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Perrine Caillet-Fauquet Laboratoire de Virologie Moléculaire Université Libre de Bruxelles

B19 production in hepatocarcinoma cells and neutralization. Perrine Caillet-Fauquet Laboratoire de Virologie Moléculaire Université Libre de Bruxelles. R&D. B19 BIOLOGY. Predilection for infecting dividing cells

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Perrine Caillet-Fauquet Laboratoire de Virologie Moléculaire Université Libre de Bruxelles

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  1. B19 production in hepatocarcinoma cells and neutralization Perrine Caillet-Fauquet Laboratoire de Virologie Moléculaire Université Libre de Bruxelles

  2. R&D 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 (b integrin)

  3. R&D • 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-07 B1647 • 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 lines

  4. R&D KU812F pluripotent cells Elimination of the remaining input and 3X wash addition of medium B19 Virus (WHO 99/800, NISBC) INCUBATION 3 days INCUBATION 2 hours Infected cells KU812F CENTRIFUGATION 6%O2 or 20%O2 6%O2 or 20%O2 + EPO CENTRIFUGATION Supernatant: DNA extraction Nested-PCR cells

  5. R&D B19 production in KU812F cells (+epo) Hypoxia compared to Normoxia 3 2 B19 detectable end-point (logdilution) 1 0 0 1 10 100 1000 10000 Virus input (IU) 6%O2 20%O2

  6. R&D HepG2(or HuH7) Cellular model for B19 production WHO 99/800, NISBC B19 2 hours 37°C Cells Cells Washing 3x Centrifugation HepG2 is Human hepatoblastoma cell line 24, 48, 72 hours 37°C Cells DNA Extraction PCR Amplification PCR POSITIVE Supernatant Erythrovirus B19

  7. R&D B19 production in HepG2 cells and HuH7 A 0.1 IU 10 IU 100 IU 0 IU HepG2 7 6 5 (log dilution) Detectable end-point 4 3 2 1 0 24 48 72 Post infection time (h) A 0.1 IU 10 IU 100 IU 0 IU HuH7 7 6 5 Detectable end-point (log dilution) 4 3 2 1 0 24 48 72 Post infection time (h)

  8. R&D Measure of apotosis: % annexin V positive hepatocarcinoma cells

  9. R&D B19 transcription in HepG2 72h 48h 24h RT-PCR:spliced mRNA VP 183bp P6 100 0 Map units NS1 B19: input IU 102 Hep NI Hep NI 1 102 1 102 1 VP1 VP1 72h 48h 24h VP2 VP2 Actin Protein: 11 kDa B19L21:2082-2067 B19L20:2066-2050 B19L2:381-404 (Brunstein et al 2000) Unspliced 1702 Splice donor: 406 splice acceptors : 1910 183bp amplicon sizes

  10. C HepG2 5 HuH7 4 Detectable end-point 3 (log dilution) 2 1 HepG2 HuH7 0 ? CONTROL + ANTI-P R&D Inhibition of B19 infection in HepG2 by antibodies anti-P blood group antigen 2 hour 4°C B19 1 hour 4°C + Cells Cells Washing 3x Anti-P 48 hours 37°C DNA Extraction Nested-PCR Supernatant

  11. R&D B19 Neutralization B19 ANTIBODIES 16 hours at RT + 48 hours at 37°C Washing 3x Culture Supernatant ? HepG2 HepG2 DNA extraction NESTED PCR

  12. R&D 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

  13. R&D 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 • IVIGconcentrations : 3x10-4 to 300 µg/ml

  14. R&D • Conclusions • New assay for parvovirus B19 infectivity and neutralization. • Use of B19 as relevant model for virus inactivation methods .

  15. R&D Aknowlegments DCF Red Cross M. Di Giambattista V. Hougardy R. Laub Université Libre de Bruxelles M.-L. Draps Y. de Launoit

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