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 RetrovirologyNIBSC, UK
(In collaboration with Indira Hewlett, CBER/FDA)
Current HIV Reference Current HIV Reference Reagents Available from Reagents Available from
NIBSCNIBSC• 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
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
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
Amplification of HIV2 ROD log dilution series
Standard Curve of HIV2 ROD standards
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
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
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
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
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