Perrine Caillet-Fauquet

Laboratoire de Virologie Moléculaire Université Libre de Bruxelles

B19 production in hepatocarcinoma cells and neutralization

B19 BIOLOGY

Predilection for infecting dividing cells

Low genetic complexity of B19 links its replication highly to cell factors (S phase and differentiation)

Strong tropism for haematopoietic cells such as marrow or fetal liver erythroïd progenitor cell.

Endocytosis via the P blood group antigen (B19 receptor) and a co-receptor ( integrin)

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Primary cultures Bone marrow cells (human-monkey) CFU-e peripheral blood Umbilical cord blood cells Fetal liver cells

Cell lines Blast cell lines UT-7 TF-1

M-07B1647

Megacaryotic leukemia cell line MB-02

Megacaryotic JK-1

Erythroid cell line KU812

Erythroid cell line KU812 Ep6

Erythroid cell line KU812F in hypoxia (6%O2)

Hepatocarcinoma cell linesR&D

INCUBATION 2 hours

CENTRIFUGATION

Elimination of the remaining inputand 3X wash

addition of medium

INCUBATION3 days

CENTRIFUGATIONSupernatant:DNA extractionNested-PCR

B19 Virus(WHO 99/800, NISBC )

+ EPO

KU812F

cells

Infected cells

6%O2 or 20%O26%O2 or 20%O2

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KU812F pluripotent cells

0

1

2

3

0 1 10 100 1000 10000

Virus input (IU)

B19

det

ecta

ble

end

-poi

nt

(log

dil

utio

n)

B19 production in KU812F cells (+epo) Hypoxia compared to Normoxia

6%O2 20%O2

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HepG2 is Human hepatoblastoma cell line

HepG2(or HuH7) Cellular model for B19 production

Erythrovirus B19

B19

2 hours

37°CCells

Washing 3xCentrifugation

24, 48, 72 hours

37°C

Supernatant PCRPOSITIVE

DNA Extraction

PCR Amplification

Cells

Cells

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WHO 99/800, NISBC

B19 production in HepG2 cells and HuH7

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

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Apoptosis: Infection of HepG2 cells by B19 (1000IU/mL)

0

20

40

60

80

NI 8 24 41

time post-infection (h)

% a

nn

exin

po

siti

ve

cells

(%) annexinV

Apoptosis: Infection of HuH7 cells by B19 (1000IU/mL)

0

10

20

30

40

NI 8 24 41

time post-infection (h)

ann

exin

V p

osi

tive

ce

lls (

%)

AnnexinV

Measure of apotosis: % annexin V positive hepatocarcinoma cells

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183bp

RT-PCR:spliced mRNA VP

Actin

B19 transcription in HepG2

1000

NS1

VP1

VP2VP2

P6

VP1

Map units

Protein:11 kDa

B19L2:381-404(Brunstein et al 2000)

B19L21:2082-2067B19L20:2066-2050

Unspliced 1702Splice donor: 406splice acceptors : 1910 183bp amplicon sizes

72h 48h 24h

B19: input IU 10211 1021 102Hep NI

Hep NI

72h 48h 24h

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0

1

2

3

4

5

CONTROL + ANTI-P

Det

ecta

ble

en

d-p

oin

t (l

og

dil

uti

on

)

HepG2

HepG2

HuH7

HuH7

C

Inhibition of B19 infection in HepG2 by antibodies anti-P blood group antigen

Cells +

Anti-P

1 hour

4°C

B19

Cells

2 hour4°C

Washing 3x

48 hours37°C

SupernatantDNA ExtractionNested-PCR

?

R&D

B19

48 hours at 37°C

HepG2

B19 Neutralization

ANTIBODIES

+16 hours

at RT

Culture Supernatant

DNA extractionNESTED PCR

Washing 3x?

HepG2

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RABBIT MODEL OF ANTI-B19 NEUTRALIZING ANTIBODIES

Method : Selection of potential VP/NS epitopes Rabbit immunization Purification of rabbit IgG Elisa on peptides and Commercial Kit (Biotrin) Testing in the cellular model

0

25

50

75

100

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

Log rabbit IgG (µg/ml)

Inh

ibit

ion

(%

) Ser 48 - Ser 57

Ser 285 - Lys 300

Ser 554 - Tyr 572

Lys 720 - His 740

Control Peptide

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0

25

50

75

100

-4 -2 0 2 4

Log Human IgG (µg/ml)

INH

IBIT

ION

(%

)

NIBSC

SANDO

MULTI

B19 NEUTRALIZATION by ANTIBODIES IN IVIG

Concentration of IVIG to obtain 50% virus neutralization- 10 ng/ml for MULTIGAM- 300 ng/ml for SANDOGLOBULIN

Method:•B19 DNA (103 IU) from a single plasma donation •incubation Over Night at room temperature with • IVIG concentrations : 3x10-4 to 300 µg/ml

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Conclusions

1. New assay for parvovirus B19 infectivity

and neutralization.

2. Use of B19 as relevant model for virus

inactivation methods .

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DCF Red Cross

M. Di Giambattista

V. Hougardy

R. Laub

Université Libre de Bruxelles

M.-L. Draps

Y. de Launoit

Aknowlegments

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