Center for Biologics Evaluation and Research

1
Standardization CBER update
June 12, 2007 XX SoGAT Indira Hewlett,
Ph.D. Chief, Lab. of Molecular Virology DETTD/CBER
/FDA
2
Current status of NAT in United States

• Donor screening NAT assays licensed for HIV-1,
  HBV, HCV, WNV
• FDA guidance published in 2004 recommending
  implementation of HIV-1 and HCV NAT for blood and
  plasma
• In-process quality control NAT for Parvo virus
  B19, Hepatitis A virus (HAV) testing of plasma
  for further manufacturing

3
Current status of CBER NAT standards

• HIV-1, HBV, HCV and WNV NAT panels currently
  available for lot release of licensed NAT
• HIV-1 subtype NAT panel formulated
• HIV-2 NAT panel being formulated

4
CBER HIV-1 RNA Panel

• HIV-1 subtype B panel for routine lot release
• Cultured patient isolate, heat inactivated and
  diluted with defibrinated Ab-ve plasma
• Gag, pol and env regions sequenced
• Virus dilutions tested by 15 labs in
  collaborative study
• 8 positives 10, 50, 100, 500, 2500, 5000, 2.5 x
  104, and 2.5 x 105 copies/mL and 2 negatives
• CBER standard is 100 IU/ml for pool test and
  10,000 IU/ml for original donation

5
NIBSC/SoGAT Collaborative Study Calibration of
HIV-1 Working reagents

• Candidate Log IU/mL
• Preparation
• QC105 (NRL, Australia) 4.04
• B5 (CBER, USA) 2.21
• B10 (CBER, USA) 3.82
• Pelispy (CLB, The Netherlands) 4.43
• PWS-1 (NIBSC, UK) 3.56
• PWS-3 (NIBSC, UK) 2.72
• IRC (Utrecht, The Netherlands) 4.27
• Ref Davis et al (2003) J Virol Methods 10737-44

6
Genetic diversity of HIV

• Two major types of HIV HIV-1 and HIV-2
• 3 distinct HIV-1 groups identified to date M
  (major), O (outlier) and N (non-M, non-O)
• Group M consists of many subtypes of viruses
  (A-H) and group O (3 prototype classes)
• 5 major HIV-2 subtypes increasing number being
  identified
• Increasing numbers of circulating recombinant
  forms (CRFs) of HIV-1, up to 34 in the
  literature 3 additional as yet unreported

7
Worldwide distribution of predominant HIV-1 group
M subtypes and CRFs
CRF14_BG
B
Adapted from Thomson et al. Lancet Infect Dis
2002.
8
Diagnostic implications

• NAT assays based on oligonucleotides representing
  limited regions of the viral genome
• Potential impact on sensitivity for new variants
• HIV genetic diversity evolving globally at a
  fairly rapid rate, new variants
• Different rates of disease progression, clinical
  outcomes for different subtypes
• Accurate and sensitive detection of subtypes may
  be clinically important
• Need for surveillance for variants and reference
  reagents for detection of major, new variants

9
CBER HIV-1 subtype RNA Panel

• HIV-1 subtype panel
• 7 subtypes of HIV-1 group M A, B, C, D, E, F, G
  group N, and group O
• Pilot-scale prototype panels were tested in
  collaborative study involving 5 NAT manufacturers
  at various dilutions
• Data analyzed at FDA and consensus values
  assigned to viral stocks
• Full-scale final panel formulated 250 vials per
  member at log 4 to log 2
• Storage at -70 C degrees at BBI BTRL, stability
  data for 3 years.

10
HIV-1 Subtype Isolates used in current CBER
Panel
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Current status of CBER HIV-2 panel development

• Seven isolates of HIV-2 belonging to subtype A
  from Spain
• Isolates were tested by three manufacturers at
  different serial dilutions
• Statistical analysis of data for value assignment
• Panel being formulated with 2 isolates to include
  log ranges of 2 4

12
CBER HIV-2 Panel Isolate Testing Summary (Log 10)
13
Current HIV-1 panel efforts

• Current CBER panel expanded to include major new
  variants CRF_02 AG and CRF_01 AE
• CBER has characterized isolates of CRF_02 AG
• CRF_01 AE strains and current strains of major
  subtypes acquired through international
  collaborations

