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1
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 4th 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
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
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
6
Viral Diversity Program Workflow 7
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 • TCID50, Final VL testing (multiple platforms), sequencing,
coreceptor usage, sterility testing
Distribute HIV to Research Laboratories • Inventory available through EQAPOL web-based system
Two-step culture process
8
Source Viral
Specimen Plasma, PBMCs,
previously-cultured
supernatant
Feeder Cells Pooled
cryopreserved
PBMCs
Master Lot • ≈40mL
• average titer >4.90e+09 cp/mL
• average culture time is 8.2 days
Aliquot of
master lot
Culture
Step 1:Small-Scale
Culture
Step 2:Large-Scale
Culture
Feeder Cells Pooled
cryopreserved
PBMCs
Culture
High Titer Culture Supernatant • ≈250 1mL aliquots
• average titer >4.48e+09 cp/mL
• 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
Characterization Performed on All Final
Viral Products
9
Viral Products
(Culture supernatant and spiked
plasma)
Sterility Testing
• Endotoxin
• Mycoplamsa
• Bacterial Inoculation
TCID50
• TZM-bl cell-based assay
Viral Load
• Roche 2.0
• Abbott (FDA)
• Future: bDNA
Sequencing
• Data uploaded to GenBank
Coreceptor Usage
• Phenotype
• NP-2 Cell Assay
Pre-culture Testing
• Fiebig staging
• VL and p24
• Sterility
• Pre-culture sequencing
10
Sample Certificate of Analysis (COA)
EQAPOL Duke University Medical Center 2 Genome Court Durham, NC, 27710 [email protected]
CERTIFICATE OF ANALYSIS
Product Information
Virus Name: DEMB94ZA001.01
Product Type (HIV-spiked Serum or Cell Culture Supernatant): Cell Culture Supernatant
Clade: B
Final Harvest Date: 08/30/2011
Viral Load of Product: 1.5 *1010 copies/mL
Co-receptor Usage (determined using cell culture supernatant): CCR5
TCID50 of Product (cell culture supernatants only): 2.5 * 104 TCID50/mL
Sterility Information
Mycoplasma Testing: PASS
Endotoxin Testing: Concentration: 0.05 EU/mL PASS
Bacterial Testing:
Soybean Casein Digest Medium PASS
Fluid Thioglycolate Medium PASS
Source Specimen Information
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
• All viral products
generated for EQAPOL
will have a COA
• COA will be signed by
EQAPOL Central Quality
Assurance Unit
• COA will be available for
download on EQAPOL
web-based system
11
0.02
A1.AU.03.PS1044_Day0
DEMF210CM007
DEMB10CN002
F2.CM.02.02CM_0016BBY F2.CM.97.CM53657
D.TZ.A280 DE04708ES004
DEMB99DE001
DEMG10CM008
C.IN.95.95IN21068
01_AE.AF.07.569M
H.GB.00.00GBAC4001
DEMB09BO001
DEMC06ES003
F2.CM.95.95CM_MP257
02_AG.CM.99.pBD6_15
DEMC09ZA008
47_BF.ES.08.P1942
DEMA105TZ001 A1.RW.92.92RW008
B.TH.90.BK132
05_DF.BE.93.VI961
DEURF10DZ001
DEMC08NG001
DEMB08ES001
DE01405BR001
A3.DDJ360
06_cpx.EE.EE0359
F1.BR.93.93BR020_1
A2.CY.94.94CY017_41
A1.UG.92.92UG037
DEURF07ES002
DEMG09ES002
A3.DDI579
DEMB05FR001
F1.BE.93.VI850
D.CD.83.ELI.K03454
24_BG.ES.08.X2456_2
DEMBF09ES003
K.CM.96.96CM_MP535
05_DF.ES.99.X492
02_AG.NG.x.IBNG
04_cpx.GR.97.GR84_97PVMY
DEMB08UY001
DEMC10ZA001
14_BG.ES.00.X605
G.NG.92.92NG083
D.CMCM_4412HAL
A2.CD.97.97CDKTB48
01_AE.CN.05.05GX001
47_BF.ES.08.X2457_2
G.PT.x.PT2695
N.CM.95.YBF30
DEMC09ZA009
05_DF.BE.x.VI1310
DEMB99PL001
01_AE.TH.90.CM240
DEMF110ES001
G.KE.93.HH8793_12_1
DEURF09ES005
DEURF10US008
DE00109CN004 DE00110CN001
J.CD.97.J_97DC_KTB147
N.CM.97.YBF106
F1.FR.96.96FR_MP411
24_BG.CU.03.CB378
C.ET.86.ETH2220
DE00109CN003
DE00400GR002 04_cpx.GR.91.GR11_97PVCH
C.BR.92.BR025_d
04_cpx.CY.94.94CY032_3
A2.CMCM_1445MV
D.UG.94.94UG114
DE02408ES002
DE00711CN003
DEMC07BR003
DE00206AO001
DEMC07AO001
DEMB10VE001
DEMA106ES002
06_cpx.AU.96.BFP90
F1.FI.93.FIN9363
DEMC08ZA011
G.BE.96.DRCBL
J.SE.93.SE9280_7887
DEMG05ES001
K.CD.97.97ZR_EQTB11
F2.CM.95.95CM_MP255
H.CF.90.056
24_BG.CU.03.CB471
06_cpx.GH.03.03GH173_06
14_BG.PT.00.00PTHDE10
B.FR.83.HXB2_LAI_IIIB_BRU
02_AG.LR.x.POC44951
C.ZA.04.04ZASK146
J.CM.04.04CMU11421
DE00208CM001
DE00208CM004
A3.DDJ369
DEMF210CM001
DEMBF09ES006
N.CM.02.DJO0131
DEMD10CM009
B.US.98.1058_11
B.NL.00.671_00T36
DEMB08FR002
DEMC07ZA011
DEMB09US002
14_BG.ES.00.X623
B A1/B
D
F1
C
CRF01_A
E
A1
CRF02_AG/A3
CRF02_A
G
CRF14_BG
G
CRF04_cpx
CRF02_AG/CRF06_cpx
F2
CRF24_BG
CRF47_BF B/F
B/F
A1/B
B
B
B B, C,
BF, 14
B
C
A
C
C D, G, 02, F2
B, BF, C,
G, F1, 02
B, 01, 07
04
B
02, 02/06
B, C
• Algeria
• Angola
• Bolivia
• Brazil
• Cameroon
• China
• France
• Germany
• Greece
• Nigeria
• Poland
• South Africa
• Spain
• Tanzania
• Uruguay
• US
• Venezuela
Current Diversity of Panel
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:
12
13
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15
17
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