1. XVIII SoGAT Washington 24 May 2005 SoGAT and HIV NAT Standards - 2 nd International Standard for HIV-1 RNA Harvey Holmes*, Clare Davis* and Alan Heath** Divisions of Retrovirology* and Informatics** National Institute for Biological Standards and Control (NIBSC) UK

2. XVIII SoGAT Washington 24 May 2005 SoGAT and NAT Standards available from NIBSC SoGAT and NIBSC have collaborated in establishing: International Standards: – 1997: 1 st International Standard for HCV RNA (96/790) – 1999: 1 st International Standard for HIV-1 RNA (97/656) – 1999: 1 st international Standard for HBV DNA (97/746) – 2000: 1 st International Standard for B19 DNA (99/800) – 2002: 1 st International Standard for HAV RNA (00/560) – 2003: 1 st International Reference Panel for HIV-1 Genotypes (01/466) – genotypes A-H – 2003: 2 nd International Standard for HCV RNA (96/798) Working Reagents: HIV-1 RNA PWS-1, PWS-2, PWS-3 HCV RNA HBV DNA HAV RNA B19 DNA Multiplex (HCV, HBV, HIV, HAV, B19) HCV Genotype Panel (genotypes 1 -6)

3. XVIII SoGAT Washington 24 May 2005 Retrovirology Standards and Working Reagents

4. XVIII SoGAT Washington 24 May 2005 Replacement of 1 st International Standard for HIV-1 RNA (NIBSC Code 97/656) Sample YY established as 1 st International Standard (code 97/656) in 1999 Assigned a concentration of 100,000 IU per vial/ml Standard made from HIV-1 PCR-positive, antibody-negative, plasmapheresis donation diluted in defibrinated plasma 2300 vials freeze-dried and stored at -20 0 C SoGAT 2003 - agreement to replace standard due to: – low Stocks (~ 450) – presence of HBV DNA – may affect suitability for multiplex assays Interim report on collaborative study presented by Clare Davis at SoGAT 2004

5. XVIII SoGAT Washington 24 May 2005 Candidate 2 nd International Standard SoGAT recommended that sample XX from first collaborative study be re-evaluated as candidate 2 nd International Standard (IS) Characteristics: – HIV-1 subtype B low passage field isolate (CCR5/NSI) from Edinburgh (P Simmonds) – Propagated on human PBMCs - stock stored under LN – Virus diluted in human cryosupernatant to suitable concentration – 2200 vials of material freeze-dried – Fill CV = 0.2%

6. XVIII SoGAT Washington 24 May 2005 International Collaborative Study to Establish 2 nd International Standard for HIV-1 RNA Aims of study:- – Calibrate XX against 1st International Standard (1 st IS) – Confirm stability of XX – Determine values in current assays 10 laboratories participated Candidate XX and the 1 st International Standard recoded (samples 1 and 2 respectively) Participants asked to test samples in 3 independent assays. Initially at 10-fold dilutions Subsequently at half log dilutions around the end point 8 data sets received, 5 from quantitative assays and 3 from qualitative assays NIBSC collated and analysed data

7. XVIII SoGAT Washington 24 May 2005 0 1 2 3 4 5 6 7 8 9 10 Estimated RNA Copies (log10/ml) 3.003.253.503.754.004.254.504.755.005.255.505.756.006.256.506.757.00 43 3 41 44 19 14 47 16 17 18 2 22 33 48 10 6 5 45 24 46 28 34 4 9 IHMQIH M AS B3 IH M M M M B3 IH M A M M NU M Monitor assaysNuclisensbDNA 3Abbott LCxIn-HouseAmpliscreen Original Collaborative Study 1st International Standard for HIV-1 RNA (YY) 0 1 2 3 4 5 6 7 8 9 10 Estimated RNA Copies (log10/ml) 3.003.253.503.754.004.254.504.755.005.255.505.756.006.256.506.757.00 163 41 43 19 44 43 34 33 47 48 10 17 18 2 24 5 28 45 14 6 22 46 9 IH AS M MQ NU B3 IH M M M M M NU A B3 IH M M M Monitor assays NuclisensbDNA 3Abbott LCxIn-HouseAmpliscreen Sample XX

