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RUTH LAUB SOGAT XIX Bern, 14-15 June 2006

DOES THE PRESENCE OF B19 DNA IN DONATIONS CORRELATE WITH VIRUS INFECTIVITY ?. RUTH LAUB SOGAT XIX Bern, 14-15 June 2006. B19 DNA detection is a major improvement that increases the margin of safety of plasma derivatives.

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RUTH LAUB SOGAT XIX Bern, 14-15 June 2006

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  1. DOES THE PRESENCE OF B19 DNA IN DONATIONS CORRELATE WITH VIRUS INFECTIVITY ? RUTH LAUB SOGAT XIX Bern, 14-15 June 2006

  2. B19 DNA detection is a major improvement that increases the margin of safety of plasma derivatives. • A limit of 104 IU/ml in plasma pools is recommended (European Pharmacopeia) on the basis of observations. • How a level of B19 DNA translates into infectivity is largely unknown, especially for low titres of B19 DNA found in donations. • What is the infectious dose in terms of geq or virus particles ? • Parvoviruses: genetic diversity (variants and defective particles). • Neutralisation of infectivity by specific antibodies. There is thus a need for an easy, validated cell model. Slide 2

  3. B19 MULTIPLICATION • Multiplication depends on host-cell-specific factors and so B19 is fastidious to propagate in cells. • It occurs mainly in the red blood cell progenitor lineage (cfu-e) where it produces lytic infection by apoptosis. • Entry into red cell progenitor cells involves specific receptors at the cell surface, such as globoside (P-blood group antigen) and/or KU80 and/or 53 integrin. • B19 can enter as a virus-Ig complex into mononuclear-derived cells. • Pathologies are linked to its presence in tissues such foetal liver, B and T cells, synovial tissues ... Hence, liver-derived cells with P-antigen could be used to produce infectious B19 viral particles. Slide 3

  4. HepG2 (or HuH7) Cellular model for B19 production Adherent human hepatoblastoma cell line HepG2 Erythrovirus B19 Slide 4

  5. B19 PRODUCTION IN THE HepG2 CELL LINE : INFECTIVITY PERSPECTIVE 1. First-round culture – production as a function of culture time • B19 : plasma WHO 99/800 • Multiplicity of infection (MOI) : 0.1-1000 IU/2 105 cells. • Minimal infectious dose : 0.1 to 1 IU in HepG2 1 to 100 geq of virus Are the produced particles infectious ? 2. Progeny production in 3 successive rounds The supernatant containing B19 from the first 48 h culture was collected, diluted (to 1000 IU/ml) and added to fresh cells (2nd round). Again, after 48 h of culture, the second culture supernatant was diluted and added to fresh cells (3rd round). Again the 48 h B19 production was collected. B19 DNA was quantified in all three culture supernatants. Slide 5

  6. 3. First- and second-round cultures and defective particles A. Control • B19 (C39) is inoculated in HepG2. • The supernatant containing B19 (1st round) is added to fresh cells (2nd round). B. B19 UVC treated Treatment : UVC irradiation (40 -> 960 J/m²) addition to fresh cells culture for 48 h (1st round) culture supernatant added to fresh cells (2nd round) Slide 6

  7. B19 NEUTRALISATION • Inhibition of B19 multiplication by • anti-P monoclonal antibody. 2.Inhibition of B19 multiplication by polyvalent antibodies from rabbit immunised with B19 capsid epitope peptides 3. Decrease of B19 production in the presence of intravenous immunoglobulins Slide 7

  8. B19 INFECTIVITY AND SPECIFIC ANTIBODIES IN B19-DNA-POSITIVE DONORS A COLLABORATIVE FOLLOW-UP STUDY • Selection of 17 donors with an initial level >105 IU/ml (in-house Real Time PCR). • 12 Males (42.9 ± 8.8 Y) – 5 Females (40.2 ± 15.8 Y). • Interviewing for clinical symptoms. • Monitored for 28 weeks. • Samples collected and analysed for B19 DNA and for specific IgM and IgG antibodies. • Anti-B19 antibodies specific to different linear and conformational B19 epitopes were quantified by 2 ELISAs. • Infectivity was monitored in parallel. Slide 8

  9. Conformational epitopes DONOR 1 (GAL) • Method • Samples are diluted to 1000 IU in medium and added to HepG2 cells. • Progeny was monitored by NAT. Linear epitopes ++ = Highly infectious + = Moderately infectious - = No infectious Slide 9

  10. Conformational epitopes DONOR 2 (HER) ++ = Highly infectious + = Moderately infectious - = No infectious Linear epitopes Slide 10

  11. Conformational epitopes DONOR 3 (SUC) ++ = Highly infectious + = Moderately infectious - = No infectious Linear epitopes Slide 11

  12. CONCLUSIONS CELL MODEL VALIDATION • HepG2, an adherent antigen-P-positive cell line, is validated as a cell model for monitoring in vivo infectivity and neutralisation by specific anti-B19. • One IU is infectious in HepG2 (about 10-100geq based on a plasmid with an integrated NS gene). • The virus particles (progeny) produced in vitro are infectious. This remains true through successive rounds of cell infection. • Defective viruses can be identified by measuring the infectivity after several rounds. • B19 Neutralisation by antibodies with different specificities. Slide 12

  13. B19-DNA-POSITIVE DONORS AND B19 INFECTIVITY • No correlation between symptoms and levels of B19 DNA. • A low B19 DNA titre can be detected for over one year. • Antibodies neutralise B19 infectivity. • Donors can be infective even in presence of anti-B19. • Donors can be not infective despite a low B19 DNA level . Slide 13

  14. DRK • Blutspende- • dienst • W.K. Roth • Themann • E. Seifried • KM Hourfar • M. Schmidt CAF-DCF Red Cross M. Di Giambattista T. Branckaert R. Laub Université Libre de Bruxelles M-L. Draps Y. de Launoit P. Caillet Slide 14

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