DEVELOPMENT OF AN INTERNATIONAL REFERENCE PREPARATION FOR HEPATITIS - - PowerPoint PPT Presentation

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DEVELOPMENT OF AN INTERNATIONAL REFERENCE PREPARATION FOR HEPATITIS - - PowerPoint PPT Presentation

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DEVELOPMENT OF AN INTERNATIONAL REFERENCE PREPARATION FOR HEPATITIS D VIRUS RNA AREVIR Meeting 3 May 2012, Bonn Michael Chudy Paul-Ehrlich-Institut Federal Institute for Vaccines and Biomedicines WHO Collaborating Centre for Quality

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DEVELOPMENT OF AN INTERNATIONAL REFERENCE PREPARATION FOR HEPATITIS D VIRUS RNA

WHO Collaborating Centre for Quality Assurance of Blood Products and in vitro Diagnostic Devices

Michael Chudy Paul-Ehrlich-Institut Federal Institute for Vaccines and Biomedicines AREVIR Meeting 3 May 2012, Bonn

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1

Hepatitis D Virus (HDV)

  • Member of the genus Deltavirus
  • 8 major clades (HDV-1 to HDV-8)
  • Genetic variability ranges from 20 to 35%
  • 36 nm defective viral particle;

needs HBV to replicate (coinfection or superinfection)

  • Virion is encapsidated by HBsAg (L, M, S)
  • Circular ss (-) RNA genome of ~ 1.7 kb

which is encased in 60 molecules HDAg

  • Transmitted via infected blood or blood products; sex contact
  • Individuals at risk are HBV carriers, IDUs, hemodialysis patients and highly

promiscuous groups

  • Worldwide ~ 5% of HBV carriers are anti-HDV positive (10 - 15 million people)
  • Mortality rate lies between 2 and 20% (ten times higher than for HBV)

Gudima et al., J Virol 81:3608-3617 (2007)

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Global Epidemiology of HDV Infection

Wedemeyer & Manns, Nature Reviews, Gastroenterol & Hepatol 7, 2010.

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HDV Genome - Three HDV RNAs

Both genome and antigenome are circles with rod-like folding and 74% base-pairing

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HDV Infection - Prevention and Treatment

  • No specific vaccine or post-exposure prophylaxis
  • HBV-HDV coinfection can be prevented with HBV vaccine or

anti-HBs-Ig

  • Prevention of HBV-HDV superinfection depends primarily on

education to reduce high-risk behaviour

  • No effective antiviral therapy is currently available
  • Nucleosid(t)e analogues and other antiviral agents are

inefficient

  • Approved treatment is PEG- αIFN
  • In cases of end-stage chronic HDV infection liver

transplantation could be an option

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Clinical Utility of HDV RNA Quantification

  • Serological tests often lacks on sensitivity
  • The most sensitive method is NAT
  • Identify individuals with active HDV infection
  • Decide to initiate treatment
  • Monitor antiviral treatment efficacy
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HDV NAT and Standardization

  • Despite the high sequence diversity, primers and probe of

NAT assays has to be selected from highly conserved regions to cover all 8 HDV clades

  • NAT assays are usually in-house developed
  • No standardization
  • No proficiency testing program in place
  • HDV RNA quantification currently is unreliable
  • HDV RNA IS is particularly important for…

− assay comparison − development and calibration of diagnostic assays − calibration of secondary references and working standards − evaluation of standardized preparations used in quality control and quality assurance

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HDV NAT – Calibration

  • HDV cDNA plasmid
  • Synthetic HDV RNA (transcripts)
  • Armored RNA*
  • No reference method for quantification

(simple OD measurement is not sufficient!)

  • HDV RNA Reference Preparation (preparation of whole virus

in human plasma)

  • IU (arbitrary unitage) ≠ geq ≠ copies

_______________________________________________________________ *Armored RNA, complex of MS2 bacteriophage coat protein and RNA; RNA sequences are completely protected from RNase digestion; developed by Ambion company

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Development of the 1st International Standard for HDV RNA (1)

  • Proposal at 2nd WHO CCs IVD Meeting, Feb 2009, Langen
  • Discussion proposal at Clinical SoGAT II, Sep 2009, Istanbul
  • Adoption of proposal by WHO ECBS in October 2009
  • EASL* Monothematic Conference on Delta Hepatitis

