Download - CDER Meeting: Surrogate Markers of Immunity Presenter: Judith A. Britz, Ph.D. October 16, 2000
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CDER Meeting: Surrogate Markers of Immunity
Presenter: Judith A. Britz, Ph.D.
October 16, 2000
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HIV Pathogenesis • Cellular Immune Function Tests
have Prognostic Value:– independent of CD4+ Count– Up to 1 year before CD4 Count Decline
• Progressive Loss of Function as measured by response to:– Mitogens– Alloantigens– Recall Antigens
Clerici et al. 1989. J. Clin. Invest. 84: 1892Dolan et al. 1995. J. Infect. Dis.172:79
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Immune Reconstitution
• LPA Reactivity to HIV p24 rAg enhanced:– Long Term Non-Progressors– Following early treatment of acute
HIV infection– Strengthened by treatment
interruption (in some patients)
Rosenberg, E.S et al. 1997. Science 278: 1447Rosenberg, E.S. et al. 2000. Nature 408: 523
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Lymphoproliferation (LPA)Traditional method for evaluating cell-mediated immune function:
• Time-consuming: 5-7 Days• Radioactive• Labor-intensive• Not widely available• Not standardized• Shipping impact• Not practical for clinical use
Betensky et al. 2000. Clin. Diag. Lab. Immunol. 7(5): 759
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Objectives
• Clinical Correlate of Cell-Mediated Immunity
• 24 Hours or less• Non-Radioactive• Whole Blood or PBMCs• Cost Effective• Adaptable to Test with Multiple
Antigens• Standardized
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ATPATP ATPATPATPATP
Cell lysis to release ATP
ATP detection reagents
Luminometer
Measure light intensity
LymphocyteStimulation
WashIncubate
Magnetic separationof CD4 cells.
4 ho
urs
to o
vern
ight
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0
500
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3000
0 0.3 0.6 1.2 2.5 5 10 20 40microgram PHA-L/mL
0
10
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LPA in vitro CMI
DPM x 100LPA assay 96 HRS ATP ng/mL
In vitro CMI Overnight
Comparison with LPA
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Reference
Sottong, P.R., J.A. Rosebrock, J.A. Britz, and T.R. Kramer. 2000. Measurement of T-lymphocyte responses in whole blood cultured using newly synthesized DNA and ATP. Clinical and Diagnostic Laboratory Immunology 7(2): 307-311
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PHA Distribution of Normal and HIV+ Patients
0
100
200
300
400
500
600
700
800
900
AT
P (
ng/m
L)
Normal HIV+
481
140
Normal HIV+
Homi Farzadegan. Johns Hopkins. 2000. IHV Meeting.
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Therapy Change, August
0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
9-Jul 24-Sep 11-Feb
Date
Sti
mu
lati
on
In
de
x
0
1
2
3
4
5
6
Lo
g V
L,
CD
4/ u
L*1
0-2
PHA SI CD4 log VL
Measurement Of Cell Activation By Mitogens And Recall Antigens
Therapy Changed
B.J. Loechelt, M. Chan Children’s National Medical Center, Washington, D.C; 2000. Manuscript in Preparation.
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Non-Compliant Patient
0.0
5.0
10.0
15.0
20.0
25.0
30.0
6-Aug 5-Nov 21-Jan
Date
Sti
mu
lati
on
In
de
x
0
1
2
3
4
5
6
Lo
g V
L,C
D4/ u
L*1
0-2
PHA SI CD4 log VL
Measurement Of Cell Activation By Mitogens And Recall Antigens
B.J. Loechelt, M. Chan Children’s National Medical Center, Washington, D.C; 2000. Manuscript in preparation
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COMPARISON OF RESPONDERS AND NON RESPONDERS TO P24 ANTIGEN IN HIV INFECTION
COMPARED TO CONTROLS
0
20
40
60
80
100
120
p24Control
p24 Antigen
HIV-Controls
HIV+Non Responders
HIV+Responders
p<0.01
Cunningham-Rundles, S. and J. Cervia. Cornell University School of Medicine. 2000. Manuscript in preparation.
ATP
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ATP Response in gp160 Vaccinated HIV+ Patients
0
10
20
3040
50
60
70
80
4058 253 3484
HIV+ Patient
ATP
(ng/
mL)
Non-Stim
gp 160
p24
CMV
Wahren, B. et al. Karolinska Institute. 2000. IHV Meeting
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LymphocyteStimulation
6 - 24 hoursInflux of ions Surface receptor clusteringRNA synthesis Cytokine production & release DNA replication
3 – 7 daysClonal ExpansionProliferation
ATP1/2 - 6 hours
Lymphocyte ResponseCylex In Vitro CMI
Cytokine Assays
Lympho-proliferati
on
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Analytical Parameters• Accuracy• Precision• Sensitivity• Specificity• Reproducibility• Stability• Standardization
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AIDS Patient Management
CMI
Viral Load
CD4
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Acknowledgements
• Susanna Cunnigham-Rundles: Cornell• Brett Loechelt: George Washington• Homi Farzadegan: Johns Hopkins• Britta Wahren: Karolinska• Tim Kramer: USDA• Peter Sottong: Cylex• Richard Kowalski: Cylex• Julie Woodcock: Cylex