Status of Analyses & Publications of Previous and Active - PowerPoint PPT Presentation

Status of Analyses & Publications of Previous and Active Virology WG Collaborative Studies • Published: • International Survey on NAT Testing of Blood Donations: Expanding Implementation and Yield from 1999 to 2009. Vox Sang International Forum; 2011 • Pilot Studies for Development of an HIV Subtype Panel for Surveillance of Global Diversity. AIDS Res Hum Retrovirology; 2012 • Studies completed, manuscripts drafted: • NAT and HCV Ag Testing Performance for Reducing the HCV Window Phase (Egypt, France, Germany, Japan, Lithuwania, Poland, USA), led by Syria Laperche • Distribution of HIV Viral Loads in NAT Yield Donations and Detection by 4 th Generation HIV Ag/Ab assays in Donors from France, Germany, Japan, Poland, South Africa and the United States, led by Leslie Tobler and Mike Busch • Rates, Demographics and Virologic Profiles of HIV Elite Controllers Detected Through Donor Screening in the US, France, Germany, South Africa and Australia, led by Mike Busch • Data compiled, 2 presentations at ISBT Cancun, manuscripts in development: • International ID-NAT Study Group (17 countries) Nico Lelie, Steve Kleinman, Brian Custer, Mike Busch • HIV Transmission Risk and Efficacy of Screening Strategies • HCV Transmission Risk and Efficacy of Screening Strategies • Efficacy of HBV Screening Assays in an International Survey • Cost Effectiveness of Alternative NAT and Serological Screening Strategies for HIV, HCV & HBV 1

Global Distribution of HIV-1 Genotypes

HIV Viral Panels Project Requirements • Well characterized HIV reference panels encompassing epidemic • Full length single genome sequencing • Verified RNA concentration • Fiebig staging and serological profiles for current assays/platforms including rapid POC assays • Comparisons of VL from different FDA approved commercially available platforms • Panels with larger volumes for use on newer diagnostic platforms • Pilot study completed and published in 2010; Duke awarded 7 year contract for full scale study

5

6 Overview of EQAPOL Viral Diversity Program • EQAPOL (External Quality Assurance Oversight Laboratory) • Seven year NIAID contract • Encompasses multiple EQA programs (ELISpot, Flow Cytometry Luminex) and the Viral Diversity Program • Viral Diversity Program Goals • Create HIV panels of plasma and viral isolates representative of worldwide viral diversity • Grow 50 high-titer/high-volume cultures per year • Characterize viruses • Conduct work in a GCLP environment • Make viruses available for order through a web-based system

7 Viral Diversity Program Workflow Collect viral specimens • Sources include National Blood Collection origanization (BSRI), FDA, Instituto de Salud Carlos III and First Affiliated Hospital of China Medical University Perform Initial Characterization • Fiebig staging, VL, p24, pre-culture sequencing, sterility testing Culture to High-titer, High-volume • Two step culture process • Results in culture supernatant and HIV-spiked plasma Characterize Virus • TCID 50 , Final VL testing (multiple platforms), sequencing, coreceptor usage, sterility testing Distribute HIV to Research Laboratories • Inventory available through EQAPOL web-based system

8 Two-step culture process Step 1:Small-Scale Step 2:Large-Scale Culture Culture Source Viral Feeder Cells Feeder Cells Aliquot of Specimen Pooled Pooled master lot Plasma, PBMCs, cryopreserved cryopreserved previously-cultured PBMCs PBMCs supernatant Culture Culture Master Lot High Titer Culture Supernatant • ≈40mL • ≈250 1mL aliquots • average titer >4.90e+09 cp/mL • average titer >4.48e+09 cp/mL • average culture time is 8.2 days • Culture time generally 4-7 days HIV Spiked-plasma • ≈100 1mL aliquots at 1e+07 cp/mL • ≈20 1mL aliquots at 5e+07 cp/mL

9 Characterization Performed on All Final Viral Products Viral Load Sequencing • Roche 2.0 • Data uploaded to • Abbott (FDA) GenBank • Future: bDNA TCID 50 Coreceptor Usage • TZM-bl cell-based • Phenotype assay • NP-2 Cell Assay Viral Pre-culture Sterility Testing Testing Products • Endotoxin • Fiebig staging (Culture • Mycoplamsa • VL and p24 • Bacterial • Sterility supernatant Inoculation • Pre-culture and spiked sequencing plasma)

10 CERTIFICATE OF ANALYSIS Sample Product Information Certificate of Virus Name: DEMB94ZA001.01 Product Type (HIV-spiked Serum or Cell Culture Supernatant): Cell Culture Supernatant Analysis Clade: B (COA) Final Harvest Date: 08/30/2011 Viral Load of Product: 1.5 *10 10 copies/mL Co-receptor Usage (determined using cell culture supernatant): CCR5 • TCID 50 of Product (cell culture supernatants only): 2.5 * 10 4 TCID 50 /mL All viral products generated for EQAPOL Sterility Information will have a COA Mycoplasma Testing: PASS • COA will be signed by Endotoxin Testing: Concentration: 0.05 EU/mL PASS EQAPOL Central Quality Bacterial Testing: Assurance Unit Soybean Casein Digest Medium PASS • COA will be available for Fluid Thioglycolate Medium PASS download on EQAPOL Source Specimen Information web-based system Fiebig Stage of Source Plasma (if available): II Country of Origin for Source Specimen: South Africa Year of Donation for Source Specimen: 1994 Additional Information about Source Specimen: ______________________________ ______________ Quality Assurance Signature Date

