Update on HIV Variants Panel Indira Hewlett, Ph.D LMV/DETTD/CBER/FDA 23 rd SoGAT meeting, 2012
Update on HIV Variants Panel
Indira Hewlett, Ph.D
LMV/DETTD/CBER/FDA
23rd SoGAT meeting, 2012
EQAPOL VIRAL DIVERSITY PROJECT
FOR GLOBAL HIV PANELS
2
4/16/2012
NIAID- Duke Global HIV Viral Panels
Project: Purpose
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
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 in 2010; Duke awarded 7 year
contract for full scale study
Presentation Overview
5
Introduction to EQAPOL Viral Diversity Program
Workflow for Viral Diversity Program
Current Viral Diversity Panel
Web-based Application
Request for Samples
Overview of EQAPOL Viral Diversity
Program • EQAPOL (External Quality Assurance Oversight
Laboratory)
• Seven year, $52.8 million NIAID contract
• Encompasses multiple EQA programs (ELISpot, Flow Cytometry
Luminex) and Viral Diversity Program
• Viral Diversity Program Goals
• Create HIV panels 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 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
International Collaborators for Viral
Specimens • Mike Busch, Blood Systems Research Institute
• Acquisition of Nat-Yield and recently infected plasma
• Collaboration with multiple international and domestic blood donor sites
• Lucia Perez, Instituto de Salud Carlos III (Madrid, Spain)
• Culture supernatants from wide-range of geographic and genetic subtypes
• Dr. Hong Shang, First Affiliated Hospital of China Medical University (Shengyang, China)
• Culture supernatants from China
• Indira Hewlett, FDA (USA)
• Collaboration to receive culture supernatants and source plasma from Cameroon
• Testing products on Abbott Viral Load assay
8
Two-step culture process
9
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
Sequence and Coreceptor Analysis
Whole Genome Analysis
• RNA extraction
• Plasma: pre-culture screening
(bulk PCR)
• Virus stock: final
characterization (SGA)
• Amplification of two overlapping
half genomes by PCR
• Genotyping
• Similarity plot
• Bootstrap plot
• Phylogenetic tree
Coreceptor Usage
• Determination of coreceptor usage by cell culture (NP-2 cells)
• CCR5
• CXCR4
• Prediction of coreceptor usage by sequence analysis
• Web PSSM
• Geno2pheno
• 11/25 rule
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Summary of Current Viral Diversity Panel
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As of March 2012, 44 viruses cultured
250-300 1mL vials of culture supernatant
100 1mL vials of HIV-spiked plasma (1 *107cp/mL)
30 1mL vials of HIV-spiked plasma (5 *107cp/mL)
17 countries represented
13 genetic subtypes represented
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Current Geographic Diversity of Panel
• Algeria
• Angola
• Bolivia
• Brazil
• Cameroon
• China
• France
• Germany
• Greece
• Nigeria
• Poland
• South Africa
• Spain
• Tanzania
• Uruguay
• US
• Venezuela
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Current Genetic Diversity of Panel
Spain (1)
Tanzania (1)
A Bolivia (1)
France (1)
Germany (1)
Poland (1)
Spain (1)
Uruguay (1)
US (2)
Venezuela (1)
China (1)
B
Spain (2)
BF
Angola (1)
Brazil (1)
France (1)
Nigeria (1)
South Africa (5)
Spain (1)
C
Cameroon (1)
D
Cameroon (1)
Spain (2)
G
China (3)
CRF01
Cameroon (2)
Spain (1)
Algeria (1)
CRF02
Greece (1)
CRF04
China (1)
CRF07
Brazil (1)
CRF14
Spain (1)
F1
Cameroon (2)
F2
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:
15
EQAPOL Web-based Application: Inventory Page
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https://eqapolapp.dhvi.duke.edu
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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
Request for Samples
• EQAPOL is looking for additional viral source specimens
• Plasma, PBMCs, culture supernatants
• Virus characteristics desired
• Acute infections
• Genetic Diversity
• Geographic Diversity
• Samples from recent years (last five years)
• Please contact [email protected] to contribute
samples
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Acknowledgements
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EQAPOL Viral Culture Team
• Thomas Denny (PI)
• Holly Alley
• Christie Brinkley
• Sara Brown
• Todd DeMarco
• Sarah Keinonen
• Kyle Liebl
• Brook Liebl
• Laura Racz
• Linda Walker
• Dongning (Daisy) Wang
EQAPOL Viral Molecular Characterization Team
• Feng Gao
• Yue Chen
• Bhavna Hora
EQAPOL Central Quality Assurance Unit •Marcella Sarzotti-Kelsoe
•Chris Todd
BSRI •Mike Busch
•Leslie Tobler
FDA •Indira Hewlett
•Viswanath Ragupathy
Instituto de Salud Carlos III •Lucia Perez
NIAID Jim Lane
Marco Schito
First Affiliated Hospital of China Medical University •Hong Shang
National Institute of Allergy and Infectious Disease
CBER HIV RNA genotype panels
CBER has an ongoing effort to develop HIV-1 and HIV-2 RNA
genotype reference panels for assay standardization and kit
lot testing.
CBER currently has reference panels consisting of one
primary isolate each of group M subtypes A-G, groups O, N,
and HIV-2 subtype A cultured in PBMC and characterized by
sequencing.
Recent panels include CRF02_AG, CRF01_AE, recombinants
BF and BC.
Virus isolates are inactivated by heat treatment at 60 C for 60
mins.
Panel composed of 3 members of each subtype at 103, 104
and 105 copies/ml spiked in negative plasma.
HIV-1 BC and BF NAT Panel development
• CBER obtained viruses from NIBSC, UK and Carlos de Salud institute, Spain and in-house project in Cameroon
• Viruses were cultured in PBMC to high titers.
• Viral stocks were heat-treated to inactivate the virus, no loss of RNA copy number
• Aliquots were sent to multiple collaborating labs for copy number determination
• Data analysis has been completed and panel has been formulated and is available for use
B/C – B/F Isolate Titer Testing Summary(log10)
Isolate ID Lab A Lab B Lab C Mean Standard
Deviation
P1942 9.11 9.09 8.81 9.0 0.14
92023 9.0 9.16 8.63 8.93 0.22
X531-2 8.25 8.29 7.92 8.15 0.17
2754-2 8.67 8.78 8.23 8.56 0.24
2457-2 9.16 9.54 8.93 9.21 0.25
475-2/754-
2
8.93 9.21 8.66 8.93 0.22
Future efforts
• CBER has obtained virus isolates of Group P
and novel Group N from the Laboratoire de
Virologie, Institut de Biologie Clinique, Rouen
• Viruses will be cultured in PBMC to high titers.
• Viral stocks will be heat-treated to inactivate the virus, no loss of RNA copy number
• Aliquots will be sent to multiple collaborating labs for copy number determination.
• Panel will be formulated and made available for use in the future
Acknowledgments
• DETTD/CBER
Sherwin Lee
Owen Wood
Viswanath Ragupathy
Stephen Kerby
Collaborators
• Lucia Perez, Carlos de Salud institute, Spain
• Phillipe Nyambi, NYU, US
• Test manufacturers
Roche Molecular Systems
Gen-Probe Inc.
Abbott Diagnostics