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Update on CBER HIV-1 Subtype panel PowerPoint PPT Presentation


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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 10 4 , 10 3 , 10 2 per ml for all subtypes

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Update on CBER HIV-1 Subtype panel

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Update on cber hiv 1 subtype panel l.jpg

Update on CBER HIV-1 Subtype panel

Indira Hewlett, Ph.D

CBER/FDA


Cber hiv 1 subtype panel l.jpg

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


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Virus isolates for HIV-1 subtype panel


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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


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Multi-lab testing


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Consensus Values of subtype panel samples


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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).


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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


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West Nile virus lot release and validation panel development


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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


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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


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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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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


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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


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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

    • HuWNV20029.5 x106/mL


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Quantitation of virus isolates: copies/mL


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Correlation between Copy/mL and PFU/mL


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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


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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


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Acknowledgements

CBER

  • Owen Wood

  • Sherwin LeeNelson Michael, DoD

  • Rolf TaffsHarvey Holmes, NIBSC

  • Jinjie Hu

  • Ana MachucaRobert Lanciotti, CDC

  • Maria RiosLaura Kramer, NYDOH


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Acknowledgements


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