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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@duke.edu.

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slide1

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

slide2

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.
slide3

Key Parameters of the Neutralizing Antibody Response to Monitor

  • Magnitude
  • Breadth
  • Duration
  • Kinetics
  • Epitope specificity
  • Escape
  • Systemic & mucosal
  • Correlate of immunity
slide4

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

slide5

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
slide6

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
slide7

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)
slide8

Latest Technology

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

slide9

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
slide10

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

slide11

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

slide12

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
slide13

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
slide14

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

slide15

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)

slide16

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

slide17

Examples of Intra-Assay Variation:

Comparison of Two Luciferase Kits (PerkinElmer vs Promega)

SF162.LS

slide18

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
slide19

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

slide20

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
slide21

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.

slide22

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)