1. Nucleic Acid Testing vs Infectivity Michael P. Busch, M.D., Ph.D. Blood Centers of the Pacific Blood Systems, Inc. EPFA, Lisbon Portugal May, 2001

2. NAT vs Infectivity Overview of stages of infections and importance of understanding stage-specific gEq:infectivity ratios (concentration vs volume infused) Review of NA dynamics (pre/peri/post-SC) for HIV, HCV and HBV Review infectivity data for each stage Need for future studies animal transmission models lookback studies (NAT/SC donors; recipient cases)

3. Stages of TTVIs and infectivity Pre-ramp-up viremia Ramp-up viremia Plateau phase / peak viremia Peri-SC viremia Post-SC viremia –Persistent carrier (viral load set point) –“Resolved” infection? Immunosilent carrier Transient Viremia w/o SC

4. Stages of TTVIs and infectivity Pre-ramp-up Viremia –Low-level, intermittent “blips” of RNA/DNA detected prior to quantifiable ramp-up phase –Innoculum vs focal replication that seeds dissemination vs abortive replication –Can this occur transiently w/o subsequent ramp-up/SC? –Is this viremia infectious? –Kinetic, viral sequencing & infectivity studies in progress

5. Testing Algorithm for HCV NAT-conversion Panels Alpha Therapeutic / NGI screening (Antibody & 512 Pool PCR) 63 HCV NAT-conversion Panels ( 774 Donations ) 5 Control Panels ( 54 Donations ) dHCV TMA Testing Quantitative Viral Load ( Roche Monitor ) Intermittent Pre-Ramp-Up Viremia * in 25/41 (61%) panels >3 pre-r/u units Ramp-Up & Plateau Phase Replicate dTMA ( x 3 ) & NGI UltraQual PCR Testing * Ramp-Up = consistent RNA detection with progressive increase in viral load.

6. Low-Level Intermittent HCV Viremia Preceding Ramp-Up Phase Assay sensitivity (6 x 10 2 ) -60 -40-20 0 10 20 % dTMA Positive (4 replicates) Days Pre/Post 1st Quantitative RNA+ Donation x10 3 x10 2 x10 4 x10 6 x10 5 x10 8 HCV Monitor PCR (gEq/mL) 100 50 75 25 NGI 512 PCR Neat PCR x10 7 (BCP ID 10083)

7. Representative HIV Conversion Panels with Pre-Ramp-Up “Blip” Viremia (RNA “blip” observed in 7/19 informative panels) Days from First MP-PCR+ Test p24 AgHIV-1 Ab HIV-1 RNA (copies / mL) # Pos / # Replicate PCR - 21Neg < 1001 / 10 - 19Neg < 1007 / 8 - 14Neg < 1000 / 8 - 11Neg < 1000 / 8 - 7Neg < 1000 / 8 - 4Neg < 1003 / 8 0Neg 2605 / 5 3Neg 27,000 7PosNeg370,000 9PosNeg2,800,000 16PosREACT410,000 Alpha / BCP Case 1012

8. 10 100 1,000 10,000 100,000 1,000,000 -37-35-29-26-21-18-11-8038 0.00 20.00 40.00 60.00 80.00 100.00 120.00 140.00 HBV DNA Load (gEq/mL) Representative HBV Panel with Pre-Ramp-Up Viremia (pre- ramp-up viremia observed in 12/23 informative panels) HBsAg S/C Day from 1st + HBsAg test Pre-Ramp-Up Ramp-Up + -

9. Stages of TTVIs and Infectivity Ramp-up viremia –Persistent viremia with progressive increase in viral load leading to peak or plateau viremia –Doubling time used to project WP differences of ID-NAT, MP-NAT and Ag assays –Presumed infectious (chimp study in progress)

10. HCV RNA Production Rate and Linear Regression Model during Ramp-up Viremia -2 0 2 4 6 8 10 12 -20-15-10-505101520 Day from first HCV RNA positive sample N = 101 samples from 37 donors Log HCV RNA [gEq/mL] DT = 14.9 hrs (95% CI: 12.9-17.8)

11. Individual Rates and Linear Regression Model of HIV RNA Production during Early Infection. LOG HIV RNA [gEq/mL] 1 2 3 4 5 6 7 8 9 -10-505101520 Day N = 97 Samples from 44 Plasma donors DT: 21.5 hrs (95% CI: 19.2-24.6)

