Neutralization of HIV by Antibodies

  • Ilia Prado
  • Timothy R. Fouts
  • Antony S. Dimitrov
Part of the Methods in Molecular Biology™ book series (MIMB, volume 525)


Antibodies can neutralize HIV-1 with potency and cross-reactivity that varies widely and is related but not correlated to their antigen-binding affinity. Therefore, in addition to measuring binding affinity, an evaluation of the antibody neutralizing activity in tissue cultures is important for development of antibody-based therapeutics, design of candidate vaccine immunogens, and understanding the mechanisms of virus entry, neutralization, and evasion of immune responses. The development of a standardized assay for measurement of the in vitro neutralizing activities of the antibody has remained a challenging goal in the last two decades. There are two types of widely used assays, which vary in details between different laboratories – assays based on cell line/pseudovirus and assays based on infection of peripheral blood mononuclear cells (PBMCs). Here we describe in detail the PBMC-based assay, which is more laborious but in our opinion represents a closer approximation of the in vivo situation. As with all other in vitro assays the results of such measurements are only an indication of the antibody potency in vivo, and animal studies and ultimately clinical trials are needed for the development of such antibodies as potential prophylactics and therapeutics.

Key words

HIV-1 PBMC neutralization p24 infection inhibition of infection IC50 


  1. 1.
    Gallant, J. E., DeJesus, E., Arribas, J. R., Pozniak, A. L., Gazzard, B., Campo, R. E., et al. (2006) Tenofovir DF, emtricitabine, and efavirenz vs. zidovudine, lamivudine, and efavirenz for HIV. N. Engl. J. Med. 354, 251–260.PubMedCrossRefGoogle Scholar
  2. 2.
    Shafer, R. W., Smeaton, L. M., Robbins, G. K., De Gruttola, V., Snyder, S. W., D'Aquila, R. T., et al. (2003) Comparison of four-drug regimens and pairs of sequential three-drug regimens as initial therapy for HIV-1 infection. N. Engl. J. Med. 349, 2304–2315.PubMedCrossRefGoogle Scholar
  3. 3.
    Robbins, G. K., De Gruttola, V., Shafer, R. W., Smeaton, L. M., Snyder, S. W., Pettinelli, C., et al. (2003) Comparison of sequential three-drug regimens as initial therapy for HIV-1 infection. N. Engl. J. Med. 349, 2293–2303.PubMedCrossRefGoogle Scholar
  4. 4.
    Ledergerber, B., Egger, M., Opravil, M., Telenti, A., Hirschel, B., Battegay, M., et al. (1999) Clinical progression and virological failure on highly active antiretroviral therapy in HIV-1 patients: a prospective cohort study. Swiss HIV Cohort Study. Lancet 353, 863–868.PubMedCrossRefGoogle Scholar
  5. 5.
    Palella, F. J., Jr., Delaney, K. M., Moorman, A. C., Loveless, M. O., Fuhrer, J., Satten, G. A., et al. (1998) Declining morbidity and mortality among patients with advanced human immunodeficiency virus infection. HIV Outpatient Study Investigators. N. Engl. J. Med. 338, 853–860.PubMedCrossRefGoogle Scholar
  6. 6.
    Fellay, J., Boubaker, K., Ledergerber, B., Bernasconi, E., Furrer, H., Battegay, M., et al. (2001) Prevalence of adverse events associated with potent antiretroviral treatment: Swiss HIV Cohort Study. Lancet 358, 1322–1327.PubMedCrossRefGoogle Scholar
  7. 7.
    Friis-Moller, N., Sabin, C. A., Weber, R., d'Arminio Monforte, A., El-Sadr, W. M., Reiss, P., et al. (2003) Combination antiretroviral therapy and the risk of myocardial infarction. N. Engl. J. Med. 349, 1993–2003.PubMedCrossRefGoogle Scholar
  8. 8.
    Friis-Moller, N., Weber, R., Reiss, P., Thiebaut, R., Kirk, O., d’Arminio Monforte, A., et al. (2003) Cardiovascular disease risk factors in HIV patients – association with antiretroviral therapy. Results from the DAD study. AIDS 17, 1179–1193.PubMedCrossRefGoogle Scholar
  9. 9.
    Hirsch, M. S., Brun-Vezinet, F., Clotet, B., Conway, B., Kuritzkes, D. R., D'Aquila, R. T., et al. (2003) Antiretroviral drug resistance testing in adults infected with human immunodeficiency virus type 1: 2003 recommendations of an International AIDS Society-USA Panel. Clin. Infect. Dis. 37, 113–128.PubMedCrossRefGoogle Scholar
  10. 10.
