IgG3 enhances neutralization potency and Fc effector function of an HIV V2-specific broadly neutralizing antibody

English version

Autoři: Simone I. Richardson ^aff001; Bronwen E. Lambson ^aff001; Andrew R. Crowley ^aff003; Arman Bashirova ^aff004; Cathrine Scheepers ^aff001; Nigel Garrett ^aff006; Salim Abdool Karim ^aff006; Nonhlanhla N. Mkhize ^aff001; Mary Carrington ^aff004; Margaret E. Ackerman ^aff003; Penny L. Moore ^aff001; Lynn Morris ^aff001
Působiště autorů: Centre for HIV and STI’s, National Institute for Communicable Diseases, Johannesburg, Gauteng, South Africa ^aff001; Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, Gauteng, South Africa ^aff002; Thayer School of Engineering, Dartmouth College, Hanover, New Hampshire, United States of America ^aff003; Ragon Institute of Massachusetts General Hospital, MIT, and Harvard University, Boston, Massachusetts, United States of America ^aff004; Basic Science Program, Frederick National Laboratory for Cancer Research, Frederick, Maryland, United States of America ^aff005; Centre for the AIDS Programme of Research in South Africa (CAPRISA), University of KwaZulu-Natal, Congella, KwaZulu-Natal, South Africa ^aff006; Department of Public Health Medicine, School of Nursing and Public Health, University of KwaZulu-Natal, Durban, South Africa ^aff007; Department of Epidemiology, Columbia University, New York, NY, United States of America ^aff008
Vyšlo v časopise: IgG3 enhances neutralization potency and Fc effector function of an HIV V2-specific broadly neutralizing antibody. PLoS Pathog 15(12): e32767. doi:10.1371/journal.ppat.1008064
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.ppat.1008064

Souhrn

Broadly neutralizing antibodies (bNAbs) protect against HIV infection in non-human primates and their efficacy may be enhanced through interaction with Fc receptors on immune cells. Antibody isotype is a modulator of this binding with the IgG3 subclass mediating potent Fc effector function and is associated with HIV vaccine efficacy and HIV control. BNAb functions are typically assessed independently of the constant region with which they are naturally expressed. To examine the role of natural isotype in the context of a bNAb lineage we studied CAP256, an HIV-infected individual that mounted a potent V2-specific bNAb response. CAP256 expressed persistently high levels of plasma IgG3 which we found mediated both broad neutralizing activity and potent Fc function. Sequencing of germline DNA and the constant regions of V2-directed bNAbs from this donor revealed the expression of a novel IGHG3 allele as well as IGHG3*17, an allele that produces IgG3 antibodies with increased plasma half-life. Both allelic variants were used to generate CAP256-VRC26.25 and CAP256-VRC26.29 IgG3 bNAbs and these were compared to IgG1 versions. IgG3 variants were shown to have significantly higher phagocytosis and trogocytosis compared to IgG1 versions, which corresponded to increased affinity for FcγRIIa. Neutralization potency was also significantly higher for IgG3 bNAbs, particularly against viruses lacking the N160 glycan. By exchanging hinge regions between subclass variants, we showed that hinge length modulated both neutralization potency and Fc function. This study showed that co-operation between the variable and natural IgG3 constant regions enhanced the polyfunctionality of antibodies, indicating the value of leveraging genetic variation which could be exploited for passive immunity.

Klíčová slova:

AIDS – Antibodies – Enzyme-linked immunoassays – Fc receptors – HIV infections – Phagocytosis – Antigenic variation

Zdroje

1. Walker LM, Burton DR. Passive immunotherapy of viral infections: 'super-antibodies' enter the fray. Nat Rev Immunol. 2018;18(5):297–308. doi: 10.1038/nri.2017.148 29379211

2. Bournazos S, Klein F, Pietzsch J, Seaman MS, Nussenzweig MC, Ravetch JV. Broadly neutralizing anti-HIV-1 antibodies require Fc effector functions for in vivo activity. Cell. 2014;158(6):1243–53. doi: 10.1016/j.cell.2014.08.023 25215485

3. Halper-Stromberg A, Lu CL, Klein F, Horwitz JA, Bournazos S, Nogueira L, et al. Broadly neutralizing antibodies and viral inducers decrease rebound from HIV-1 latent reservoirs in humanized mice. Cell. 2014;158(5):989–99. doi: 10.1016/j.cell.2014.07.043 25131989

