Neutralization-guided design of HIV-1 envelope trimers with high affinity for the unmutated common ancester of CH235 lineage CD4bs broadly neutralizing antibodies

English version

Autoři: Celia C. LaBranche ^aff001; Rory Henderson ^aff002; Allen Hsu ^aff003; Shay Behrens ^aff001; Xuejun Chen ^aff004; Tongqing Zhou ^aff004; Kevin Wiehe ^aff002; Kevin O. Saunders ^aff001; S. Munir Alam ^aff002; Mattia Bonsignori ^aff002; Mario J. Borgnia ^aff003; Quentin J. Sattentau ^aff006; Amanda Eaton ^aff001; Kelli Greene ^aff001; Hongmei Gao ^aff001; Hua-Xin Liao ^aff002; Wilton B. Williams ^aff002; James Peacock ^aff002; Haili Tang ^aff001; Lautaro G. Perez ^aff001; Robert J. Edwards ^aff002; Thomas B. Kepler ^aff007; Bette T. Korber ^aff008; Peter D. Kwong ^aff004; John R. Mascola ^aff004; Priyamvada Acharya ^aff001; Barton F. Haynes ^aff002; David C. Montefiori ^aff001
Působiště autorů: Department of Surgery, Duke University Medical Center, Durham, NC, United States of America ^aff001; Duke Human Vaccine Institute, Duke University Medical Center, Durham, NC, United States of America ^aff002; Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, NC, United States of America ^aff003; Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America ^aff004; Department of Medicine, Duke University Medical Center, Durham, NC, United States of America ^aff005; The Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom ^aff006; Department of Microbiology, Boston University School of Medicine, Boston, MA, United States of America ^aff007; Los Alamos National Laboratory, Theoretical Biology & Biophysics, Los Alamos, NM, United States of America ^aff008
Vyšlo v časopise: Neutralization-guided design of HIV-1 envelope trimers with high affinity for the unmutated common ancester of CH235 lineage CD4bs broadly neutralizing antibodies. PLoS Pathog 15(9): e1008026. doi:10.1371/journal.ppat.1008026
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1008026

Souhrn

The CD4 binding site (CD4bs) of the HIV-1 envelope glycoprotein is susceptible to multiple lineages of broadly neutralizing antibodies (bnAbs) that are attractive to elicit with vaccines. The CH235 lineage (VH1-46) of CD4bs bnAbs is particularly attractive because the most mature members neutralize 90% of circulating strains, do not possess long HCDR3 regions, and do not contain insertions and deletions that may be difficult to induce. We used virus neutralization to measure the interaction of CH235 unmutated common ancestor (CH235 UCA) with functional Env trimers on infectious virions to guide immunogen design for this bnAb lineage. Two Env mutations were identified, one in loop D (N279K) and another in V5 (G458Y), that acted synergistically to render autologous CH505 transmitted/founder virus susceptible to neutralization by CH235 UCA. Man₅-enriched N-glycans provided additional synergy for neutralization. CH235 UCA bound with nanomolar affinity to corresponding soluble native-like Env trimers as candidate immunogens. A cryo-EM structure of CH235 UCA bound to Man₅-enriched CH505.N279K.G458Y.SOSIP.664 revealed interactions of the antibody light chain complementarity determining region 3 (CDR L3) with the engineered Env loops D and V5. These results demonstrate that virus neutralization can directly inform vaccine design and suggest a germline targeting and reverse engineering strategy to initiate and mature the CH235 bnAb lineage.

Klíčová slova:

Biology and life sciences – Genetics – Biochemistry – Organisms – Research and analysis methods – Proteins – Molecular biology – Molecular biology techniques – Medicine and health sciences – Mutation – Point mutation – Microbiology – Medical microbiology – Microbial pathogens – Pathology and laboratory medicine – Pathogens – Physiology – Health care – Viral pathogens – Immunodeficiency viruses – HIV – Retroviruses – Lentivirus – Viruses – RNA viruses – Immunology – Immune physiology – Immune system proteins – HIV-1 – Antibodies – Transfection – Biological cultures – Cell lines – 293T cells – Microscopy – Electron microscopy – Electron cryo-microscopy – Antigens

Zdroje

1. Kwong PD, Mascola JR, and Nabel GJ. Broadly neutralizing antibodies and the search for an HIV‑1 vaccine: the end of the beginning. Nat Rev Immunol. 2013; 9:693–701.

