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 J. Clin. Invest. 84: 1892 Dolan et al 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 Science 278: 1447 Rosenberg, E.S. et al 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 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
ATP ATP ATP Cell lysis to release ATP ATP detection reagents Luminometer Measure light intensity Lymphocyte Stimulation Wash Incubate Magnetic separation of CD4 cells. 4 hours to overnight
DPM x 100 LPA 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 Measurement of T-lymphocyte responses in whole blood cultured using newly synthesized DNA and ATP. Clinical and Diagnostic Laboratory Immunology 7(2):
PHA Distribution of Normal and HIV+ Patients NormalHIV+ Homi Farzadegan. Johns Hopkins IHV Meeting.
Measurement Of Cell Activation By Mitogens And Recall Antigens Therapy Changed B.J. Loechelt, M. Chan Children’s National Medical Center, Washington, D.C; Manuscript in Preparation.
Measurement Of Cell Activation By Mitogens And Recall Antigens B.J. Loechelt, M. Chan Children’s National Medical Center, Washington, D.C; Manuscript in preparation
COMPARISON OF RESPONDERS AND NON RESPONDERS TO P24 ANTIGEN IN HIV INFECTION COMPARED TO CONTROLS HIV- Controls HIV+ Non Responders HIV+ Responders p<0.01 Cunningham-Rundles, S. and J. Cervia. Cornell University School of Medicine Manuscript in preparation. ATPATP
Wahren, B. et al. Karolinska Institute IHV Meeting
Lymphocyte Stimulation hours Influx of ions Surface receptor clustering RNA synthesis Cytokine production & release DNA replication 3 – 7 days Clonal Expansion Proliferation ATP 1/2 - 6 hours Lymphocyte Response Cylex In Vitro CMI Cytokine Assays Lympho- proliferation
Analytical Parameters Accuracy Precision Sensitivity Specificity Reproducibility Stability Standardization
AIDS Patient Management
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