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Update on CBER HIV NAT panels and International panels

NYU. Phillipe Nyambi. 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. Group O 1993. Subtypes. HIV. Nomenclature. Co-infection, dual- & super-infections. CRF. 1998. 1983.

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Update on CBER HIV NAT panels and International panels

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  1. NYU Phillipe Nyambi 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

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

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

  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 600C for 60 mins) Panel composed of 3 members of each subtype at 103, 104 and 105 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 (600C 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)

  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 103, 104 and 105 copies/ml (log)

  9. Isolate ID Lab A Lab B Lab C Lab D Lab E Mean SD AE-1 8.71 8.65 8.19 8.06 8.47 8.42 0.25 AE-2 8.87 8.84 8.51 8.31 8.68 8.64 0.21 AE-3 8.70 8.69 8.34 8.12 8.32 8.43 0.23 AE-9 8.92 8.85 8.52 8.34 8.52 8.63 0.22 AE-10 8.69 8.61 8.24 8.15 8.47 8.43 0.21 NYU 360 8.57 8.79 8.08 8.71 9.27 8.68 0.38 NYU2395 9.86 9.53 9.33 n/a 9.15 9.47 0.26 NYU4730 9.36 9.43 9.03 n/a 9.19 9.25 0.16 NYU5203 9.30 8.84 9.35 8.81 9.23 9.11 0.23 NYU5466 8.58 9.59 8.90 8.73 9.21 9.0 0.36 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. number % CRF02 26 68.42 URF 9 23.7 CRF06 1 2.6 F2 1 2.6 Unknown 1 2.6 Total samples 38 CRF06_cpx (2.6%) F2 (2.6%) Unknown (2.6%) URF (23.7%) URF % CRF02 + F2 22 CRF02 + CRF35 11 CRF02 + CRF37 11 CRF02 + B 22 CRF11 + A1 11 CRF11 + CRF13 11 CRF04 + U 12 CRF02_AG (68.4%) HIV subtypes in Cameroon blood donors – 2006 to present

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

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