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

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

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

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., 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

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

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

CBER HIV-2 Panel Isolate Testing Summary (Log 10) Isolate IDManufacturer A Manufacturer B Manufacturer C Mean Standard Deviation B B B B B B B

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)

Isolate IDLab ALab BLab CLab DLab EMean SD AE AE AE AE AE NYU NYU n/a NYU n/a NYU NYU CRF01_AE and CRF02_AG Isolate Testing Summary

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

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%

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% CRF URF CRF F Unknown 12.6 Total samples 38 URF % CRF02 + F222 CRF02 + CRF3511 CRF02 + CRF3711 CRF02 + B22 CRF11 + A111 CRF11 + CRF1311 CRF04 + U12

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

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

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

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 CBER Critical Path Grant Intl. Collab study group M. Busch, BSRI M. Schito,NIAID S. Peel, WRAIR M. Manak, Seracare S.Stovanabutra and others