2. Sok D, and Burton DR. Recent progress in broadly neutralizing antibodies to HIV. Nature Immunology. 2018; 19:1179–1188. doi: 10.1038/s41590-018-0235-7 30333615

3. Kwong PD, Doyle ML, Casper DJ, Cicala C, Leavitt SA, Majeed S. et al. HIV-1 evades antibody-mediated neutralization through conformational masking of receptor-binding sites. Nature. 2002; 420:678–682. doi: 10.1038/nature01188 12478295

4. Wei X, Decker JM, Wang S, Hui H, Kappes JC, Wu X, et al. Antibody neutralization and escape by HIV-1. Nature. 2003; 422:307–312. doi: 10.1038/nature01470 12646921

5. Jardine JG, Ota T, Sok D, Pauthner M, Kulp DW, Kalyuzhniy O, et al. Priming a broadly neutralizing antibody response to HIV-1 using a germline-targeting immunogen. Science. 2015; 349:156–161. doi: 10.1126/science.aac5894 26089355

6. McGuire AT, Gray MD, Dosenovic P, Gitlin AD, Freund NT, Petersen J, et al. Specifically modified Env immunogens activate B-cell precursors of broadly neutralizing HIV-1 antibodies in transgenic mice. Nat. Commun. 2016; 7:10618. doi: 10.1038/ncomms10618 26907590

7. Haynes BF, Fleming J, St Clair EW, Katinger H, Stiegler G, Kunert R, et al. Cardiolipin polyspecific autoreactivity in two broadly neutralizing HIV-1 antibodies. Science. 2005; 308:1906–1908. doi: 10.1126/science.1111781 15860590

8. Haynes BF, and Verkoczy L. AIDS/HIV. Host controls of HIV neutralizing antibodies. Science. 2014; 344:588–589. doi: 10.1126/science.1254990 24812389

9. Kelsoe G, and Haynes BF. Host controls of HIV broadly neutralizing antibody development. Immunological Reviews. 2017; 275:79–88. doi: 10.1111/imr.12508 28133807

10. Klein F, Mouquet H, Dosenovic P, Scheid JF, Scharf L, and Nussenzweig MC. Antibodies in HIV-1 vaccine development and therapy. Science. 2013; 341:1199–1204. doi: 10.1126/science.1241144 24031012

11. Kwong PD, and Mascola JR. Human antibodies that neutralize HIV-1: identification, structures, and B cell ontogenies. Immunity. 2012; 37:412–425. doi: 10.1016/j.immuni.2012.08.012 22999947

12. Bricault CA, Yusim K, Seaman MS, Yoon H, Theiler J, Giorgi EE, et al. HIV-1 neutralizing antibody signatures and application to epitope-targeted vaccine design. Cell Host & Microbe. 2019; 25:59–72.

13. Pauthner M, Havenar-Daughton C, Sok D, Nkolola JP, Bastidas R, Boopathy AV, et al. Elicitation of robust tier 2 neutralizing antibody responses in nonhuman primates by HIV envelope trimer immunization using optimized approaches. Immunity. 2017; 46:1073–1088. doi: 10.1016/j.immuni.2017.05.007 28636956

14. 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 Rep. 2017; 18:2175–2188. doi: 10.1016/j.celrep.2017.02.003 28249163

15. Xu K, Acharya P, Kong R, Cheng C, Chuang GY, Liu K, et al. Epitope-based vaccine design yields fusion peptide-directed antibodies that neutralize diverse strains of HIV-1. Nat Med. 2018; 6:857–867.

16. Haynes BF, and Mascola JR. The quest for an antibody-based HIV vaccine. Immunol Rev. 2017; 275:5–10. doi: 10.1111/imr.12517 28133795

17. Kwong PD, and Mascola JR. HIV-1 vaccines based on antibody identification, B cell ontogeny, and epitope structure. Immunity. 2018; 48:855–871. doi: 10.1016/j.immuni.2018.04.029 29768174

18. Jardine J, Julien JP, Menis S, Ota T, Kalyuzhniy O, McGuire A, et al. Rational HIV immunogen design to target specific germline B cell receptors. Science. 2013; 340(6133):711–6. doi: 10.1126/science.1234150 23539181; PubMed Central PMCID: PMC3689846.

