CDER Meeting: Surrogate Markers of Immunity Presenter: Judith A. Britz, Ph.D. October 16, 2000.
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Transcript of CDER Meeting: Surrogate Markers of Immunity Presenter: Judith A. Britz, Ph.D. October 16, 2000.
CDER Meeting: Surrogate Markers of Immunity
Presenter: Judith A. Britz, Ph.D.
October 16, 2000
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
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
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
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
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
0
500
1000
1500
2000
2500
3000
0 0.3 0.6 1.2 2.5 5 10 20 40microgram PHA-L/mL
0
10
20
30
40
50
60
70
80
90
100
LPA in vitro CMI
DPM x 100LPA assay 96 HRS ATP ng/mL
In vitro CMI Overnight
Comparison with LPA
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
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.
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.
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
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
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
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
Analytical Parameters• Accuracy• Precision• Sensitivity• Specificity• Reproducibility• Stability• Standardization
AIDS Patient Management
CMI
Viral Load
CD4
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