Plans for International Standard for HIV-2 RNA and 2nd Second International Reference Panel for HIV-1 RNA Genotypes

1
Plans for International Standard for HIV-2 RNA
and 2nd Second International Reference Panel for
HIV-1 RNA Genotypes

• Harvey Holmes, Clare Morris and Neil Berry
• Division of Retrovirology
• NIBSC, UK

(In collaboration with Indira Hewlett, CBER/FDA)
2
Current HIV Reference Reagents Available from
NIBSC

• 2nd International Standard for HIV-1 RNA.
• (code 97/650)
• Unitage 5.56 IU Log 10/ml
• International Reference Panel for HIV-1 RNA
  Genotypes (code 01/466) established in 2003
• No unitage assigned
• Working reagents for NAT
• Working Reagent 1 medium copy number (3.56
  log10 IU/ml)
• Working Reagent 2 high copy number (4.56
  log10 IU/ml)
• Working Reagent 3 low copy number (2.56
  log10 IU/ml)
• Working Reagents and Proficiency Samples can also
  be made at NIBSC under contract to a pre-defined
  concentration and using various HIV-1 strains and
  genotypes

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International Standard for HIV-2 RNA

• Rationale
• Global HIV pandemic due to HIV-1
• Most NAT assays aimed at HIV-1 detection and
  quantification
• HIV-2 mainly found in West Africa and European
  countries with close links eg Portugal, France,
  Belgium.
• Assays capable of detecting HIV-2 RNA or both
  HIV-1/HIV-2 are either available (eg Roche Cobas
  Taqscreen MPX) or in development
• International Standard for the HIV-2 RNA
  component would be valuable once assays in wider
  use
• Anticipated need/usage
• Currently limited demand
• Once commercial assays that can detect HIV-2 more
  available, demand may be greater
• WHO Status
• New project endorsed by ECBS in October 2006
• Discussed at meeting of WHO Collaborating Centres
  in Jan 07

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Key Scientific issues

• Few commercial assays available that can
  detect/quantitate HIV-2 RNA
• At NIBSC, in house real-time PCR assay based on
  LTR sequence used for HIV-2 quantification
• Source/Type of Material
• To be based on HIV-2 grown in culture and diluted
  in negative plasma
• Currently assessing representatives of HIV-2
  subtype A and subtype B
• Suitable strain(s) to be selected and stock laid
  down
• Virus stock to be characterised and sequence
  confirmed
• Heat inactivation to be evaluated
• Batch to be freeze-dried
• International collaborative study to be organised

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Amplification of HIV2 ROD log dilution series
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Standard Curve of HIV2 ROD standards
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1st International Reference Panel for HIV-1 RNA
Genotypes (code 01/466)

• Established by ECBS in 2003
• 500 panels prepared and frozen at -80C
• Contains representatives of HIV-1
• Subtypes A, B, C, D, AE, F, G, AGH
• Group N and O
• 120 panels remain
• Stability at -80C very good over 5 years
• Proposal for 2nd IRP

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Second Genotype Panel for HIV-1 RNA

• Rationale
• Many inter-subtype recombinant forms now
  circulating (CRF01 CRF32)
• Increasingly being encountered important
  pandemic strains
• Less common subtypes may be difficult to detect
• Proposal to prepare extended panel containing
• Subtypes G, H, J and K, group N and O
• Range of CRFs
• Will give kit manufacturers and others access to
  rare and challenging strains of HIV to enable
  them to assess their ability to detect these
  viruses
• Anticipated need/usage
• Discussed at SoGAT working group meeting
• Limited need
• Provides a source of well characterised diverse
  CRFs and isolates
• WHO Status
• Project endorsed by ECBS October 2006
• Discussed at meeting of WHO Collaborating Centres
  in Jan 07

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Second Genotype Panel for HIV-1 RNA

• Source/Type of Material
• Only relatively small number of characterised
  viruses belonging to uncommon subtypes or CRFs
  available as infectious virus
• many only available as cloned DNA
• Will source viruses from CFAR/NIBSC, CBER and NIH
  ARRRP and from scientists who have described them
  in the literature
• Viruses to be grown in PBMC culture and seed
  stocks stored down
• Viruses will be characterised including sequence
  confirmation
• Viruses will be spiked into HIV-negative plasma
• Heat inactivation and freeze-drying to be
  evaluated
• International collaborative study to be organised

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HIV field isolates, reference strains and CRFs
currently available

• Subtype G
• Subtype H
• Subtype J
• Subtype K
• Group N (to be sourced)
• Group O
• HIV-2 Subtype A
• HIV-2 subtype B

• CRF01_AE
• CRF02_AG
• CRF04_AGHKU (plasma only)
• CRF07_BC
• CRF11_A01GJ
• CRF13_A01GJU
• CRF14_BG