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Neutralizing Antibody Assays for HIV-1, SIV and SHIV: Recent Advances in Technology

Neutralizing Antibody Assays for HIV-1, SIV and SHIV: Recent Advances in Technology. David C. Montefiori, Ph.D. Laboratory for AIDS Vaccine Research & Development Duke University Medical Center Durham, NC monte@duke.edu.

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Neutralizing Antibody Assays for HIV-1, SIV and SHIV: Recent Advances in Technology

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  1. Neutralizing Antibody Assays for HIV-1, SIV and SHIV: Recent Advances in Technology David C. Montefiori, Ph.D. Laboratory for AIDS Vaccine Research & Development Duke University Medical Center Durham, NC monte@duke.edu

  2. Why Neutralizing Antibodies are Considered Important to HIV/AIDS Vaccines • Pre-existing neutralizing antibodies (active and passive immunization) can prevent AIDS virus infection through intravenous, vaginal, rectal and oral routes of challenge in nonhuman primates. • A rapid secondary responses to infection that is primed by prior vaccination might control virus replication, prevent early immunologic damage, prolong survival and reduce the probability of transmitting virus.

  3. Key Parameters of the Neutralizing Antibody Response to Monitor • Magnitude • Breadth • Duration • Kinetics • Epitope specificity • Escape • Systemic & mucosal • Correlate of immunity

  4. Separate components of fusion Fusion-competent intermediate T cell Virus-cell fusion T cell CCR5 CD4 T cell HIV-1 gp120 HIV-1 gp41 HIV-1 Stages of HIV-1 Entry as Targets for Neutralization NAbs are entry inhibitors

  5. Assay Requirements • Sensitive, quantitative, reproducible, high throughput and have correlative value • Optimized and validated to meet GCLP requirements for human clinical trials • Reagents need to be standardized and traceable • Assay needs to be transferable to multiple labs

  6. Various Assays Formats days 1 hr Add cells Virus + Ab Measure infection • TCLA • Primary isolates • TCLA and primary isolates • CD4+ cell lines • PBMC • Genetically engineered cell lines expressing HIV entry receptors and containing reporter genes • Syncytia • Cell-killing • Plaques • Gag Ag ELISA or FACS • RT activity • luciferase • green fluorescence protein • secreted alk. phosphatase • B-gal

  7. PBMC Assay • Advantages: • Gold standard for many years • Broadly susceptible to infection by primary isolates • Correlative value in passive Ab studies • Disadvantages: • Time consuming and labor intensive • Expensive • Lacks precision • Difficult to validate (e.g., PBMC from different donors, mixed cell population, viral quasispecies)

  8. Latest Technology Tat-Regulated Reporter Gene Assays in Genetically Engineereed Cell Lines Using Molecularly Cloned Env-Pseudotyped Viruses

  9. Luciferase Reporter Gene Assay in TZM-bl Cells Based on Single-Round Infection with Molecularly Cloned Env-Pseudotyped Viruses • TZM-bl (JC53-bl) is a genetically engineered HeLa cell line that expresses CD4, CXCR4 and CCR5 and contains Tat-inducible Luc and -Gal reporter genes: • High success rate in single-round infections • Increased assay capacity (2-day assay) • Increased precision (accurately measure 50% neutralization) • Improved level of standardization (stable cell line) • Optimized and validated

  10. Lights “ON” Molecular cloning LUC Tzm-bl cell + pEnv DNA pHIVEnv DNA Infection Transfection 293T cell Pseudovirus SEQUENTIAL EVENTS IN DETECTING NEUTRALIZATION OF ENV-PSEUDOTYPED VIRUSES IN TZM-BL CELLS

  11. Lights “OFF” Molecular cloning Tzm-bl cell + pEnv DNA No infection pHIVEnv DNA LUC Y Y Y Antibody Y Y Transfection 293T cell Pseudovirus SEQUENTIAL EVENTS IN DETECTING NEUTRALIZATION OF ENV-PSEUDOTYPED VIRUSES IN TZM-BL CELLS

  12. OPTIMIZATION OF THE TZM-BL ASSAY • Cell culture conditions • Range of isolates that infect adequately • Cell number • Virus dose • Incubation time • Choice of 96-well plates for luminescence • Luminescence readings • DEAE-dextran • Indinavir • Uncloned vs cloned virus

  13. VALIDATION OF THE TZM-BL ASSAY • Specificity: • Background activity of normal human serum and plasma • Accuracy: • Comparisons have been made to other in-house assays and assays performed in other labs • Precision: • Well-to-well variability in cell control, virus control and test wells • Intra- and inter-assay variability • Intra- and inter-operator variability • Limits of Quantitation: • Upper and lower limits established • Linearity & Range: • Neutralization curves generated with positive serum samples and mAbs show a consistent pattern of linearity over a range of 20-85% reductions in RLU. Values in this range are directly proportional to the concentration of neutralizing antibodies in the sample. • Ruggedness & Robustness: • Stability of CD4, CCR5 and CXCR4 expression • Stability of TZM-bl infectivity after multiple passages • Effect of DEAE-dextran on neutralizing antibody activity • Effect of heat-inactivation on neutralizing antibody activity • Serum vs plasma • Uniformity of multiple luminometers

