www.pei.de
Hepatitis E Virus Issues for Plasma Products
20th IPFA/PEI Workshop Helsinki, 23rd-24th April 2013
Sally A. Baylis, Division of Virology, Paul-Ehrlich-Institut, Langen, Germany
Virology Division
Identification/Characterization of HEV in pooled plasma
Transmission of HEV by S/D plasma, concentrates
Clearance of HEV
WHO International Standard for HEV for NAT
Development of HEV reference panels
Update on introduction of HEV NAT for S/D plasma
Virology Division
Adlhoch et al., Vox Sang. 2009
A regular plasma donor was diagnosed with acute hepatitis and elevated levels of ALT Negative for HAV, HBV, HCV, EBV, CMV and adenovirus Tested for anti-HEV - IgM positive
Robert Koch-Institut was notified and a look-back study was performed
A genotype 3f virus was identified - donor worked in a slaughter house
HEV Infection in a German Plasma Donor
Virology Division
The HEV Window Period
Adlhoch et al., Vox Sang 2009
Virology Division
Canada, Andonov et al., ISBT July 2012
Retrospective study of thrombotic thrombocytopenic purpura patients (TTP) treated with large volumes of pooled plasma Solvent/detergent (S/D)-treated plasma (2500 donors) Cryosupernatant plasma (150 donors)
38 patients received 20-40 litres of plasma 17 - S/D plasma 19 - cryosupernatant plasma 2 - FFP and Pentaspan or albumin
Samples taken at 0, 1 and 6 months post-treatment
HEV and Plasma Products – TTP Patients
Virology Division
No clinical signs of viral hepatitis in any of the patients
No serological evidence of HEV infection at 0 and 1 month post-treatment in any patients
4 out of 17 patients treated with S/D plasma seroconverted at 6 months for anti-HEV IgG, 2 also anti-HEV IgM +ve
The 2 patients positive for anti-HEV IgM and IgG were HEV RNA +ve at 1 month post-treatment - gt 3a
Indirect evidence of HEV transmission by pooled plasma autochthonous HEV in Canada, 4 cases in last 5 years
Annual incidence of HEV infection in German blood donors 0.34% (Juhl et al., Transfusion, Epub ahead of print)
HEV and Plasma Products – TTP Patients Contd.
Virology Division
Japan, Toyoda et al., Intervirology, 2008 Evaluation of haemophiliacs, haemodialysis patients vs.
blood donors
16.3% of haemophiliacs were anti-HEV positive vs. blood donors (3.7%)
Parental transmission of HEV likely in recipients of non-virally inactivated concentrates
HEV and Plasma Products - Haemophiliacs
Virology Division
Hepeviridae family
Humans, birds, pigs, wild boar, deer, rabbits, rats, ferrets, bats, fish
Small non-enveloped viruses 27-34 nm 7.2 kb RNA genome (5´ cap and 3´ poly A tail) ORF1 – non-structural protein ORF2 – virus capsid protein ORF3 – protein involved in morphogenesis (overlaps ORF2)
HEV strains infecting humans represent a single serotype
Hepeviridae Family of Viruses
Virology Division
Yunoki et al., Vox Sang. 2008
Investigation of:
Liquid heat treatment - 25% albumin, 60°C Dry heat treatment - 60°C, 80°C Virus filtration
Viruses used for spiking studies – swine faeces (gt 3 or 4)
A549 cells (lung carcinoma) used for virus titration or detection of HEV RNA genome by qRT-PCR
Inactivation/Removal of HEV
Virology Division
Virus titres determined by infectivity assays
Residual virus was detected after heat treatment in the presence of albumin
HEV Reduction Factors (log10) - Liquid-Heat Treatment
Heat treatment 3JPα swJB-N2
3US swJB-M5
3SP swJB-E10
4JP swJB-H1
Liquid heating, 60°C, 30 min
≥ 2.7 ≥ 3.7 ≥ 3.7 ≥ 2.4
Liquid heating, 60°C, 5h, 25% albumin
2.0 1.0 2.0 ≥ 2.2
Yunoki et al., Vox Sang. 2008
Virology Division
Virus titres determined by infectivity assays
Residual moisture was < 0.3%
Matrix may affect sensitivity to inactivation method
HEV Reduction Factors (log10) - Dry-Heat Treatment
Heat treatment 3US swJB-M5
3SP swJB-E10
Dry-heat, 80°C, 24h, fibrinogen ≥ 4.0 ≥ 4.0 Dry-heat, 60°C, 72h, fibrinogen 2.0 3.0
Yunoki et al., Vox Sang. 2008
Virology Division
Virus spiked into PBS → 0.2 µm filtration/75 nm filtration
Dead end filtration using Planova 35N, 20N and 15N virus filters
Virus titres determined by qRT-PCR
HEV Reduction Factors (log10) - Virus Filtration
Filter 3JPα swJB-N2
3US swJB-M5
3SP swJB-E10
3SP cultured HEV
4JP swJB-H1
35N 1.3 ≥ 3.6 2.6 ≥ 2.8 1.1 20N ≥ 3.8 ≥ 3.6 ≥ 3.2 ≥ 2.8 ≥ 2.6 15N ≥ 3.8 ≥ 3.6 ≥ 3.2 ≥ 2.8 ≥ 2.6
Yunoki et al., Vox Sang. 2008
Virology Division
Intermediates spiked HEV derived from swine faeces or human plasma
Evaluation using real-time PCR
