Update on status of West Nile virus test, lot release and validation panel development

1. Update on status of West Nile virus test, lot release and validation panel development Indira Hewlett, Ph.D CBER/FDA Blood Products Advisory Committee Meeting June 19, 2003

2. Previous FDA Actions • March BPAC discussion of FDA proposal for : - clinical study design - unit and donor management - FDA efforts in panel development

3. FDA Actions – con’t Study design for test sensitivity • Repository specimens, including transfusion and community acquired WNV illness • Positive cases from prospective studies • Seroconversion panels

4. Clinical sensitivity • Testing a common set of pedigreed specimens by all candidate investigational tests to determine whether assays have equivalent sensitivity • Testing all reactive specimens identified during IND studies by all manufacturer’s assays

5. Analytical sensitivity • FDA’s current standard for WNV NAT assays is 100 copies/ml for the individual donation • Standard may be revised as assay sensitivity improves and additional data on viremia and infectivity become available in future studies

6. Unit and donor management FDA proposed foll: scheme for donor and unit management - Reactive invest. NAT results on the individual donation could be confirmed by F/U testing with invest. NAT, alternate NAT and IgM - If F/U sample is positive by invest. NAT or alt. NAT, donor remains deferred for an additional 28 days - Donor may be eligible for reinstatement if F/U sample prior to 28 days is NAT-ve, and IgM +ve

7. Progress on test development • Multiple IND studies are in progress • Two manufacturers have publicly acknowledged existing INDs: Gen-Probe Inc. and Roche Molecular Systems Inc. • IND tests are based on NAT using pooled or individual samples • Intended use for whole blood, blood components, source plasma, bone marrow, cord blood, hematopoietic progenitor cells, tissue and organ donors

8. Progress on test development • Expected start date for testing is early July, 2003 • All samples will be collected under approved IRBs with necessary informed consent • Analytical sensitivity of IND tests is comparable and between 5-15 copies/ml

9. Procleix® WNV Assay • TMA-based assay for screening blood donations for West Nile virus RNA • Uses existing instrument platform as Gen-Probe’s licensed NAT blood screening assay • Procleix Semi-automated System (eSAS) currently used with Procleix HIV-1/HCV Assay • Uses existing formulations as much as possible

10. Procleix® WNV Assay • Analytical sensitivity: 95% detection rate between 7-15 copies/ml • Specificity in pre-clinical studies evaluated by testing 1180 blood donations • No cross reactivity to other blood borne viruses • HTLV, HIV-1/-2, HCV, HBV, HGV, Rubella, HAV, CMV, EBV, HCV, Parvo B19 • No cross reactivity to other flaviviruses: Dengue (1-4), Yellow Fever Virus, and St. Louis Encephalitis virus

11. WNV IND Two Phased Clinical Protocols • Phase I: Retrospective prevalence study • 89,000 archived American Red Cross samples from 6 high incidence areas during the 2002 season • Phase II: Prospective donor screening • Voluntary donations of whole blood and source plasma at 25 testing sites : IDT or pools (site dependent) • Nation-wide testing expected to begin by July 1, 2003

12. Procleix WNV AssayEarly TestingContingency Plan • Upon WNV regional outbreak, samples will be shipped and prospective testing initiated at Phase I ARC site(s) • Current testing capability limited • Archiving samples from ~June 1st onward • Testing of samples based on regional prevalence Contingent on IRB-approval, WNV informed consent in place

13. Roche WNV NAT: Pre-clinical Performance Studies • PCR-based screening assay for use with pooled samples • Analytical sensitivity between 5-7 copies/ml • No cross-reactivity seen with non-WNV microorganisms: HTLV-I/II, HIV, HCV, HBV, CMV, HSV, HAV, HPV, Varicella, Adenovirus • Clinical specificity - 400 random volunteer samples from WNV low- and high- prevalence areas

14. FDA Panel Development Efforts • Lot release panel for licensure and post-market surveillance of NAT and IgM tests • Qualification panel for evaluation of relative sensitivities of investigational NAT and IgM assays

15. FDA NAT Panels • FDA NY99 and FDA-Hu2002 isolates characterized by genetic sequencing • Viral infectivity determination • PFUdetermined at both FDA and NY Dept. of Health Laboratories, and by cytopathic assays at FDA • RNA concentration measurements • Fluorescence and Optical density determination • TaqMan • Final panel specifications are being established through collaborative studies

16. PFU Results on FDA Isolates • At FDA • NY99 (CDC Flamingo Isolate) 108/mL • HuWNV2002 108/mL • At NY State Dept. of Health • NY99 (CDC Flamingo Isolate) 5.5 x107/mL • HuWNV2002 9.5 x106/mL

17. Viral Titer Determination Copy/mL

18. Correlation between Copy/mL and PFU/mL

19. FDA Plan for Qualification Panel • At least 100 pedigreed clinical specimens • RNA positive only • IgM positive only • Dual RNA and IgM positive • FDA also recommends that all reactive specimens identified in IND clinical trials be made available to all manufacturers through sharing of samples

20. FDA IgM panel • Panel will consist of clinical specimens containing varying titers of antibodies to WNV and some members that are also NAT positive • Panel will be evaluated in collaborative studies using various candidate IgM assays • Specifications for NAT and IgM panels will be established based on results of collaborative studies

21. Summary • Both NY99 and FDA-Hu2002 stocks have a viral titer of 1010 copies/mL • PFU titers at both NY State Dept of Health Laboratory and at FDA were three logs lower than copy numbers • Heat treatment virus results in loss of infectivity and 2 to 3 log reduction in copy number determined by TaqMan

22. Acknowledgements • Gen-Probe, Inc • Roche Molecular Systems, Inc. • Maria Rios, CBER, FDA • Robert Lanciotti, CDC • Laura Kramer, New York Department of Health