(September 24 – 26, 2010, Istanbul, Turkey)

  • Global Disease
  • Should be on the agenda of WHO
  • Standardized HDV RNA testing (IS) - HDV RNA quantification is a crucial

tool to diagnose, treat and manage HDV infections

  • Project update at Clinical SoGAT III, Jan 2011, London
  • Update at 3rd WHO CCs IVD Meeting, Mar 2011, Potters Bar
  • Project update at Blood SoGAT III, Apr 2012, Vilnius

_______________________________________________________________ *European Association for the Study of the Liver

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Development of the 1st International Standard for HDV RNA (2)

  • Collaboration

− Institute of Hepatology, Ankara University, Turkey (Prof. Bozdayi) − Institute of Med. Virol., Justus-Liebig University Gießen, Nat. Ref. Centre for HBV and HDV (PD Dr. Glebe/Dr. Schüttler)

  • Seven HDV-positive plasma samples are available

− Volume 250 – 300 mL − Viral load 105 – 107 cps/mL (pre-analyzed) − All samples represent genotype HDV-1 (sequenced)

  • Further characterization (quant: HDV RNA, HBV DNA, HBsAg;

qual: anti-HDV total; anti-HBc total, HBeAg/anti-HBe; anti-HCV, anti-HIV1/2)

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HBV Markers / Anti-HCV / Anti-HIV 1/2

Sample HBV DNA (IU/ml)

1

HBV DNA (IU/ml)

2

HBsAg (IU/ml)

3

Anti-HBc total

4

HBeAg

5

Anti-HBe

6

Anti-HCV

7

Anti-HIV 1/2

8

N6356 228 203 5.570 pos neg pos neg neg N6357 <120 20 15.625 pos neg pos neg neg N6358 <120 102 16.190 pos pos pos neg neg N6359 9.140 4.250 1.600 pos neg pos neg neg N6360 1.470 1.323 14.545 pos neg pos neg neg N6361 <120 10 18.945 pos neg pos neg neg N6362 <120 10 22.730 pos neg pos neg neg

1CAP/CTM, 2Abbott RealTime , 3Architect, 4Architect, 5Elecsys, 6Elecsys, 7Murex, 8Axsym Ag/Ab Combo

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HDV NAT – Titration of synthetic HDV RNA (Transcripts)

HDV RNA Real-time RT-PCR - RoboGene HDV RNA Quant Kit*

Plot of fluorescence against cycle numbers Cycle threshold plotted against logarithmic concentration of serial dilutions

*AJ Roboscreen GmbH, Leipzig, Germany

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HDV RNA and Anti-HDV

1In-house Taqman, Ankara; 2RoboGene HDV RNA Quantification Kit, PEI; 3Murex Anti-Delta, PEI.

Sample HDV RNA (log10 copies/ml) Anti-HDV total3 NAT Assay 11 NAT Assay2 ∆ N6356 5,58 6,78 1,20 15,24 N6357 7,38 8,72 1,34 15,75 N6358 6,40 7,33 0,93 15,75 N6359 5,14 6,21 1,07 15,75 N6360 6,24 7,70 1,46 14,77 N6361 6,82 7,60 0,78 14,77 N6362 5,95 6,33 0,38 14,77

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Comparison of Direct HDV / HBV Markers

WHO IS Candidate Materials

HDV RNA (copies/ml) HBV DNA / HBsAg (IU/ml)

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Feasibility study (1)

Feasibility study

  • Two candidate materials (N6357 and N6360)
  • To assess the suitability of candidate materials
  • Parallel testing with armored RNA (N6363)
  • Different labs and different NATs
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Feasibility Study (2)

HDV RNA (log10 copies/ml) Sample ID Lab 1

Ankara

Lab 2

Langen

Lab 3

Ffm

Lab 4

Gießen

Lab 5

Leipzig

Lab 6

Berlin

Mean SD N6357 7,38 8,72 8,25 7,35 5,48 7,44 1,24 N6360 6,24 7,70 7,81 6,54 5,43 6,74 1,01 N6363 9,75 11,11 8,46 9,99 8,43 9,55 1,13

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Feasibility Study (3)