14
Future HIV panel efforts cont

• Collect viral strains representing different
  HIV-1 and HIV-2 subtypes, different geographic
  regions through collaborations
• Determine and assign copy number of each
  candidate viral strain through collaborative
  studies
• Dilute selected viral strains to chosen copy
  number
• Determine stability of final panel

15
CBER HCV RNA Panel

• A 10-member HCV panel derived from the HCV stock
  diluted with anti-HCV negative, defibrinated
  pooled plasma, genotype 1b
• 8 positives with target levels of 5, 10, 50, 100,
  500, 103, 104, and 105 copies/mL, 2 negatives
• Current HCV standard 100 IU/ml and 5,000 IU/ml
  for the original donation
• (Ref Yu et al, Hepatology 1998 28566A)

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HCV NAT Standard

• Sample IU/mL Genotype Anti-HCV
• International Std 100,000 1a
  Pos
• NIBSC 96/586 710 3
  Pos
• CLB/Pelispy 1,000 1a
  Neg
• PEI Ref 5 (Germany) 25,000 1
  Neg
• ISS 0498 (Italy) 1,700 1
  Pos
• CBER member 1 250 1b
  Neg
• (1000 copies/mL)
• Ref Saldanha et al, Vox Sang 2000 78 (4)
  217-24

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HBV NAT panel

• CBER HBV DNA panel derived from a window period
  specimen genotype A, serotype adw2
• Panel members are 0, 10 and 100 copies/ml
• Panel tested by 3 NAT manufacturers

18
WNV testing

• WNV transmission by transfusion identified in
  2002
• All reported cases due to donations collected in
  acute, viremic phase
• NAT most appropriate strategy to interdict
  infectious donations
• Virus titer in blood low compared to other
  transmissible viruses (1-5x103 copies/ml) and
  the viremia is transient.
• Need for standards to evaluate sensitivity and
  correlate infectivity with NAT

19
WNV NAT Panel

• FDA NY99 and FDA-Hu2002 (patient derived)
  isolates inactivated by heat treatment
  characterized by genetic sequencing
• Viral infectivity determination
• RNA concentration measurements
• Heat treatment of the virus resulted in loss of
  infectivity by PFU and 2 to 3 log reduction of
  copy number as determined by TaqMan
• The correlation between PFU and RNA copy number
  is 1500
• Final panel specifications established through
  collaborative studies

20
WNV Panel Formulation and Evaluation in
Collaborative Studies

• Panel formulated using NY99-FDA and FDA-Hu2002
  strains (patient isolate)
• composed of 14 coded members (1000, 500, 100, 50,
  10, 5 and 0 viral copies/mL, one from each
  isolate)
• Distributed to 7 independent laboratories
• Final panel has been formulated FDA standard
  for WNV NAT is 100 copies/ml
• Stability studies panel stable for at least 17
  months at 4oC

21
Dengue

• Most common viral disease transmitted by
  arthropod vectors worldwide
• Endemic in tropics and subtropics 50-100 million
  annual cases worldwide
• 250,000-500,000 annual cases dengue hemorrhagic
  fever (DHF)
• CDC investigated 199 suspected of clinical dengue
  cases in 2005 Travel?
• 78/199 (39) had laboratory diagnosis of dengue
• 70/78 (90) had elevated anti-dengue IgM
  antibodies
• 8/78 (10) had viremia by PCR or viral isolation.
• 18/199 (9) patients without reported travel risk
  diagnosed by elevated anti-dengue IgM antibodies
  suggesting autochthonous transmission.

