Harvey Holmes, Clare Morris and Neil Berry Division of Retrovirology 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)

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

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

500 panels prepared and frozen at -80C 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