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Development of an HIV-2 RNA International Standard Harvey Holmes, Clare Morris, Neil Berry, Alan Heath and Collaborative Study Group. 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
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Development of an HIV-2 RNA International StandardHarvey Holmes, Clare Morris, Neil Berry, Alan Heath and Collaborative Study Group
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
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
HIV2 ALI HIV2 MD2 HIV2 BEN HIV2 CAM HIV2 GH1 NIBSC CAM-2 HIV2 D194 HIV1 ISY HIV2 UC2 HIV2 MCR35 HIV2 MCN13 HIV2 ROD SIV MMH4 NIBSC HIV-2 ROD SIV 32H SIV 239 SIV 251 1A11 HIV2 D205 HIV2 UC1 HIV2 KR020 HIV2 EHO HIV-2 7312A 0.02 HIV-2 gag sequences HIV-2 subtype A SIVmac/smm HIV-2 subtype B SoGAT XXI
Preparation of candidate standards • Virus cultured in CEM cells and stocks stored down at ≤ -80oC • RNA concentration determined using in house real time PCR assay (LTR) • Virus heat inactivated at 600C for 60 minutes • Inactivation confirmed using tissue culture • No growth with inactivated samples • 2500 vials of each virus freeze dried
RNA concentration • Virus stocks tested pre and post heat inactivation and pre and post freeze drying NIBSC real-time PCR assay - values shown as copy number (log10)
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)
HIV-2 CAM2 HIV-2 CAM2 SoGAT XXI
HIV-2 ROD HIV-2 ROD SoGAT XXI
HIV-2 CAM2 HIV-2 CAM2 SoGAT XXI
HIV-2 ROD HIV-2 ROD SoGAT XXI
(CAM2 relative to CAM2) SoGAT XXI
(ROD relative to CAM2) SoGAT XXI
(ROD relative to CAM2) SoGAT XXI
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
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 1st 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
Acknowledgements • NIBSC Project Team • Dr. Indira Hewlett, CBER/FDA, USA • Dr Ana Padilla, WHO, Switzerland • Collaborative Study Group • 29 international participants – Thank You !!!!!
Other HIV-1 Standards Current HIV-1 2nd 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 2nd genotype panel – work is underway Collaborative study: 2010