    Hirsch, M. S., Brun-Vezinet, F., D'Aquila, R. T., Hammer, S. M., Johnson, V. A., Kuritzkes, D. R., et al. (2000) Antiretroviral drug resistance testing in adult HIV-1 infection: recommendations of an International AIDS Society – USA Panel. JAMA 283, 2417–2426.PubMedCrossRefGoogle Scholar
  11. 11.
    Hirsch, M. S., Conway, B., D'Aquila, R. T., Johnson, V. A., Brun-Vezinet, F., Clotet, B., et al. (1998) Antiretroviral drug resistance testing in adults with HIV infection: implications for clinical management. International AIDS Society – USA Panel. JAMA 279, 1984–1991.PubMedCrossRefGoogle Scholar
  12. 12.
    Wit, F. W., van Leeuwen, R., Weverling, G. J., Jurriaans, S., Nauta, K., Steingrover, R., et al. (1999) Outcome and predictors of failure of highly active antiretroviral therapy: one-year follow-up of a cohort of human immunodeficiency virus type 1-infected persons. J. Infect. Dis. 179, 790–798.PubMedCrossRefGoogle Scholar
  13. 13.
    Katsnelson, A. (2006) Biotech jostles with pharma for slice of HIV market. Nat. Biotechnol. 24, 3–4.PubMedCrossRefGoogle Scholar
  14. 14.
    McClure, M. O., Sattentau, Q. J., Beverley, P. C., Hearn, J. P., Fitzgerald, A. K., Zuckerman, A. J., et al. (1987) HIV infection of primate lymphocytes and conservation of the CD4 receptor. Nature 330, 487–489.PubMedCrossRefGoogle Scholar
  15. 15.
    Sattentau, Q. J. (1992) CD4 activation of HIV fusion. Int. J. Cell Cloning 10, 323–332.PubMedCrossRefGoogle Scholar
  16. 16.
    Sattentau, Q. J., and Moore, J. P. (1993) The role of CD4 in HIV binding and entry. Philos. Trans. R Soc. Lond. B Biol. Sci. 342, 59–66.PubMedCrossRefGoogle Scholar
  17. 17.
    Broder, C. C., and Collman, R. G. (1997) Chemokine receptors and HIV. J. Leukoc. Biol. 62, 20–29.PubMedGoogle Scholar
  18. 18.
    Clapham, P. R., and McKnight, A. (2001) HIV-1 receptors and cell tropism. Br. Med. Bull. 58, 43–59.PubMedCrossRefGoogle Scholar
  19. 19.
    Dimitrov, A. S., Xiao, X., Dimitrov, D. S., and Blumenthal, R. (2001) Early intermediates in HIV-1 envelope glycoprotein-mediated fusion triggered by CD4 and co-receptor complexes. J. Biol. Chem. 276, 30335–30341.PubMedCrossRefGoogle Scholar
  20. 20.
    Gallo, S. A., Finnegan, C. M., Viard, M., Raviv, Y., Dimitrov, A., Rawat, S. S., et al. (2003) The HIV Env-mediated fusion reaction. Biochim. Biophys. Acta 1614, 36–50.PubMedCrossRefGoogle Scholar
  21. 21.
    Pierson, T. C., and Doms, R. W. (2003) HIV-1 entry and its inhibition. Curr. Top. Microbiol. Immunol. 281, 1–27.PubMedGoogle Scholar
  22. 22.
    Dimitrov, A. S., Louis, J. M., Bewley, C. A., Clore, G. M., and Blumenthal, R. (2005) Conformational changes in HIV-1 gp41 in the course of HIV-1 envelope glycoprotein-mediated fusion and inactivation. Biochemistry 44, 12471–12479.PubMedCrossRefGoogle Scholar
  23. 23.
    Bayry, J., Lacroix-Desmazes, S., Kazatchkine, M. D., and Kaveri, S. V. (2004) Intravenous immunoglobulin for infectious diseases: back to the pre-antibiotic and passive prophylaxis era? Trends Pharmacol. Sci. 25, 306–310.PubMedCrossRefGoogle Scholar
  24. 24.
    Dezube, B. J., Proper, J., Zhang, J., Choy, V. J., Weeden, W., Morrissey, J., et al. (2003) A passive immunotherapy, (PE) HRG214, in patients infected with human immunodeficiency virus: a phase I study. J. Infect. Dis. 187, 500–503.PubMedCrossRefGoogle Scholar
  25. 25.
    Rusert, P., Kuster, H., Joos, B., Misselwitz, B., Gujer, C., Leemann, C., et al. (2005) Virus isolates during acute and chronic human immunodeficiency virus type 1 infection show distinct patterns of sensitivity to entry inhibitors. J. Virol. 79, 8454–8469.PubMedCrossRefGoogle Scholar
  26. 26.
    Choudhry, V., Zhang, M. Y., Dimitrova, D., Prabakaran, P., Dimitrov, A. S., Fouts, T. R., et al. (2006) Antibody-based inhibitors of HIV infection. Expert Opin. Biol. Ther. 6, 523–531.PubMedCrossRefGoogle Scholar
  27. 27.
    Mc Cann, C. M., Song, R. J., and Ruprecht, R. M. (2005) Antibodies: can they protect against HIV infection? Curr. Drug Targets Infect. Disord. 5, 95–111.PubMedCrossRefGoogle Scholar
  28. 28.