8. XVIII SoGAT Washington 24 May 2005 0 1 2 3 4 5 6 7 8 9 10 Estimated IU (log10/ml) 3.003.253.503.754.004.254.504.755.005.255.505.756.006.256.506.757.00 1619 41 24 28 34 4 45 33 10 3 46 5 14 47 48 6 17 18 2 44 9 43 22 43 IHAS IH M NU A B3 IH M M B3 IH M M M M M M MQ Monitor assaysNuclisensbDNA 3Abbott LCxIn-HouseAmpliscreen Original Collaborative Study Sample XX relative to 1st IS in IU

9. XVIII SoGAT Washington 24 May 2005 Sample XX in Current Study (coded Sample 1) Current Collaborative Study 1 st IS (coded Sample 2)

10. XVIII SoGAT Washington 24 May 2005 Current Collaborative Study Potency of XX (Sample 1) relative to 1 st IS (IU)

11. XVIII SoGAT Washington 24 May 2005 MethodTypeNo. LabsMean AmplicorQuantitative15.64 bDNA V3Quantitative15.61 MonitorQuantitative15.70 NuclisensQuantitative15.09 TaqmanQuantitative15.27 OverallQuantitative55.46 In HouseQualitative15.00 NGIQualitative16.25 ProcleixQualitative14.91 OverallQualitative35.39 Overall85.44 Current Study: Relative Potency of XX to 1 st IS – Log10 IU/ml

12. XVIII SoGAT Washington 24 May 2005 MethodTypeN LabsMeanMinMax AbbottQuantitative15.52 bDNA v3Quantitative45.715.625.80 In-HouseQuantitative25.675.595.76 MonitorQuantitative95.655.305.90 NuclisensQuantitative35.225.165.32 OverallQuantitative195.59 All LabsQualitative55.665.356.10 Overall245.60 Relative Potency of XX to 1 st IS Original Study (Log10 IU/ml)

13. XVIII SoGAT Washington 24 May 2005 2 nd International Standard for HIV-1 RNA No evidence of any drift in values between the two studies - no stability issues Four options for assigning value to XX: – 1: Use results from original study (5.60 log 10 IU/ml) – 2: Use results from current study (5.44 log 10 IU/ml) – 3: Pool data from both studies (5.56 log 10 IU/ml) – 4: Determine overall mean for each method and obtain compromise between methods – pragmatic rather than statistically based Option 3 favoured by NIBSC statistician

14. XVIII SoGAT Washington 24 May 2005 International Collaborative Study to Establish 2 nd International Standard for HIV-1 RNA Participants T CuypersSanquin – CLB, The Netherlands M ChudyPaul Ehrlich Institute, Germany J DockerGen Probe, USA K FransenInstitute of Tropical Medicine, Belgium I HewlettCBER, FDA, USA F IntelmannBaxter, Austria D JardineNational Reference Laboratory, Australia J Turczyn Bayer Testing lab, USA Richard SmithNGI, USA C Davis/H Holmes NIBSC, UK

15. XVIII SoGAT Washington 24 May 2005 HIV-1 Variability – Genotype panels WHO 1 st International Reference Panel for HIV-1 Genotypes (Code 01/466) established in 2003 Contains 11 members: – Group M – Subtypes A - G, and AG-GH recombinant – Group N and O representative HIV diversity increasing, with greater frequency of unusual subtypes, outlier groups and circulating recombinant forms (CRFs) being encountered especially in cities eg London, Paris Greater diversity presents continuing challenge to NAT technology Would a second genotype panel be of value containing: – More diverse subtypes, group N, group O representatives? – More CRFs? – HIV-2 representatives?

16. XVIII SoGAT Washington 24 May 2005 Conclusions Report on candidate 2 nd International Standard for HIV-1 RNA to WHO ECBS Recommendation on unitage – option 3?? Need for 2 nd HIV Genotype Panel containing more diverse field isolates??