RUTH LAUB

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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
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• 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.
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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.
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HepG2 (or HuH7) Cellular model for B19 production
Adherent human hepatoblastoma cell line HepG2
Erythrovirus B19
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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.
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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 -gt 960 J/m²)
addition to fresh cells culture for 48 h (1st
round)
culture supernatant added to fresh cells (2nd
round)
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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
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B19 INFECTIVITY AND SPECIFIC ANTIBODIES IN
B19-DNA-POSITIVE DONORS A COLLABORATIVE
FOLLOW-UP STUDY

• Selection of 17 donors with an initial level gt105
  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.

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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
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Conformational epitopes
DONOR 2 (HER)
Highly infectious Moderately
infectious - No infectious
Linear epitopes
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Conformational epitopes
DONOR 3 (SUC)
Highly infectious Moderately
infectious - No infectious
Linear epitopes
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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.

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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 .

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• 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