1. Development of an HIV-2 RNA International Standard Harvey Holmes, Clare Morris, Neil Berry, Alan Heath and Collaborative Study Group

2. HIV-2: Background
HIV-2 is a diverse group of viruses closely related to and thought to be derived from the SIV of sooty mangabey monkeys
First isolated in 1986 from AIDS patient in West Africa
Generally confined to W Africa and in countries with close links such as Portugal.
Different subtypes exist; A and B main subtypes infecting humans
Different disease progression to HIV-1
Lower virulence – slower progression to AIDS
Lower viral load – lower transmission rates
Poorly or not detected by most HIV-1 assays.
International Standard for HIV-2 RNA would be valuable for assays that detect HIV-2

3. HIV-2 Virus Isolates
Genotype A most common HIV-2 subtype affecting humans
Although most subtype A strains have been grown in culture, subtype B strains less easily cultured
In consultation with WHO and CBER/FDA, two subtype A strains were identified that were available and for which full length sequences had been published:
HIV-2 ROD – isolated 1985 in Cape Verde Islands, Senegal
HIV-2 CAM2 – isolated 1987 in Guinea Bissau
Low passage isolates acquired that grew well in T-cell lines

4. HIV-2 gag sequences HIV-2 subtype A SIVmac/smm HIV-2 subtype B
NIBSC HIV-2 ROD
HIV2 ROD
HIV2 MCN13
HIV2 MCR35
HIV1 ISY
NIBSC CAM-2
HIV2 CAM
HIV2 MD2
HIV2 ALI
HIV2 BEN
HIV2 GH1
HIV2 D194
HIV2 UC2
SIV MMH4
SIV 32H
SIV 239
SIV 251 1A11
HIV A
HIV2 KR020
HIV2 EHO
HIV2 UC1
HIV2 D205
0.02
HIV-2 subtype A
SIVmac/smm
HIV-2 subtype B
27-28 May 2009
SoGAT XXI

5. Preparation of candidate standards
Virus cultured in CEM cells and stocks stored down at ≤ -80oC
RNA concentration determined using in house real time PCR assay (LTR)
Virus heat inactivated at 600C for 60 minutes
Inactivation confirmed using tissue culture
No growth with inactivated samples
2500 vials of each virus freeze dried

6. Post heat inactivation
RNA concentration
Virus stocks tested pre and post heat inactivation and pre and post freeze drying
Reagent Status
HIV-2 CAM-2
HIV-2 ROD
Pre heat inactivation
9.62
9.51
Post heat inactivation
9.25
9.05
Pre freeze dried
3.66
4.26
Post freeze dried
3.47
4.06
NIBSC real-time PCR assay - values shown as copy number (log10)

7. International Collaborative Study
29 laboratories took part in the collaborative study.
Including Europe, USA, Canada, Japan, Australia, South Africa
Each Lab was sent 4 vials of each of candidate labelled S1-S4
S1 and S2 were HIV-2 CAM2
S3 and S4 were HIV-2 ROD
Requested to test in at least 3 assays, with the first assay containing 10 fold dilutions, then 0.5 log dilution around the end point
Majority of results were from qualitative assays – from which end-point dilutions were determined
9 labs provided quantitative data from in-house assays
Quantitative estimates used where all results positive or limited range of dilutions used
Both quantitative and qualitative estimates used where full set of set of dilutions across end-point provided
Results analysed by NIBSC statistician (Alan Heath)

8. HIV-2 CAM2
HIV-2 CAM2
27-28 May 2009
SoGAT XXI

9. HIV-2 ROD
HIV-2 ROD
27-28 May 2009
SoGAT XXI

10. HIV-2 CAM2
HIV-2 CAM2
27-28 May 2009
SoGAT XXI

11. HIV-2 ROD
HIV-2 ROD
27-28 May 2009
SoGAT XXI

12. (CAM2 relative to CAM2)
27-28 May 2009
SoGAT XXI

13. (ROD relative to CAM2)
27-28 May 2009
SoGAT XXI

14. (ROD relative to CAM2)
27-28 May 2009
SoGAT XXI

15. Stability - Accelerated Degradation Studies
Vials of freeze dried CAM-2 and ROD stored at range of elevated temperatures
Tested at intervals of 4, 8, 12 months, 2,3,4,5 years
Can predict stability using Arrhenius equation

16. Conclusions and proposal
Considerable variation between the results from different labs and different assays
Use of relative potency improves agreement between labs and assay methods
Good agreement between S1 and S2 (HIV-2 CAM2) and between S3 and S4 (HIV-2 ROD)
Statistically, results were similar and there was no preference for either S1/S2 or S3/S4
We suggest that HIV-2 CAM2 (S1/S2) be proposed to WHO ECBS as 1st International Standard for HIV-2 RNA with a unitage of 10,000 IU/vial/ml
We welcome feedback and comments from the SoGAT Working Group

17. Acknowledgements NIBSC Project Team Dr. Indira Hewlett, CBER/FDA, USA
Dr Ana Padilla, WHO, Switzerland
Collaborative Study Group
29 international participants – Thank You !!!!!

18. Other HIV-1 Standards
Current HIV-1 2nd IS has stocks that will last 3-4 years
Need to start planning replacement now
Virus HIV-1 genotype B stock used for previous IS still available – shall we use this??
Should we heat-inactivate the virus? This makes processing and shipping more straight-forward
2nd genotype panel – work is underway
Collaborative study: 2010