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

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 Assurance of Blood Products and in vitro Diagnostic Devices

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 Gudima et al., J Virol 81:3608-3617 (2007)  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) 1

Global Epidemiology of HDV Infection Wedemeyer & Manns, Nature Reviews, Gastroenterol & Hepatol 7, 2010.

HDV Genome - Three HDV RNAs Both genome and antigenome are circles with rod-like folding and 74% base-pairing 3

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- α IFN  In cases of end-stage chronic HDV infection liver transplantation could be an option 4

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

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

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 7

Development of the 1 st International Standard for HDV RNA (1)  Proposal at 2 nd 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 3 rd 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 8

Development of the 1 st 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 10 5 – 10 7 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) 9

HBV Markers / Anti-HCV / Anti-HIV 1/2 HBV DNA HBV DNA HBsAg Anti-HBc 1 2 3 4 5 6 7 8 Sample (IU/ml) (IU/ml) (IU/ml) total HBeAg Anti-HBe Anti-HCV Anti-HIV 1/2 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 1 CAP/CTM, 2 Abbott RealTime , 3 Architect, 4 Architect, 5 Elecsys, 6 Elecsys, 7 Murex, 8 Axsym Ag/Ab Combo 10

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 11 *AJ Roboscreen GmbH, Leipzig, Germany

HDV RNA and Anti-HDV Sample HDV RNA Anti-HDV total 3 (log 10 copies/ml) ∆ NAT Assay 1 1 NAT Assay 2 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 5,14 N6359 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 1 In-house Taqman, Ankara; 2 RoboGene HDV RNA Quantification Kit, PEI; 3 Murex Anti-Delta, PEI. 12

Comparison of Direct HDV / HBV Markers WHO IS Candidate Materials HDV RNA (copies/ml) HBV DNA / HBsAg (IU/ml) 13

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 14

Feasibility Study (2) HDV RNA (log 10 copies/ml) Sample ID Lab 1 Lab 2 Lab 3 Lab 4 Lab 5 Lab 6 Mean SD Ankara Langen Ffm Gießen Leipzig Berlin 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 15

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 16

Limiting dilution of Sample N6357 RoboGene HDV RNA Quantification Kit* PCR Assay (probit method) for data in: HDV.roboscreen1 1 0.95 Dilution NAT Positive 0.75 10 -5.0 10/10 0.63 #positive/#trials 10 -5.5 8/8 0.5 10 -6.0 9/10 10 -6.5 3/8 10 -7.0 1/10 0.25 10 -7.5 0/8 0 -7.5 -7.0 -6.5 -6.344 -6.0 -5.5 -5.0 log10(dose) PEI, Thu Apr 5 13:12:34 2012 1.845 x 10 8 HDV RNA copies/ml (log 10 8.27) quant NAT ∆0.15 1.325 x 10 8 HDV RNA copies/ml (log 10 8.12) NAT 63% *AJ Roboscreen GmbH, Leipzig, Germany

Preparation of the HDV RNA Standard  Material N6357 dilution of 1:50 (final concentration about 10 6 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 (log 10 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) 18 * estimated by the RoboGene HDV RNA Quantification Kit

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) 19

Development of WHO Biological Reference Preparations PEI Projects Marker WHO BRP PEI-Code IVD Establishment HBV genotype 1 st Ref panel 5086/08 NAT ECBS 2009 Panel HBV genotype 1 st Ref panel 6100/09 HBsAg ECBS 2011 Panel HEV RNA 1 st IS NAT ECBS 2011 1 st IS HDV RNA 7657/12 NAT Proposed 2013 Transfusion Bacterial 1 st Ref relevant PEI-B-06 detection ECBS 2011 bacterial strain Repository methods panel For more information: www.pei.de/who-reference-material

Thank you for your attention! 21

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HDV Genome – Three HDV RNAs* *Kindly provided by John Taylor, Fox Chase Cancer Center, Philadelphia, U.S.A.