19. McGuire AT, Hoot S, Dreyer AM, Lippy A, Stuart A, Cohen KW, et al. Engineering HIV envelope protein to activate germline B cell receptors of broadly neutralizing anti-CD4 binding site antibodies. The Journal of experimental medicine. 2013; 210(4):655–63. doi: 10.1084/jem.20122824 23530120; PubMed Central PMCID: PMC3620356.

20. Sliepen K, Medina-Ramírez M, Yasmeen A, Moore JP, Klasse PJ, and Sanders RW. Binding of inferred germline precursors of broadly neutralizing HIV-1 antibodies to native-like envelope trimers. Virology. 2015; 486:116–120. doi: 10.1016/j.virol.2015.08.002 26433050

21. de Taeye S.W., Moore J.P., and Sanders R.W. (2016) HIV-1 envelope trimer design and immunization strategies to induce broadly neutralizing antibodies. Trends in Immunology. 37, 221–232. doi: 10.1016/j.it.2016.01.007 26869204

22. Bale S, Martiné A, Wilson R, Behrens A-J, Le Fourn V, de Val N. et al. Cleavage-independent HIV-1 trimers from CHO cell lines elicit robust autologous tier 2 neutralizing antibodies. Front Immunol. 2018; 9:1116. doi: 10.3389/fimmu.2018.01116 29881382

23. de Taeye SW, Ozorowski G, Torrents de la Pen˜ a A, Guttman M, Julien JP, van den Kerkhof TL, et al. Immunogenicity of stabilized HIV-1 envelope trimers with reduced exposure of non-neutralizing epitopes. Cell. 2015; 163:1702–1715. doi: 10.1016/j.cell.2015.11.056 26687358

24. Joyce MG, Georgiev IS, Yang Y, Druz A, Geng H, Chuang GY, et al. Soluble prefusion closed DS-SOSIP.664-Env trimers of diverse HIV-1 strains. Cell Rep. 2017; 21:2992–3002. doi: 10.1016/j.celrep.2017.11.016 29212041

25. Medina‑Ramírez M, Garces F, Escolano A, Skog P, de Taeye SW, Moral‑Sanchez ID, et al. Design and crystal structure of a native-like HIV-1 envelope trimer that engages multiple broadly neutralizing antibody precursors in vivo. J Exp Med. 2017; 214:2573–2590. doi: 10.1084/jem.20161160 28847869

26. Sanders RW, Derking R, Cupo A, Julien J-P, 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; Sep; 9(9): e1003618. doi: 10.1371/journal.ppat.1003618 24068931

27. Torrents de la Peña A, Julien JP, de Taeye SW, Garces F, Guttman M, Ozorowski G, et al. Improving the immunogenicity of native-like HIV-1 envelope trimers by hyperstabilization. Cell Rep. 2017; 20:1805–1817. doi: 10.1016/j.celrep.2017.07.077 28834745

28. Munro JB, Gorman J, Ma X, Zhou Z, Arthos J, Burton DR, et al. Conformational dynamics of single HIV-1 Env trimers on the surface of native virions. Science. 2014; 346:759–763. doi: 10.1126/science.1254426 25298114

29. LaBranche CC, McGuire AT, Gray MD, Behrens S, Chen X, Zhou T, et al. HIV-1 envelope glycan modifications that permit neutralization by germline-reverted VRC01-class broadly neutralizing antibodies. PLoS Pathog. 2018; 14(11):e1007431. doi: 10.1371/journal.ppat.1007431 30395637

30. Liao HX, Lynch R, Zhou T, Gao F, Alam SM, Boyd SD, et al. Co-evolution of a broadly neutralizing HIV-1 antibody and founder virus. Nature. 2013; 496(7446):469–76. doi: 10.1038/nature12053 23552890; PubMed Central PMCID: PMC3637846.

