1. TMA Assay for Detection of West Nile Virus BPAC Meeting - March 13, 2003 Cristina Giachetti, Ph.D. J.Linnen; A.Broulik; W. Wu; J.Cline; M.Lewis; G.Dennis; M.Cass; M.Alden; V.Casas; and S.Miller Gen-Probe Incorporated, San Diego, CA

2. Objectives of the West Nile Virus Assay Development Program n To develop and manufacture a TMA-based assay for detection of West Nile virus in blood, plasma and organ/tissue donor specimens n Phase 1 clinical study to determine RNA reactivity in archived linked samples collected in areas of potentially high WNV incidence rate during 2002 n Phase 2 clinical protocol to allow for widespread donor screening. It is anticipated that this trial will be initiated July 2003.

3. WNV Assay Development Goals n Analytical sensitivity: at least 95% detection at 50 copies/mL n Detection of genetic variants of WNV (e.g Lineage 1, including Kunjin virus, and Lineage 2 strains) with similar sensitivity n Analytical specificity: > 99.5 % n Internal Control to validate each reaction n Completely compatible with Procleix ® eSAS (semi- automated) and TIGRIS TM (fully automated) instrument platforms

4. Amplification (TMA) Amp Rgt., Oil, and Enzyme Water baths Procleix ® Semi-Automated System: Assay Protocol Pipetting Calibrators, Specimens, and TCR TECAN Sample Processing Viral Lysis, RNA Capture, and Washes TCS Detection (HPA) Probe Hybridization Selection Water bath Results Automatic RLU Reading Luminometer TECANTarget Capture System (TCS)Luminometer

5. Specimen Processing Target Capture/Magnetic Microparticle Separation Viral Lysis n Treat specimens with heat and detergent n Release nucleic acid Nucleic Acid Capture n Hybridize target sequence to capture probes n Hybridize capture probe to oligomer sequence bind to magnetic particle Removal of unwanted specimen n Apply magnetic field to separate target from residual sample n Remove residual specimen by washing Magnetic Microparticle TTTTTTTTTTTTTT Magnet 3’ AAAAAA…AAACAUCUAAC…CGU 5’ 5’ GUAGAUUG…GCA 3’ Capture Oligo Target RNA or DNA TTTTTTTTTTTTTT

6. Amplification Transcription-Mediated Amplification (TMA) n Utilizes two enzymes: Reverse Transcriptase T7 RNA Polymerase n Amplifies RNA or DNA n Produces RNA amplicon n Exponential amplification (> billion fold amplification in less than one hour) n Isothermal, simplifies automation

7. Detection Hybridization Protection Assay (HPA) n Utilizes Acridinium Ester (AE) labeled probes n Reaction Steps: 1. Hybridization AE-labeled probe hybridizes to target RNA in solution 2. Selection Label on unhybridized probe is hydrolyzed, label on hybridized probe is protected 3. Detection Label on protected hybridized probe is detected by chemiluminescence

8. Detection (cont.) Dual Kinetics Analysis (DKA) n Used to differentiate Internal Control (IC) signal from target signal n Utilizes Acridinium Ester (AE) labeled probes with differential kinetics of light-off n Ortho Fluoro Acridinium Ester labeled probe = flasher probe, hybridizes to IC n 2’Methyl Acridinium Ester labeled probes = Glower probes, hybridize to West Nile virus amplicon n ETF Algorithm deconvolutes light-off and calculates each signal 0 2000 4000 6000 8000 10000 12000 14000 16000 147 1013161922252831343740434649 Interval RLU Flasher Background Glower

9. Current Performance of WNV Assay n Specificity in normal blood donor specimens n Specificity in the presence of other blood borne viruses n Analytical sensitivity n Clinical sensitivity

10. Specificity in normal blood donor specimens

11. Specificity in the presence of other blood borne viruses

12. Analytical Sensitivity Limit of detection using in vitro transcript

13. Analytical Sensitivity Detection of WNV in CDC viral panel* * Provided by Dr. Robert Lanciotti, CDC-DVBID

14. Analytical Sensitivity Detection of WNV in Qualification Panel QWN701 from BBI

15. Analytical Sensitivity Limit of detection in Lineage 2 Panel

16. Clinical Sensitivity Detection of WNV in Specimens from CDC Case Investigations * Reactive in either plasma or RBC segment; ** IgM positive; *** Reactive with increased sample volume

17. Summary Specificity n No cross-reactivity with other blood borne viruses n Initial reactive rate in normal blood donor population was 0% [n=575], for 100% specificity

18. Summary Analytical Sensitivity n 95% Detection rate at n 6.6-11.5 copies/mL Lineage 1 transcript n 7.1-12.8 copies/mL Lineage 2 virus n 0.005-0.042 pfu/mL CDC virus panel n 50% Detection rate at n 2.2-3.9 copies/mL Lineage 1 transcript n 3.5-6.1 copies/mL Lineage 2 virus n 0.001-0.003 PFU/mL CDC virus panel

19. Summary Clinical Sensitivity n Procleix WNV assay has a clinical sensitivity equal or better than CDC Taqman Assay n All reactive results with Procleix WNV Assay were confirmed with TMA assay using an alternative amplification region

20. Conclusions n Performance to date meets design goals for specificity and sensitivity n Results support feasibility of pool testing

21. Acknowledgments This project is funded in part with federal funds from the National Heart, Lung and Blood Institute under Contract