Development of an HIV2 RNA International Standard Harvey Holmes, Clare Morris, Neil Berry, Alan Heat

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Development of an HIV-2 RNA International
StandardHarvey Holmes, Clare Morris, Neil
Berry, Alan Heath and Collaborative Study Group
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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

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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

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HIV-2 gag sequences
HIV-2 subtype A
SIVmac/smm
HIV-2 subtype B
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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

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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)
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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)

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HIV-2 CAM2
HIV-2 CAM2
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HIV-2 ROD
HIV-2 ROD
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HIV-2 CAM2
HIV-2 CAM2
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HIV-2 ROD
HIV-2 ROD
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(CAM2 relative to CAM2)
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(ROD relative to CAM2)
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(ROD relative to CAM2)
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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

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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

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Acknowledgements

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

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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