1. www.pei.de Hepatitis E Virus – Progress in Standardization of NAT-Based Assays Blood Products Advisory Committee Rockville, 20 th September 2012 Sally A. Baylis, Division of Virology, Paul-Ehrlich-Institut, Langen, Germany

2. Virology Division  Initial investigation of laboratory performance of NAT assays for the detection of HEV RNA  Evaluation of a candidate WHO International Standard for HEV RNA for NAT-based assays  Review of current NAT assays; commercial, in-house  Progress in development of an HEV genotype panel  Update on proposal to introduce HEV NAT for S/D plasma

3. Virology Division  HEV NAT standardization project proposed by the Paul- Ehrlich-Institut (PEI) at the 2 nd World Health Organization Collaborating Centres Meeting (Langen, Feb. 2009)  Presented at SoGAT XX in Brussels, May 2009 and flagged for development in SoGAT survey  Project proposal endorsed by WHO Expert Committee on Biological Standardization - Oct. 2009 (WHO/BS/09.2126)  Anticipated users  Clinical laboratories (hepatitis reference centres)  Blood banks/plasma centres  Research laboratories and vaccine developers  IVD manufacturers Standardizing HEV RNA Assays - Background

4. Virology Division  To investigate HEV NAT assay performance for the first time using blinded panel of samples – Q4 2009/Q1 2010  To determine an appropriate strain to develop into a candidate IS  The panel comprised 22 HEV positive samples (10-fold serial dilutions) and 2 negative plasma controls  genotypes 3a, 3b, 3f, 4c (zoonotic genotypes)  Positive plasma samples obtained from blood donors  Japan and Germany 1 st Collaborative Study – Aim & Approach

5. Virology Division HEV Strains Investigated in 1 st Study GenotypeVirus strain HEV RNA (copies/ml) Anti-HEV IgM/IgG ALT (IU/L) 3aHRC-HE1041.6 x 10 7 -/-36 3bJRC-HE32.5 x 10 7 +/-398 3fRKI1.3 x 10 6 -/-Negative 4cHRC-HE151.0 x 10 6 -/-505

6. Virology Division Analysis based upon partial ORF2 sequence

7. Virology Division  20 participating laboratories, from 10 countries  Participants with expertise in molecular analysis of HEV  Requested to use regular assays for HEV RNA  report results as either positive or negative i.e. HEV RNA detected or not detected  Data was returned from 24 different assays  10 labs returned quantitative data (optional)  All assays, except one, were developed in-house using conventional or real-time RT-PCR methodologies 1 st Collaborative Study – Labs & Methods

8. Virology Division Nominal concentration (log 10 copies/ml) 6.25.24.23.22.21.2 Lab no. 1++++/--- 2 a+++++- 2 b+++++/-- 3+++++- 4++++- 5+++++- 6++++-- 7++++-- 8+++-+- 9++++-- 10+++-+/-- 11 a++---- 11 b+++/---- 12++++++ 13 † ++++-- 14++++++ 15 a++++-- 15 b++++-- 16++++-- 17++++-- 18 a+++--- 18 b++++-- 19------ 20+++-+/-- Total number of tests24 Percentage positive96 92/8875/6738/2513/8 Example - Qualitative Analysis of HRC-HE104 (Genotype 3a)

9. Virology Division Nominal concentration (log 10 copies/ml) 5.04.03.02.01.0 Lab no. 1+++/--- 2 a++--- 2 b++--- 3++++- 4+/-+ -- 5++--- 6+++ - 7++ -- 8+-+-- 9++++- 10++--+ 11 a++--- 11 b++--- 12++++- 13++++/- 14+++++ 15 a----- 15 b++--- 16++++- 17++++- 18 a++--- 18 b++--- 19----- 20+---- Total number of tests24 Percentage positive 92/888350/3833/254/0 Example - Qualitative Analysis of HRC-HE15 (Genotype 4c)