4. Hessell AJ, Hangartner L, Hunter M, Havenith CE, Beurskens FJ, Bakker JM, et al. Fc receptor but not complement binding is important in antibody protection against HIV. Nature. 2007;449(7158):101–4. doi: 10.1038/nature06106 17805298

5. Lu CL, Murakowski DK, Bournazos S, Schoofs T, Sarkar D, Halper-Stromberg A, et al. Enhanced clearance of HIV-1-infected cells by broadly neutralizing antibodies against HIV-1 in vivo. Science. 2016;352(6288):1001–4. doi: 10.1126/science.aaf1279 27199430

6. Hessell AJ, Shapiro MB, Powell R, Malherbe DC, McBurney SP, Pandey S, et al. Reduced cell-associated DNA and improved viral control in macaques following passive transfer of a single anti-V2 monoclonal antibody and repeated SHIV challenges. J Virol. 2018.

7. Horwitz JA, Bar-On Y, Lu CL, Fera D, Lockhart AAK, Lorenzi JCC, et al. Non-neutralizing Antibodies Alter the Course of HIV-1 Infection In Vivo. Cell. 2017;170(4):637–48 e10. doi: 10.1016/j.cell.2017.06.048 28757252

8. Moog C, Dereuddre-Bosquet N, Teillaud JL, Biedma ME, Holl V, Van Ham G, et al. Protective effect of vaginal application of neutralizing and nonneutralizing inhibitory antibodies against vaginal SHIV challenge in macaques. Mucosal Immunol. 2014;7(1):46–56. doi: 10.1038/mi.2013.23 23591718

9. Santra S, Tomaras GD, Warrier R, Nicely NI, Liao HX, Pollara J, et al. Human Non-neutralizing HIV-1 Envelope Monoclonal Antibodies Limit the Number of Founder Viruses during SHIV Mucosal Infection in Rhesus Macaques. PLoS Pathog. 2015;11(8):e1005042. doi: 10.1371/journal.ppat.1005042 26237403

10. Ackerman ME, Mikhailova A, Brown EP, Dowell KG, Walker BD, Bailey-Kellogg C, et al. Polyfunctional HIV-Specific Antibody Responses Are Associated with Spontaneous HIV Control. PLoS Pathog. 2016;12(1):e1005315. doi: 10.1371/journal.ppat.1005315 26745376

11. Forthal DN, Landucci G, Daar ES. Antibody from patients with acute human immunodeficiency virus (HIV) infection inhibits primary strains of HIV type 1 in the presence of natural-killer effector cells. J Virol. 2001;75(15):6953–61. doi: 10.1128/JVI.75.15.6953-6961.2001 11435575

12. Lambotte O, Ferrari G, Moog C, Yates NL, Liao HX, Parks RJ, et al. Heterogeneous neutralizing antibody and antibody-dependent cell cytotoxicity responses in HIV-1 elite controllers. AIDS. 2009;23(8):897–906. doi: 10.1097/QAD.0b013e328329f97d 19414990

13. Chung AW, Ghebremichael M, Robinson H, Brown E, Choi I, Lane S, et al. Polyfunctional Fc-effector profiles mediated by IgG subclass selection distinguish RV144 and VAX003 vaccines. Sci Transl Med. 2014;6(228):228ra38. doi: 10.1126/scitranslmed.3007736 24648341

14. Yates NL, Liao HX, Fong Y, deCamp A, Vandergrift NA, Williams WT, et al. Vaccine-induced Env V1-V2 IgG3 correlates with lower HIV-1 infection risk and declines soon after vaccination. Sci Transl Med. 2014;6(228):228ra39. doi: 10.1126/scitranslmed.3007730 24648342

15. Perez LG, Martinez DR, deCamp AC, Pinter A, Berman PW, Francis D, et al. V1V2-specific complement activating serum IgG as a correlate of reduced HIV-1 infection risk in RV144. PLoS One. 2017;12(7):e0180720. doi: 10.1371/journal.pone.0180720 28678869

16. Pollara J, Bonsignori M, Moody MA, Liu P, Alam SM, Hwang KK, et al. HIV-1 vaccine-induced C1 and V2 Env-specific antibodies synergize for increased antiviral activities. J Virol. 2014;88(14):7715–26. doi: 10.1128/JVI.00156-14 24807721

