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National Institute for Biological Standards and Control Assuring the quality of biological medicines Development of an HIV-2 RNA International Standard.

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Presentation on theme: "National Institute for Biological Standards and Control Assuring the quality of biological medicines Development of an HIV-2 RNA International Standard."— Presentation transcript:

1 National Institute for Biological Standards and Control Assuring the quality of biological medicines Development of an HIV-2 RNA International Standard Harvey Holmes, Clare Morris, Neil Berry, Alan Heath and Collaborative Study Group

2 HIV-2: Background HIV-2 is a diverse group of viruses closely related to and thought to be derived from the SIV of sooty mangabey monkeys First isolated in 1986 from AIDS patient in West Africa Generally confined to W Africa and in countries with close links such as Portugal. Different subtypes exist; A and B main subtypes infecting humans Different disease progression to HIV-1 –Lower virulence – slower progression to AIDS –Lower viral load – lower transmission rates Poorly or not detected by most HIV-1 assays. International Standard for HIV-2 RNA would be valuable for assays that detect HIV-2

3 HIV-2 Virus Isolates Genotype A most common HIV-2 subtype affecting humans Although most subtype A strains have been grown in culture, subtype B strains less easily cultured In consultation with WHO and CBER/FDA, two subtype A strains were identified that were available and for which full length sequences had been published: –HIV-2 ROD – isolated 1985 in Cape Verde Islands, Senegal –HIV-2 CAM2 – isolated 1987 in Guinea Bissau Low passage isolates acquired that grew well in T-cell lines

4 NIBSC HIV-2 ROD HIV2 ROD HIV2 MCN13 HIV2 MCR35 HIV1 ISY NIBSC CAM-2 HIV2 CAM HIV2 MD2 HIV2 ALI HIV2 BEN HIV2 GH1 HIV2 D194 HIV2 UC2 SIV MMH4 SIV 32H SIV 239 SIV 251 1A11 HIV-2 7312A HIV2 KR020 HIV2 EHO HIV2 UC1 HIV2 D205 0.02 HIV-2 gag sequences HIV-2 subtype A HIV-2 subtype B SIVmac/smm 27-28 May 20094SoGAT XXI

5 Preparation of candidate standards Virus cultured in CEM cells and stocks stored down at ≤ - 80 o C RNA concentration determined using in house real time PCR assay (LTR) Virus heat inactivated at 60 0 C for 60 minutes Inactivation confirmed using tissue culture –No growth with inactivated samples 2500 vials of each virus freeze dried

6 RNA concentration Reagent StatusHIV-2 CAM-2HIV-2 ROD Pre heat inactivation9.629.51 Post heat inactivation9.259.05 Pre freeze dried3.664.26 Post freeze dried3.474.06 Virus stocks tested pre and post heat inactivation and pre and post freeze drying NIBSC real-time PCR assay - values shown as copy number (log 10 )

7 International Collaborative Study 29 laboratories took part in the collaborative study. –Including Europe, USA, Canada, Japan, Australia, South Africa Each Lab was sent 4 vials of each of candidate labelled S1-S4 –S1 and S2 were HIV-2 CAM2 –S3 and S4 were HIV-2 ROD Requested to test in at least 3 assays, with the first assay containing 10 fold dilutions, then 0.5 log dilution around the end point Majority of results were from qualitative assays – from which end-point dilutions were determined 9 labs provided quantitative data from in-house assays –Quantitative estimates used where all results positive or limited range of dilutions used –Both quantitative and qualitative estimates used where full set of set of dilutions across end-point provided Results analysed by NIBSC statistician (Alan Heath)

8 27-28 May 20098SoGAT XXI HIV-2 CAM2

9 27-28 May 20099SoGAT XXI HIV-2 ROD

10 27-28 May 200910SoGAT XXI HIV-2 CAM2

11 27-28 May 200911SoGAT XXI HIV-2 ROD

12 27-28 May 200912SoGAT XXI (CAM2 relative to CAM2)

13 27-28 May 200913SoGAT XXI (ROD relative to CAM2)

14 27-28 May 200914SoGAT XXI (ROD relative to CAM2)

15 Stability - Accelerated Degradation Studies Vials of freeze dried CAM-2 and ROD stored at range of elevated temperatures Tested at intervals of 4, 8, 12 months, 2,3,4,5 years Can predict stability using Arrhenius equation

16 Conclusions and proposal Considerable variation between the results from different labs and different assays Use of relative potency improves agreement between labs and assay methods Good agreement between S1 and S2 (HIV-2 CAM2) and between S3 and S4 (HIV-2 ROD) Statistically, results were similar and there was no preference for either S1/S2 or S3/S4 We suggest that HIV-2 CAM2 (S1/S2) be proposed to WHO ECBS as 1 st International Standard for HIV-2 RNA with a unitage of 10,000 IU/vial/ml We welcome feedback and comments from the SoGAT Working Group

17 Acknowledgements NIBSC Project Team Dr. Indira Hewlett, CBER/FDA, USA Dr Ana Padilla, WHO, Switzerland Collaborative Study Group –29 international participants – Thank You !!!!!

18 Other HIV-1 Standards Current HIV-1 2 nd IS has stocks that will last 3-4 years Need to start planning replacement now Virus HIV-1 genotype B stock used for previous IS still available – shall we use this?? Should we heat-inactivate the virus? This makes processing and shipping more straight-forward 2 nd genotype panel – work is underway Collaborative study: 2010


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