31. Bonsignori M, Zhou T, Sheng Z, Chen L, Gao F, Joyce MG, et al. Maturation Pathway from Germline to Broad HIV-1 Neutralizer of a CD4-Mimic Antibody. Cell. 2016; 165(2):449–63. doi: 10.1016/j.cell.2016.02.022 26949186; PubMed Central PMCID: PMC4826291

32. Gao F, Bonsignori M, Liao HX, Kumar A, Xia SM, Lu X, et al. Cooperation of B cell lineages in induction of HIV-1-broadly neutralizing antibodies. Cell. 2014; 158(3):481–91. doi: 10.1016/j.cell.2014.06.022 25065977; PubMed Central PMCID: PMC4150607.

33. Zhou T, Doria-Rose NA, Cheng C, Stewart-Jones GBE, Chuang GY, Chambers M, et al. Quantification of the impact of the HIV-1-glycan shield on antibody elicitation. Cell Rep. 2017; 19:719–732. doi: 10.1016/j.celrep.2017.04.013 28445724

34. Blattner C, Lee JH, Sliepen K, Derking R, Falkowska E, de la Pena AT, et al. Structural delineation of a quaternary, cleavage-dependent epitope at the gp41-gp120 interface on intact HIV-1 Env trimers. Immunity. 2014; 40(5):669–80. doi: 10.1016/j.immuni.2014.04.008 24768348; PubMed Central PMCID: PMC4057017.

35. Falkowska E, Le KM, Ramos A, Doores KJ, Lee JH, Blattner C, et al. Broadly neutralizing HIV antibodies define a glycan-dependent epitope on the prefusion conformation of gp41 on cleaved envelope trimers. Immunity. 2014; 40(5):657–68. doi: 10.1016/j.immuni.2014.04.009 24768347; PubMed Central PMCID: PMC4070425.

36. Reeves PJ, Callewaert N, Contreras R, Khorana HG. Structure and function in rhodopsin: high-level expression of rhodopsin with restricted and homogeneous N-glycosylation by a tetracycline-inducible N-acetylglucosaminyltransferase I-negative HEK293S stable mammalian cell line. Proceedings of the National Academy of Sciences of the United States of America. 2002; 99(21):13419–24. doi: 10.1073/pnas.212519299 12370423; PubMed Central PMCID: PMC129688.

37. Binley JM, Ban YE, Crooks ET, Eggink D, Osawa K, Schief WR, et al. Role of complex carbohydrates in human immunodeficiency virus type 1 infection and resistance to antibody neutralization. Journal of virology. 2010; 84(11):5637–55. doi: 10.1128/JVI.00105-10 20335257; PubMed Central PMCID: PMC2876609.

38. Crooks ET, Grimley SL, Cully M, Osawa K, Dekkers G, Saunders K, et al. Glycoengineering HIV-1 Env creates ‘supercharged’ and ‘hybrid’ glycans to increase neutralizing antibody potency, breadth and saturation. PLoS pathogens. 2018; 14(5):e1007024. Epub 2018/05/03. doi: 10.1371/journal.ppat.1007024 29718999; PubMed Central PMCID: PMC5951585.

39. Wyatt R, and Sodroski J. The HIV-1 envelope glycoproteins: fusogens, antigens, and immunogens. Science. 1998; 280:1884–1888. doi: 10.1126/science.280.5371.1884 9632381

40. Montefiori DC, Roederer M, Morris L, and Seaman MS. Neutralization tiers of HIV-1. Curr Opin HIV AIDS. 2018; 13:1–9. doi: 10.1097/COH.0000000000000422

41. Diskin R, Klein F, Horwitz JA, Halper-Stromberg A, Sather DN, Marcovecchio PM, et al. Restricting HIV-1 pathways for escape using rationally designed anti-HIV-1 antibodies. J Exp Med. 2013; 210:1235–1249. doi: 10.1084/jem.20130221 23712429

42. Klein F, Halper-Stromberg A, Horwitz JA, Gruell H, Scheid JF, Bournazos S, et al. HIV therapy by a combination of broadly neutralizing antibodies in humanized mice. Nature. 2012; 492:118–122 doi: 10.1038/nature11604 23103874

43. Li Y, O’Dell S, Walker LM, Wu X, Guenaga J, Feng Y, et al. Mechanism of neutralization by the broadly neutralizing HIV-1 monoclonal antibody VRC01. Journal of virology. 2011; 85(17):8954–67. doi: 10.1128/JVI.00754-11 21715490; PubMed Central PMCID: PMC3165784.

