All human immunodeficiency virus type-1 (HIV-1) viruses use CD4 to enter cells. Consequently, the viral envelope CD4-binding site (CD4bs) is relatively conserved, making it a logical neutralizing antibody target. It is important to understand how CD4-binding site variation allows for escape from neutralizing antibodies. Alanine scanning mutagenesis identifies residues in antigenic sites, whereas escape mutant selection identifies viable mutants. We selected HIV-1 to escape CD4bs neutralizing mAbs b12, A12 and HJ16. Viruses that escape from A12 and b12 remained susceptible to HJ16, VRC01 and J3, whilst six different viruses that escape HJ16 remained sensitive to A12, b12 and J3. In contrast, their sensitivity to VRC01 was variable. Triple HJ16/A12/b12-resistant virus proved that HIV-1 could escape multiple broadly neutralizing monoclonal antibodies, but still retain sensitivity to VRC01 and the llama-derived J3 nanobody. This antigenic variability may reflect that occurring in circulating viruses, so studies like this can predict immunologically relevant antigenic forms of the CD4bs for inclusion in HIV-1 vaccines.


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  1. Balla-Jhagjhoorsingh S. S., Corti D., Heyndrickx L., Willems E., Vereecken K., Davis D., Vanham G.(2013). The N276 glycosylation site is required for HIV-1 neutralization by the CD4 binding site specific HJ16 monoclonal antibody. PLoS ONE 8, e68863. [View Article][PubMed] [Google Scholar]
  2. Barbas C. F. III, Björling E., Chiodi F., Dunlop N., Cababa D., Jones T. M., Zebedee S. L., Persson M. A., Nara P. L., Norrby E.(1992). Recombinant human Fab fragments neutralize human type 1 immunodeficiency virus in vitro. Proc Natl Acad Sci U S A 89, 93399343. [View Article][PubMed] [Google Scholar]
  3. Burton D. R., Barbas C. F. III, Persson M. A., Koenig S., Chanock R. M., Lerner R. A.(1991). A large array of human monoclonal antibodies to type 1 human immunodeficiency virus from combinatorial libraries of asymptomatic seropositive individuals. Proc Natl Acad Sci U S A 88, 1013410137. [View Article][PubMed] [Google Scholar]
  4. Burton D. R., Pyati J., Koduri R., Sharp S. J., Thornton G. B., Parren P. W., Sawyer L. S., Hendry R. M., Dunlop N. et al.(1994). Efficient neutralization of primary isolates of HIV-1 by a recombinant human monoclonal antibody. Science 266, 10241027. [View Article][PubMed] [Google Scholar]
  5. Corti D., Langedijk J. P., Hinz A., Seaman M. S., Vanzetta F., Fernandez-Rodriguez B. M., Silacci C., Pinna D., Jarrossay D. et al.(2010). Analysis of memory B cell responses and isolation of novel monoclonal antibodies with neutralizing breadth from HIV-1-infected individuals. PLoS ONE 5, e8805. [View Article][PubMed] [Google Scholar]
  6. Dreja H., O’Sullivan E., Pade C., Greene K. M., Gao H., Aubin K., Hand J., Isaksen A., D’Souza C. et al.(2010). Neutralization activity in a geographically diverse East London cohort of human immunodeficiency virus type 1-infected patients: clade C infection results in a stronger and broader humoral immune response than clade B infection. J Gen Virol 91, 27942803. [View Article][PubMed] [Google Scholar]
  7. Falkowska E., Ramos A., Feng Y., Zhou T., Moquin S., Walker L. M., Wu X., Seaman M. S., Wrin T. et al.(2012). PGV04, an HIV-1 gp120 CD4 binding site antibody, is broad and potent in neutralization but does not induce conformational changes characteristic of CD4. J Virol 86, 43944403. [View Article][PubMed] [Google Scholar]
  8. Forsman A., Beirnaert E., Aasa-Chapman M. M., Hoorelbeke B., Hijazi K., Koh W., Tack V., Szynol A., Kelly C. et al.(2008). Llama antibody fragments with cross-subtype human immunodeficiency virus type 1 (HIV-1)-neutralizing properties and high affinity for HIV-1 gp120. J Virol 82, 1206912081. [View Article][PubMed] [Google Scholar]
  9. Gao F., Morrison S. G., Robertson D. L., Thornton C. L., Craig S., Karlsson G., Sodroski J., Morgado M., Galvao-Castro B. et al.(1996). Molecular cloning and analysis of functional envelope genes from human immunodeficiency virus type 1 sequence subtypes A through G. The WHO and NIAID Networks for HIV Isolation and Characterization. J Virol 70, 16511667.[PubMed] [Google Scholar]
  10. Gaschen B., Taylor J., Yusim K., Foley B., Gao F., Lang D., Novitsky V., Haynes B., Hahn B. H. et al.(2002). Diversity considerations in HIV-1 vaccine selection. Science 296, 23542360. [View Article][PubMed] [Google Scholar]
  11. Gray L., Sterjovski J., Ramsland P. A., Churchill M. J., Gorry P. R.(2011). Conformational alterations in the CD4 binding cavity of HIV-1 gp120 influencing gp120-CD4 interactions and fusogenicity of HIV-1 envelopes derived from brain and other tissues. Retrovirology 8, 42. [View Article][PubMed] [Google Scholar]
