Download A HepG2

Active B19 virions production in
hepatoblastoma and hepatocarcinoma cell
lines:
amplification and genomic stability.
Op de beeck A.1, Draps M.-L.1, Baurin S.2, Timmerman D. 2,
Branckaert Th. 2, Caillet-Fauquet P.1, Laub R.2
Laboratory of Virology, Medicine Faculty, Free University of Brussels1.
R&D- Central Department for Fractionation, Brussels2
Quantification of B19
• Direct qPCR in patient samples
• Infection model : Quantification of B19
mRNA and/or DNA in infected cells (red
blood cell progenitor lineage)
• Infectivity model : Measure production
of infectious B19 new particles in cell
culture (HepG2, Huh-7)
HepG2 and Huh-7 cellular models for B19 production
WHO 99/800, NIBSC
B19
HepG2 Human hepatoblastoma cell line
2 hours
Huh-7 human hepatocarcinoma cell line
Cells
37°C
Cells
Washing 3x
24, 48, 72 hours
37°C
Erythrovirus B19
DNA Extraction
Supernatant
PCR Amplification
Caillet-Fauquet et al, Transfusion 2004; 44:1340-3.
Cells
PCR
POSITIVE
7
A
0.1 IU
HepG2
10 IU
100 IU
0 IU
7
Detectable end-point
(log dilution)
Detectable end-point
(log dilution)
Detection of B19 in the supernatant
of HepG2 and Huh-7
6
5
4
3
2
1
A
0.1 IU
HuH7
10 IU
100 IU
6
5
4
3
2
1
0
0
24
48
Post infection time (h)
72
24
48
Post infection time (h)
B19 : plasma WHO 99/800
Multiplicity of infection (MOI) : 0.1-100 IU/5 105 cells : low M.O.I. !
Minimal infectious dose : 0.1 to 1 IU in HepG2
Detection by Nested-PCR (Finkel et al., 1994 Lancet 343 (8908), 1255–1258)
72
0 IU
Viral progeny is infectious
B19 : C39 positive donation devoid of anti-B19 IgG or IgM
Input of each run : 100 IU/ 5 105 cells (m.o.i. = 0.002)
Quantif qPCR (Roche kit ) versus standard WHO
Control + = Run1
Genomic stability
The sequence of the input (run 0) and of the viral progeny
at run 5 are IDENTICAL
C39 : 5594 bp sequenced
99,3 % identity with stain HV (Genbank)
Specific Inhibition of B19 infectivity by
anti-receptor (globoside)
1 hour
+
Cells
4°C
2 hour
4°C
B19
Cells
Washing 3x
Anti-globoside Ab
48 hours
37°C
Detectable end-point
(log dilution)
HepG2
5
HuH7
4
3
2
1
HepG2
HuH7
DNA Extraction
Nested-PCR
?
Supernatant
0
CONTROL
+ ANTI-P
B19 Neutralisation by specific
anti-VP2 capsid IgG
B19
16 hours
+
at RT
Anti-capsid ANTIBODIES
Culture
Supernatant
?
Washing 3x
48 hours at 37°C
HepG2
HepG2
DNA extraction
NESTED PCR
23
Inhibition of B19 infectivity by specific anti-VP2
capsid protein antibodies
Inhibition (%)
100
Ser 48 - Ser 57
75
Ser 285 - Lys 300
Ser 554 - Tyr 572
50
Lys 720 - His 740
Control Peptide
25
0
-5
-4
-3
-2
-1
0
1
2
Log rabbit IgG (µg/ml)
B19 : C39 positive donation devoid of anti-B19 IgG or IgM
Multiplicity of infection (MOI) : 100 IU/2 105 cells.
Detection by end-point dilution and Nested-PCR (Finkel et al., 1994 Lancet 343
(8908), 1255–1258)
Measure of infectivity : an assay
more sensitive than qPCR!
7
6
7
7
5
7
6
6
HepG2
0.1 IU
10 IU
100 IU
HepG2
HepG2
0.1
0.1 IU
IU
10
10 IU
IU
100
100 IU
IU
0.1 IU
10 IU
100 IU
4
6
5
5
3
(log
5 dilution)
4
2
Detectable end-point
4
1
3
(log
dilution)
0
3
2
24
(log
dilution)
Detectable
end-point
2
1
48
72
Post infection time (h)
Detectable end-point
1
0
0
Minimal infectious dose : 0,1 IU
24
48
72
Input 0,1
IU Post
gives
a 48viral
progeny 72
infection
time (h)
24
Post infection time (h)
=> 1 IU is more than 10 infectious particles
Applications
Validation of IVIG neutralisation capacity (1)
INHIBITION (%)
Concentration of IVIG to obtain 50% virus neutralisation
- 10 ng/ml for IVIG 2 (MULTIGAM)
- 300 ng/ml for IVIG 1 (SANDOGLOBULIN)
100
75
NIBSC
50
IVIG 1
25
IVIG 2
0
-4
-2
0
2
Log Human IgG (µg/ml)
Method:
- B19 DNA (103 IU) from a single plasma donation
- Incubation overnight at room temperature with
IVIG at concentrations 3x10-4 to 300 µg/ml
4
Applications
Validation of IVIG neutralisation capacity (2)
Product
Batch
Reduction factor (log)
Nanogam
1
Nanogam
2
Nanogam
3
Nanogam
4
Gammaquin
1
Gammaquin
2
Gammaquin
3
>4.15
>3.81
>3.95
>4.02
>5.32
>5.36
>5.23
Applications
450
425
400
375
350
325
300
275
250
225
200
175
150
125
100
75
50
25
0
DONOR 05
7,00
IgG IBL
IgM IBL
DNA B19 Log IU/ml
5,00
4,00
3,00
2,00
1,00
0,00
0
2
4
6
8
10
12
16
20
Week
B19
multiplication
6,00
++
+
-
-
-
-
-
-
-
Each sample was diluted to obtain 1000 IU B19-DNA before infecting the cells.
B19 DNA (log IU/ml)
Antibody (Index)
Measure B19 infectivity from donor plasma
Applications
B19 PRODUCTION (Log IU/ml)
Validation of UVC efficiency for inactivation of B19
10
9
8
7
6
5
4
3
2
1
0
FIRST ROUND
SECOND ROUND
0
40
100
240
480
960
2
UVC DOSE (J/m²)
B19 (C39) is inoculated into HepG2 cultures.
The supernatant containing B19 (1st round) is added to fresh cells (2nd round).
UVC induces defective viruses
Conclusions
HepG2 cells efficiently produce infectious B19 virus
(5 successive runs)
The sequence of the viral progeny is identical to the
input : genomic stability
Infectivity assay highly sensitive : 0,1 IU of B19 gives
a viral progeny
Excellent tool for B19 virus validations
ULB
Op de beeck A.
Draps M.-L.
Caillet-Fauquet P.
German Red Cross
Schmidt M.
CAF-DCF
Branckaert Th.
Baurin S.
Timmerman D.
Laub R.
Sanquin
Over J.
Sanquin Oye
Tolo H.