12. Individual Rates and Linear Regression Model of HBV DNA Production during Early HBV Infection 0 1 2 3 4 5 6 7 8 -60-50-40-30-20-100102030 Day from first HBsAg positive sample Log HBV DNA [gEq/mL] N = 70 samples from 21donors DT: 2.6 days (95% CI: 2.6-3.2)

13. Window Period Viremia Parameters Derived from NAT Analysis of Plasma Donor Panels * Based on follow-up of 30 plasma donors detected as MP- NAT-pos by Alpha and Bayer (95% CI 29-85 days)

14. Projections of HCV Window Period Reductions by MP / ID NAT Using the Linear Regression Model of HCV Production -2 0 2 4 6 8 10 12 -20-15-10-505101520 Day from first HCV RNA positive sample Log HCV RNA [gEq/mL] MP NAT [1,000 gEq/mL] ID NAT [50 gEq/mL] a b c d HCV RNA

15. Incremental WP Closure and Projected Yield (U.S.) of ID over MP-NAT Screening Assumes sensitivity of ID NAT = 50 gEq / mL, and MP NAT = 1000 gEq / mL Does not include detection by ID-NAT of intermittent, low-level viremic episodes preceding ramp-up viremia

16. Detection of HIV in antibody negative window phase geq/ml Days infectious window NucliSens-Ampliscreen 1:48 TMA single TMA 1:16

17. Relationship of Infectivity & Viral Load pre-SC Animal Models Analyses of Plasma Stocks with Defined CID 50. 1.Correlate limit of detection of NAT assays to 1 CID 50. 2.Characterize viral load at several CID dilutions. Calculate viral load : CID 50 ratio. 3.For HBV/HCV ~10 gEq/CID 50. Primary Infection Transmission Studies (Alter et al. Transfusion 1999) 1Infect 1 O animals, draw samples through SC. 2Characterize serial samples by NAT, Ag, Ab assays. 3Serially transfuse eclipse and NAT(+) blood into 2 O animals. 4Correlate detection of viremia with development of infectivity. Transfuse Human WP Samples into Test Animal 1Plasma donor SC panels. 2Pre-SC units from blood donors with documented transmission.

18. Infectivity Early in HCV Infection Assessed by Transmission to Chimpanzees Phase I (12 Weeks) Phase II (12 Weeks) Phase III (?) (12 Weeks) Phase IV (Control) CHIMP I Select 10 Donors, time “A” 50 mL each 250 mL / Day X2 Same 10 Donsrs, Time “C” Same volume Same 10 Donors, Time “B” Same volume Same 10 Donsrs, Time “D” Same volume CHIMP II 10 Different Donors with no “B” peak at time “C” Same 10 donors at time “D” HCV RNA Level AC B D3 D2D2 D1D1 Time

19. Relationship of Infectivity & Viral Load pre-SC Human Transmission Data 1.Investigate Recipients of Prior Donations by NAT / Ab SC Donors (Donor directed lookback) Model Duration of "Infectious WP" Based on Rate of Recipient Infection Relative to ID Intervals Petersen, et al. Transfusion 1993;33:552-7 For Cases with Documented Transmission and Available Plasma, Characterize Viral Load and Detection by MP/SD NAT Tests Sch  ttler, et al. Lancet 2000 Robbins, et al. JAMA 2000 Roth, et al. submitted Transfuse human infectious plasma with low viral load into animals to validate sensitivity of the models

20. HIV Test Results for Donor, Recipients in Singapore Transmission Case Robbins, et al. JAMA, 2000

21. Reactivity (+ / Total) of Donor Screening NAT assays on a Coded Plasma Panel of Singapore transmission case and controls Robbins, et al. JAMA, 2000

22. HCV Transmission by Blood Donation Negative by NAT Schüttler CG, et al. Lancet 2000; 355:41-2 Donation 8 weeks prior to SC donation HCV transmission by platelet concentrate (~50mL plasma) but not RBC (~5mL plasma) NAT studies of FFP, incl. “enhanced input” PCR assays, negative for HCV RNA Conclusion: “Even a negative NAT test cannot completely prevent transmission of HCV.”

