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

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. Stages of HIV-1 Entry as Targets for Neutralization
T cell
gp120
gp41
CCR5
CD4
Separate components of fusion
Fusion-competent intermediate
Virus-cell fusion
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
1 hr
days
Virus + Ab
Add cells
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. SEQUENTIAL EVENTS IN DETECTING NEUTRALIZATION OF ENV-PSEUDOTYPED VIRUSES IN TZM-BL CELLS
Lights “ON”
Tzm-bl cell
Infection
Molecular cloning
pEnv
DNA
LUC
pHIVEnv
DNA +
Transfection
Pseudovirus
293T cell

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

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 QH
IgG1b12 - circle
2G12 - triangle
2F5 - square
200 TCID50
10,000 cells/well
30 g/ml DEAE dextran
RLU measured after 48 hrs
% Reduction in RLU
Control RLU = 197,433
Background RLU = 1,029
Range = 196,404 RLU
Concentration (g/ml)

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

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 = % of mean titers

19. Intra-Laboratory Variability in the TZM-bl Assay: Results of 3 independent operatorsQH AC
PVO.4 -
WITO.33 -
THRO.18 -
CAAN.A2 -
IgG1b12
Pool C
2F5
4E10 QH AC
PVO.4 -
WITO.33 -
THRO.18 -
CAAN.A2 - QH AC
PVO.4 -
WITO.33 -
THRO.18 -
CAAN.A2 - QH AC
PVO.4 -
WITO.33 -
THRO.18 -
CAAN.A2 -
TriMab
Pool B
2G12
Neg. Serum QH AC
PVO.4 -
WITO.33 -
THRO.18 -
CAAN.A2 - QH AC
PVO.4 -
WITO.33 -
THRO.18 -
CAAN.A2 - QH AC
PVO.4 -
WITO.33 -
THRO.18 -
CAAN.A2 - QH AC
PVO.4 -
WITO.33 -
THRO.18 -
CAAN.A2 -
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 Antibody neutralization and escape. Nature 422:
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,
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:
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:
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)