Update on CBER HIV-1 Subtype panel Indira Hewlett, Ph.D CBER/FDA

CBER HIV-1 subtype panel 20 specimens representing subtypes A-G of group M, N and O 2 isolates of each subtype of group M, 1 of group N and 5 of group O Dilutions 104, 103, 102 per ml for all subtypes Samples tested by five manufacturers

Virus isolates for HIV-1 subtype panel

Prototype Panel Composition Each panel was comprised of 2 isolates Seven members including one negative control per panel; Panel O has nineteen members containing five distinct isolates and four negative controls. Diluted to targeted ranges of Log 2 to Log 4 (copies/ml). Tested by 5 different manufacturer’s assays

Multi-lab testing

Consensus Values of subtype panel samples

Expected final formulation for panel One isolate of each subtype representing subtypes A-G of group M, N and O Four members including one negative control per panel Diluted to targeted ranges of Log 2 to Log 4 (copies/ml).

Summary CBER has developed a well characterized panel for qualitative and quantitative detection of HIV-1 subtype RNA Panel may be used 1) to establish LOD and 2) for lot release of HIV NAT assays for blood and plasma donor screening

West Nile virus lot release and validation panel development

Background The 2002 US outbreak of WNV identified transmission by Blood transfusion, Transplantation, Breast-feeding, Transplacental and Occupational by percutaneus injury During Aug 28, 2002-March 1, 2003, 61 possible Transfusion-Transmitted cases reported 21 confirmed from 14 blood donations 19 are not transfusion related, 21 inconclusive due to incomplete donor follow-up

WNV testing All reported cases of WNV transmission by blood transfusion have occurred during the acute, viremic phase NAT may be the most appropriate strategy to interdict infectious donations Virus titer in blood is low compared to other transmissible viruses (~1-5x103 copies/ml) and the viremia is transient. Impact of pooling on sensitivity of WNV NAT assays is of concern

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

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

FDA NAT Panels FDA NY99 and FDA-Hu2002 isolates characterized by genetic sequencing Viral infectivity determination PFU determined 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

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

Quantitation of virus isolates: copies/mL Sample Lab 1 Lab 2 Lab 3 Lab 4 Average HuWNV 10-1 109 HuWNV 10-4 106 ND HuWNV 10-7 102 HuWNV 10-1 60oC/2hr 107 NY99 10-1 NY99 10-4 NY99 10-7 NY99 10-1 60oC/2hr 108 104 106.5

Correlation between Copy/mL and PFU/mL Sample Av. copy PFU HuWNV 1010 107/mL HuWNV 60oC/2 hr 107 NY99 108/mL NY99 60oC/2 hr 106.5

FDA Plan for Qualification Panel At least 100 pedigreed clinical specimens RNA positive only IgM positive only Dual RNA and IgM positive Panel will be evaluated in collaborative studies using various candidate NAT assays Specifications for NAT panel will be established based on results of collaborative studies

Summary Both NY99 and FDA-Hu2002 stocks have a viral titer of 1010 copies/mL PFU titers determined at NY State Dept of Health Laboratory and at FDA were three logs lower than copy numbers Heat treatment of virus resulted in loss of infectivity and 2 to 3 log reduction in copy numbers as determined by TaqMan Prototype panel is being formulated to evaluate performance of panel in a larger collaborative study

Acknowledgements Owen Wood Sherwin Lee Nelson Michael, DoD CBER Owen Wood Sherwin Lee Nelson Michael, DoD Rolf Taffs Harvey Holmes, NIBSC Jinjie Hu Ana Machuca Robert Lanciotti, CDC Maria Rios Laura Kramer, NYDOH

Acknowledgements