op de beeck a. 1 , draps m.-l. 1 , baurin s. 2 , timmerman d. 2 ,
DESCRIPTION
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 - PowerPoint PPT PresentationTRANSCRIPT
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 Human hepatoblastoma cell line
Huh-7 human hepatocarcinoma cell line
HepG2 and Huh-7 cellular models for B19 production
Erythrovirus B19
B19
2 hours
37°CCells
Washing 3x
24, 48, 72 hours
37°C
Supernatant PCRPOSITIVE
DNA Extraction
PCR Amplification
Cells
Cells
WHO 99/800, NIBSC
Caillet-Fauquet et al, Transfusion 2004; 44:1340-3.
Detection of B19 in the supernatant of HepG2 and Huh-7
B19 : plasma WHO 99/800Multiplicity of infection (MOI) : 0.1-100 IU/5 105 cells : low M.O.I. ! Minimal infectious dose : 0.1 to 1 IU in HepG2Detection by Nested-PCR (Finkel et al., 1994 Lancet 343 (8908), 1255–1258)
0
1
2
3
4
5
6
7
24 48 72
Post infection time (h)
Det
ecta
ble
en
d-p
oin
t (l
og
dil
uti
on
)
0.1 IU 10 IU 100 IU 0 IUA HepG2
0
1
2
3
4
5
6
7
24 48 72
Post infection time (h)
Det
ecta
ble
en
d-p
oin
t (l
og
dil
uti
on
)
0.1 IU 10 IU 100 IU 0 IUA HuH7
Viral progeny is infectious
B19 : C39 positive donation devoid of anti-B19 IgG or IgMInput of each run : 100 IU/ 5 105 cells (m.o.i. = 0.002) Quantif qPCR (Roche kit ) versus standard WHOControl + = Run1
Genomic stabilityThe sequence of the input (run 0)and of the viral progeny
at run 5 are IDENTICAL
C39 : 5594 bp sequenced99,3 % identity with stain HV (Genbank)
Cells +
Anti-globoside Ab
1 hour
4°C
B19
Cells
2 hour4°C
Washing 3x
48 hours37°C
Supernatant
DNA ExtractionNested-PCR
?
0
1
2
3
4
5
CONTROL + ANTI-P
Detectable end-point
(log dilution)
HepG2
HepG2
HuH7
HuH7
Specific Inhibition of B19 infectivity by anti-receptor (globoside)
23
B19
48 hours at 37°C
HepG2
B19 B19 NeutralisationNeutralisation by specific by specific anti-VP2 capsid IgGanti-VP2 capsid IgG
Anti-capsid ANTIBODIES
+16 hours
at RT
Culture Supernatant
DNA extractionNESTED PCR
Washing 3x?
HepG2
0
25
50
75
100
-5 -4 -3 -2 -1 0 1 2
Log rabbit IgG (µg/ml)
Inhibition (%)
Ser 48 - Ser 57
Ser 285 - Lys 300
Ser 554 - Tyr 572
Lys 720 - His 740
Control Peptide
B19 : C39 positive donation devoid of anti-B19 IgG or IgMMultiplicity 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)
Inhibition of B19 infectivity by specific anti-VP2 capsid protein antibodies
Measure of infectivity : an assay more sensitive than qPCR!
Minimal infectious dose : 0,1 IUInput 0,1 IU gives a viral progeny
=> 1 IU is more than 10 infectious particles1 IU is more than 10 infectious particles
0
1
2
3
4
5
6
7
24 48 72
Post infection time (h)
Detectable end-point
(log dilution)
0.1 IU 10 IU 100 IUHepG2
0
1
2
3
4
5
6
7
24 48 72
Post infection time (h)
Detectable end-point
(log dilution)
0.1 IU 10 IU 100 IUHepG2
0
1
2
3
4
5
6
7
24 48 72
Post infection time (h)
Detectable end-point
(log dilution)
0.1 IU 10 IU 100 IUHepG2
0
1
2
3
4
5
6
7
24 48 72
Post infection time (h)
Detectable end-point
(log dilution)
0.1 IU 10 IU 100 IUHepG2
Det
ecta
b le
end-
poin
t (lo
g d i
lutio
n)
Concentration of IVIG to obtain 50% virus neutralisation- 10 ng/ml for IVIG 2 (MULTIGAM)- 300 ng/ml for IVIG 1 (SANDOGLOBULIN)
0
25
50
75
100
-4 -2 0 2 4
Log Human IgG (µg/ml)
INH
IBIT
ION
(%
)
NIBSC
IVIG 1
IVIG 2
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
Validation of IVIG neutralisation capacity (1)
Applications
Applications
Product Batch Reduction factor (log)
Nanogam 1 >4.15Nanogam 2 >3.81Nanogam 3 >3.95Nanogam 4 >4.02
Gammaquin 1 >5.32Gammaquin 2 >5.36Gammaquin 3 >5.23
Validation of IVIG neutralisation capacity (2)
DONOR 05
0255075
100125150175200225250275300325350375400425450
0 2 4 6 8 10 12 16 20Week
Antibody (Index)
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
B19 DNA (log IU/ml)
IgG IBL
IgM IBL
DNA B19 Log IU/ml
B19 multiplication
++ + - - - - - - -
Each sample was diluted to obtain 1000 IU B19-DNA before infecting the cells.
ApplicationsMeasure B19 infectivity from donor plasma
Applications
Validation of UVC efficiency for inactivation of B19
B19 (C39) is inoculated into HepG2 cultures.The supernatant containing B19 (1st round) is added to fresh cells (2nd round).
UVC induces defective viruses
0
1
2
3
4
5
6
7
8
9
10
0 40 100 240 480 960
UVC DOSE (J/m_)
B19 PRODUCTION (Log IU/ml)
FIRST ROUND
SECOND ROUND
2
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.
CAF-DCFBranckaert Th. Baurin S. Timmerman D.Laub R.
German Red CrossSchmidt M.
SanquinOver J.
Sanquin OyeTolo H.