10. Virology Division Quantitative Analysis of HEV Panel Virus strain Nominal concentration log 10 copies/ml N Geometric mean MedianMin.Max. HRC-HE104 6.2 125.845.774.827.48 5.2 124.744.723.636.40 4.2 113.853.843.115.64 3.2 93.042.962.404.49 JRC-HE3 6.4 126.166.154.437.70 5.4 125.075.142.157.00 4.4 124.214.272.605.58 3.4 103.403.202.925.00 RKI 5.1 124.634.573.916.26 4.1 103.773.633.205.26 3.1 92.832.631.774.28 HRC-HE15 5.0 124.564.443.286.28 4.0 103.403.442.634.04 3.0 81.832.464.20

11. Virology Division Quantitative Analysis HRC-HE104 gt 3a JRC-HE3 gt 3b RKI gt 3f HRC-HE15 gt 4c

12. Virology Division  Qualitative data  ~100- to 1000-fold difference in sensitivity - majority of assays, independent of strain  real-time RT-PCR methods were most sensitive  ORF1 directed assays were least sensitive  Quantitative data  at least two thirds of the data sets fell within ± 0.5 log 10 copies/ml of the geometric mean value for the different HEV strains  All negative plasma samples were correctly reported (single equivocal result for one replicate sample)  One false positive result, genotyping by the lab in question detected gt 1 (not included in the panel) 1 st Collaborative Study – Conclusions

13. Virology Division  Project progress report submitted to WHO in Q2, 2010; recommendation to take forward the high titre genotype 3 samples as candidate standards  well detected in study  represent globally distributed genotype  Baylis et al., J Clin Micro 49,1243-9  The following strains were lyophilized in September 2010  HRC-HE104 (genotype 3a) – WHO International Standard  JRC-HE3 (genotype 3b) – Japanese National Standard  Diluted in citrated plasma used in 1 st study which tested negative for  HIV-1/2 RNA, HCV RNA, HBV DNA – Roche TaqScreen MPX  HEV RNA and anti-HEV (IgM and IgG) 1 st Collaborative Study – Outcome

14. Virology Division WHO Candidate AB630970 NIID Candidate AB630971

15. Virology Division  Genotype 3a strain - candidate WHO standard  Coefficient of variation of fill volume 1.1%  Residual moisture 0.73%  4251 vials filled  Titre of HEV RNA ~5.0-5.5 log 10 copies/ml (no loss post-lyophilization)  Full length sequence determined  Candidate WHO standard evaluated together with the genotype 3b strain in a further collaborative study Candidate WHO Standard

16. Virology Division  Study was run in conjunction with the Japanese National Institute for Infectious Diseases (NIID)  Developing national standard (genotype 3b)  24 participating laboratories, from 10 countries  Each laboratory was sent 4 vials of each candidate  Sample 1 + Sample 2 - HRC-HE104 (genotype 3a)  Sample 3 + Sample 4 - JRC-HE3 (genotype 3b)  Samples shipped at ambient temperature  Labs tested samples in 4 separate asays runs (qual./quant.)  Data returned by 23 laboratories, all in house assays  21 qualitative data sets, 14 quantitative data sets 2 nd Collaborative Study

17. Virology Division Overall Mean Estimates from Qualitative Assays (log 10 NAT-detectable units/ml) Samplenmeansdlower clupper clmedianminmaxcv_geo 1195.250.515.015.505.324.426.20150% 2205.260.624.975.565.294.006.37179% 3205.270.794.905.645.273.727.42226% 4205.310.645.025.615.304.426.87183% Candidatenmeansdlower clupper clmedianminmaxcv_geo WHO395.260.565.085.445.324.006.37163% NIID405.290.715.075.525.303.727.42202%

18. Virology Division Overall Mean Estimates from Quantitative Assays (log 10 copies/ml) Samplenmeansdlower clupper clmedianminmaxcv_geo 11235.580.295.325.855.464.366.8598% 21255.600.285.335.875.464.436.6994% 31245.660.205.405.935.504.496.6377% 41255.660.205.405.935.484.646.7776% Candidatenmeansdlower clupper clmedianminmaxcv_geo WHO2485.590.305.335.865.464.366.8599% NIID2495.660.205.405.935.484.496.7776%

