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Immunology Core: Neutralizing Antibody Assays
The CFAR NAb Core 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 service.
Brief descriptions of our two validated neutralization assays are given included below. Additional information, including detailed non-document controlled assay protocols, macros, educational materials and related links may be found at:
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 (1) and was later optimized and validated at Duke University (2, 3). 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).
A3R5 Assay Description
Like the TZM-bl assay, this assay also measures neutralization as a function of a reduction in Luc reporter gene expression and has been formally validated (10). A3R5 cells (A3.01/R5.7) were provided by Col. Jerome Kim and Dr. Robert McLinden of the US Medical HIV Research Program (USMHRP). This is a derivative of the human lymphoblastoid cell line, CEM, that naturally expresses CD4 and CXCR4 (11) and was engineered by Dr. Robert McLinden in Col. Kim’s laboratory to express CCR5 (12). The cells are maintained in growth medium containing 1 mg/ml geneticin to preserve this receptor. The cells are moderately permissive to infection by most strains of HIV-1. DEAE-Dextran is used in the medium during neutralization assays to enhance infectivity. Because the cell line does not contain a reporter gene, molecularly cloned viruses that carry a reporter gene in the viral genome must be used. Env-expressing infectious molecular clones carrying a Renilla Luc reporter gene (Env.IMC.LucR viruses) provide suitable infection for neutralization assays (13). Expression of the reporter genes is induced in cis by viral Tat protein soon after infection. DEAE dextran is added to the medium to enhance infection and has been found to have no obvious effects on NAb activity. Luc activity is quantified as 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 (9).
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- Montefiori, D.C. (2004) Evaluating neutralizing antibodies against HIV, SIV and SHIV in luciferase reporter gene assays. Current Protocols in Immunology (Coligan, J.E., A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W. Strober, and R. Coico, eds.), John Wiley & Sons, 12.11.1-12.11.15.
- Sarzotti-Kelsoe, M., Bailer, R.T., Turk, E., Lin, C.L., Bilska, M., Greene, K.M., Gao, H., Todd, C.A., Ozaki, D.A., Seaman, M.S., Mascola, J.R. and Montefiori, D.C. (2013) Optimization and validation of the TZM-bl assay for standardized assessments of neutralizing antibodies against HIV-1. J. Immunol. Methods, S0022-1759(13)00359-1.
- Platt, E. J., Wehrly, K., Kuhmann, S. E., Chesebro, B. and Kabat, D. (1998) Effects of CCR5 and CD4 cell surface concentrations on infection by macrophage tropic isolates of human immunodeficiency virus type 1. J. Virol. 72, 2855-2864.
- Wei, X., Decker, J. M., Liu, H., Zhang, Z., Arani, R. B., Kilby, J. M., Saag, M. S., Wu, X., Shaw, G. M. and Kappes, J.C. (2002) Emergence of resistant human immunodeficiency virus type 1 in patients receiving fusion inhibitor (T-20) monotherapy. Antimicrob. Agents Chemother. 46, 1896-1905.
- Mascola, J. R., D'Souza, P., Gilbert, P., Hahn, B., Haigwood, N. L., Morris, L., Petropoulos, J. C., Polonis, V. R., Sarzotti-Kelsoe, M. and Montefiori, D. C. (2005) Recommendations for the design and use of standard virus panels to assess the neutralizing antibody response elicited by candidate human immunodeficiency virus type 1 vaccines. J. Virol. 79, 10103-10107.
- 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.
- 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.
- 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.
- 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.
- Folks, T., Benn, S., Rabson, A., Theodore, T., Hoggan, M.D., Martin, M., Lightfoote, M., and Sell, K. (1985) Characterization of a continuous T-cell susceptible to the cytopathic effects of the acquired immunodeficiency syndrome (AIDS)-associated retrovirus. Proc. Natl. Acad. Sci., 82, 4539-4543.
- McLinden, R.J., LaBranche, C.C., Chenine, A.L., Polonis, V.R., Eller, M.A., Wieczorek, L., Ochsenbauer, C., Kappes, J.C., Perfetto, S., Montefiori, D.C., Michael, N.L., and Kim, J.H. (2013) Detection of HIV-1 neutralizing antibodies in a human CD4+/CXCR4+/CCR5+ T-lymphoblastoid cell assay system. PLoS One, 8(11), e77756.
- 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.
Core Staff Contact
David Charles Montefiori, PhD