  12. Haynes B. F., Montefiori D. C.(2006). Aiming to induce broadly reactive neutralizing antibody responses with HIV-1 vaccine candidates. Expert Rev Vaccines 5, 347363. [View Article][PubMed] [Google Scholar]
  13. Klein F., Halper-Stromberg A., Horwitz J. A., Gruell H., Scheid J. F., Bournazos S., Mouquet H., Spatz L. A., Diskin R. et al.(2012). HIV therapy by a combination of broadly neutralizing antibodies in humanized mice. Nature 492, 118122. [View Article][PubMed] [Google Scholar]
  14. Li Y., O’Dell S., Walker L. M., Wu X., Guenaga J., Feng Y., Schmidt S. D., McKee K., Louder M. K. et al.(2011). Mechanism of neutralization by the broadly neutralizing HIV-1 monoclonal antibody VRC01. J Virol 85, 89548967. [View Article][PubMed] [Google Scholar]
  15. McCoy L. E., Weiss R. A.(2013). Neutralizing antibodies to HIV-1 induced by immunization. J Exp Med 210, 209223. [View Article][PubMed] [Google Scholar]
  16. McCoy L. E., Quigley A. F., Strokappe N. M., Bulmer-Thomas B., Seaman M. S., Mortier D., Rutten L., Chander N., Edwards C. J. et al.(2012). Potent and broad neutralization of HIV-1 by a llama antibody elicited by immunization. J Exp Med 209, 10911103. [View Article][PubMed] [Google Scholar]
  17. Mo H., Stamatatos L., Ip J. E., Barbas C. F., Parren P. W., Burton D. R., Moore J. P., Ho D. D.(1997). Human immunodeficiency virus type 1 mutants that escape neutralization by human monoclonal antibody IgG1b12. off. J Virol 9, 68696874.[PubMed] [Google Scholar]
  18. Pace C. S., Song R., Ochsenbauer C., Andrews C. D., Franco D., Yu J., Oren D. A., Seaman M. S., Ho D. D.(2013). Bispecific antibodies directed to CD4 domain 2 and HIV envelope exhibit exceptional breadth and picomolar potency against HIV-1. Proc Natl Acad Sci U S A 110, 1354013545. [View Article][PubMed] [Google Scholar]
  19. Pancera M., Majeed S., Ban Y. E., Chen L., Huang C. C., Kong L., Kwon Y. D., Stuckey J., Zhou T. et al.(2010). Structure of HIV-1 gp120 with gp41-interactive region reveals layered envelope architecture and basis of conformational mobility. Proc Natl Acad Sci U S A 107, 11661171. [View Article][PubMed] [Google Scholar]
  20. Pietzsch J., Scheid J. F., Mouquet H., Klein F., Seaman M. S., Jankovic M., Corti D., Lanzavecchia A., Nussenzweig M. C.(2010). Human anti-HIV-neutralizing antibodies frequently target a conserved epitope essential for viral fitness. J Exp Med 207, 19952002. [View Article][PubMed] [Google Scholar]
  21. Ratner L., Haseltine W., Patarca R., Livak K. J., Starcich B., Josephs S. F., Doran E. R., Rafalski J. A., Whitehorn E. A. et al.(1985). Complete nucleotide sequence of the AIDS virus, HTLV-III. Nature 313, 277284. [View Article][PubMed] [Google Scholar]
  22. Roben P., Moore J. P., Thali M., Sodroski J., Barbas C. F. III, Burton D. R.(1994). Recognition properties of a panel of human recombinant Fab fragments to the CD4 binding site of gp120 that show differing abilities to neutralize human immunodeficiency virus type 1. J Virol 68, 48214828.[PubMed] [Google Scholar]
  23. Salahuddin S. Z., Markham P. D., Wong-Staal F., Franchini G., Kalyanaraman V. S., Gallo R. C.(1983). Restricted expression of human T-cell leukemia–lymphoma virus (HTLV) in transformed human umbilical cord blood lymphocytes. Virology 129, 5164. [View Article][PubMed] [Google Scholar]
  24. Saphire E. O., Parren P. W., Pantophlet R., Zwick M. B., Morris G. M., Rudd P. M., Dwek R. A., Stanfield R. L., Burton D. R., Wilson I. A.(2001). Crystal structure of a neutralizing human IGG against HIV-1: a template for vaccine design. Science 293, 11551159. [View Article][PubMed] [Google Scholar]
  25. Walker L. M., Bowley D. R., Burton D. R.(2009). Efficient recovery of high-affinity antibodies from a single-chain Fab yeast display library. J Mol Biol 389, 365375. [View Article][PubMed] [Google Scholar]
  26. Wu X., Yang Z. Y., Li Y., Hogerkorp C. M., Schief W. R., Seaman M. S., Zhou T., Schmidt S. D., Wu L. et al.(2010). Rational design of envelope identifies broadly neutralizing human monoclonal antibodies to HIV-1. Science 329, 856861. [View Article][PubMed] [Google Scholar]
  27. Zhou T., Xu L., Dey B., Hessell A. J., Van Ryk D., Xiang S. H., Yang X., Zhang M. Y., Zwick M. B. et al.(2007). Structural definition of a conserved neutralization epitope on HIV-1 gp120. Nature 445, 732737. [View Article][PubMed] [Google Scholar]
  28. Zhou T., Georgiev I., Wu X., Yang Z. Y., Dai K., Finzi A., Kwon Y. D., Scheid J. F., Shi W. et al.(2010). Structural basis for broad and potent neutralization of HIV-1 by antibody VRC01. Science 329, 811817. [View Article][PubMed] [Google Scholar]

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