Download Slide 2 B19 DNA detection is a major improvement that

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 53 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