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

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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

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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

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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

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Sample Certificate of Analysis (COA)

EQAPOL Duke University Medical Center 2 Genome Court Durham, NC, 27710 EQAPOL@duke.edu

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

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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:

EQAPOL@duke.edu

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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