Partitioning of HEV (different spike preparations) during ethanol fractionation is variable - ? lipid, Ab effects
HEV Partitioning During Ethanol Fractionation
M. Yunoki, Pers. Comm. 2013
Virology Division
Anti-HEV may not always effectively neutralize virus
Possible to propagate infectious HEV in culture using viraemic serum containing anti-HEV – Takahashi et al., J Clin Microbiol 2010
Vaccine studies – protective levels of anti-rHEV immunoglobulin of at least 20 WR U/ml (~ 2.5 WHO units/ml) Shrestha et al., NEJM 2008
Transfusion Transmission of HEV - Japan
M. Yunoki, Pers. Comm. 2013
Year HEV Markers in Donor Plasma HEV RNA HEV IgG HEV IgM
Hepatitis E severity in recipient
2005 + - - -
2005 + - - -
2008 + - - -
2008 + + + -
2008 + - - -
Virology Division
HEV RNA Positive Japanese Donations
M. Yunoki, Pers. Comm. 2013
Area Dates HEV Positive Ratio
Hokkaido (Japanese Red Cross) 01/2005 - 12/2012 254 / 2,207,772
(1 / 8,692)
Tokyo (Japanese Red Cross) 05/2006 - 07/2006 3 / 44,332
(1 / 14,777)
Japan excl. Hokkaido (Benesis – source plasma) 07/2007 – 03/2013 24 / 378,718
(1 / 15,800)
Virology Division
Analysis of HEV RNA in Europe and the US
Country Rate Reference
England 1: 7,040 Ijaz et al. Vox Sang 2012 – extrapolated*
Germany 1: 4,415 Baylis et al.,
Vox Sang 2012** Sweden 1: 8,278
USA <1: 50,456
* Blood donors ** S/D plasma donors
Similar data in Germany - Vollmer et al., J Clin Micro, 2012 - Hourfar et al., ISBT abstract, 2012 - Corman et al., Vox Sang, 2013
Virology Division
HEV RNA Positive German & Swedish Donations
Viraemic titers usually ~2 to >5 log10 IU/ml However, viraemic titers may exceed 7 log10 IU/ml Only 3 of 12 viraemic donations were ALT positive All viruses genotype 3 (3a, 3c, 3e, 3f etc.)
Virology Division
HEV in Plasma Pools for Fractionation
Source of Pools No. Positive / No. Analysed
Europe 3/34
Europe/North America 0/3
North America 1/4
Middle East 0/11
Asia 4/23
Overall 8/75
RNA concentration: ≤ 1000 copies/ml Low antibody levels in fractionation pools (except Asia) Genotype 4 HEV – Asia; genotype 3 Europe and USA
Virology Division
HEV Strains Developed into Reference Materials for NAT-Based Assays
Genotype Virus strain HEV RNA
(copies/ml)
Anti-HEV
IgM/IgG
ALT (IU/L) Reference
preparation
3a HRC-HE104 1.6 x 107 -/- 36
WHO
International
Standard
3b JRC-HE3 2.5 x 107 +/- 398
Japanese
National
Standard
Virology Division
Nominal concentration (log10 copies/ml) 6.2 5.2 4.2 3.2 2.2 1.2
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 tests 24 24 24 24 24 24 Percentage positive 96 96 92/88 75/67 38/25 13/8
Example - Qualitative Analysis of HRC-HE104 (Genotype 3a)
Virology Division
Quantitative assays (white - copies/ml); qualitative assays (blue - NAT-detectable /ml).
Histograms of Participants Results
Virology Division
Quantitative assays (white); qualitative assays (blue).
Potency relative to candidate IS = difference in estimated log10 units/ml + assigned value of candidate IS (5.39 log10 IU/ml)
Potencies Expressed Relative to Sample 1
Virology Division
1st 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)
WHO/BS/2011.2175
Establishment of the 1st WHO IS for HEV RNA
Virology Division
The WHO Expert Committee on Biological Standardization endorsed the proposal to prepare an HEV RNA genotype panel at the annual meeting in October 2011 (WHO/BS/2011.2179)
The panel is intended to contain representative of all genotypes and important sub-genotypes
Candidate samples for the preparation of the panel include materials evaluated in the original collaborative study, strains detected in blood/plasma donors & clinical isolates
Future, re-evaluate the WHO anti-HEV IRR
HEV Reference Panels
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 published in Pharmeuropa (issue 25.1) in January 2013
– 5 month consultation period If agreed, referral to the Ph. Eur. Commision – November 2013 If adopted, publication of revised monograph – June 2014 Implementation, January 2015
Proposal to Amend the Ph. Eur. Monograph 1646
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 log10 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
Virology Division
Acknowledgments
JRCS Keiji Matsubayashi Hidekatsu Sakata
Benesis Corp. Mikihiro Yunoki
NIID, Japan Saeko Mizusawa Yoshiaki Okada
Thomas Gärtner
Anton Andonov
WHO Ana Padilla and 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
UK Harry Dalton Linda Scobie/Claire Crossan