19. Virology Division Quantitative Data – Box Plots

20. Virology Division Quantitative assays (blue - copies/ml); qualitative assays (white - NAT-detectable /ml). Histograms of Participants Results

21. Virology Division Quantitative assays (blue); qualitative assays (white). Potency relative to candidate IS = difference in estimated log 10 units/ml + assigned value of candidate IS (5.39 log 10 IU/ml) Potencies Expressed Relative to Sample 1

22. Virology Division Potency Relative to Candidate (Sample 1) SampleAssayNo. dataGeoMean 95%-Confidence Limits %GCV S2 quantitative195.465.35 – 5.583% qualitative135.425.38 – 5.461% combined325.455.38 – 5.512% S3 quantitative205.455.27 – 5.655% qualitative135.485.37 – 5.592% combined335.465.35 – 5.584% S4 quantitative205.515.38 – 5.643% qualitative135.475.36 – 5.592% combined335.495.41 – 5.583%

23. Virology Division  All assays were able to detect both candidate standards  Combined mean estimates for the 2 candidate standards  5.60 log 10 copies/ml (quant. NAT)  5.26 and 5.29 log 10 NAT-detectable units (qual. NAT - end points)  Combined data - potency of preps - 5.39 log 10 units/ml  Participants standards:  Plasmid DNA  Synthetic oligonucleotides  In vitro transcribed RNA  Calibrated plasma/stool samples  No standard controls - reflected in the observed variation  Expressing results relative to Sample 1, as a standard, improved agreement between different labs/methods Conclusions 2 nd Collaborative Study

24. Virology Division  1 st WHO International Standard (IS) for Hepatitis E Virus RNA was established in October 2011  Japanese NIID – simultaneously establishing a national standard  The IS contains a blood donor-derived genotype 3a HEV strain, diluted in plasma, and lyophilized  The IS has a unitage of 250,000 International Units/ml  The IS is available from the PEI (code # 6329/10)  Baylis et al. WHO/BS/2011.2175 Establishment of the 1 st WHO IS for HEV RNA

25. Virology Division  The availability of an IS for HEV will facilitate  Comparison of results of different HEV NAT assays  Defining analytical sensitivity  Clinical diagnostics  Blood/plasma screening  Viral load testing – chronic infection  Validation of (new) assays Conclusions 2 nd Collaborative Study Contd.

26. Virology Division HEV NAT - Commercial Assays ManufacturerAssay nameTechnologyNotes altona DIAGNOSTICSRealStar® HEV RT-PCR kit 1.0 Real-time PCRCE-mark* 95% cut-off; Vollmer et al., JCM, 5 IU/ml Corman et al., Vox, 260 IU/ml Beijing Kinghawk Pharmaceutical Co. Ltd HEV RNA (FQ-PCR)Real-time PCRIVD gt 1, 4 CEERAM S.A.S.hepatitisE@ceeramTool®Real-time PCRCE-mark* Genome Diagnostics Pvt. Ltd Geno-Sen’s HEV Real Time PCR Kit Real-time PCRCE-mark* 95% cut-off – 80 cps/ml Hologic/Gen-ProbeIn developmentTMA95% cut-off – 22 IU/ml Liferiver (Gentaur)HEV Real Time RT-PCR Kit RNA Real-time PCRCE-mark* gt 4 Mediagnost GmbHHEVGene®-Detection kitPCRRUO MIKROGEN GmbHampliCUBE HEVReal-time PCRCE-mark* PrimerDesign LtdPath-HEVReal-time PCRRUO, <100 cps Roche Molecular Systems Inc. Cobas® HEV Test In development - Japan Real-time PCR95% cut-off – 50 cps/ml gt 1-4 *In compliance with Directive 98/79/EC on In Vitro Diagnostic Medical Devices (Annex III, manufacturer's self-declaration)

27. Virology Division  Jothikumar et al., A broadly reactive one-step real-time RT-PCR assay for rapid and sensitive detection of hepatitis E virus. J Virol Meth 131, 65-71  Targets a conserved region in ORF2/ORF3  Probe is very short, Tm ~10°C lower than normal  Database - small number of HEV strains with polymorphisms Widely Used HEV Real-Time PCR – Issues

