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

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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:

EQAPOL@duke.edu

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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 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

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 EQAPOL@duke.edu 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