23. HCV Transmission by Blood Donation Negative by NAT Schüttler CG, et al. Lancet 2000; 355:41-2 Follow-up study in collaboration w/ Dr. Gerlich Coded panel with 3 aliquots (2.5 mL) of implicated plasma and 2 positive (low-level HCV RNA ramp-up) and 1 negative controls. GenProbe, NGI Controls correctly identified by both assays GenProbe HCV dTMA (+) in 2 of 3 replicates; NGI Ultraqual PCR (-) on all 3 replicates Sequencing and chimp transmission studies in progress

24. Donor directed lookback studies from NAT+/SC donors Roth et al. Lancet (submitted) Virus# units/recips evaluated # units/ recipients infected HCV220 HIV110 HBV130

25. Relationship of Infectivity & Viral Load pre-SC Human Transmission Data 2. Investigate Donors to Recipients with reported post-transfusion infections (Recipient traceback) Retest stored aliquots of donation plasma Recall implicated donors Perform sequencing/phylogenetic analysis to confirm linkage

26. Analysis of Reported Post-Tx Cases in Japan Matsumoto et al. Transfusion 2001 141 “TA-HBV” cases reported ’97-99 –103 fully investigated: donation samples & f/u available –15 HBV DNA(+) donations linked to 14 cases 9 WP units - donors SC to anti-HBc/anti-HBc on f/u –5 tested HBsAg EIA+, all w/ >4,000 gEq/mL 2 units anti-HBc+ at donation and f/u 3 low-level HBV DNA(+) donations w/ HBV(-) donors on f/u –1 case w/ 43 HBV DNA- donations, 1 donor SCd on f/u 92 reported “TA-HCV” cases: all donations NAT(-) and all donors (-) on f/u 0 cases of “TA-HIV” reported

27. Stages of TTVIs and infectivity Plateau phase (HCV) –Prolonged, stable high-titer viremia preceding SC –Minor fluctuations in viral load may reflect host cell capacity to support viral replication Peak viremia (HIV & HBV) –Rapid rise then fall in viral load corresponding to immune control and clearance of virus –Results in clearance or post-SC steady state viremia

28. Stages of TTVIs and infectivity Peri-SC phase – cellular and humoral immune response results in down-modulation of viral production and, in some cases, clearance –Smooth decline in viral load to clearance or steady state viremia in most cases –Some cases evidence marked fluctuation in viremia including intermittent neg-pos ID NAT results

29. Virologic/Serologic Profile – HCV Panel 6211 DaysDaysS/COS/CO HCV PCR Quantification 46 Days 100 1,000 10,000 100,000 1,000,000 10,000,000 0 0 20 40 60 80 100 120 140 160 180 200 1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 10 PCR

30. Time to Detection of Transfusion- Transmitted HCV Infection by Test Time toMean95% CI RNA12.61.5-23.7 ALT51.518.5-84.5 EIA3.070.732.8-128.6

31. HEPATOLOGY Vol. 29, No. 3, 1999

32. HCV NAT-Positive Donor HCV NAT-Positive Donor with fluctuating viremia during plateau-SC phase 0 250 500 750 1,000 1,250 1,500 0306090120 ALT 0 5 10 15 20 25 30 (S/CO) -147 10 2 3 4 5 6 -365 Viral load RIBA––––––Ind ++ ALT TMA EIA Viral load

33. Stages of TTVIs and infectivity Immunosilent carriers –Persistent viremia in absence of detectable SC Case reports for HIV, HCV and HBV Recipient infection documented for HCV –Durand et al. Ann Intern Med 2000 –Peoples et al. Transfusion 2000 Transient Viremia without SC –Confirmed by alternate sample and f/u with serotyping –Frequency in HR populations detectable by high sensitivity NAT is now under study.

34. Seroreactive Seronegative Seroreactive, NAT Negative Seronegative, NAT Negative ARC HCV NAT Reactive Donations Confirmed with Follow up Sample(s) N = 25 Number of Days

35. Stages of TTVIs and infectivity Post-SC –Chronic seropositive carrier state Set-point viral load (NA vs Ag levels) Variable infectivity to NA/Ag ratio –Defective viral particles –Excess Ag production –Neutralizing Ab –Impact of unit storage and recipient susceptibility –“Resolved” infection Sensitivity of NAT vs infectivity in blood? Persistence of infections virus in liver or other tissues Waning Ab titers w/ persistent cellular immune responses

36. Rate of HCV Viremia (dHCV TMA) among Seropositive Subjects with Divergent Risk Factors and Immune Status StudyCategorySero+ No. RNA -pos No. ( % ) ARCDonors 4,5653,653 (80%) BSIDonors 1,5141,193 (79%) UHSIVDU 300255 (85%) VATSHIV+ Patients 11293 (83%)