28. Virology Division  Garson et al., Minor groove binder modification of widely used TaqMan probe for hepatitis E virus reduces risk of false-negative real-time PCR results. J Virol Meth, in press  Serologically confirmed hepatitis E cases reinvestigated using the modified probe, identified additional HEV RNA positive samples Probe 5´-TGA TTC TCA GCC CTT CGC UK patient 5´-TGA TTC TCA GCC CTT TGC  MGB modification ↑ Tm of probe and restored detection  Polymorphism seen in UK patients, caucasians Widely Used HEV Real-Time PCR – Issues Contd.

29. Virology Division  Analysis of plasma donors by the PEI in collaboration with Octapharma has identified a further polymorphism Probe 5´-TGA TTC TCA GCC CTT CGC UK patient 5´-TGA TTC TCA GCC CTT TGC Swedish donor 5´-TGA TTC CCA GCC CTT CGC Widely Used HEV Real-Time PCR – Issues Contd. WHO IS HEV RNA positive plasma donors NTC

30. Virology Division  The WHO ECBS endorsed a proposal by the PEI to prepare a genotype panel for HEV at the annual meeting in October 2011 (WHO/BS/2011.2179)  The panel is intended to contain representatives of all genotypes and important sub-genotypes  The panel will be lyophilized  Candidate samples for the preparation of the panel include materials evaluated in the original collaborative study, strains detected in blood/plasma donors & clinical isolates HEV Genotype Panel Proposal

31. Virology Division  Samples are currently being sourced/characterized :  Gt 1 - strains sourced from India  Gt 1 - China (Xinjiang epidemic strain)  Gt 3b - JRC-HE3 (Japanese)  Gt 3c - European plasma donor samples  Gt 3e - European and Japanese samples  Gt 3f - RKI window period and s/c samples; other strains  Gt 3 - strains which challenge current assays; rabbit strain  Gt 4c - HRC-HE15 (Japanese)  Gt 4i - Japanese plasma donor  Gt 1/4 prospectively sourced, Sudan (1e), Bangladesh, China and clinical cases - Europe  Gt 2 – problematic to source; inclusion of cloned nucleic acid? Sourcing of Samples

32. Virology Division b Analysis based upon partial RdRp sequence

33. Virology Division  The proposal is to amend monograph 1646 - Human plasma (pooled and treated for virus inactivation)  HEV detected in respective European plasma donations  Amendment would see the introduction of HEV NAT  The proposal was discussed at the group 6B (Human Blood and Blood Products) meeting at EDQM - March 2012  Report to be published in Pharmeuropa (issue 25.1) in January 2013 – 3 month consultation period  Comments expected to be reviewed by group 6B in April 2013  If agreed, referral to the Ph. Eur. Commision – June 2013  If adopted, publication of revised monograph – January 2014  Implementation, July 2014 Proposal to Amend the Ph. Eur. Monograph 1646

34. Virology Division  The Hepatitis E virus RNA: The plasma pool is tested using a validated nucleic acid amplification technique (2.6.21). A positive control with 2.5 log 10 IU of hepatitis E virus RNA per mililitre and, to test for inhibitors, an internal control prepared by addition of a suitable marker to a sample of the plasma pool are included in the test. The test is invalid if the positive control indicates the presence of inhibitors. The pool complies with the test if it is found non-reactive for hepatitis E virus RNA. Proposed Text

35. Virology Division Acknowledgments  JRCS  Keiji Matsubayashi  NIID, Japan  Saeko Mizusawa  Yoshiaki Okada   Thomas Gärtner  WHO  Ana Padilla  Collaborative study participants  PEI  Johannes Blümel  Kay-Martin Hanschmann  Roswitha Kleiber  Sigrid Nick  Micha Nübling  Gudrun Winskowsky  Institute of Virology, Bonn  Felix Drexler  Victor Corman

36. Virology Division Our Focus is on Health