17. Bruhns P, Iannascoli B, England P, Mancardi DA, Fernandez N, Jorieux S, et al. Specificity and affinity of human Fcgamma receptors and their polymorphic variants for human IgG subclasses. Blood. 2009;113(16):3716–25. doi: 10.1182/blood-2008-09-179754 19018092

18. Janda A, Bowen A, Greenspan NS, Casadevall A. Ig Constant Region Effects on Variable Region Structure and Function. Front Microbiol. 2016;7:22. doi: 10.3389/fmicb.2016.00022 26870003

19. Parsons MS, Chung AW, Kent SJ. Importance of Fc-mediated functions of anti-HIV-1 broadly neutralizing antibodies. Retrovirology. 2018;15(1):58. doi: 10.1186/s12977-018-0438-x 30134945

20. Recke A, Konitzer S, Lemcke S, Freitag M, Sommer NM, Abdelhady M, et al. The p.Arg435His Variation of IgG3 With High Affinity to FcRn Is Associated With Susceptibility for Pemphigus Vulgaris-Analysis of Four Different Ethnic Cohorts. Front Immunol. 2018;9:1788. doi: 10.3389/fimmu.2018.01788 30116249

21. Oxelius VA, Pandey JP. Human immunoglobulin constant heavy G chain (IGHG) (Fcgamma) (GM) genes, defining innate variants of IgG molecules and B cells, have impact on disease and therapy. Clin Immunol. 2013;149(3):475–86. doi: 10.1016/j.clim.2013.10.003 24239836

22. Damelang T, Rogerson SJ, Kent SJ, Chung AW. Role of IgG3 in Infectious Diseases. Trends Immunol. 2019;40(3):197–211. doi: 10.1016/j.it.2019.01.005 30745265

23. Yates NL, Lucas JT, Nolen TL, Vandergrift NA, Soderberg KA, Seaton KE, et al. Multiple HIV-1-specific IgG3 responses decline during acute HIV-1: implications for detection of incident HIV infection. AIDS. 2011;25(17):2089–97. doi: 10.1097/QAD.0b013e32834b348e 21832938

24. Dugast AS, Stamatatos L, Tonelli A, Suscovich TJ, Licht AF, Mikell I, et al. Independent evolution of Fc- and Fab-mediated HIV-1-specific antiviral antibody activity following acute infection. Eur J Immunol. 2014;44(10):2925–37. doi: 10.1002/eji.201344305 25043633

25. Dugast AS, Tonelli A, Berger CT, Ackerman ME, Sciaranghella G, Liu Q, et al. Decreased Fc receptor expression on innate immune cells is associated with impaired antibody-mediated cellular phagocytic activity in chronically HIV-1 infected individuals. Virology. 2011;415(2):160–7. doi: 10.1016/j.virol.2011.03.012 21565376

26. Chung AW, Kumar MP, Arnold KB, Yu WH, Schoen MK, Dunphy LJ, et al. Dissecting Polyclonal Vaccine-Induced Humoral Immunity against HIV Using Systems Serology. Cell. 2015;163(4):988–98. doi: 10.1016/j.cell.2015.10.027 26544943

27. Lai JI, Licht AF, Dugast AS, Suscovich T, Choi I, Bailey-Kellogg C, et al. Divergent antibody subclass and specificity profiles but not protective HLA-B alleles are associated with variable antibody effector function among HIV-1 controllers. J Virol. 2014;88(5):2799–809. doi: 10.1128/JVI.03130-13 24352471

28. Sadanand S, Das J, Chung AW, Schoen MK, Lane S, Suscovich TJ, et al. Temporal variation in HIV-specific IgG subclass antibodies during acute infection differentiates spontaneous controllers from chronic progressors. AIDS. 2018;32(4):443–50. doi: 10.1097/QAD.0000000000001716 29239894

29. Kardava L, Sohn H, Youn C, Austin JW, Wang W, Buckner CM, et al. IgG3 regulates tissue-like memory B cells in HIV-infected individuals. Nat Immunol. 2018;19(9):1001–12. doi: 10.1038/s41590-018-0180-5 30104633