22
Future efforts - collaborative study

• 1- Acquire viremic specimen from all 4 serotypes
• 2- Isolate virus by cultivation
• 3- Perform genetic characterization of viral
  isolates
• 4- Determine viral load in culture supernatant in
  collaborative studies
• 3- Define final panel formulation in
  collaborative studies
• 4- Determine the stability the final panel (test
  various storage and shipping conditions)

23
Parvovirus B19 NAT as an In-Process Control

• Require validation as an analytical test and
  approve it under relevant products license
• Proposed limit lt104 IU of B19 DNA per mL in all
  manufacturing pools
• B19 transmissions associated with S/D Treated
  Pooled Plasma in a phase 4 study in healthy
  donors
• lt104 GE/mL in non-transmitting lots
• Viral neutralization by anti-B19 in pools
• Viral clearance by manufacturing procedures

24
CBER B19 DNA Standard

• Derived from a window-period plasma unit, 1012
  GE/mL
• Diluted with pooled, cryo-poor plasma negative
  for HBsAg, anti-HIV, anti-HCV, anti-B19, HIV RNA,
  HCV RNA, HBV DNA, B19 DNA, and HAV RNA
• 106 IU/mL (1 mL/vial) stored at ? -70 C

25
WHO/NIBSC Collaborative Study International
Standard for B19 DNA

• Candidate Log GE/mL Log
  IU/mL
• Preparation Targeted Mean
• AA (NIBSC, FD) 6 5.92 6
• BB (NIBSC, FD) 6 5.82
• CC (CBER, Liquid) 6 5.89 6
• DD (CLB, Liquid) 7 - 8 7.7

26
CBER HAV RNA Standard (I)

• Derived from a window-period plasma unit, 106
  copies/mL
• Diluted with a pooled, cryo-poor plasma negative
  for anti-HAV, HBsAg, anti-HIV, anti-HCV, HIV RNA,
  HCV RNA, HBV DNA, B19 DNA and HAV RNA
• ca. 104 copies/mL consensus level determined by
  the WHO/NIBSC collaborative study

27
Summary

• FDA has established panels for HIV, HCV, HBV,
  WNV, B19 and HAV and standards for licensing
  tests
• Panel for HIV-2 being formulated
• Panel for major emerging HIV variants (CRF 02 AG
  and CRF 01 AE) being developed
• Future efforts include Dengue panel development

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Standards for New Emerging Diagnostic
Technologies

• Gene Chips, microarrays, nanotechnology
• Fusion of micro- and nanotechnologies
• Reduce time, improve sensitivity, simplify assay
  procedure and costs
• Miniaturization technologies for low cost chips

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General features/applications of
microarrays/nanoparticles

• Platform for detection of amplified products or
  oligonucleotides i.e. NAT
• Microscopic spots of immobilized nucleic acid
  sequences
• Samples that react with the arrays
• Detection system that quantitates hybridization
  or binding events
• Computer assisted data analysis
• Gene expression, genotyping, SNP, comparative
  genome hybridization
• Potential for multiplexing allowing detection of
  different pathogens on the same array

30
Common Nanoscale Particles in Biological Use
McNeil, (2005), J. Leuk. Biol., 78585-594
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Nanoparticle/microarray detection of avian
influenza virus subtypes
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Issues for assay standardization

• Generally the same as for NAT for gene arrays
• RNA purification, Quality of RNA
• Amplification linear amplification
• Slide Printing
• Variation in spot intensities
• Labeling probe, direct labeling
• Hybridization efficacy
• Data analysis

33
Reference reagents

• Currently no reference materials available for
  microrray/nanotechnology pathogen detection
  assays
• Pooled RNA (or proteins for protein arrays)
  representing various pathogens/targets spotted on
  array under evaluation for expression arrays
• Reference materials for gene arrays/nano-assays
  would likely be similar to those used for current
  methods i.e. NAT, e.g. virus preparations for a
  viral detection assay
• Collaborative study efforts to evaluate
  suitability of current standards for
  microarray/nanotechnology based assays are needed
  as they are developed

34
Summary

• Microarrays increasingly evaluated for multiplex
  pathogen detection
• Nanotechnologies useful for both individual and
  multiplex detection, protein and nucleic acid
  detection simultaneously
• Reference materials needed to facilitate
  comparison of different microarray and
  nanotechnology assays
• Regardless of technology or platform, reference
  preparations would generally be the same for a
  pathogen detection assay i.e to allow accurate
  detection of the pathogen

35
Acknowledgements

• CBER/FDA WRAIR
• S. Lee N. Michael
• M. Yu ARC
• O. Wood S. Stramer
• M. Rios Carlos Salud
• S. Kerby V. Soriano
• R. Biswas NYDOH
• R. Duncan L. Kramer
• C. Hsia NYU
• J. Zhao P. Nyambi