Post on 23-Dec-2015
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
O.3
3 -
TH
RO
.18
-
CA
AN
.A2
-
IgG1b12
10
100
1000
10000
QH
0692
.42
-
AC
10.0
.29
-
PV
O.4
-
WIT
O.3
3 -
TH
RO
.18
-
CA
AN
.A2
-
Pool C
10
100
1000
10000
QH
0692
.42
-
AC
10.0
.29
-
PV
O.4
-
WIT
O.3
3 -
TH
RO
.18
-
CA
AN
.A2
-
2F5
10
100
1000
10000
QH
0692
.42
-
AC
10.0
.29
-
PV
O.4
-
WIT
O.3
3 -
TH
RO
.18
-
CA
AN
.A2
-
4E10
10
100
1000
10000
QH
0692
.42
-
AC
10.0
.29
-
PV
O.4
-
WIT
O.3
3 -
TH
RO
.18
-
CA
AN
.A2
-
TriMab
10
100
1000
10000
QH
0692
.42
-
AC
10.0
.29
-
PV
O.4
-
WIT
O.3
3 -
TH
RO
.18
-
CA
AN
.A2
-
Pool B
10
100
1000
10000
QH
0692
.42
-
AC
10.0
.29
-
PV
O.4
-
WIT
O.3
3 -
TH
RO
.18
-
CA
AN
.A2
-
2G12
Intra-Laboratory Variability in the TZM-bl Assay: Results of 3 independent operators
10
100
1000
10000
QH
0692
.42
-
AC
10.0
.29
-
PV
O.4
-
WIT
O.3
3 -
TH
RO
.18
-
CA
AN
.A2
-
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)