30. Lefranc MP, Lefranc G. Human Gm, Km, and Am allotypes and their molecular characterization: a remarkable demonstration of polymorphism. Methods Mol Biol. 2012;882:635–80. doi: 10.1007/978-1-61779-842-9_34 22665258

31. Stapleton NM, Andersen JT, Stemerding AM, Bjarnarson SP, Verheul RC, Gerritsen J, et al. Competition for FcRn-mediated transport gives rise to short half-life of human IgG3 and offers therapeutic potential. Nat Commun. 2011;2:599. doi: 10.1038/ncomms1608 22186895

32. Liu H, May K. Disulfide bond structures of IgG molecules: structural variations, chemical modifications and possible impacts to stability and biological function. MAbs. 2012;4(1):17–23. doi: 10.4161/mabs.4.1.18347 22327427

33. Rispens T, Davies AM, Ooijevaar-de Heer P, Absalah S, Bende O, Sutton BJ, et al. Dynamics of inter-heavy chain interactions in human immunoglobulin G (IgG) subclasses studied by kinetic Fab arm exchange. J Biol Chem. 2014;289(9):6098–109. doi: 10.1074/jbc.M113.541813 24425871

34. Roux KH, Strelets L, Michaelsen TE. Flexibility of human IgG subclasses. J Immunol. 1997;159(7):3372–82. 9317136

35. Mkhize NN, Durgiah R, Ashley V, Archary D, Garrett NJ, Karim QA, et al. Broadly neutralizing antibody specificities detected in the genital tract of HIV-1 infected women. AIDS. 2016;30(7):1005–14. doi: 10.1097/QAD.0000000000001038 26836790

36. Doria-Rose NA, Schramm CA, Gorman J, Moore PL, Bhiman JN, DeKosky BJ, et al. Developmental pathway for potent V1V2-directed HIV-neutralizing antibodies. Nature. 2014;509(7498):55–62. doi: 10.1038/nature13036 24590074

37. Doria-Rose NA, Bhiman JN, Roark RS, Schramm CA, Gorman J, Chuang GY, et al. New Member of the V1V2-Directed CAP256-VRC26 Lineage That Shows Increased Breadth and Exceptional Potency. J Virol. 2015;90(1):76–91. doi: 10.1128/JVI.01791-15 26468542

38. Brown EP, Licht AF, Dugast AS, Choi I, Bailey-Kellogg C, Alter G, et al. High-throughput, multiplexed IgG subclassing of antigen-specific antibodies from clinical samples. J Immunol Methods. 2012;386(1–2):117–23. doi: 10.1016/j.jim.2012.09.007 23023091

39. van Loggerenberg F, Mlisana K, Williamson C, Auld SC, Morris L, Gray CM, et al. Establishing a cohort at high risk of HIV infection in South Africa: challenges and experiences of the CAPRISA 002 acute infection study. PLoS One. 2008;3(4):e1954. doi: 10.1371/journal.pone.0001954 18414658

40. Abdool Karim Q, Abdool Karim SS, Frohlich JA, Grobler AC, Baxter C, Mansoor LE, et al. Effectiveness and safety of tenofovir gel, an antiretroviral microbicide, for the prevention of HIV infection in women. Science. 2010;329(5996):1168–74. doi: 10.1126/science.1193748 20643915

41. Ackerman ME, Moldt B, Wyatt RT, Dugast AS, McAndrew E, Tsoukas S, et al. A robust, high-throughput assay to determine the phagocytic activity of clinical antibody samples. J Immunol Methods. 2011;366(1–2):8–19. doi: 10.1016/j.jim.2010.12.016 21192942

42. Richardson SI, Crowther C, Mkhize NN, Morris L. Measuring the ability of HIV-specific antibodies to mediate trogocytosis. J Immunol Methods. 2018.

