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: 1892
Dolan et al. 1995. J. Infect.

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:
1447
Rosenberg, E.S. et al. 2000. Nature 408:

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

4 hours to overnight

Wash

Incubate

Lymphocyte
Stimulation

ATP

ATP

ATP

ATP detection
reagents

Cell lysis to release ATP

Magnetic separation
of CD4 cells.
Luminometer

Measure light intensity

Comparison with LPA

3000

100
90

2500

80
70

ATP ng/mL
In vitro CMI Overnight

2000

DPM x 100
LPA assay 96 HRS

60

1500

50
40

1000

30
20

500

10

0
0

0.3 0.6 1.2 2.5 5 10 20

m icrogram PHA-L/m L

LPA

40

in vitro CMI

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

900
Normal

800

ATP (ng/mL)

700
600
500

481

400
300
200
140

100
0

Normal

HIV+

Homi Farzadegan. Johns Hopkins. 2000. IHV Meeting.

HIV+

Measurement Of Cell Activation By Mitogens And Recall Antigens

Therapy Change, August
35.0

Therapy Changed
30.0

4
20.0
3
15.0
2
10.0
1

5.0

0.0

0
9-Jul

24-Sep

11-Feb

Date
PHA SI

CD4

log VL

B.J. Loechelt, M. Chan Childrens National Medical Center, Washington, D.C; 2000.
Manuscript in Preparation.

Log VL, CD4/ uL*10-2

Stimulation Index

25.0

Measurement Of Cell Activation By Mitogens And Recall Antigens

30.0

25.0

20.0

15.0

10.0

5.0

0.0

0
6-Aug

5-Nov

21-Jan

Date
PHA SI

CD4

log VL

B.J. Loechelt, M. Chan Childrens National Medical Center, Washington, D.C; 2000.
Manuscript in preparation

Log VL,CD4/ uL*10-2

Stimulation Index

Non-Compliant Patient

COMPARISON OF RESPONDERS AND NON RESPONDERS

TO P24 ANTIGEN IN HIV INFECTION
COMPARED TO CONTROLS
120

p<0.01

100

A
T
P

p24Control

80

p24 Antigen

60
40
20
0

HIVControls

HIV+
Non Responders

HIV+
Responders

Cunningham-Rundles, S. and J. Cervia. Cornell

University School of Medicine. 2000. Manuscript in
preparation.

ATP(ng/mL)

ATP Response in gp160 Vaccinated HIV+ Patients

80
70
60
50
40
30
20
10
0

Non-Stim
gp 160
p24
CMV

4058

253

3484

HIV+ Patient

Wahren, B. et al. Karolinska Institute.

2000. IHV Meeting

Lymphocyte Response
Cylex
In Vitro CMI

Cytokine
Assays

Lymphoproliferati
on

ATP
1/2 - 6 hours

Lymphocyte
Stimulation

6 - 24 hours
Influx of ions
Surface receptor clustering
RNA synthesis
Cytokine production & release
DNA replication

3 7 days
Clonal Expansion
Proliferation

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