ruth laub sogat xix bern, 14-15 june 2006
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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. - PowerPoint PPT PresentationTRANSCRIPT
RUTH LAUB
SOGAT XIX Bern, 14-15 June 2006
DOES THE PRESENCE OF B19 DNAIN DONATIONS CORRELATE WITH
VIRUS INFECTIVITY ?
Slide 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 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 53 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.
Adherent human hepatoblastoma cell line
HepG2
HepG2 (or HuH7) Cellular model for B19 production
Erythrovirus B19 Slide 4
B192 hours
37°CCell
Washing 3x
24, 48, 72 hours
37°C
Supernatant PCRPOSITIVE
DNA Extraction
Real-Time PCR
WHO 99/800, NISBCB19
2 hours
37°CCell
Washing 3x
37°C
Supernatant PCRPOSITIVE
DNA Extraction
Real-Time PCR
WHO 99/800, NISBCB19
2 hours
37°CCell
Washing 3x
24, 48, 72 hours
37°C
Supernatant PCRPOSITIVE
DNA Extraction
Real-Time PCR
WHO 99/800, NISBCB19
2 hours
37°CCell
Washing 3x
37°C
Supernatant PCRPOSITIVE
DNA Extraction
Real-Time PCR
WHO 99/800, NISBC
Slide 5
B19 PRODUCTION IN THE HepG2 CELL LINE :INFECTIVITY PERSPECTIVE
0
1
2
3
4
5
6
7
24 48 72
Post infection time (h)
Det
ecta
ble
end-
poin
t (lo
g di
lutio
n)
0.1 IU 10 IU 100 IUHepG2
0
1
2
3
4
5
6
7
8
9
10
1 2 3ROUND OF INFECTION
B19
PR
OD
UC
TIO
N (L
og IU
/mL)
1. First-round culture – production as a function of culture time
2. Progeny production in 3 successive rounds
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 HepG21 to 100 geq of virus
Are the produced particles infectious ?
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 6
3. First- and second-round cultures and defective particles
B19 (C39) is inoculated in HepG2.
The supernatant containing B19 (1st round) is added to fresh cells (2nd round).
Treatment :UVC irradiation(40 -> 960 J/m²)
addition to fresh cellsculture for 48 h (1st round)
culture supernatant addedto fresh cells (2nd round)
A. Control
B. B19 UVC treated
0123456789
10
0 40 100 240 480 960UVC DOSE (J/m²)
B19
PR
OD
UC
TIO
N (L
og IU
/ml)
FIRST ROUNDSECOND ROUND
Slide 7
0
25
50
75
100
-5 -4 -3 -2 -1 0 1 2Log rabbit IgG (µg/ml)
Inhi
bitio
n (%
) Ser 48 - Ser 57
Ser 285 - Lys 300
Ser 554 - Tyr 572
Lys 720 - His 740
Control Peptide
0
1
2
3
4
5
CONTROL + ANTI-P
Det
ecta
ble
end-
poin
t (lo
g di
lutio
n)
