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

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.

Key Parameters of the Neutralizing Antibody Response to Monitor

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

HIV-1

HIV-1

HIV-1

T cellT cell

T cell

gp120

gp41

CCR5

CD4

Separate components of fusion Fusion-competent

intermediate

Virus-cell fusion

Stages of HIV-1 Entry as Targets for Neutralization

NAbs are entry inhibitors

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

Virus + Ab Add cells Measure infection1 hr days

• 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

Various Assays Formats

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)

Latest Technology

Tat-Regulated Reporter Gene Assays in Genetically Engineereed Cell Lines Using

Molecularly Cloned Env-Pseudotyped Viruses

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

Tzm-bl cell

InfectionpHIVEnv DNA

pEnv DNA

293T cell

++++Transfection

Lights “ON”

Molecular cloning

Pseudovirus

LUC

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

Tzm-bl cell

LUC

pHIVEnv DNA

pEnv DNA

293T cell

Molecular cloning

++++Transfection

YY

Y

YY Antibody

Lights “OFF”

Pseudovirus

No infection

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

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

OPTIMIZATION 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

VALIDATION OF THE TZM-BL ASSAY

1,000

10,000

100,000

1,000,000

0 1 100 10000

QH0692.42SS1196.16101.1O

Rel

ativ

e lu

min

esce

nce

un

its

(RL

U)

TCID50 added per well

Env-pseudotyped virus

Linear Range of Infection in TZM-bl Cells

Cell killing at high virus input

0

10

20

30

40

50

60

70

80

90

100

0.01 0.10 1.00 10.00 100.00

% R

edu

ctio

n i

n R

LU

Concentration (g/ml)

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

Control RLU = 197,433

Background RLU = 1,029

Range = 196,404 RLU

Examples of Inter-Assay and Inter-Operator Variability in the TZM-bl Assay: Neutralizing Activity of TriMab

0.001 0.01 0.1 1 10 1000

20

40

60

80

100HG, ID50 = 0.35HG, ID50 = 0.31NH, ID50 = 0.29HG, ID50 = 0.45BW, ID50 = 0.41

TriMab Concentration (g/ml)

3988.25 QH0692.42

SS1196.1

0.01 0.1 1 10 1000

20

40

60

80

100HG, ID50 = 5.0HG, ID50 = 4.7NH, ID50 = 2.9BW , ID50 = 2.9

TriMab Concentration (g/ml)

BG1168.1

% R

edu

ctio

n in

RL

U%

Red

uct

ion

in R

LU

0.001 0.01 0.1 1 10 1000

20

40

60

80

100HG, ID50 = 0.36NH, ID50 = 0.77HG, ID50 = 0.55

TriMab Concentration (g/ml)

0.001 0.01 0.1 1 10 1000

20

40

60

80

100HG, ID50 = 0.55HG, ID50 = 0.43NH, ID50 = 0.60HG, ID50 = 0.54HG, ID50 = 0.43

TriMab Concentration (g/ml)

Three operators: HG, NH and BW

Examples of Intra-Assay Variation:

Comparison of Two Luciferase Kits (PerkinElmer vs Promega)

sCD4

0.001 0.01 0.1 1 10 1000

20

40

60

80

100PerkinElmerPromega

sCD4 (ug/ml)

ID50

(u

g/m

l)

IgG1b12

0.01 0.1 1 10 1000

20

40

60

80

100PerkinElmerPromega

IgG1b12 (ug/ml)

ID50

(u

g/m

l)

2G12

0.01 0.1 1 10 1000

20

40

60

80

100PerkinElmerPromega

2G12 (ug/ml)

ID50

(u

g/m

l)

2F5

0.01 0.1 1 10 1000

20

40

60

80

100PerkinElmerPromega

2F5 (ug/ml)

ID50

(u

g/m

l)4E10

0.01 0.1 1 10 1000

20

40

60

80

100PerkinElmerPromega

4E10 (ug/ml)

ID50

(u

g/m

l)

TriMab

0.001 0.01 0.1 1 10 1000

20

40

60

80

100PerkinElmer

Promega

TriMab (ug/ml)

ID50

(u

g/m

l)

SF162.LS

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

10

100

1000

10000

QH

0692

.42

-

AC

10.0

.29

-

PV

O.4

-

WIT

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3 -

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RO

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-

CA

AN

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-

IgG1b12

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QH

0692

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AN

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Pool C

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0692

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2F5

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4E10

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TriMab

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0692

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Pool B

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2G12

Intra-Laboratory Variability in the TZM-bl Assay: Results of 3 independent operators

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QH

0692

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Neg. Serum

Inside bar = 2-fold variation from mean; Outside bar = 3-fold variation from mean

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

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.

REFERENCES

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)