1. 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)

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

3. 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

4. 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

5. Amplification of HIV2 ROD log dilution series

6. Standard Curve of HIV2 ROD standards

7. 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

8. 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

9. 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

10. 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