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

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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_at_duke.edu
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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.

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Key Parameters of the Neutralizing Antibody
Response to Monitor

• Magnitude
• Breadth
• Duration
• Kinetics
• Epitope specificity
• Escape
• Systemic mucosal
• Correlate of immunity

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Stages of HIV-1 Entry as Targets for
Neutralization
NAbs are entry inhibitors
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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

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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

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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)

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

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SEQUENTIAL EVENTS IN DETECTING NEUTRALIZATION OF
ENV-PSEUDOTYPED VIRUSES IN TZM-BL CELLS
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SEQUENTIAL EVENTS IN DETECTING NEUTRALIZATION OF
ENV-PSEUDOTYPED VIRUSES IN TZM-BL CELLS
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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

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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

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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
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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)
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Examples of Inter-Assay and Inter-Operator
Variability in the TZM-bl Assay Neutralizing
Activity of TriMab
Three operators HG, NH and BW
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Examples of Intra-Assay Variation Comparison of
Two Luciferase Kits (PerkinElmer vs Promega)
SF162.LS
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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

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

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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 422307-312. 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,
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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
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K.M. Greene, M. Bilska, D.L. Kothe, J.F.
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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.,
7910108-10125. Li, M,. J.F. Salazar-Gonzalez,
C.A. Derdeyn, L. Morris, C. Williamson, J.E.
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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.
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• Dr. Montefioris 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)