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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. 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 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 53 integrin. B19 can enter as a virus-Ig complex into mononuclearderived 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 HepG2 (or HuH7) Cellular model for B19 production Adherent human hepatoblastoma cell line HepG2 Erythrovirus B19 Slide 4 B19 PRODUCTION IN THE HepG2 CELL LINE : INFECTIVITY PERSPECTIVE 1. First-round culture – production as a function of culture time Detectable end-point (log dilution) 0.1 IU HepG2 7 10 IU 100 IU 6 5 4 3 2 1 0 24 48 72 Post infection time (h) 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 B19 PRODUCTION (Log IU/mL) 10 9 8 7 6 5 4 3 2 1 0 1 2 3 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. ROUND OF INFECTION Slide 5 3. First- and second-round cultures and defective particles B19 PRODUCTION (Log IU/ml) A. Control 10 9 8 7 6 5 4 3 2 1 0 FIRST ROUND SECOND ROUND 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) 0 40 100 240 480 960 addition to fresh cells culture for 48 h (1st round) UVC DOSE (J/m²) culture supernatant added to fresh cells (2nd round) Slide 6 B19 NEUTRALISATION 1. 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 HepG2 Detectable end-point (log dilution) 5 HuH7 4 3 2 100 HepG2 HuH7 Inhibition (%) 1 0 CONTROL + ANTI-P 3. Decrease of B19 production in the presence of intravenous immunoglobulins Ser 48 - Ser 57 75 Ser 285 - Lys 300 Ser 554 - Tyr 572 50 Lys 720 - His 740 Control Peptide 25 0 -5 INHIBITION (%) -4 -3 -2 -1 0 Log rabbit IgG (µg/ml) 100 75 NIBSC 50 IVIG A SANDO 25 IVIG B MULTI 0 -4 -2 0 Log Human IgG (µg/ml) 2 4 Slide 7 1 2 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 DONOR 1 (GAL) Conformational epitopes 160 - IgG Biotrin 140 10 - 141 9 132 IgM Biotrin 8 7 - IgG (IU/ml) IgM (Index) 100 60 40 75 - 80 + 37 ++ - - 85 - 6 5 73 4 50 3 37 B19 DNA (log IU/ml) DNA B19 120 2 20 11,5 1 4,2 0,5 2,9 2,8 2,7 0,8 0,5 0,3 0,2 0 0 0 2 4 6 8 10 12 16 20 WEEK Method Samples 1000 IU added to Progeny by NAT. are diluted to in medium and HepG2 cells. was monitored Linear epitopes 450 7,0 - 401 IgG IBL - IgM IBL 6,0 343 350 DNA B19 5,0 IgG (U/ml) IgM (U/ml) 300 - 250 244 - 4,0 209 - 200 150 100 + - - 83 ++ 38 24 2,0 70 67 64 41 50 3,0 140 B19 DNA (log IU/ml) 400 1,0 24 18 1 15 10 9 0 0,0 0 2 4 6 8 10 12 16 20 WEEK Slide 9 ++ = Highly infectious + = Moderately infectious - = No infectious DONOR 2 (HER) Conformational epitopes 200 10 + IgM Biotrin 160 DNA B19 + IgG (IU/ml) IgM (Index) 140 + 168 155 8 132 120 + 93 100 7 6 + 97 5 + 80 60 + 40 20 4 3 49 32 + + 2 14 8 5,4 0,4 9 B19 DNA (log IU/ml) IgG Biotrin 180 1 5,3 2,5 1,8 0,8 0,6 0,3 0,3 0 0 0 2 4 6 8 10 12 16 20 ++ = Highly infectious WEEK Linear epitopes 450 10,0 + IgG IBL 350 8,0 DNA B19 + + 290 300 IgG (U/ml) IgM (U/ml) 9,0 375 IgM IBL 7,0 275 6,0 250 + 5,0 186 200 4,0 150 + 100 50 83 + 0,9 13 6,7 + + 34 22,5 52,5 20 + 3,0 67,1 19 2,0 11 B19 DNA (log IU/ml) 400 1,0 10 9 9 0 0,0 0 2 4 6 8 10 12 16 20 WEEK Slide 10 + = Moderately infectious - = No infectious DONOR 3 (SUC) Conformational epitopes 180 7,0 IgG Biotrin IgM Biotrin 160 DNA B19 6,0 5,0 IgG (IU/ml) IgM (Index) 120 100 - - - 54,5 57,2 57,4 80 60 4,0 + ++ 42,2 ++ 72 - - 72 + 74 + 73 3,0 54 53 46,9 2,0 B19 DNA (log IU/ml) 140 40 1,0 20 5,3 2,28 1,2 0,7 0,45 0,9 0,7 0,7 0,2 0,1 0,1 0 ++ = Highly infectious 0,0 0 2 4 6 8 10 12 16 20 24 28 WEEK Linear epitopes - 500 490 7,0 IgG IBL IgG (U/ml) IgM (U/ml) 6,0 IgM IBL 450 392 400 - 350 336 DNA B19 5,0 - - 300 4,0 274 249 250 3,0 - 200 179 150 - + ++ 100 ++ 59 91 + 86 83 1,0 32 50 2,0 + 106 B19 DNA (log IU/ml) 550 16 14 15 14 15 14 15 14 15 10 0 0,0 0 2 4 6 8 10 12 16 20 24 28 WEEK Slide 11 + = Moderately infectious - = No infectious 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 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 CAF-DCF Red Cross Université Libre de Bruxelles DRK Blutspendedienst M. Di Giambattista T. Branckaert R. Laub M-L. Draps Y. de Launoit P. Caillet W.K. Roth A. Themann E. Seifried KM Hourfar M. Schmidt Slide 14