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