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