Neutralizing Antibody Assays

The Neutralizing Antibody Component provides standardized assessments of the magnitude, breadth, kinetics and duration of vaccine-elicited neutralizing antibody responses in preclinical and clinical trials of candidate HIV and SIV vaccines using validated assays and standardized reference strains in a GCLP-compliant environment. These same assays are used to assess the magnitude and breadth of neutralizing activity of serum samples and monoclonal antibodies from infected individuals. We also offer neutralizing antibody epitope mapping, virus infectivity measurements and neutralization tier phenotyping services.

TZM-bl Assay Description

Antibody-mediated neutralization of HIV, SIV and SHIV is measured as a function of reductions in Tat-regulated Firefly luciferase (Luc) reporter gene expression after a single round of infection in TZM-bl cells. This assay was first developed by Dr. George Shaw and colleagues at the University of Alabama, Birmingham and was later optimized and validated at Duke University. TZM-bl (also known as JC53BL-13) is a CXCR4-positive HeLa cell clone that was engineered to express CD4 and CCR5 (4). The cells were further engineered to contain integrated reporter genes for firefly luciferase and Escherichia coli b-galactosidase under control of an HIV long-terminal repeat sequence (5). TZM-bl cells are permissive to infection by a wide variety of HIV, SIV and SHIV strains, including primary HIV isolates and molecularly cloned Env-pseudotyped viruses. Assay stocks of Env-pseudotyped viruses are produced in 293T/17 cells by co-transfection with an Env expression plasmid and a second plasmid expressing the entire HIV-1 genome except Env. Only the latter env-minus plasmid replicates in 293T/17 cells; this plasmid is packaged by the pseudovirions for delivery of the tat gene to TZM-bl cells. Thus, co-transfection generates pseudovirus particles that are infectious but are unable to produce infectious progeny virions for subsequent rounds of infection. Reporter gene expression is induced in trans by viral Tat protein soon after single cycle infection. DEAE dextran is added to the medium to enhance infection and has been found to have no obvious effects on NAb activity. Luciferase activity is quantified as relative luminescence units (RLU) and is directly proportional to the number of infectious virus particles present in the initial inoculum over a wide range of values. Neutralization titers are the dilution at which RLU are reduced by 50% compared to virus control wells after subtraction of background RLUs. The assay is performed in 96-well plates for high throughput capacity, and utilizes well-characterized reference strains for uniformity across studies (6-9).

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  7. Li, M., Gao, F., Mascola, J.R., Stamatatos, L., Polonis, V.R., Koutsoukos, M., Voss, G., Goepfert, P., Gilbert, P., Greene, K.M., Bilska, M., Kothe, D.L., Salazar-Gonzalez, J.F., Wei, X., Decker, J.M., Hahn, B.H. and Montefiori, D.C. (2005) Human immunodeficiency virus type 1 env clones from acute and early subtype B infections for standardized assessments of vaccine-elicited neutralizing antibodies.  J. Virol. 79, 10108-10125.
  8. Li, M., Salazar-Gonzalez, J. F., Derdeyn, C. A., Morris, L., Williamson, C., Robinson, J. E., Decker, J. M., Li, Y., Salazar, M. G., Polonis, V. R., Mlisana, K., Karim, S. A., Hong, K., Greene, K. M., Bilska, M., Zhou, J. T., Allen, S., Chomba, E., Mulenga, J., Vwalika, C., Gao, F., Zhang, M., Korber, B. T. M., Hunter, E., Hahn, B. H. and Montefiori, D. C. (2006) Genetic and neutralization properties of subtype C human immunodeficiency virus type 1 molecular env clones from acute and early heterosexually acquired infections in southern Africa. J. Virol., 80,11776-11790.
  9. DeCamp, A., Hraber, P., Bailer, R.T., Seaman, M.S., Ochsenbauer, C., Kappes, J., Gottardo, R., Edlefsen, P., Self, S., Tang, H., Greene, K., Gao, H., Daniell, X., Sarzotti-Kelsoe, M., Gorny, M.K., Zolla-Pazner, S., LaBranche, C.C., Mascola, J.R., Korber, B.T., and Montefiori, D.C. (2014) Global panel of HIV-1 Env reference strains for standardized assessments of vaccine-elicited neutralizing antibodies. J. Virol., 88(5), 2489-2507.
  10. Sarzotti-Kelsoe, M., Daniell, X., Todd, C.A., Bilska, M., Martelli, A., LaBranche, C., Perez, L.G., Ochsenbauer, C., Kappes, J.C., Rountree, W., Denny, T.N. and Montefiori, D.C. (2014) Optimization and validation of a neutralizing antibody assay for HIV-1 in A3R5 cells.  J. Immunol. Methods, S0022-1759(14)00072-6.
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  13. Edmonds, T.G., Ding H., Yuan X., Wei Q., Smith K.S., Conway J.A., Wieczorek L., Brown B., Polonis V., West J.T., Montefiori D.C., Kappes J.C., and Ochsenbauer C. (2010)  Replication competent molecular clones of HIV-1 expressing Renilla luciferase facilitate the analysis of antibody inhibition in PBMC. Virol., 408:1-13.
  14. Wagh, K., Seaman, M.S., Zingg, M., Fitzsimmons, T., Barouch, D., Burton, D., Connors, M., Ho, D., Mascola, J., Nussenzweig, M., Ravetch, J., Gautam, R., Martin, M.A., Montefiori, D., and Korber, B. (2018) Potential of conventional and bispecific broadly neutralizing antibodies for prevention of HIV-1 subtype A, C and D infections. PLoS Pathog., 14(3): e1006860.
  15. Bilska, M., Tang, H., and Montefiori, D.C. (2017) Potential risk of replication-competent virus in HIV-1 Env-pseudotyped virus preparation. AIDS Res. Hum. Retroviruses, 33(4): 368-372.
  16. Hraber, P., Rademeyer, C., Williamson, C., Seaman, M.S., Gottardo, R., Tang, H., Greene, K., Gao, H., LaBranche, C., Mascola, J.R., Morris, L., Montefiori, D.C., and Korber, B. (2017) Panels of HIV-1 subtype C Env reference strains for standardized neutralization assessments. J. Virol.,91(19), pii: e00991-17.
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David Charles Montefiori, PhD, Director

Amanda Eaton, MBA, Research Project Manager: