Parvovirus B19 DNA Genotype Panel

Sally Baylis1, Alan Heath2, Mei-ying Yu3

1Viral Safety Section, PEI; 2Informatics Laboratory, NIBSC-HPA; 3Division of Hematology, CBER/FDA

SoGAT XXI,Brussels, 28th-29th May 2009

Parvovirus B19 and Plasma Screening

Parvovirus B19 (B19V) viraemia can exceed 1012 copies/ml in asymptomatic blood & plasma donors

Fractionation plasma pools can contain >109 copies/ml of B19V DNA

B19V transmission has occurred in patients treated with S/D plasma, clotting factor concentrates & fibrin sealants; also blood components

For S/D plasma, no B19V seroconversion was observed in patients receiving <104 copies/ml B19V DNA, used to define threshold concentrations of B19V in start pools for certain products

Regulatory Requirements for B19V DNA NAT Testing in Europe

In 2004 introduction of Ph Eur requirements for B19V DNA testing for plasma pools used in the manufacture of: Human plasma (pooled and treated for virus

inactivation) Human anti-D immunoglobulin Human anti-D immunoglobulin for IV

administration Human albumin/normal immunoglobulin added

to anti-D immunoglobulin

Quantitative test limit of 10 IU/l

Genetic Variation of Parvovirus B19

Previously it was believed that the B19V genome varied only by 1-2%

Identification of more divergent variant viruses (V9, A6, LaLi)

Classification into three distinct genotypes, with further sub-groups identified for genotypes 1 and 3

Overall the sequences differ by ~10%, with the promoter region differing by >20%

In the VP1/2 region variation is ~1% at the amino acid level between virus genotypes, representing a single serotype

Parsyan et al. 2007

D91.1

V9

A6

Genetic Diversity of Parvovirus B19

IM81

LaLi

Regulatory Requirements for Detection of Variants of B19V

8th Report of the International Committee on the Taxonomy of Viruses, 2005 classified A6, LaLi and V9 as strains of B19V

Now included in mandatory Ph Eur test requirement

But problems of implementation

Issues with specificity of commercial kits

No reference materials available

Assays for the Detection of Parvovirus B19 DNA

LightCycler Parvovirus B19 Quantification Kit (Roche) No detection of gt 2 and gt 3

RealArt Parvo B19 LC Kit (Qiagen/artus), alternative kits available for the ABI and Rotorgene platforms Under detection of gt 3b (LC) Under detection of gt 2 and gt 3 (TM)

In-house tests Several are unable to detect gt 2 and gt 3

Sensitivity of Detection of Different Genotypes of Parvovirus

B19

0

1

2

3

4

5

6

7

8

Roche Artus

Lo

g C

on

cen

tra

tio

n I

U/m

l

Genotype 1 (B19)

Genotype 2 (A6)

Genotype 3 (V9)

Genotype 3 (D91.1)

Batch Release - Issues

The detection of different genotypes of B19V is still an ongoing issue, although presence of genotypes 2 and 3 is extremely rare in plasma from Europe and North America

Published studies have reviewed the ability of different assays (in-house and commercial) to detect different genotypes

Proficiency Testing Schemes (PTSs) co-ordinated by EDQM for the OMCLs and plasma fractionators highlighted discrepant results when variants of B19V have been includes in the panels

Discrepant results have occurred between OMCLs and plasma fractionators – failure of batch release test

Addressing the Issues

Meeting at EDQM in November 2006 to discuss commercial NAT assays for B19V

Extraordinary Meeting of SoGAT at NIBSC in March 2007 - standardization of B19V for different genotypes Classification of B19V Epidemiology Suitability of test procedures Survey of blood products Sources of reference materials Recommendations and way forward

Preparation of a plasma genotype panel

Current Status of Parvovirus B19 NAT Screening by U.S.

Manufacturers (I) In 1999, safety of pooled plasma S/D treated

correlated with those lots having <104 geq/ml of B19V DNA in manufacturing pool (a phase 4 study)

Since Sep 1999 BPAC, B19V NAT for plasma for further manufacturing is an in-process test and is reviewed under Biologics Licensing Applications (BLAs) or their supplements for plasma derivatives

Proposed B19V DNA limit: ≤104 IU/ml for manufacturing pools destined for all plasma derivatives

In-date blood components be quarantined and destroyed when possible

Current Status of Parvovirus B19 NAT Screening by U.S.

Manufacturers (II) B19V NAT methods for testing minipools

and manufacturing pools In-house procedures, mostly quantitative NAT

Minipool testing (384 – 512 donations) & sensitivity for excluding original donations of ~106 IU/ml

The need to detect all 3 genotypes Detection is indirectly based upon sequence

alignment analysis of primers and probes with known isolates

Both WHO 1st /2nd International Standards and CBER working standard for B19V DNA are all genotype 1

Validation as an analytical procedure according to ICH and OMCL guidelines

Current Status of Parvovirus B19 NAT Screening by U.S.

Manufacturers (III) Currently several weeks can elapse between

donation collection & identification of B19V NAT-positive donation. Hence, no meaningful notification or retrieval is feasible within the dating period of any cellular blood component intended for use in transfusion FDA encourages steps to shorten this time lapse

When a donor management procedure is in place, such as identification & deferral of individual B19V-reactive Whole Blood (WB) donors, it is no longer considered in-process testing. The threshold level to withhold WB donations or components from use for transfusion needs to be evaluated in clinical trials

WHO Collaborative Study 1st International Standard for B19V DNA

Candidate Preparation*

Log10 geq/ml Log10 IU/ml Anti-B19

AA (lyophilized)

NIBSC 99/800

5.76 6.00 Pos

BB (lyophilized)

NIBSC 99/802

5.73 5.96 Pos

CC (liquid)CBER

5.82 6.06 Neg

DD (liquid)CLB

7.70 7.94 Neg

* All are genotype 1 B19V99/800=WHO 1st IS (5 x 105 IU/vial) established in Oct 2000 (Saldanha et al, Vox Sang 2002)99/802=WHO 2nd IS in Oct 2008CC = CBER working standard, liquid frozen stored at ≤−70 ºC since 1999

Genotype Panel Preparation (I)

3 window-period plasma donations for preparing 3 positive members Gt 1: same originating plasma (NIBSC-UK-ENG-

3QG), ~1012 IU/ml, for WHO 1st IS and 2nd IS for B19V DNA from NIBSC

Gt 2: ~1011 IU/ml, IM-81 strain (GenBank AY903437) from Baxter BioScience under MTA [Blümel et al, J Virol 2005]

Gt 3: ~5 x 1011 IU/ml (P1, GenBank FJ265736) from Talecris under MTA [Rinckel et al, Transfusion, in press]

Genotype Panel Preparation (II) One negative member derived from

pooled plasma (also used as diluent for viral stocks) Formulation of a defibrinated negative human

plasma pool (Basematrix, Lot 113985, SeraCare) derived from 25 screened Source Plasma units (~20 L in total) kindly provided by National Genetics Institute (NGI). All donations were found negative for the following markers:

Anti-HIV 1/2, anti-HCV, HBsAg, anti-HBc (IgG and IgM, Abbott Corzyme), & anti-B19V (IgG and IgM, Biotrin)

ID-NAT: HIV-1, HCV, HBV, B19V, HAV, & WNV

Pooled plasma testing ID-NAT for HIV-1, HCV, HBV, HAV, & B19V by both NGI

and Talecris; negative for all viral markers

Genotype Panel Preparation (III)

Formulation of intermediate viral stocks, ~1010 & ~108 IU/ml, for all 3 genotypes

Quantification of 3 intermediate stocks, ~108

IU/ml, by 4 laboratories (PEI, NGI, Talecris, and CBER) 6 quantitative NAT methods

Formulation of 3 final bulks targeted to contain ~106 IU/ml Each bulk monitored before fill by CBER-

TaqMan

Filling of 4-member genotype panel 3000 vials filled per member (1.1 ml fill per

vial) per week and stored at ≤−70 ºC (filled under contract by SeraCare)

Testing Summary of Intermediate Viral Stocks, ~108 IU/ml, for 3

Genotypes

B19V DNA, IU/mlAssayGt 1 Gt 2 Gt 3

Talecris

8.6 x 107 2.8 x 107

2.0 x 108

PEI/ LC

PEI/ TaqMan

NGI*

PEI/Artus kit

CBER/TaqMan

GeoMean

1.2 x 108 4.7 x 108 3.4 x 108

1.3 x 108 6.0 x 108 3.1 x 108

1.3 x 108 7.0 x 108 2.2 x 108

1.2 x 108 4.1 x 1081.4 x 108

9.8 x 107 3.5 x 108 1.7 x 108

1.1 x 108 4.5 x 108 2.2 x 108

* Average of two runs, removing the outlier results

The plasma panel (Members 1-4) has been evaluated in parallel with the 2nd WHO IS (genotype 1) 99/802

Member 1 – gt 1 Member 2 – gt 2 Member 3 – gt 3 Member 4 – negative plasma

The collaborative study commenced in October 2008

Participants were requested to use assays targeting conserved sequences of B19V and to test samples in four independent assays

B19V Genotype Panel Collaborative Study

B19V Genotype Panel Collaborative Study contd.

35 laboratories, from 13 different countries participated in the collaborative study

33 sets of data from quantitative assays and 9 sets from qualitative assays were returned for analysis

The majority of data sets are from in-house real-time PCR assays (quantitative)

Variety of different extraction methods; assays cover different regions of the B19V genome

The negative panel member (Member 4) has been consistently reported as non-reactive

Estimated IU/ml (log10) from Quantitative Assays

Sample N Mean 95% CI Min.-Max. Range

IS 32 5.99 5.95-6.03 5.75-6.22 0.47

M1 33 5.98 5.92-6.03 5.61-6.32 0.71

M2 31 5.94 5.85-6.04 5.43-6.52 1.09

M3 30 5.97 5.86-6.08 5.21-6.54 1.33

N - Number of laboratory estimates

Absolute Estimates of IS 99/802

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Estimated IU (log10/ml)

4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00 6.25 6.50 6.75 7.00 7.25 7.50 7.75 8.00

09

29A

29B

34A

34B

01A

01B1

01B2

02

03

05

06

07

10

12

18

19

20

21A

21B

21C

22

23

24

27

28

32

35

08

16

30

Absolute Estimates of 2nd IS 99/802

Absolute Estimates of Panel Member M1

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Estimated IU (log10/ml)

4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00 6.25 6.50 6.75 7.00 7.25 7.50 7.75 8.00

09 01A

01B1

13

19

22

29A

29B

01B2

03

05

10

12

18

20

21A

21B

21C

23

24

26

27

28

32

34A

34B

35

02

06

07

08

16

30

Absolute Estimates of Panel Member M1

Absolute Estimates of Panel Member M2

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Estimated IU (log10/ml)

4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00 6.25 6.50 6.75 7.00 7.25 7.50 7.75 8.00

28 09 01A

02

18

19

29A

29B

34A

01B1

32

34B

01B2

03

05

08

10

13

20

21A

21B

22

24

26

35

06

07

12

21C

23

27

30

16

Absolute Estimates of Panel Member M2

Absolute Estimates of Panel Member M3

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Estimated IU (log10/ml)

4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00 6.25 6.50 6.75 7.00 7.25 7.50 7.75 8.00

18 09

22

01A

01B1

03

08

13

28

29A

34B

01B2

05

07

10

20

21A

24

27

29B

30

32

35

12

21B

23

26

06

16

21C

Absolute Estimates of Panel Member M3

Labs 02 & 34A found M3 negative. Lab 19 found M3 positive, but unable to quantify the B19V DNA content. Results excluded.

Overall Means of Potencies (log10 IU/ml) Relative to Concurrently Tested IS for Quantitative and Qualitative Assays

Sample AssayType

NOverallMean

log10 IU/ml

95% CIlog10 IU/ml Min.-Max. Range

M1Qual. 10 5.95 5.68 6.22 5.10-6.57 1.47

Quant. 34 5.98 5.94 6.02 5.74-6.20 0.46

M2Qual. 10 5.84 5.26 6.41 3.78-6.68 2.90

Quant. 34 5.87 5.74 5.99 4.52-6.36 1.83

M3Qual. 9 5.47 4.75 6.18 3.69-6.32 2.63

Quant. 31 5.97 5.87 6.07 5.18-6.58 1.40

N - Number of laboratory estimates

Potency of Panel Member M1 Relative to the IS

Potency of Panel Member M1 Relative to IS

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IU (log10/ml)

3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00 6.25 6.50 6.75 7.00 7.25 7.50

11 01A

01B1

09

13

19

25

01B2

01C2

02

03

05

07

08

10

12

14

15

17

18

20

21A

21B

21C

22

23

24

26

27

28

29A

29B

30

32

33

34B

34C

35

01C1

06

16

31

34A

04

Potency of Panel Member M2 Relative to the IS

Potency of Panel Member M2 Relative to IS

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IU (log10/ml)

3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00 6.25 6.50 6.75 7.00 7.25 7.50

04 28 09 01A

02

15

18

19

01B1

08

11

29A

29B

32

34A

34C

01B2

03

05

07

10

12

13

14

20

21A

21B

22

23

24

26

27

30

31

33

34B

35

01C2

06

16

17

21C

25 01C1

Potency of Panel Member M3 Relative to the IS

Potency of Panel Member M3 Relative to IS

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IU (log10/ml)