44. Lynch RM, Wong P, Tran L, O’Dell S, Nason MC, Li Y, et al. HIV-1 fitness cost associated with escape from the VRC01 class of CD4 binding site neutralizing antibodies. Journal of virology. 2015; 89(8):4201–13. doi: 10.1128/JVI.03608-14 25631091; PubMed Central PMCID: PMC4442379.

45. 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 Rep. 2017; 21:3681–3690. doi: 10.1016/j.celrep.2017.12.028 29281818

46. 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; 329:811–817. doi: 10.1126/science.1192819 20616231

47. Zhou T, Zhu J, Wu X, Moquin S, Zhang B, Acharya P, et al. Multidonor analysis reveals structural elements, genetic determinants, and maturation pathway for HIV-1 neutralization by VRC01-class antibodies. Immunity. 2013; 39:245–58. doi: 10.1016/j.immuni.2013.04.012 23911655

48. Zhou T, Lynch RM, Chen L, Acharya P, Wu X, Doria-Rose NA, et al. Structural repertoire of HIV-1-neutralizing antibodies targeting the CD4 supersite in 14 donors. Cell. 2015; 161:1280–1292. doi: 10.1016/j.cell.2015.05.007 26004070

49. Wu X, Zhou T, Zhu J, Zhang B, Georgiev I, Wang C, et al. Focused evolution of HIV-1 neutralizing antibodies revealed by structures and deep sequencing. Science. 2011; 333:1593–1602. doi: 10.1126/science.1207532 21835983

50. Liu Q, Acharya P, Dolan MA, Zhang P, Guzzo C, Lu J, et al. Quaternary contact in the initial interaction of CD4 with the HIV-1 envelope trimer. Nat Struct Mol Biol. 2017; 24:370–378. doi: 10.1038/nsmb.3382 28218750

51. Gorman J, Soto C, Yang MM, Davenport TM, Guttman M, Bailer RT, et al. Structures of HIV-1 Env V1V2 with broadly neutralizing antibodies reveal commonalities that enable vaccine design. Nat Struct Mol Biol 2016; 23:81–90. doi: 10.1038/nsmb.3144 26689967

52. 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. doi: 10.1016/j.celrep.2016.02.058 26972002; PubMed Central PMCID: PMC4805854.

53. Cao L, Diedrich JK, Kulp DW, Pauthner M, He L, Park SR, et al. Global site-specific N-glycosylation analysis of HIV envelope glycoprotein. Nature communications. 2017; 8:14954. doi: 10.1038/ncomms14954 28348411; PubMed Central PMCID: PMC5379070.

54. Wu X, Yang ZY, Li Y, Hogerkorp CM, Schief WR, Seaman MS, et al. Rational design of envelope identifies broadly neutralizing human monoclonal antibodies to HIV-1. Science. 2010; 329:856–861. doi: 10.1126/science.1187659 20616233

55. Kong R, Xu K, Zhou T, Acharya P, Lemmin T, Liu K, et al. Fusion peptide of HIV-1 as a site of vulnerability to neutralizing antibody. Science. 2016; 352:828–833. doi: 10.1126/science.aae0474 27174988

56. Huang J, Ofek G, Laub L, Louder MK, Doria-Rose NA, Longo NS, et al. Broad and potent neutralization of HIV-1 by a gp41-specific human antibody. Nature. 2012; 491(7424):406–12. doi: 10.1038/nature11544 23151583; PubMed Central PMCID: PMC4854285.

57. Huang J, Kang BH, Ishida E, Zhou T, Griesman T, Sheng Z, et al. Identification of a CD4-Binding-Site Antibody to HIV that Evolved Near-Pan Neutralization Breadth. Immunity. 2016; 45(5):1108–21. doi: 10.1016/j.immuni.2016.10.027 27851912.

58. 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; PubMed Central PMCID: PMC3351836.

59. 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.

60. 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. Journal of virology. 2012; 86(8):4688–92. doi: 10.1128/JVI.07163-11 22301150; PubMed Central PMCID: PMC3318651.

