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 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

3. 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

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 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. 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

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. Adapted from Thomson et al. Lancet Infect Dis 2002. Worldwide distribution of predominant HIV-1 group M subtypes and CRFs CRF14_BG CRF01_AE B

8. 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

9. 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.

10. HIV-1 Subtype Isolates used in current CBER Panel Clade Origin ID #Viral copies/ml A Djbouti DJ/258/91 6.6 x 10 6 B United States 9697 1.9 x 10 9 C Senegal SE/364/90 1.9 x 10 7 D Uganda UG/021/92 4.2 x 10 7 E Thailand TH/022/92 1.9 x 10 7 F Brazil BZ/162/90 1.6 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

11. 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

12. CBER HIV-2 Panel Isolate Testing Summary (Log 10) Isolate ID Manufacture A Manufacture B Manufacture C Mean Standard Deviation B2 8.7782 8.4116 9.3424 8.8441 0.4689 B3 8.5798 8.6702 9.3424 8.8642 0.4167 B4 8.6128 8.6263 9.4133 8.8841 0.4583 B5 8.6812 8.5988 9.4425 8.9075 0.4651 B7 8.8451 8.3522 9.1732 8.7902 0.4133 B8 8.6721 8.1847 9.1732 8.6767 0.4943 B9 8.2788 7.7924 9.0969 8.3894 0.6593

13. 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

14. 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. 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. 16 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]

17. 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

18. 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

19. 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

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 4 o C

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 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

24. 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. 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) 65.82 CC (CBER, Liquid) 65.89 6 DD (CLB, Liquid) 7 - 87.7

26. 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. 10 4 copies/mL; consensus level determined by the WHO/NIBSC collaborative study

27. 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

28. 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

29. 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

30. McNeil, (2005), J. Leuk. Biol., 78:585-594 Common Nanoscale Particles in Biological Use

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

32. 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

33. 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

34. 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

35. 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