Standardization – CBER update June 12, 2007 XX SoGAT Indira Hewlett, Ph.D. Chief, Lab. of Molecular Virology DETTD/CBER/FDA

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

Current status of CBER NAT standards u HIV-1, HBV, HCV and WNV NAT panels currently available for lot release of licensed NAT u HIV-1 subtype NAT panel formulated u HIV-2 NAT panel being formulated

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 10 4, and 2.5 x 10 5 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 107:37-44

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

Adapted from Thomson et al. Lancet Infect Dis Worldwide distribution of predominant HIV-1 group M subtypes and CRFs CRF14_BG CRF01_AE B

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

CBER HIV-1 subtype RNA Panel u 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.

HIV-1 Subtype Isolates used in current CBER Panel Clade Origin ID #Viral copies/ml A Djbouti DJ/258/ x 10 6 B United States x 10 9 C Senegal SE/364/ x 10 7 D Uganda UG/021/ x 10 7 E Thailand TH/022/ x 10 7 F Brazil BZ/162/ x 10 7 G Nigeria G3/Nigeria 1.2 x 10 7 N Cameroon Sinnousi 2.0 x 10 7 O Spain 1422/German 8.1 x 10 6

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

CBER HIV-2 Panel Isolate Testing Summary (Log 10) Isolate ID Manufacture A Manufacture B Manufacture C Mean Standard Deviation B B B B B B B

Current HIV-1 panel efforts u Current CBER panel expanded to include major new variants CRF_02 AG and CRF_01 AE u CBER has characterized isolates of CRF_02 AG u CRF_01 AE strains and current strains of major subtypes acquired through international collaborations

Future HIV panel efforts– con’t u Collect viral strains representing different HIV-1 and HIV-2 subtypes, different geographic regions through collaborations u Determine and assign copy number of each candidate viral strain through collaborative studies u Dilute selected viral strains to chosen copy number u Determine stability of final panel

15 CBER HCV RNA Panel u 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, 10 3, 10 4, and 10 5 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; 28:566A)

16 HCV NAT Standard Sample IU/mL Genotype Anti-HCV International Std 100,000 1a Pos NIBSC 96/ Pos CLB/Pelispy 1,000 1a Neg PEI Ref 5 (Germany) 25,000 1 Neg ISS 0498 (Italy) 1,700 1 Pos CBER member # b Neg (1000 copies/mL) [Ref: Saldanha et al, Vox Sang 2000; 78 (4) ]

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

WNV testing u WNV transmission by transfusion identified in 2002 u All reported cases due to donations collected in acute, viremic phase u NAT most appropriate strategy to interdict infectious donations u Virus titer in blood low compared to other transmissible viruses (~1-5x10 3 copies/ml) and the viremia is transient. u Need for standards to evaluate sensitivity and correlate infectivity with NAT

WNV NAT Panel u FDA NY99 and FDA-Hu2002 (patient derived) isolates inactivated by heat treatment characterized by genetic sequencing u Viral infectivity determination u RNA concentration measurements u 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 u The correlation between PFU and RNA copy number is 1:500 u Final panel specifications established through collaborative studies

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 4 o C

Dengue Most common viral disease transmitted by arthropod vectors worldwide Endemic in tropics and subtropics; million annual cases worldwide 250, ,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.

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)

Parvovirus B19 NAT as an In-Process Control u Require validation as an analytical test and approve it under relevant product’s license u Proposed limit: <10 4 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 »<10 4 GE/mL in non-transmitting lots –Viral neutralization by anti-B19 in pools –Viral clearance by manufacturing procedures

CBER B19 DNA Standard u Derived from a window-period plasma unit, ~10 12 GE/mL u 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 –~10 6 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)* BB (NIBSC, FD) CC (CBER, Liquid) DD (CLB, Liquid)

CBER HAV RNA Standard (I) u Derived from a window-period plasma unit, ~10 6 copies/mL u 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 copies/mL; consensus level determined by the WHO/NIBSC collaborative study

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

Standards for New Emerging Diagnostic Technologies u Gene Chips, microarrays, nanotechnology u Fusion of micro- and nanotechnologies u Reduce time, improve sensitivity, simplify assay procedure and costs u Miniaturization technologies for low cost chips

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

McNeil, (2005), J. Leuk. Biol., 78: Common Nanoscale Particles in Biological Use

Nanoparticle/microarray detection of avian influenza virus subtypes RNA target Hybridization viral RNA Au-probe Hybridization H1H3MH5N1N2 control

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

Reference reagents u Currently no reference materials available for microrray/nanotechnology pathogen detection assays u Pooled RNA (or proteins for protein arrays) representing various pathogens/targets spotted on array under evaluation for expression arrays u 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 u Collaborative study efforts to evaluate suitability of current standards for microarray/nanotechnology based assays are needed as they are developed

Summary u Microarrays increasingly evaluated for multiplex pathogen detection u Nanotechnologies useful for both individual and multiplex detection, protein and nucleic acid detection simultaneously u Reference materials needed to facilitate comparison of different microarray and nanotechnology assays u 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

Acknowledgements CBER/FDAWRAIR S. LeeN. Michael M. YuARC O. WoodS. Stramer M. RiosCarlos Salud S. KerbyV. Soriano R. BiswasNYDOH R. DuncanL. Kramer C. HsiaNYU J. Zhao P. Nyambi