Active B19 virions production in hepatoblastoma and

hepatocarcinoma cell lines:amplification and genomic stability.

Op de beeck A.1, Draps M.-L.1, Baurin S.2, Timmerman D. 2, Branckaert Th. 2, Caillet-Fauquet P.1, Laub R.2

Laboratory of Virology, Medicine Faculty, Free University of Brussels1.

R&D- Central Department for Fractionation, Brussels2

Quantification of B19

• Direct qPCR in patient samples

• Infection model : Quantification of B19 mRNA and/or DNA in infected cells (red blood cell progenitor lineage)

• Infectivity model : Measure production of infectious B19 new particles in cell culture (HepG2, Huh-7)

HepG2 Human hepatoblastoma cell line

Huh-7 human hepatocarcinoma cell line

HepG2 and Huh-7 cellular models for B19 production

Erythrovirus B19

B19

2 hours

37°CCells

Washing 3x

24, 48, 72 hours

37°C

Supernatant PCRPOSITIVE

DNA Extraction

PCR Amplification

Cells

Cells

WHO 99/800, NIBSC

Caillet-Fauquet et al, Transfusion 2004; 44:1340-3.

Detection of B19 in the supernatant of HepG2 and Huh-7

B19 : plasma WHO 99/800Multiplicity of infection (MOI) : 0.1-100 IU/5 105 cells : low M.O.I. ! Minimal infectious dose : 0.1 to 1 IU in HepG2Detection by Nested-PCR (Finkel et al., 1994 Lancet 343 (8908), 1255–1258)

0

1

2

3

4

5

6

7

24 48 72

Post infection time (h)

Det

ecta

ble

en

d-p

oin

t (l

og

dil

uti

on

)

0.1 IU 10 IU 100 IU 0 IUA HepG2

0

1

2

3

4

5

6

7

24 48 72

Post infection time (h)

Det

ecta

ble

en

d-p

oin

t (l

og

dil

uti

on

)

0.1 IU 10 IU 100 IU 0 IUA HuH7

Viral progeny is infectious

B19 : C39 positive donation devoid of anti-B19 IgG or IgMInput of each run : 100 IU/ 5 105 cells (m.o.i. = 0.002) Quantif qPCR (Roche kit ) versus standard WHOControl + = Run1

Genomic stabilityThe sequence of the input (run 0)and of the viral progeny

at run 5 are IDENTICAL

C39 : 5594 bp sequenced99,3 % identity with stain HV (Genbank)

Cells +

Anti-globoside Ab

1 hour

4°C

B19

Cells

2 hour4°C

Washing 3x

48 hours37°C

Supernatant

DNA ExtractionNested-PCR

?

0

1

2

3

4

5

CONTROL + ANTI-P

Detectable end-point

(log dilution)

HepG2

HepG2

HuH7

HuH7

Specific Inhibition of B19 infectivity by anti-receptor (globoside)

23

B19

48 hours at 37°C

HepG2

B19 B19 NeutralisationNeutralisation by specific by specific anti-VP2 capsid IgGanti-VP2 capsid IgG

Anti-capsid ANTIBODIES

+16 hours

at RT

Culture Supernatant

DNA extractionNESTED PCR

Washing 3x?

HepG2

0

25

50

75

100

-5 -4 -3 -2 -1 0 1 2

Log rabbit IgG (µg/ml)

Inhibition (%)

Ser 48 - Ser 57

Ser 285 - Lys 300

Ser 554 - Tyr 572

Lys 720 - His 740

Control Peptide

B19 : C39 positive donation devoid of anti-B19 IgG or IgMMultiplicity of infection (MOI) : 100 IU/2 105 cells.Detection by end-point dilution and Nested-PCR (Finkel et al., 1994 Lancet 343 (8908), 1255–1258)

Inhibition of B19 infectivity by specific anti-VP2 capsid protein antibodies

Measure of infectivity : an assay more sensitive than qPCR!

Minimal infectious dose : 0,1 IUInput 0,1 IU gives a viral progeny

=> 1 IU is more than 10 infectious particles1 IU is more than 10 infectious particles

0

1

2

3

4

5

6

7

24 48 72

Post infection time (h)

Detectable end-point

(log dilution)

0.1 IU 10 IU 100 IUHepG2

0

1

2

3

4

5

6

7

24 48 72

Post infection time (h)

Detectable end-point

(log dilution)

0.1 IU 10 IU 100 IUHepG2

0

1

2

3

4

5

6

7

24 48 72

Post infection time (h)

Detectable end-point

(log dilution)

0.1 IU 10 IU 100 IUHepG2

0

1

2

3

4

5

6

7

24 48 72

Post infection time (h)

Detectable end-point

(log dilution)

0.1 IU 10 IU 100 IUHepG2

Det

ecta

b le

end-

poin

t (lo

g d i

lutio

n)

Concentration of IVIG to obtain 50% virus neutralisation- 10 ng/ml for IVIG 2 (MULTIGAM)- 300 ng/ml for IVIG 1 (SANDOGLOBULIN)

0

25

50

75

100

-4 -2 0 2 4

Log Human IgG (µg/ml)

INH

IBIT

ION

(%

)

NIBSC

IVIG 1

IVIG 2

Method:- B19 DNA (103 IU) from a single plasma donation - Incubation overnight at room temperature with

IVIG at concentrations 3x10-4 to 300 µg/ml

Validation of IVIG neutralisation capacity (1)

Applications

Applications

Product Batch Reduction factor (log)

Nanogam 1 >4.15Nanogam 2 >3.81Nanogam 3 >3.95Nanogam 4 >4.02

Gammaquin 1 >5.32Gammaquin 2 >5.36Gammaquin 3 >5.23

Validation of IVIG neutralisation capacity (2)

DONOR 05

0255075

100125150175200225250275300325350375400425450

0 2 4 6 8 10 12 16 20Week

Antibody (Index)

0,00

1,00

2,00

3,00

4,00

5,00

6,00

7,00

B19 DNA (log IU/ml)

IgG IBL

IgM IBL

DNA B19 Log IU/ml

B19 multiplication

++ + - - - - - - -

Each sample was diluted to obtain 1000 IU B19-DNA before infecting the cells.

ApplicationsMeasure B19 infectivity from donor plasma

Applications

Validation of UVC efficiency for inactivation of B19

B19 (C39) is inoculated into HepG2 cultures.The supernatant containing B19 (1st round) is added to fresh cells (2nd round).

UVC induces defective viruses

0

1

2

3

4

5

6

7

8

9

10

0 40 100 240 480 960

UVC DOSE (J/m_)

B19 PRODUCTION (Log IU/ml)

FIRST ROUND

SECOND ROUND

2

Conclusions

HepG2 cells efficiently produce infectious B19 virus(5 successive runs)

The sequence of the viral progeny is identical to the input : genomic stability

Infectivity assay highly sensitive : 0,1 IU of B19 gives a viral progeny

Excellent tool for B19 virus validations

ULB Op de beeck A.Draps M.-L.Caillet-Fauquet P.

CAF-DCFBranckaert Th. Baurin S. Timmerman D.Laub R.

German Red CrossSchmidt M.

SanquinOver J.

Sanquin OyeTolo H.