Standardized Assessments of Neutralizing Antibodies for HIV/AIDS Vaccine Development

David C. Montefiori, Duke University Medical Center

Introduction

This site provides detailed information for making standardized assessments of neutralizing antibodies against HIV, SIV and SHIV using molecularly cloned Env-pseudotyped viruses in an optimized and validated luciferase (Luc) reporter gene assay. Access is provided to assay protocols and macros currently in use at the Duke Central Reference Laboratory. This site also contains educational slides, tutorials and links to relevant publications and assay reagents. Special attention is given to HIV-1 reference strains that are available as molecularly cloned Env-pseudotyped viruses.

Assay Protocol and Support Documents:

• Protocol for Neutralizing Antibody Assay for HIV-1 in TZM-bl Cells
• Protocol for Neutralizing Antibody Screening Assay for HIV-1 in TZM-bl Cells
• Protocol for the Preparation and Titration of HIV-1 Env-Pseudotyped Viruses
• Protocol for Heat-Inactivation of Serum and Plasma Samples
• Protocol for the Preparation of Cells for Detection of Mycoplasma Species
• Protocol for Reagent Preparation for Use in the Neutralizing Antibody Assay for HIV-1 in TZM-bl Cells
• Protocol for Thawing Cryopreserved TZM-bl and 293T/17 Cells
• Protocol for Trypsin EDTA Treatment for Disruption of Cell Monolayers
• Protocol for HIV-1 Isolation by PBMC Co-Culture
• Protocol for Preparation of Cell-Free Stocks of TCLA HIV-1 in Cell Lines
• Protocol for Preparation of Cell-Free Stocks of HIV-1 in PBMC
• Protocol for Neutralizing Antibody Assay in PBMC
• Protocol for the Determination of Optimal Concentration of DEAE-Dextran
• Protocol for Neutralizing Antibody Assay Reagent Bridging Studies
• Assay Excel Macros (note: change the filetype to 'xlt' before using):
  • Screening for Neutralization at a Single Sample Dilution
  • Measurement of Neutralization Titers
  • Calculation of TCID50
  • Calculation of TCID-Cutoff
  • User Guide To the Luminescence Macro
  • User Guide to the TCID50 Macro
  • User Guide to the TCID - Cutoff Macro

Key Publications:

• Mascola et al. 2005. Recommendations for the Design and Use of Standard Virus Panels To Assess Neutralizing Antibody Responses Elicited by Candidate Human Immunodeficiency Virus Type 1 Vaccines. J Virol. 2005 Aug;79(16):10103-7. PMID 16051803
• Li et al. 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. Aug;79(16):10108-25. PMID 16051804
• Li et al. 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. PMID 16971434. (Link to PDF.)
• Kulkarni et al. 2009. Highly Complex Neutralization Determinants on a Monophyletic Lineage of Newly Transmitted Subtype C HIV-1 Env Clones from India. Virol., 385(2): 505-20. PMID: 19167740
• Gnanakaran et al. 2010. Genetic Signatures in the Envelope Glycoproteins of HIV-1 that Associate with Broadly Neutralizing Antibodies. PLoS Comput. Biol., 6(10): E1000955. PMID: 20949103
• Seaman et al. 2010. Tiered Categorization of a Diverse Panel of HIV-1 Env Pseudoviruses for Assessment of Neutralizing Antibodies. PMID 19939925. (Link to PDF.)
• Ozaki et al. 2012. International technology transfer of a GCLP-compliant HIV-1 neutralizing antibody assay for human clinical trials. PLoS One, 7(1): e30963. PMID: 22303476
• Todd et al. 2012. Development of implementation of an international proficiency testing program for a neutralizing antibody assay for HIV-1 in TZM-bl cells. J. Immunol. Methods, 375(1-2): 57-67. PMID: 21968254
• Sarzotti-Kelsoe et al. 2014. Optimization and Validation of the TZM-bl Assay for Standardized Assessments of Neutralizing Antibodies Against HIV-1. J. Immunol. Methods, 409: 131-46. PMID: 24291345
• Hraber et al. 2014. Prevalence of Broadly Neutralizing Antibody Responses During Chronic HIV-1 Infection. AIDS, 28(2): 163-9. PMID: 24361678
• deCamp et al. 2014. Global Panel of HIV-1 Env Reference Strains for Standardized Assessments of Vaccine-Elicited Neutralizing Antibodies. J. Virol., 88(5): 2489-507. PMID: 24352443
• Hraber et al. 2014. Impact of Clade, Geography, and Age of the Epidemic on HIV-1 Neutralization by Antibodies. J. Virol., 88(21): 12623-43. PMID: 25142591
• Wagh et al. 2016. Optimal combinations of broadly neutralizing antibodies for prevention and treatment of HIV-1 clade C infection. PLoS Pathog., 12(3):e1005520. PMID: 27028935
• Rademeyer et al. 2016. Features of recently transmitted HIV-1 clade C viruses that impact antibody recognition: Implications for active and passive immunization. PLoS Pathog, 12(7):e1005742. PMID: 28945784
• Hraber et al. 2017. Panels of HIV-1 subtype C Env reference strains for standardized neutralization assessments. J. Virol., 91(19), pii: e00991-17. PMID: 28747500

Reference Reagents:

• Standard Reference Panel of Subtype B HIV-1 Env Clones (NIH AIDS Research and Reference Reagent Program)
• Subtype C HIV-1 Reference Panel of Env Clones (NIH AIDS Research and Reference Reagent Program)
• Standard Reference Panel of Indian Subtype C Panel of HIV-1 Env Clones (NIH AIDS Research and Reference Reagent Program)
• Global Reference Panel of HIV-1 Env Strains (NIH AIDS Research and Reference Reagent Program)
• Notes on the sequence alignment

Please send questions, comments and suggestions to Kelli Greene at: kelli.greene@duke.edu
We thank the Bill & Melinda Gates Foundation and their support of the Collaboration for AIDS Vaccine Discovery (CAVD) - a component of the Global HIV Vaccine Enterprise.
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