61. Williams LD, Ofek G, Schatzle S, McDaniel JR, Lu X, Nicely NI, et al. Potent and broad HIV-neutralizing antibodies in memory B cells and plasma. Science Immunology 2017; 2(7):eaal2200. doi: 10.1126/sciimmunol.aal2200 28783671

62. Corti D, Langedijk JP, Hinz A, Seaman MS, Vanzetta F, Fernandez-Rodriguez BM, et al. Analysis of memory B cell responses and isolation of novel monoclonal antibodies with neutralizing breadth from HIV-1-infected individuals. PloS one. 2010; 5(1):e8805. doi: 10.1371/journal.pone.0008805 20098712; PubMed Central PMCID: PMC2808385.

63. Burton DR, Pyati J, Koduri R, Sharp SJ, Thornton GB, Parren PW, et al. Efficient neutralization of primary isolates of HIV-1 by a recombinant human monoclonal antibody. Science. 1994; 266(5187):1024–7. doi: 10.1126/science.7973652 7973652.

64. Sok D, van Gils MJ, Pauthner M, Julien JP, Saye-Francisco KL, Hsueh J, et al. Recombinant HIV envelope trimer selects for quaternary-dependent antibodies targeting the trimer apex. Proceedings of the National Academy of Sciences of the United States of America. 2014; 111(49):17624–9. doi: 10.1073/pnas.1415789111 25422458; PubMed Central PMCID: PMC4267403.

65. 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. doi: 10.1038/nature10373 21849977; PubMed Central PMCID: PMC3393110.

66. Gorny MK, Williams C, Volsky B, Revesz K, Cohen S, Polonis VR, et al. Human monoclonal antibodies specific for conformation-sensitive epitopes of V3 neutralize HIV-1 primary isolates from various clades. J Virol. 2002; 76:9035–9045. doi: 10.1128/JVI.76.18.9035-9045.2002 12186887

67. Gorny MK, Revesz K, Williams C, Volsky B, Louder MK, Anyangwe CA, et al. The V3 loop is accessible on the surface of most human immunodeficiency virus type 1 primary isolates and serves as a neutralization epitope. J Virol. 2004; 78:2394–2404. doi: 10.1128/JVI.78.5.2394-2404.2004 14963135

68. Gorny MK, Williams C, Volsky B, Revesz K, Wang XH, Burda S, et al. Cross-clade neutralizing activity of human anti-V3 monoclonal antibodies derived from the cells of individuals infected with non-B clades of HIV-1. J Virol. 2006; 80:6865–6872. doi: 10.1128/JVI.02202-05 16809292

69. Gorny MK, Wang XH, Williams C, Volsky B, Revesz K, Witover B, et al. Preferential use of the VH5-51 gene segment by the human immune response to code for antibodies against the V3 domain of HIV-1. Mol Immunol. 2009; 46:917–926. doi: 10.1016/j.molimm.2008.09.005 18952295

70. Gorny MK, Xu J-Y, Karwowska S, Buchbinder A, and Zolla-Pazner S. Repertoire of neutralizing human monoclonal antibodies specific for the V3 domain of HIV-1 gp120. J Immunol. 1993; 150:635–643. 7678279

71. Gorny MK, VanCott TC, Hioe C, Israel Z, Michael NL, Conley AJ, et al. Human monoclonal antibodies to the V3 loop of HIV-1 with intra- and interclade cross-reactivity. J Immunol. 1997; 159:5114–5122. 9366441

72. Nyambi PN, Gorny MK, Bastiani L, van der Groen G, Williams C, and Zolla-Pazner S. Mapping of epitopes exposed on intact human immunodeficiency virus type 1 (HIV-1) virions: a new strategy for studying the immunologic relatedness of HIV-1. J Virol. 1998; 72:9384–9391. 9765494

73. Jeffs SA, Gorny MK, Williams C, Revesz K, Volsky B, Burda S, et al. Characterization of human monoclonal antibodies selected with a hypervariable loop-deleted recombinant HIV-1(IIIB) gp120. Immunol Lett. 2001; 79:209–213. doi: 10.1016/s0165-2478(01)00289-9 11600200

