Update on CBER HIV-1 Subtype Panel AND Update on West Nile virus lot release and validation panel development

Update on CBER HIV-1 Subtype panel Indira Hewlett, Ph.D CBER/FDA C B E R 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 C B E R Virus isolates for HIV-1 subtype panel Isolate A A2 C C2 D D2 E E2 F F2 G1 G2 N O1 O2 O3 O4 O5 B B ID DJ/258/91 UG/029/92 SE/364/90 192431 UG/035/92 UG/021/92 TH/022/92 TH/1465/95 BZ/162/90 BZ/126/89 G3/Nigeria HH8793 NA NA NA NA NA NA 10207 9697 Phenotype NSI SI NSI SI NSI SI NSI SI NSI SI Not known Not known Not known NSI NSI Not known Not known Not known Not known Not known Source WRAMC NIBSC WRAMC CDC NIBSC NIBSC NIBSC WRAMC WRAMC WRAMC NIBSC NIBSC Sinnousi Spain Spain German German German US US C B E R 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 C B E R Multi-lab testing Sample A A2 C C2 D D2 E E2 F F2 G1 G2 N O1 O2 O3 O4 O5 p24 (ng/ml) 0.47 0.3 75.3 21.1 7.4 75 5.5 37 1.5 166 5.6 56 79 1.7 0.55 2.3 0.67 0.39 MANUFACTURE (viral load - logs) A 7.65 5.92 9.15 8.67 8.32 8.91 8.23 8.51 8.18 9.43 6.92 9.11 8.95 2.75 B 7.26 6.41 8.89 8.61 8.15 9.46 7.85 9.40 7.90 10.08 8.18 9.65 8.76 7.32 7.32 9.46 8.92 8.93 C 6.49 5.41 7.20 7.70 6.76 8.57 7.20 8.38 7.11 8.85 5.58 8.28 8.26 8.26 8.65 8.74 8.51 D 6.52 5.45 8.38 7.73 7.36 8.62 7.15 8.45 7.11 9.18 7.11 8.48 7.40 6.00 5.92 6.32 6.61 6.36 E 6.60 4.40 8.30 9.15 5.88 8.67 7.11 8.43 7.00 8.30 C B E R Consensus Values of subtype panel samples ID A A2 C C2 D D2 E E2 F F2 G G2 N O1 O2 O3 O4 O5 Consensus Values 6.90 5.52 8.38 8.37 7.29 8.85 7.51 8.63 7.46 9.17 6.95 8.88 8.37 7.19 7.17 8.15 8.09 7.93 SD 0.52 0.75 0.75 0.63 1.01 0.37 0.50 0.43 0.54 0.66 1.07 0.63 0.85 1.13 1.18 1.63 1.28 1.38 CV (%) 7.6 13.5 8.9 7.6 13.9 4.2 6.7 5.0 7.2 7.2 15.3 7.1 10.1 15.8 16.4 20.0 15.9 17.4 C B E R 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). C B E R 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 C B E R West Nile virus lot release and validation panel development C B E R 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 C B E R 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 C B E R 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 C B E R FDA Panel Development Efforts • Lot release panel for licensure and postmarket surveillance of NAT tests • Qualification panel for evaluation of relative sensitivities of investigational NAT assays C B E R 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 studiesC B E R 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 C B E R Quantitation of virus isolates: copies/mL Sample HuWNV 10-1 HuWNV 10-4 HuWNV 10-7 HuWNV 10-1 60oC/2hr NY99 10-1 NY99 10-4 NY99 10-7 NY99 10-1 60oC/2hr Lab 1 109 106 102 107 109 106 102 107 Lab 2 109 ND ND 107 109 ND ND 108 Lab 3 109 106 102 107 109 106 102 106 Lab 4 109 106 102 107 109 106 102 104 Average 109 106 102 107 109 106 102 106.5 C B E R Correlation between Copy/mL and PFU/mL Sample HuWNV HuWNV 60oC/2 hr NY99 Av. copy 1010 107 1010 PFU 107/mL 0 108/mL NY99 60oC/2 hr 106.5 0 C B E R 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 C B E R 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 C B study E R Acknowledgements • • • • • • CBER Owen Wood Sherwin Lee Rolf Taffs Jinjie Hu Ana Machuca Maria Rios Nelson Michael, DoD Harvey Holmes, NIBSC Robert Lanciotti, CDC Laura Kramer, NYDOH C B E R Acknowledgements C B E R