HepG2
HepG2
HuH7
HuH7
B19 NEUTRALISATION
2. Inhibition of B19 multiplication by polyvalent antibodies from rabbit immunised with B19 capsid epitope peptides
1. Inhibition of B19 multiplication by anti-P monoclonal antibody.
3. Decrease of B19 production in the presence of intravenous immunoglobulins
0
25
50
75
100
-4 -2 0 2 4
Log Human IgG (µg/ml)
INH
IBIT
ION
(%)
NIBSC
SANDO
MULTI
IVIG A
IVIG B
0
25
50
75
100
-4 -2 0 2 4
Log Human IgG (µg/ml)
INH
IBIT
ION
(%)
NIBSC
SANDO
MULTI
IVIG A
IVIG B
Slide 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 9
DONOR 1 (GAL)
37 37
50
75
141132
85
73
2,911,5
4,2 2,8 2,7 0,8 0,5 0,3 0,20,50
20
40
60
80
100
120
140
160
0 2 4 6 8 10 12 16 20
WEEK
IgG
(IU/
ml)
IgM
(Ind
ex)
0
1
2
3
4
5
6
7
8
9
10
B19
DNA
(log
IU/m
l)
IgG Biotrin
IgM Biotrin
DNA B19
+++
--
-
- -
--
Conformational epitopes
Linear epitopes
++ = Highly infectious
+ = Moderately infectious
- = No infectious
Method
Samples are diluted to 1000 IU in medium and added to HepG2 cells.
Progeny was monitored by NAT.
1
4167
140
209
401
343
244
70
24
8364
3824 18 15 10 9
0
50
100
150
200
250
300
350
400
450
0 2 4 6 8 10 12 16 20
WEEK
IgG
(U/m
l)Ig
M (U
/ml)
0,0
1,0
2,0
3,0
4,0
5,0
6,0
7,0
B19
DNA
(log
IU/m
l)
IgG IBL
IgM IBL
DNA B19
+++ -
--
-
-
-
-
Slide 10
DONOR 2 (HER)Conformational epitopes
Linear epitopes
++ = Highly infectious
+ = Moderately infectious
- = No infectious
0,9 6,722,5
52,567,1
186
290
375
275
13
83
3411 10 9 9
1920
0
50
100
150
200
250
300
350
400
450
0 2 4 6 8 10 12 16 20
WEEK
IgG
(U/m
l)Ig
M (U
/ml)
0,0
1,0
2,0
3,0
4,0
5,0
6,0
7,0
8,0
9,0
10,0
B19
DNA
(log
IU/m
l)
IgG IBL
IgM IBL
DNA B19
++
++ +
+
+
+
+
814
32
49
93 97
132
155168
0,4 5,3 2,5 1,8 0,8 0,6 0,3 0,35,40
20
40
60
80
100
120
140
160
180
200
0 2 4 6 8 10 12 16 20
WEEK
IgG
(IU/
ml)
IgM
(Ind
ex)
0
1
2
3
4
5
6
7
8
9
10
B19
DNA
(log
IU/m
l)
IgG Biotrin
IgM Biotrin
DNA B19
+ ++
+
+ +
+
+ +
Slide 11
DONOR 3 (SUC)
42,2 46,954,5 57,2 57,4
72 72
54
74 73
53
5,3 2,28 1,2 0,45 0,9 0,7 0,7 0,2 0,1 0,10,70
20
40
60
80
100
120
140
160
180
0 2 4 6 8 10 12 16 20 24 28
WEEK
IgG
(IU/
ml)
IgM
(Ind
ex)
0,0
1,0
2,0
3,0
4,0
5,0
6,0
7,0
B19
DN
A (lo
g IU
/ml)
IgG BiotrinIgM BiotrinDNA B19
++++
+ - - - - --
- + +
Conformational epitopes
Linear epitopes
++ = Highly infectious
+ = Moderately infectious
- = No infectious
5991
179
336
490
392
274249
10686 83
1015151415141414151632
0
50
100
150
200
250
300
350
400
450
500
550
0 2 4 6 8 10 12 16 20 24 28
WEEK
IgG
(U/m
l)Ig
M (U
/ml)
0,0
1,0
2,0
3,0
4,0
5,0
6,0
7,0
B19
DNA
(log
IU/m
l)
IgG IBL
IgM IBL
DNA B19
++++
+
-
-
-
-
- -
- + +
Slide 12
CONCLUSIONSCELL 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 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 14
CAF-DCFRed Cross
M. Di GiambattistaT. Branckaert
R. Laub
Université Libre de Bruxelles
M-L. DrapsY. de Launoit
P. Caillet
DRK Blutspende-
dienst
W.K. RothA. ThemannE. SeifriedKM HourfarM. Schmidt