74. Zolla-Pazner S, O'Leary J, Burda S, Gorny MK, Kim M, Mascola J, and McCutchan F. Serotyping of primary human immunodeficiency virus type 1 isolates from diverse geographic locations by flow cytometry. J Virol. 1995; 69:3807–3815. 7745728

75. Posner MR, Hideshima T, Cannon T, Mukherjee M, Mayer KH, and Byrn RA. An IgG human monoclonal antibody that reacts with HIV-1/gp120, inhibits virus binding to cells, and neutralizes infection. J Immunol. 1991; 146:4325–4332 1710248

76. Tang H, Robinson JE, Gnanakaran S, Li M, Rosenberg ES, Perez LG, et al. Epitopes immediately below the base of the V3 loop of gp120 as targets for the initial autologous neutralizing antibody response in two HIV-1 subtype B-infected individuals. Journal of virology. 2011; 85(18):9286–99. doi: 10.1128/JVI.02286-10 21734041; PubMed Central PMCID: PMC3165744.

77. Montefiori DC. Measuring HIV neutralization in a luciferase reporter gene assay. Methods in molecular biology. 2009; 485:395–405. doi: 10.1007/978-1-59745-170-3_26 19020839.

78. Eggink D, Melchers M, Wuhrer M, van Montfort T, Dey AK, Naaijkens BA, et al. Lack of complex N-glycans on HIV-1 envelope glycoproteins preserves protein conformation and entry function. Virology. 2010; 401(2):236–47. doi: 10.1016/j.virol.2010.02.019 20304457; PubMed Central PMCID: PMC3776475.

79. Alam SM, Liao HX, Tomaras GD, Bonsignori M, Tsao CY, Hwang KK, et al. Antigenicity and immunogenicity of RV144 vaccine AIDSVAX clade E envelope immunogen is enhanced by a gp120 N‐terminal deletion. J Virol. 2013; 87:1554–1568. doi: 10.1128/JVI.00718-12 23175357

80. Zhang K. Gctf: Real-time CTF determination and correction. J Struct Biol. 2016; 193:1–12. doi: 10.1016/j.jsb.2015.11.003 26592709

81. Zivanov J, Nakane T, Forsberg BO, Kimanius D, Hagen WJ, Lindahl E, and Scheres SH. New tools for automated high-resolution cryo-EM structure determination in RELION-3. Elife. 2018; pii: e42166. doi: 10.7554/eLife.42166 30412051

82. Punjani A, Rubinstein JL, Fleet DJ, and Brubaker MA. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat Methods. 2017; 14:290–296. doi: 10.1038/nmeth.4169 28165473

83. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, and Ferrin TE. UCSF Chimera—a visualization system for exploratory research and analysis. J Comput Chem. 2004; 25:1605–1612. doi: 10.1002/jcc.20084 15264254

84. Emsley P, Lohkamp B, Scott WG, and Cowtan K. Features and development of Coot. Acta Crystallogr D Biol Crystallogr. 2010; 66:486–501. doi: 10.1107/S0907444910007493 20383002

85. Wang RY, Song Y, Barad BA, Cheng Y, Fraser JS, and DiMaio F. Automated structure refinement of macromolecular assemblies from cryo-EM maps using Rosetta. Elife. 2016; pii: e17219. doi: 10.7554/eLife.17219 27669148

86. Adams PD, Afonine PV, Bunkóczi G, Chen VB, Davis IW, Echols N, et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Cryst. 2010; D66:213–221.

87. Chen VB, Arendall WB 3rd, Headd JJ, Keedy DA, Immormino RM, Kapral GJ, et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr D Biol Crystallogr. 2010; 66(Pt 1):12–21. doi: 10.1107/S0907444909042073 20057044

88. Barad BA, Echols N, Wang RY, Cheng Y, DiMaio F, Adams PD, and Fraser JS. EMRinger: side chain-directed model and map validation for 3D cryo-electron microscopy. Nat Methods. 2015; 12, 943–946. doi: 10.1038/nmeth.3541 26280328

89. Lütteke T, von der Lieth CW. pdb-care (PDB CArbohydrate REsidue check): a program to support annotation of complex carbohydrate structures in PDB files. BMC Bioinformatics 2004, 5: 69. doi: 10.1186/1471-2105-5-69 15180909