    Wolfe, E. J., Cavacini, L. A., Samore, M. H., Posner, M. R., Kozial, C., Spino, C., et al. (1996) Pharmacokinetics of F105, a human monoclonal antibody, in persons infected with human immunodeficiency virus type 1. Clin. Pharmacol. Ther. 59, 662–667.PubMedCrossRefGoogle Scholar
  29. 29.
    Cavacini, L. A., Samore, M. H., Gambertoglio, J., Jackson, B., Duval, M., Wisnewski, A., et al. (1998) Phase I study of a human monoclonal antibody directed against the CD4-binding site of HIV type 1 glycoprotein 120. AIDS Res. Hum. Retroviruses 14, 545–550.PubMedCrossRefGoogle Scholar
  30. 30.
    Armbruster, C., Stiegler, G. M., Vcelar, B. A., Jager, W., Koller, U., Jilch, R., et al. (2004) Passive immunization with the anti-HIV-1 human monoclonal antibody (hMAb) 4E10 and the hMAb combination 4E10/2F5/2G12. J. Antimicrob. Chemother. 54, 915–920.PubMedCrossRefGoogle Scholar
  31. 31.
    Armbruster, C., Stiegler, G. M., Vcelar, B. A., Jager, W., Michael, N. L., Vetter, N., et al. (2002) A phase I trial with two human monoclonal antibodies (hMAb 2F5, 2G12) against HIV-1. AIDS 16, 227–233.PubMedCrossRefGoogle Scholar
  32. 32.
    Dimitrov, A. (2007) Ibalizumab, a CD4-specific mAb to inhibit HIV-1 infection. Curr. Opin. Investig. Drugs 8, 653–661.PubMedGoogle Scholar
  33. 33.
    Trkola, A., Ketas, T. J., Nagashima, K. A., Zhao, L., Cilliers, T., Morris, L., et al. (2001) Potent, broad-spectrum inhibition of human immunodeficiency virus type 1 by the CCR5 monoclonal antibody PRO 140. J. Virol. 75, 579–588.PubMedCrossRefGoogle Scholar
  34. 34.
    No authors listed (2008) Phase 2 clinical trials started on PRO 140. AIDS Patient Care STDS 22, 159–160.CrossRefGoogle Scholar
  35. 35.
    Moulard, M., Phogat, S. K., Shu, Y., Labrijn, A. F., Xiao, X., Binley, J. M., et al. (2002) Broadly cross-reactive HIV-1-neutralizing human monoclonal Fab selected for binding to gp120–CD4–CCR5 complexes. Proc. Natl. Acad. Sci. USA 99, 6913–6918.PubMedCrossRefGoogle Scholar
  36. 36.
    Zhang, M. Y., Shu, Y., Phogat, S., Xiao, X., Cham, F., Bouma, P., et al. (2003) Broadly cross-reactive HIV neutralizing human monoclonal antibody Fab selected by sequential antigen panning of a phage display library. J. Immunol. Methods 283, 17–25.PubMedCrossRefGoogle Scholar
  37. 37.
    Zhang, M. Y., Shu, Y., Rudolph, D., Prabakaran, P., Labrijn, A. F., Zwick, M. B., et al. (2004) Improved breadth and potency of an HIV-1-neutralizing human single-chain antibody by random mutagenesis and sequential antigen panning. J. Mol. Biol. 335, 209–219.PubMedCrossRefGoogle Scholar
  38. 38.
    Zhang, M. Y., Xiao, X., Sidorov, I. A., Choudhry, V., Cham, F., Zhang, P. F., et al. (2004) Identification and characterization of a new cross-reactive human immunodeficiency virus type 1-neutralizing human monoclonal antibody. J. Virol. 78, 9233–9242.PubMedCrossRefGoogle Scholar
  39. 39.
    Zhang, M. Y., and Dimitrov, D. S. (2007) Novel approaches for identification of broadly cross-reactive HIV-1 neutralizing human monoclonal antibodies and improvement of their potency. Curr. Pharm. Des. 13, 203–212.PubMedCrossRefGoogle Scholar
  40. 40.
    Polonis, V. R., Brown, B. K., Borges, A. R., Zolla-Pazner, S., Dimitrov, D. S., Zhang, M. Y., et al. (2008) Recent advances in the characterization of HIV-1 neutralization assays for standardized evaluation of the antibody response to infection and vaccination. Virology 375, 315–320.PubMedCrossRefGoogle Scholar
  41. 41.
    Brown, B. K., Wieczorek, L., Sanders-Buell, E., Borges, A. R., Robb, M. L., Birx, D. L., et al. (2008) Cross-clade neutralization patterns among HIV-1 strains from the six major clades of the pandemic evaluated and compared in two different models. Virology 375, 529–538.PubMedCrossRefGoogle Scholar
  42. 42.
    Choudhry, V., Zhang, M. Y., Harris, I., Sidorov, I. A., Vu, B., Dimitrov, A. S., et al. (2006) Increased efficacy of HIV-1 neutralization by antibodies at low CCR5 surface concentration. Biochem. Biophys. Res. Commun. 348, 1107–1115.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Ilia Prado
    • 1
  • Timothy R. Fouts
    • 1
  • Antony S. Dimitrov
    • 1
  1. 1.Profectus BioSciences, Inc.BaltimoreUSA

Personalised recommendations