43. Pollara J, Hart L, Brewer F, Pickeral J, Packard BZ, Hoxie JA, et al. High-throughput quantitative analysis of HIV-1 and SIV-specific ADCC-mediating antibody responses. Cytometry A. 2011;79(8):603–12. doi: 10.1002/cyto.a.21084 21735545

44. Tay MZ, Liu P, Williams LD, McRaven MD, Sawant S, Gurley TC, et al. Antibody-Mediated Internalization of Infectious HIV-1 Virions Differs among Antibody Isotypes and Subclasses. PLoS Pathog. 2016;12(8):e1005817. doi: 10.1371/journal.ppat.1005817 27579713

45. Boesch AW, Miles AR, Chan YN, Osei-Owusu NY, Ackerman ME. IgG Fc variant cross-reactivity between human and rhesus macaque FcgammaRs. MAbs. 2017;9(3):455–65. doi: 10.1080/19420862.2016.1274845 28055295

46. Musich T, Li L, Liu L, Zolla-Pazner S, Robert-Guroff M, Gorny MK. Monoclonal Antibodies Specific for the V2, V3, CD4-Binding Site, and gp41 of HIV-1 Mediate Phagocytosis in a Dose-Dependent Manner. J Virol. 2017;91(8).

47. Mazor Y, Yang C, Borrok MJ, Ayriss J, Aherne K, Wu H, et al. Enhancement of Immune Effector Functions by Modulating IgG's Intrinsic Affinity for Target Antigen. PLoS One. 2016;11(6):e0157788. doi: 10.1371/journal.pone.0157788 27322177

48. Bournazos S, Ravetch JV. Diversification of IgG effector functions. Int Immunol. 2017;29(7):303–10. doi: 10.1093/intimm/dxx025 28472280

49. Natsume A, In M, Takamura H, Nakagawa T, Shimizu Y, Kitajima K, et al. Engineered antibodies of IgG1/IgG3 mixed isotype with enhanced cytotoxic activities. Cancer Res. 2008;68(10):3863–72. doi: 10.1158/0008-5472.CAN-07-6297 18483271

50. Perez LG, Costa MR, Todd CA, Haynes BF, Montefiori DC. Utilization of immunoglobulin G Fc receptors by human immunodeficiency virus type 1: a specific role for antibodies against the membrane-proximal external region of gp41. J Virol. 2009;83(15):7397–410. doi: 10.1128/JVI.00656-09 19458010

51. Giuntini S, Granoff DM, Beernink PT, Ihle O, Bratlie D, Michaelsen TE. Human IgG1, IgG3, and IgG3 Hinge-Truncated Mutants Show Different Protection Capabilities against Meningococci Depending on the Target Antigen and Epitope Specificity. Clin Vaccine Immunol. 2016;23(8):698–706. doi: 10.1128/CVI.00193-16 27307451

52. Scharf O, Golding H, King LR, Eller N, Frazier D, Golding B, et al. Immunoglobulin G3 from polyclonal human immunodeficiency virus (HIV) immune globulin is more potent than other subclasses in neutralizing HIV type 1. J Virol. 2001;75(14):6558–65. doi: 10.1128/JVI.75.14.6558-6565.2001 11413323

53. Barouch DH, Alter G, Broge T, Linde C, Ackerman ME, Brown EP, et al. Protective efficacy of adenovirus/protein vaccines against SIV challenges in rhesus monkeys. Science. 2015;349(6245):320–4. doi: 10.1126/science.aab3886 26138104

54. Sips M, Krykbaeva M, Diefenbach TJ, Ghebremichael M, Bowman BA, Dugast AS, et al. Fc receptor-mediated phagocytosis in tissues as a potent mechanism for preventive and therapeutic HIV vaccine strategies. Mucosal Immunol. 2016;9(6):1584–95. doi: 10.1038/mi.2016.12 26883728

55. Richardson SI, Chung AW, Natarajan H, Mabvakure B, Mkhize NN, Garrett N, et al. HIV-specific Fc effector function early in infection predicts the development of broadly neutralizing antibodies. PLoS Pathog. 2018;14(4):e1006987. doi: 10.1371/journal.ppat.1006987 29630668

56. Redpath S, Michaelsen TE, Sandlie I, Clark MR. The influence of the hinge region length in binding of human IgG to human Fcgamma receptors. Hum Immunol. 1998;59(11):720–7. doi: 10.1016/s0198-8859(98)00075-5 9796740

57. Patel KR, Roberts JT, Barb AW. Multiple Variables at the Leukocyte Cell Surface Impact Fc gamma Receptor-Dependent Mechanisms. Front Immunol. 2019;10:223. doi: 10.3389/fimmu.2019.00223 30837990

58. Cavacini LA, Emes CL, Power J, Desharnais FD, Duval M, Montefiori D, et al. Influence of heavy chain constant regions on antigen binding and HIV-1 neutralization by a human monoclonal antibody. J Immunol. 1995;155(7):3638–44. 7561063

