E992750C 1 Replacement of HIV-1 p24 Antigen Screening with HIV-1 RNA Nucleic Acid Testing (NAT) for Whole Blood Donations S.L. Stramer, R.A. Porter, J.P. Brodsky, K.J. Waldman, R.Y. Dodd, T.A. Armstrong and G.D. Griffin

E992750C 2 Background uSince the implementation of p24 HIV-1 antigen (Ag) screening in March, 1996, only 5 Ag positive window-case donations have been identified uDue to low Ag yield and the improved sensitivity of NAT screening for HIV-1, replacement of Ag with HIV-1 NAT should be possible

E992750C 3 HIV-1 p24 Antigen Testing Experience During 2 years of testing 14 million donations (ARC), 136 samples were HIV-1 p24 Ag confirmed positive u58 were false positive (based on RNA negativity, lack of seroconversion, and nonreproducible Ag neutralization) = 1:241,379 u74 were HIV Ab positive u4 were recently infected seroconverting donors uAs of November 1999, 5 seroconverting donors identified in 23 million donations –1:4.6 million (ARC) –1:9.2 million (US)

E992750C 4 Samples for Testing up24 Ag and HIV NAT reactive yield samples (N=4) –Tested undiluted and diluted 1:128 in RNA-negative defibrinated plasma uCommercial seroconversion samples (N=25) –Tested undiluted and diluted 1:128 uDiluted p24 Ag “External Control” sample that is tested on all NAT runs (N=437) –3 Ag+/Ab– units (S/CO 1-2 Coulter) pooled –Pool diluted 1:16

E992750C 5 Comparison of HIV-1 p24 Ag Positive Window-Case U.S. Blood Donors 1Index6 x 10 5 *14.0* x POS – minus p x POS – all bands 2Index2 x 10 6 *28.5* x POS – minus p17, p x POS – all bands 3Index1 x x 10 5 *26.5*908.8POS – minus p31 4Index1 x x 10 6 *17.5*892.7IND – p x POS – all bands 5Index3 x * x 10 5 *0.24.3POS – minus p x POS – minus p x POS – minus p x POS – minus p31 SC Sample Collection (days) WB RNA Copies/mL p24 Ag S/CO Percent Neut. HIV-1/ HIV-2 Ab S/CO

E992750C 6 Detection of HIV-1 p24 Ag and NAT Yield Samples by HIV-1 NAT (1:128 Dilution) ARC 2Ag6x10 5 +(16.08) ARC 3Ag1x10 5 +(16.06) ARC 5Ag3x10 5 +(29.59) MilwaukeeNAT2x10 4 +(7.86) Case Index Detected By Index RNA Conc. Undiluted (copies/mL) Multiplex 1:128* (S/CO) * Diluted in RNA screened negative defibrinated plasma

E992750C 7 HIV Panel 6240 – Virologic/Serologic Profile Days S/CO HIV PCR Quantitation PCR 5 Days 2 Days

HIV Panel 6240 – Virologic/Serologic Profile Days S/CO HIV PCR Quantitation 2 Days 1:128 7 Days Neat E992750C 8 PCR 5 Days

PCR Days S/CO HIV PCR Quantitation HIV Panel PRB932 – Virologic/Serologic Profile Cutoff E992750C 9

PCR Days S/CO HIV PCR Quantitation HIV Panel PRB932 – Virologic/Serologic Profile Cutoff E992750C Days

HIV Panel PRB943 – Virologic/Serologic Profile Days S/CO HIV PCR Quantitation PCR Cutoff E992750C Days 2 Days

PCR HIV Panel PRB943 – Virologic/Serologic Profile Days S/CO HIV PCR Quantitation Cutoff E992750C 12 5 Days 7 Days 2 Days

E992750C 13 Clinical Sensitivity, HIV Panels, Gen-Probe Multiplex Assay S/CO (Neat) p24 Antigen S/CO Cutoff Lot HIV Seronegative Bleeds (92 samples from 25 individuals) 29 NAT +/p24 Ag –

E992750C 14 Cutoff Clinical Sensitivity, HIV Panels, Gen-Probe Lot HIV Seronegative Bleeds (92 samples from 25 individuals) Multiplex Assay S/CO (1:128) p24 Antigen S/CO 21 NAT +/p24 Ag – 1:128

E992750C 15 Effect of Dilution on NAT Reactivity (Gen-Probe HIV-1/HCV Assay) Lot 355 HIV-1 (N=25 Panels) Lot 356 HCV (N=22 Panels) Lot 355 Lot 356 Undiluted Diluted 1: (90%)148 (89%)151 (93%) 151 (94%) p24 Ag82 (57%)82 (55%)—— ALT (  60)——50 (44%)*49 (43%)** * Of 114 tested; ** Of 113 tested * Of 114 tested; ** Of 113 tested

E992750C 16 Gen-Probe Linked Study – Phase I External Control Panel Member HIV-1300 ± 150 HCV 300 ± 150 NEG— Weak HIV-1 p24 Ag (S/CO 1-2 Coulter diluted 1:16)6,800 Target Copies/mL

Gen-Probe Linked Study – Phase I External Control Distributions Frequency S/CO ControlN Mean S/CO Range HIV *8.9 –37.6 HCV *2.1 –10.4 NEG –0.9 HIV-1/p24 Ag *12.1 –25.2 *p<0.05 (437 runs total) 0 E992750C 17

E992750C 18 Conclusions  High RNA titers (  10 5 copies/mL) correspond with p24 Ag positivity uNo p24 Ag reactive sample was found NAT nonreactive even using a pool size of 128 uNAT, even using pools of 128, is more sensitive than screening with the currently licensed tests for HIV-1 p24 Ag. According to these data, the Ag test could be replaced by the use of licensed pooled NAT

E992750C 19 Linear Relationship Between HIV RNA Levels and p24 S/CO Ratios 4.03 log ( ,108 copies/mL) = 1

E992750C Log HIV RNA Level [copies/mL] Log p24 S/CO N = % Prediction Limits for Relationship of HIV RNA Levels and p24 S/CO Ratios

E992750C Log HIV RNA Level [copies/mL] RNA Only (N=61)p24 Pos (N=85) P = <0.0001* * Mann-Whitney Test HIV-1 RNA Levels During “RNA Only” and “p24 Positive” Window Periods

E992750C Log RNA Level [copies/mL] Log p24 S/CO Linear Relationship Between HIV-1 RNA Levels and p24 S/CO Ratios in 32 Cases