087.713 4122075.

74. Trevino SR, Schaefer S, Scholtz JM, Pace CN. Increasing protein conformational stability by optimizing beta-turn sequence. J Mol Biol. 2007;373(1):211–8. Epub 2007/09/04. doi: 10.1016/j.jmb.2007.07.061 17765922; PubMed Central PMCID: PMC2084202.

75. Nicely NI, Dennison SM, Spicer L, Scearce RM, Kelsoe G, Ueda Y, et al. Crystal structure of a non-neutralizing antibody to the HIV-1 gp41 membrane-proximal external region. Nat Struct Mol Biol. 2010;17(12):1492–4. Epub 2010/11/16. doi: 10.1038/nsmb.1944 21076400.

76. Liao HX, Sutherland LL, Xia SM, Brock ME, Scearce RM, Vanleeuwen S, et al. A group M consensus envelope glycoprotein induces antibodies that neutralize subsets of subtype B and C HIV-1 primary viruses. Virology. 2006;353(2):268–82. Epub 2006/10/14. doi: 10.1016/j.virol.2006.04.043 17039602; PubMed Central PMCID: PMC1762135.

77. Otwinowski Z, Minor W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 1997;276:307–26. Epub 1997/01/01. 27754618.

78. Terwilliger TC, Grosse-Kunstleve RW, Afonine PV, Moriarty NW, Zwart PH, Hung LW, et al. Iterative model building, structure refinement and density modification with the PHENIX AutoBuild wizard. Acta Crystallogr D Biol Crystallogr. 2008;64(Pt 1):61–9. Epub 2007/12/21. doi: 10.1107/S090744490705024X 18094468; PubMed Central PMCID: PMC2394820.

79. Emsley P, Lohkamp B, Scott WG, Cowtan K. Features and development of Coot. Acta Crystallogr D Biol Crystallogr. 2010;66(Pt 4):486–501. Epub 2010/04/13. doi: 10.1107/S0907444910007493 20383002; PubMed Central PMCID: PMC2852313.

80. Adams PD, Afonine PV, Bunkoczi G, Chen VB, Davis IW, Echols N, et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr. 2010;66(Pt 2):213–21. Epub 2010/02/04. doi: 10.1107/S0907444909052925 20124702; PubMed Central PMCID: PMC2815670.

81. Lovell SC, Davis IW, Arendall WB 3rd, de Bakker PI, Word JM, Prisant MG, et al. Structure validation by Calpha geometry: phi,psi and Cbeta deviation. Proteins. 2003;50(3):437–50. Epub 2003/01/31. doi: 10.1002/prot.10286 12557186.

82. Jo S, Kim T, Iyer VG, Im W. CHARMM-GUI: A Web-based Graphical User Interface for CHARMM. J Comput Chem. 2008;29:1859–65. doi: 10.1002/jcc.20945 18351591

83. Saunders KO, Verkoczy LK, Jiang C, Zhang J, Parks R, Chen H, et al. Vaccine Induction of Heterologous Tier 2 HIV-1 Neutralizing Antibodies in Animal Models. Cell reports. 2017;21(13):3681–90. Epub 2017/12/28. doi: 10.1016/j.celrep.2017.12.028 PubMed Central PMCID: PMC5777169. 29281818

84. Henderson R, Watts BE, Ergin HN, Anasti K, Parks R, Xia SM, et al. Selection of immunoglobulin elbow region mutations impacts interdomain conformational flexibility in HIV-1 broadly neutralizing antibodies. Nat Commun. 2019;10(1):654. Epub 2019/02/10. doi: 10.1038/s41467-019-08415-7 30737386; PubMed Central PMCID: PMC6368608.

85. Montefiori DC. Measuring HIV neutralization in a luciferase reporter gene assay. Methods Mol Biol. 2009;485:395–405. Epub 2008/11/21. doi: 10.1007/978-1-59745-170-3_26 19020839.

86. Sanders RW, Derking R, Cupo A, Julien JP, Yasmeen A, de Val N, et al. A next-generation cleaved, soluble HIV-1 Env trimer, BG505 SOSIP.664 gp140, expresses multiple epitopes for broadly neutralizing but not non-neutralizing antibodies. PLoS pathogens. 2013;9(9):e1003618. Epub 2013/09/27. doi: 10.1371/journal.ppat.1003618 24068931; PubMed Central PMCID: PMC3777863.