Study outcome

  • Striking differences in the concentration of the samples by

different NAT assays

  • No difference between clinical samples and armored RNA
  • Sample N6357 candidate material for the standard preparation
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Dilution NAT Positive 10-5.0 10/10 10-5.5 8/8 10-6.0 9/10 10-6.5 3/8 10-7.0 1/10 10-7.5 0/8

quant NAT

1.845 x 108 HDV RNA copies/ml (log10 8.27) ∆0.15 NAT63% 1.325 x 108 HDV RNA copies/ml (log10 8.12)

Limiting dilution of Sample N6357

RoboGene HDV RNA Quantification Kit*

PCR Assay (probit method) for data in:

log10(dose) #positive/#trials

  • 7.5
  • 7.0
  • 6.5
  • 6.0
  • 5.5
  • 5.0

0.25 0.5 0.75 0.95 1

HDV.roboscreen1

PEI, Thu Apr 5 13:12:34 2012

  • 6.344

0.63

*AJ Roboscreen GmbH, Leipzig, Germany

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Preparation of the HDV RNA Standard

  • Material N6357 dilution of 1:50 (final concentration about 106 cps/ml*)
  • Dilution in negative plasma pool to a volume of 2.2 litre
  • Filling and lyophilization by a certified Swiss company

− Preparation of 4,000 vials − Filling volume 500 µl − Mean 504.3 µl / SD ±3.9 µl / CV 0.8%

  • Pre-study (PEI): Control of HDV RNA concentration before and after lyo

(log10 copies/ml) − Before lyo theoretical 6.95 practical 7.03 (SD±0.07) − After lyo 6.75 (SD±0.09)

  • Stability testing programme

− Real-time stability − Accelerated stability (-20°C, +4°C, +20°C, +37°C)

  • Residual moisture content: Requirement <1%

− 0.89% (SD±0.07)

*estimated by the RoboGene HDV RNA Quantification Kit

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Evaluation and Establishment of the HDV RNA Standard

  • Worldwide collaborative study to evaluate the candidate reference material
  • Proposed for Q2-3/2012
  • Invitation Apr 2012
  • NCLs, labs with special diagnostic expertise in viral hepatology,

kit manufacturers so far exist, others …

  • Incl. different NAT assays/platforms
  • Commutability (clinical sample)
  • Unitage assignment
  • Statistical analysis
  • Final report to WHO ECBS in 2013
  • Future steps

− Commutability (genotype inclusivity)

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Development of WHO Biological Reference Preparations

PEI Projects

Establishment IVD PEI-Code WHO BRP Marker ECBS 2009 NAT 5086/08 1st Ref Panel HBV genotype panel ECBS 2011 HBsAg 6100/09 1st Ref Panel HBV genotype panel ECBS 2011 NAT 1st IS HEV RNA Proposed 2013 NAT 7657/12 1st IS HDV RNA ECBS 2011 Bacterial detection methods PEI-B-06 1st Ref Repository Transfusion relevant bacterial strain panel

For more information: www.pei.de/who-reference-material

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Thank you for your attention!

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HDV Genome – Three HDV RNAs*

*Kindly provided by John Taylor, Fox Chase Cancer Center, Philadelphia, U.S.A.

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Establishment of WHO Biological Reference Preparations *

7. Characterization of final product 8. Stability studies (incl. statistical analysis) 9. International collaborative study (incl. statistical analyses)

  • 10. Report to WHO
  • 11. ECBS decision
  • 12. Storage and distribution

(maintenance) 1. Selection of candidate materials 2. Characterization of candidate materials 3. Feasibility studies 4. Inactivation (if needed) 5. Dilution of materials (dilution matrix) 6. Freeze-drying

*Recommendations for the preparation, characterization and establishment

  • f international and other biological reference standards (revised 2004); Annex 2, WHO TRS, No 932, 2005.
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WHO Biological Reference Preparations - IVDs

  • For blood safety-related IVDs:
  • Serological test platforms
  • NAT assays
  • Other tests
  • Network of WHO Collaborating Centres for Biological

Standards and Standardization

  • NIBSC; UK
  • CBER/FDA; USA
  • PEI; Germany
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Establishment of WHO Biological Reference Preparations for Nucleic Acid Amplification Techniques (NAT)

  • Pathogens with impact on blood safety
  • Clinical relevant pathogens
  • Preparation of Whole pathogens in human plasma (e. g. virus)
  • Not inactivated to maintain integrity of nucleic acid
  • Most preparations are lyophilized
  • At least 2,000 vials per preparation
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N6357 (TR-2) – different NAT amplicons