  14. Linear Range of Infection in TZM-bl Cells Cell killing at high virus input Env-pseudotyped virus Relative luminescence units (RLU) TCID50 added per well

  15. Neutralization Curves Under Optimal TZM-bl Assay Conditions Env-pseudotyped virus QH0692.42 • 200 TCID50 • 10,000 cells/well • 30 g/ml DEAE dextran • RLU measured after 48 hrs IgG1b12 - circle 2G12 - triangle 2F5 - square % Reduction in RLU Control RLU = 197,433 Background RLU = 1,029 Range = 196,404 RLU Concentration (g/ml)

  16. 3988.25 QH0692.42 % Reduction in RLU SS1196.1 BG1168.1 % Reduction in RLU Examples of Inter-Assay and Inter-Operator Variability in the TZM-bl Assay: Neutralizing Activity of TriMab Three operators: HG, NH and BW

  17. Examples of Intra-Assay Variation: Comparison of Two Luciferase Kits (PerkinElmer vs Promega) SF162.LS

  18. Internal Proficiency Test with an External Panel of Reagents • Six operators assayed 7 positive serologic reagents against 6 reference strains of Env-pseudotyped HIV-1 in TZM-bl cells (SOP HVTN02-A0009): • Mean variance = 32  16% of mean titers • Range = 10 - 79% of mean titers

  19. IgG1b12 PVO.4 - AC10.0.29 - WITO.33 - THRO.18 - CAAN.A2 - QH0692.42 - Intra-Laboratory Variability in the TZM-bl Assay: Results of 3 independent operators Pool C 2F5 4E10 PVO.4 - PVO.4 - PVO.4 - WITO.33 - THRO.18 - WITO.33 - THRO.18 - WITO.33 - THRO.18 - AC10.0.29 - CAAN.A2 - AC10.0.29 - CAAN.A2 - AC10.0.29 - CAAN.A2 - QH0692.42 - QH0692.42 - QH0692.42 - 2G12 TriMab Pool B Neg. Serum PVO.4 - WITO.33 - THRO.18 - AC10.0.29 - CAAN.A2 - PVO.4 - PVO.4 - QH0692.42 - AC10.0.29 - CAAN.A2 - WITO.33 - THRO.18 - WITO.33 - THRO.18 - AC10.0.29 - CAAN.A2 - PVO.4 - QH0692.42 - QH0692.42 - AC10.0.29 - CAAN.A2 - WITO.33 - THRO.18 - QH0692.42 - Inside bar = 2-fold variation from mean; Outside bar = 3-fold variation from mean

  20. Program of External Proficiency Testing for the TZM-bl Neutralizing Antibody Assay • Initial round of testing • Assess inter-laboratory variation under conditions of relaxed standardization • Subsequent rounds of testing • Confirm the key parameters that affect assay performance • Revise and validate the assay SOP • Develop an SOP for proficiency testing • Validate the proficiency testing SOP

  21. REFERENCES Wei, X., J. M. Decker, S. Wang, H. Hui, J. C. Kappes, X. Wu, J. F. Salazar-Gonzalez, M. G. Salazar, J. M. Kilby, M. S. Saag, N. L. Komarova, M. A. Nowak, B. H. Hahn, P. D. Kwong, and G. M. Shaw. 2003. Antibody neutralization and escape. Nature 422:307-312. 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. Mascola, J. R., P. D'Souza, P. Gilbert, B. Hahn, N. L. Haigwood, L. Morris, C. J. Petropoulos, V. R. Polonis, M. Sarzotti-Kelsoe, and D. C. Montefiori. (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., F. Gao, J.R. Mascola, L. Stamatatos, V.R. Polonis, M. Koutsoukos, G. Voss, P. Goepfert, P. Gilbert, K.M. Greene, M. Bilska, D.L. Kothe, J.F. Salazar-Gonzalez, X. Wei, J.M. Decker, B.H. Hahn, and D.C. Montefiori. (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,. J.F. Salazar-Gonzalez, C.A. Derdeyn, L. Morris, C. Williamson, J.E. Robinson, J.M. Decker, Y. Li, M.G. Salazar,V.R. Polonis, K. Mlisana, S.A. Karim, K. Hong, K.M. Greene, M. Bilska, J.T. Zhou, S. Allen, E. Chomba, J. Mulenga, C. Vwalika, F. Gao, M. Zhang, B.T.M. Korber, E. Hunter, B.H. Hahn, and D.C. Montefiori. (2006) Genetic and neutralization properties of acute and early subtype C human immunodeficiency virus type 1 molecular env clones from heterosexually acquired infections in southern Africa. J. Virol., in press.

  22. Dr. Montefiori’s laboratory is funded by: • Division of AIDS/NIAID/NIH: • Primate Core Immunology Laboratory for AIDS Vaccine Research and Development (PCIL) • HIV Vaccine Trials Network (HVTN) • Center for HIV/AIDS Vaccine Immunology (CHAVI) • Bill & Melinda Gates Foundation: • Collaboration for AIDS Vaccine Discovery (CAVD)

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