11 Current Diversity of Panel DEMB99DE001 DEMB09US002 B.US.98.1058_11 B.TH.90.BK132 B.FR.83.HXB2_LAI_IIIB_BRU DEMB99PL001 B DEMB08ES001 DEURF09ES005 A1/B DEMB10VE001 DEMB08UY001 DEURF10US008 A1/B DEMB08FR002 DEMB05FR001 DEMB09BO001 DEMB10CN002 B B.NL.00.671_00T36 DEMD10CM009 D D.CMCM_4412HAL D.CD.83.ELI.K03454 D.UG.94.94UG114 B, C D.TZ.A280 DE04708ES004 47_BF.ES.08.X2457_2 CRF47_BF 47_BF.ES.08.P1942 B, BF, C, 04 DEMBF09ES003 B/F B 05_DF.ES.99.X492 G, F1, 02 05_DF.BE.93.VI961 05_DF.BE.x.VI1310 DEMF110ES001 02, 02/06 F1.FI.93.FIN9363 B, 01, 07 F1.BE.93.VI850 F1 F1.BR.93.93BR020_1 F1.FR.96.96FR_MP411 B DEMBF09ES006 B/F DEMF210CM007 F2.CM.97.CM53657 D, G, 02, F2 C F2.CM.02.02CM_0016BBY F2 F2.CM.95.95CM_MP257 F2.CM.95.95CM_MP255 DEMF210CM001 DE02408ES002 A 24_BG.ES.08.X2456_2 24_BG.CU.03.CB378 CRF24_BG 24_BG.CU.03.CB471 B, C, DE01405BR001 C 14_BG.PT.00.00PTHDE10 B 14_BG.ES.00.X605 BF, 14 14_BG.ES.00.X623 CRF14_BG DEMG09ES002 G.PT.x.PT2695 G.KE.93.HH8793_12_1 G.NG.92.92NG083 G C G.BE.96.DRCBL DEMG10CM008 DEMG05ES001 B 06_cpx.GH.03.03GH173_06 06_cpx.AU.96.BFP90 06_cpx.EE.EE0359 DE00110CN001 DE00711CN003 CRF01_A DE00109CN004 DE00109CN003 01_AE.TH.90.CM240 • • 01_AE.AF.07.569M Algeria Nigeria E 01_AE.CN.05.05GX001 DEURF07ES002 CRF02_AG/A3 A3.DDI579 A3.DDJ360 • • Angola Poland A3.DDJ369 DEMA106ES002 A1 A1.AU.03.PS1044_Day0 DEMA105TZ001 • • A1.UG.92.92UG037 Bolivia South Africa A1.RW.92.92RW008 DEURF10DZ001 CRF02_AG/CRF06_cpx DE00208CM004 02_AG.NG.x.IBNG • • Brazil Spain 02_AG.LR.x.POC44951 CRF02_A DE00206AO001 02_AG.CM.99.pBD6_15 DE00208CM001 • • A2.CY.94.94CY017_41 G Cameroon Tanzania A2.CMCM_1445MV A2.CD.97.97CDKTB48 DE00400GR002 04_cpx.GR.97.GR84_97PVMY CRF04_cpx • • 04_cpx.GR.91.GR11_97PVCH China Uruguay 04_cpx.CY.94.94CY032_3 J.SE.93.SE9280_7887 J.CM.04.04CMU11421 • • J.CD.97.J_97DC_KTB147 France US K.CM.96.96CM_MP535 K.CD.97.97ZR_EQTB11 DEMC06ES003 DEMC07BR003 • • C.BR.92.BR025_d Germany Venezuela C.ET.86.ETH2220 DEMC10ZA001 DEMC09ZA009 C C.ZA.04.04ZASK146 • Greece DEMC08ZA011 C.IN.95.95IN21068 DEMC09ZA008 DEMC07ZA011 DEMC07AO001 DEMC08NG001 H.GB.00.00GBAC4001 H.CF.90.056 N.CM.95.YBF30 N.CM.97.YBF106 N.CM.02.DJO0131 0.02

12 EQAPOL Web-based Application • Web-based application developed for EQAPOL Viral Diversity: • Data regarding culture process • Viral characterization results • Inventory of viral products • Allows external users to order viral products • Tracks shipping and receipt of viral products • Track sites participating in the program • To use the system, users must request access via email: EQAPOL@duke.edu

13

14

15

Objectives of REDS-III HIV Diversity Project • Comparison, training and adoption of improved sequencing methods for pol gene and/or full genome to enable high yield of sequence data and better drug resistance classification and subtype assignment than in REDS-II • Perform detailed env diversity analysis targeting NAT yield and recent SC incident cases to characterize T/F viruses, and to provide data for validation of deep sequencing study (using 454 deep sequencing platform) • Demonstrate evolution of T/F viruses in blood donations with incident HIV infections by performing deep sequencing study on 300 acutely infected blood donor samples, 100 from the US, 100 from Brazil and 100 from SA, with 50 from 15-20 years ago and 50 recent NAT yields or very recent SCs per country 17