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

validation panel development

Indira Hewlett, Ph.DCBER/FDA

Blood Products Advisory Committee Meeting

June 19, 2003

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Previous FDA Actions

• March BPAC discussion of FDA proposal for :

- clinical study design- unit and donor management - FDA efforts in panel development

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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

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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

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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

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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

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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

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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

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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

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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

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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

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Procleix WNV Assay Early Testing Contingency 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

prevalenceContingent on IRB-approval, WNV informed

consent in place

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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

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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

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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

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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

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Viral Titer Determination Copy/mLSample Lab 1 Lab 2 Lab 3 Lab 4 AverageHuWNV 10-1 109 109 109 109 109

HuWNV 10-4 106 ND 106 106 106

HuWNV 10-7 102 ND 102 102 102

HuWNV 10-1

60oC/2hr107 107 107 107 107

NY99 10-1 109 109 109 109 109

NY99 10-4 106 ND 106 106 106

NY99 10-7 102 ND 102 102 102

NY99 10-1

60oC/2hr107 108 106 104 106.5

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Correlation between Copy/mL and PFU/mL

Sample Av. copy PFUHuWNV 1010 107/mL

HuWNV 60oC/2 hr 107 0

NY99 1010 108/mL

NY99 60oC/2 hr 106.5 0

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• 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

FDA Plan for Qualification Panel

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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

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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

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Acknowledgements

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

Health