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