59. Miranda LR, Duval M, Doherty H, Seaman MS, Posner MR, Cavacini LA. The neutralization properties of a HIV-specific antibody are markedly altered by glycosylation events outside the antigen-binding domain. J Immunol. 2007;178(11):7132–8. doi: 10.4049/jimmunol.178.11.7132 17513762

60. Astronomo RD, Santra S, Ballweber-Fleming L, Westerberg KG, Mach L, Hensley-McBain T, et al. Neutralization Takes Precedence Over IgG or IgA Isotype-related Functions in Mucosal HIV-1 Antibody-mediated Protection. EBioMedicine. 2016;14:97–111. doi: 10.1016/j.ebiom.2016.11.024 27919754

61. Tudor D, Yu H, Maupetit J, Drillet AS, Bouceba T, Schwartz-Cornil I, et al. Isotype modulates epitope specificity, affinity, and antiviral activities of anti-HIV-1 human broadly neutralizing 2F5 antibody. Proc Natl Acad Sci U S A. 2012;109(31):12680–5. doi: 10.1073/pnas.1200024109 22723360

62. Cheeseman HM, Olejniczak NJ, Rogers PM, Evans AB, King DF, Ziprin P, et al. Broadly Neutralizing Antibodies Display Potential for Prevention of HIV-1 Infection of Mucosal Tissue Superior to That of Nonneutralizing Antibodies. J Virol. 2017;91(1).

63. Molinos-Albert LM, Clotet B, Blanco J, Carrillo J. Immunologic Insights on the Membrane Proximal External Region: A Major Human Immunodeficiency Virus Type-1 Vaccine Target. Front Immunol. 2017;8:1154. doi: 10.3389/fimmu.2017.01154 28970835

64. 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 Pathog. 2013;9(9):e1003618. doi: 10.1371/journal.ppat.1003618 24068931

65. Julg B, Tartaglia LJ, Keele BF, Wagh K, Pegu A, Sok D, et al. Broadly neutralizing antibodies targeting the HIV-1 envelope V2 apex confer protection against a clade C SHIV challenge. Sci Transl Med. 2017;9(406).

66. Bournazos S, Gazumyan A, Seaman MS, Nussenzweig MC, Ravetch JV. Bispecific Anti-HIV-1 Antibodies with Enhanced Breadth and Potency. Cell. 2016;165(7):1609–20. doi: 10.1016/j.cell.2016.04.050 27315478

67. !!! INVALID CITATION !!! [50].

68. Galimidi RP, Klein JS, Politzer MS, Bai S, Seaman MS, Nussenzweig MC, et al. Intra-spike crosslinking overcomes antibody evasion by HIV-1. Cell. 2015;160(3):433–46. doi: 10.1016/j.cell.2015.01.016 25635457

69. Kratochvil S, McKay PF, Chung AW, Kent SJ, Gilmore J, Shattock RJ. Immunoglobulin G1 Allotype Influences Antibody Subclass Distribution in Response to HIV gp140 Vaccination. Front Immunol. 2017;8:1883. doi: 10.3389/fimmu.2017.01883 29326728

70. Richardson SI, Moore PL. The antibody response in HIV-1-infected donors. Curr Opin HIV AIDS. 2019;14(4):233–9. doi: 10.1097/COH.0000000000000559 31033730

71. Gray ES, Moody MA, Wibmer CK, Chen X, Marshall D, Amos J, et al. Isolation of a monoclonal antibody that targets the alpha-2 helix of gp120 and represents the initial autologous neutralizing-antibody response in an HIV-1 subtype C-infected individual. J Virol. 2011;85(15):7719–29. doi: 10.1128/JVI.00563-11 21613396

72. Scheid JF, Mouquet H, Ueberheide B, Diskin R, Klein F, Oliveira TY, et al. Sequence and structural convergence of broad and potent HIV antibodies that mimic CD4 binding. Science. 2011;333(6049):1633–7. doi: 10.1126/science.1207227 21764753

73. Tiller T, Meffre E, Yurasov S, Tsuiji M, Nussenzweig MC, Wardemann H. Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning. J Immunol Methods. 2008;329(1–2):112–24. doi: 10.1016/j.jim.2007.09.017 17996249