Presentation on theme: "Development of an HIV-2 RNA International Standard Harvey Holmes, Clare Morris, Neil Berry, Alan Heath and Collaborative Study Group."— Presentation transcript:
1 Development of an HIV-2 RNA International Standard Harvey Holmes, Clare Morris, Neil Berry, Alan Heath and Collaborative Study Group
2 HIV-2: BackgroundHIV-2 is a diverse group of viruses closely related to and thought to be derived from the SIV of sooty mangabey monkeysFirst isolated in 1986 from AIDS patient in West AfricaGenerally confined to W Africa and in countries with close links such as Portugal.Different subtypes exist; A and B main subtypes infecting humansDifferent disease progression to HIV-1Lower virulence – slower progression to AIDSLower viral load – lower transmission ratesPoorly 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 IsolatesGenotype A most common HIV-2 subtype affecting humansAlthough most subtype A strains have been grown in culture, subtype B strains less easily culturedIn 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, SenegalHIV-2 CAM2 – isolated 1987 in Guinea BissauLow passage isolates acquired that grew well in T-cell lines
5 Preparation of candidate standards Virus cultured in CEM cells and stocks stored down at ≤ -80oCRNA concentration determined using in house real time PCR assay (LTR)Virus heat inactivated at 600C for 60 minutesInactivation confirmed using tissue cultureNo growth with inactivated samples2500 vials of each virus freeze dried
6 Post heat inactivation RNA concentrationVirus stocks tested pre and post heat inactivation and pre and post freeze dryingReagent StatusHIV-2 CAM-2HIV-2 RODPre heat inactivation9.629.51Post heat inactivation9.259.05Pre freeze dried3.664.26Post freeze dried3.474.06NIBSC real-time PCR assay - values shown as copy number (log10)
7 International Collaborative Study 29 laboratories took part in the collaborative study.Including Europe, USA, Canada, Japan, Australia, South AfricaEach Lab was sent 4 vials of each of candidate labelled S1-S4S1 and S2 were HIV-2 CAM2S3 and S4 were HIV-2 RODRequested to test in at least 3 assays, with the first assay containing 10 fold dilutions, then 0.5 log dilution around the end pointMajority of results were from qualitative assays – from which end-point dilutions were determined9 labs provided quantitative data from in-house assaysQuantitative estimates used where all results positive or limited range of dilutions usedBoth quantitative and qualitative estimates used where full set of set of dilutions across end-point providedResults analysed by NIBSC statistician (Alan Heath)
15 Stability - Accelerated Degradation Studies Vials of freeze dried CAM-2 and ROD stored at range of elevated temperaturesTested at intervals of 4, 8, 12 months, 2,3,4,5 yearsCan predict stability using Arrhenius equation
16 Conclusions and proposal Considerable variation between the results from different labs and different assaysUse of relative potency improves agreement between labs and assay methodsGood 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/S4We 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/mlWe welcome feedback and comments from the SoGAT Working Group
17 Acknowledgements NIBSC Project Team Dr. Indira Hewlett, CBER/FDA, USA Dr Ana Padilla, WHO, SwitzerlandCollaborative Study Group29 international participants – Thank You !!!!!
18 Other HIV-1 StandardsCurrent HIV-1 2nd IS has stocks that will last 3-4 yearsNeed to start planning replacement nowVirus 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-forward2nd genotype panel – work is underwayCollaborative study: 2010
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