37. Systematic Review of Role of Polymerase Chain Reaction in Defining Infectiousness among People Infected with Hepatitis C Virus Dore GJ, Kaldor JM, McCaughan GW Br Med J 1997; 315:333-7 2022 people exposed to anti-HCV(+) sources 1148 exposed to PCR(+) sources: 148 transmissions 874 exposed to sources (-) for HCV: 0 transmissions Transmission rates after: Perinatal exposure 6.2% Needlestick 6.1% Solid organ / bone marrow transplantation 78% Blood components 88%

38. Results of SD-NAT Re-testing of HCV-RIBA-3-positive Specimens that Tested Negative by MP vs SD NAT Original NAT Screening # RIBA+ / NAT- Specimens Tested # (%) pos on SD-NAT (+) Minipool Single Donation 60 15 15 (25%) * 2 (13%) * Mean SD-NAT s/co = 5.0 (range 1.05 – 9.2)

39. HCV viral load in anti-HCV+ donors and outcome in recipients Operskalski et al. AABB 2001 Donation HCV RNA level # recipients exposed # (%) recipients infected Neg (<10 2.2 )156 (40%) 10 2.2 – 10 3.9 1815 (83%) 10 4.0 – 10 6.3 5349 (92%)

40. 09/08/99 – 08/31/00 Correlation of HIV NAT with Supplemental HIV Serological Data ARC 09/08/99 – 08/31/00 Western Blot Result NAT Result Pos Neg Total PosIndNeg 213 (94%) 13 226 (5.6%) 38 1,901 1,939 21 1,856 1,877 (1.5%) Total 272 3,770 4,042

41. Characteristics of WB Pos/TMA Neg Samples identified by ARC NAT Screening All samples p24 Ag negative SamplePoolNeatHIV-1/HIV-2WBHIV PCR 1NR1.4341, 120, 160Neg 2NR1.0341, 160Neg 3NR1.6241, 160Neg 4NR1.1141, 55, 160Neg 5NR2.5024, 41, 51, 61, 160Neg 6NR20.18all bandsNeg 7NR1.2224, 41, 160Neg 8NR1.1117, 41, 120, 160Neg 9NR1.8441, 160Neg 10NR1.4024, 41, 160Neg 11NR1.7017, 24, 41, 51, 160Neg 12NR20.00all bandsPos (200 copies/mL) 13NR17.84all bandsNeg 09/08/99 – 08/31/00

42. HIV-1 Transmission vs RNA Load and Storage 0103050 1 2 3 4 5 6  RBC (+)  RBC (–) Platelets (+)  FFP (+)  FFP (–) Days to Administration HIV-1 RNA Load (Log 10 ) Copies/mL Busch et al. JID 1994

43. HBV DNA Detection in anti-HBc+ HBsAg- US blood donations Kleinman et al. AABB 2001 5121 anti-HBc-only donations from 5 REDS sites tested 1231 anti-HBc+, anti-HBs <100 IU/mL by Prism 395 rep samples screened by PCR (<10 IU/mL) w/ confirmation by 2 PCR assays on sep aliquot 4 HBV DNA+ detected (4/107 anti-HBs-neg) – HBV DNA load 10-100 copies/mL Project 1 HBV DNA+ per 48,955 Tx units, similar to projected yield of HBV DNA+ pre-HBsAgWP units

44. Infectivity of HBV DNA PCR+, HBsAg-, anti-HBc+ blood Prince et al. Transfusion 2001 3 patients sampled 25-30 years post acute HBV HBsAg-/anti-HBc+/anti-HBs+ HBV DNA+ w/ 200-1300 copies/mL 1.35 mL serum + 2-4 x 10 7 PBMC inoculated Chimps monitored 15 months All chimps neg for HBsAg, HBV DNA, etc.

45. Infectivity of HBsAg (-) Hepatic Allografts with Antibodies to HBV Dodson, et al. Transplantation 1997;64:1582-4 DonorRecipientNInfected N (%) anti- HBc anti- HBs anti- HBc anti- HBs - + -- 250 + --- 18* (72%) + -- +70 + - + - 152 * 4/18 transmitting donors had low-titer anti-HBs

46. Conclusions Relationship between infectious units and RNA/DNA levels varies by stage of infection During primary (pre-SC) infection, 1-10 gEq/mL plasma appears able to transmit infection Ab+/NAT- donations must be presumed infectious (can’t d/c serology or reenter donors) Need for future studies at international level 1.animal transmission model systems 2.donor lookback studies (prior units from NAT+ and Ab SC donors) 3.investigation of donors in TA-recipient infections