3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00 6.25 6.50 6.75 7.00 7.25 7.50

11 15 25 18 01A

08

01B1

07

09

13

14

17

22

28

01B2

03

05

10

20

21A

23

24

27

29A

30

31

32

33

34B

34C

35

01C1

01C2

12

21B

26

29B

06

16

21C

Overall Means of Potencies (log10 IU/ml) Relative to Concurrently Tested IS for

Quantitative assays

Sample NOverall Mean

log10 IU/ml95% CI

log10 IU/ml

M1 34 5.98 5.94 6.02

M2 32 5.94 5.86 6.02

M3 31 5.97 5.87 6.07

Data are shown for quantitative assays (excluding Labs 9 and 28 for M2)

Stability Studies Stability studies are in progress reviewing the

real-time stability of the panel members, to date there has been no evidence for loss of titre of the panel members (~12 months at recommended storage conditions i.e. ≤−70 ºC)

A review of the potency of CC and DD from the original WHO Collaborative Study has been performed on samples stored at ≤−70 ºC for > 9 years, there is no evidence for loss of potency of these preparations

Conclude the B19V plasma positive samples are stable at recommended storage conditions

Conclusions and Recommendations

Assays used by participants have, on the whole performed extremely well with the different genotypes, the majority being in-house assays

Propose that the M1-M4 be established as the 1st International Reference Panel for B19V DNA, NIBSC code 09/110

Whilst no unitage will be assigned to M1-M3, it is proposed to include in the “instructions for use” the results of the quantitative assay absolute estimates and cite the range of reported values from the collaborative study

Study report is currently with participants for review prior to submission to ECBS by July

Parsyan et al. 2007

D91.1

V9

A6

Genetic Diversity of Parvovirus B19

IM81

LaLi

B19V DNA Collaborative Study Participants

Dr Sally Baylis Langen, Germany

Dr Johannes Blümel Langen, Germany

Dr Joan Bodiya/Maria Noedel Tempe, USA

Dr Kevin E Brown London, UK

Dr Daniel Candotti Cambridge, UK

Dr Michael Chudy/ Dr Micha Nübling Langen, Germany

Dr Theo Cuypers/ Dr Marco Koppelman Amsterdam, The Netherlands

Prof. Dr Anna-Maria Eis-Hübinger Bonn, Germany

Dr Giorgio Gallinella Bologna, Italy

Dr Thomas Gärtner Frankfurt am Main, Germany

Dr Thomas Grewing/Alke Heitmann Hamburg, Germany

Dr Yiu-Lian Fong Emeryville, USA

Dr Christoph Jochum Dreieich, Germany

Dr Marta José Barcelona, Spain

Dr Harkanwal Halait/Dr Yosh Ohhashi Pleasanton, USA

Dr Andreas Klotz/Dr Sandra Rieger Vienna, Austria

Dr Thomas Laue/Sabine Raith Hamburg, Germany

Dr Tzong-Hae Lee/Lani Montalvo San Francisco, USA

B19V DNA Collaborative Study Participants contd.

Dr German Leparc/Benjamin Reynolds St.Petersburg, USA

Dr Jeffrey M. Linnen San Diego, USA

Dr Eliza Moretti Lucca, Italy

Prof. Dr. Susanne Modrow/Dr Jürgen Wenzel Regensburg, Germany

Dr Yoshiaki Okada Tokyo, Japan

Dr Matthias Opp Luxembourg

Dr Takashi Owada Hokkaido, Japan

Mr David Padley Potters Bar, UK

Dr Lutz Pichl Hagen, Germany

Dr Giulio Pisani/Dr Francesco Marino Rome, Italy

Dr Lori Rinckel/Joshua Marley Raleigh, USA

Dr Gunnar Schalasta/Dr Martin Enders Stuttgart, Germany

Dr Annabelle Servant-Delmas Paris, France

Dr Richard Smith/Dr Jeffery Albrecht Los Angeles, USA

Dr Maria Söderlund-Venermo/Dr Maija Lappalainen

Helsinki, Finland

Dr Martin Stolz Bern, Switzerland

Dr Mei-ying W. Yu/Dr Li Ma Bethesda, USA

Ms Yi-Chen Yang Taipei, Taiwan

Acknowledgements

NGI, CSL Behring, Baxter BioScience & Talecris Biotherapeutics for materials used in the studies

Collaborative study participants

David Padley, NIBSC

Li Ma, CBER/FDA