1. Update on CBER HIV NAT panels and International panels Indira Hewlett, Ph.D Chief, Lab. of Molecular Virology DETTD/OBRR/FDA May 28, 2009 XXI SoGAT meeting Phillipe Nyambi NYU

2. HIV-1 Molecular Epidemiology: Historical Timeline and Key Milestones 1983 HIV 1998 Nomenclature 2001 CRF Variation – HIV-2 1988 A URF A D C A Kenya Uganda Tanzania 2002 URF Group O 1993 Co-infection, dual- & super-infections 2003 1995 Recombination 2004 SGRs 2 nd gen. recom binant 1992 Subtypes 14 new CRFs have been assigned in year 2007

3. Worldwide distribution of predominant HIV-1 group M subtypes and CRFs North and Central America B South America B, F1, CRF12_BF Western Europe B, A, C, G CRF14_BG Eestern Europe A CRF03_AB China B CRF07_BC, CRF08_BC Southeast Asia B CRF01_AE B South Asia C CRF01_AE AustraliaB South Africa C Central Africa Most CRFs, A, C, D, G, H, J, K, O. N Western Africa A, G CRF02_AG East Africa A, D, C

4. Impact of HIV genetic diversity on diagnosis  Schable, C., et al Lancet. (1994) Sensitivity of US licensed antibody tests for detection of HIV-1 group O infection. 344,  Amendola, et al., J AIDS, (2002) Under-evaluation of HIV -1 Plasma Viral Load by a Commercially Available Assay in a Cluster of Patients Infected With HIV - 1 A/G Circulating Recombinant Form (CRF02).  Colson, P., et al J. Clin. Virol., 2007. Impaired quantification of plasma HIV-1 RNA with a commercialized real-time PCR assay in a couple of HIV-1 infected individuals  Zouhair, S., et al JCM, (2006), Group O HIV-1 infection that escaped detection in two immunoassays  Lee, S., et al. AIDS Res and Hum Retr.( 2007) Detection of Emerging HIV variants in blood donors from urban areas of Cameroon  Yao JD, et al. J. Clin. Micro., (2008). Plasma Load Discrepancies between the Roche Cobas Amplicor Human Immunodeficiency Virus Type 1 (HIV-1) Monitor Version 1.5 and Roche Cobas AmpliPrep/Cobas TaqMan HIV-1 Assays

5. CBER Efforts in panel development CBER HIV-1 RNA subtype panel  CBER developed an HIV-1 RNA subtype panel currently in use for lot release since 2004  Panel consists of one primary isolate each of group M subtypes A-G and groups O and N cultured in PBMC and characterized by sequencing  Virus isolates are inactivated by heat treatment at 60 0 C for 60 mins)  Panel composed of 3 members of each subtype at 10 3, 10 4 and 10 5 copies/ml spiked in negative plasma

6. CBER HIV-2 RNA panel  Seven isolates of HIV-2 subtype A were cultured in PBMC cultures and characterized by partial sequencing  Viruses were inactivated by heat treatment (60 0 C for 60 mins) and spiked into negative plasma  Testing was performed in 3 laboratories  Results from labs were in good agreement  HIV-2 panel was formulated at 5, 10, 50,100 copies/ml and is currently in use for lot release testing of HIV-2 NAT assays

7. CBER HIV-2 Panel Isolate Testing Summary (Log 10) Isolate IDManufacturer A Manufacturer B Manufacturer C Mean Standard Deviation B2 8.7782 8.4116 9.3424 8.8441 0.4689 B3 8.5798 8.6702 9.3424 8.8642 0.4167 B4 8.6128 8.6263 9.4133 8.8841 0.4583 B5 8.6812 8.5988 9.4425 8.9075 0.4651 B7 8.8451 8.3522 9.1732 8.7902 0.4133 B8 8.6721 8.1847 9.1732 8.6767 0.4943 B9 8.2788 7.7924 9.0969 8.3894 0.6593

8. CBER panel for CRF02_AG and CRF01_AE  Emerging new HIV strains are circulating recombinant forms (CRFs)  CRF02_AG and CRF01_AE are currently the most prevalent CRF strains; need for standards  Five primary virus isolates each of CRF02_AG and CRF01_AE cultured in PBMCs were characterized by full genome sequencing  Heat inactivated virus isolates spiked into negative plasma were tested in five laboratories  Results were in good agreement between labs; data analyzed statistically and values assigned  Panel will consist of 3 members for each isolate spiked in negative plasma at 10 3, 10 4 and 10 5 copies/ml (log)

9. Isolate IDLab ALab BLab CLab DLab EMean SD AE-18.718.65 8.19 8.068.478.420.25 AE-28.878.84 8.51 8.318.688.640.21 AE-38.708.69 8.34 8.128.328.430.23 AE-98.928.85 8.52 8.348.528.630.22 AE-108.698.61 8.24 8.158.478.430.21 NYU 3608.578.79 8.08 8.719.278.680.38 NYU23959.86 9.53 9.33 n/a9.159.470.26 NYU47309.369.43 9.03 n/a9.199.250.16 NYU52039.308.84 9.35 8.819.239.110.23 NYU54668.589.59 8.90 8.739.219.00.36 CRF01_AE and CRF02_AG Isolate Testing Summary

10. Ongoing CBER HIV panel development  Project initiated in Cameroon in 2001 to study HIV evolution and have access to diverse HIV strains known to emerge in this region for panel development needs Study Goals:  Collect specimens, viruses; genotyping and virus tropism  Evaluate sensitivity of blood screening and diagnostic tests for ability to detect diverse strains  Identify new strains for future use as reference reagents  Develop reference panels for emerging HIV variants for lot release and assay standardization  Future panels will target B/C, B/F and A/B recombinants

11. Summary of Genotyping Findings  CRF02_AG is most prevalent strain in Cameroon (60-70%) in both study groups  Virus is continuing to evolve - pure subtypes and new CRFs, URFs, cpx strains identified in 2002  Viruses from 2006 –present are mostly recombinants of CRFs, esp. CRF02_AG with other CRFs  Only 2.6% were pure subtype in 2006 compared with 15.8% in 2002  Globally CRF02_AG prevalence is currently 6.7% compared with subtype B at 10%

12. CRF02_AG (68.4%) URF (23.7%) Unknown (2.6%) CRF06_cpx (2.6%) F2 (2.6%) HIV subtypes in Cameroon blood donors – 2006 to present number% CRF022668.42 URF 923.7 CRF06 12.6 F2 12.6 Unknown 12.6 Total samples 38 URF % CRF02 + F222 CRF02 + CRF3511 CRF02 + CRF3711 CRF02 + B22 CRF11 + A111 CRF11 + CRF1311 CRF04 + U12

13. International Collaboration for HIV diversity studies and panel development  Inter-agency PHS working group (IWG) formed in 2006 to monitor reports of HIV diversity and impact on diagnostics  Need for reference panels for emerging variants was identified  In 2008 NIAID formed an International Collaborative Group to develop studies to assure that screening, diagnostic and confirmatory assays detect circulating strains — Assays presently are based on prototype HIV strains — Numerous studies showed failure of assays to detect and accurately quantify divergent subtypes — Evidence of viral divergence in the donor pool could accelerate development of robust serological and NAT assays for donor, diagnostic and clinical management

14. Blood donor studies  Blood donors are a “convenience sample” likely to represent the larger population — Studies in donors permit population based monitoring of recently transmitted viruses, including drug resistant phenotypes. — Knowledge of virus variation is critical to public health strategies for AIDS prevention  Detection of variants in blood donors allows access to large volume plasma components for test development, evaluation and Quality Control

15. HIV Viral Panels Project: Purpose In cooperation with other HIV surveillance efforts, to establish a set of fully characterized viruses from early acute HIV infections that are consistent with the degree of viral evolution present globally, for -Developing new assays -Validating assay platforms -Assisting regulators to evaluate test kits -Monitoring HIV drug resistance -Informing vaccine development

16. Acknowledgments DETTD/CBER Owen Wood Sherwin Lee Jiangqin Zhao Ragupathy Viswanath Shixing Tang Stephen Kerby NYU Phillipe Nyambi Sherri Burda Manufacturers Collaborative Study Group Supported by NHLBI IAG- Y1-HB-5026-01 CBER Critical Path Grant Intl. Collab study group M. Busch, BSRI M. Schito,NIAID S. Peel, WRAIR M. Manak, Seracare S.Stovanabutra and others