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N IBSC
Investigation of the efficacy of
novel pathogen
removal/inactivation methods for
plasma products using B19 as a
model
Cécile El Hana
Department of Virology, NIBSC
N IBSC
B19 DNA quantification
• LightCycler real-time PCR (Roche)
• Standard curve established with the B19 international
standard (NIBSC)
N IBSC
B19 infectivity assay
•A clone of UT-7 cells: UT-7/EPO-S1, dependent on
the presence of erythropoietin in the culture medium
•2x105 cells were infected by 30ml of a virus dilution
during the S phase
•Cells were harvested 48 hours post-infection
•mRNA was extracted and amplified by one step
multiplex RT-PCR
•One set of primers detect B19-specific transcripts and
the other set detect a housekeeping gene: Actin
N I BSC
Inactivation by dry heat treatment
•BioProducts Laboratory, UK
•Factor VIII (8Y) from non-B19 screened plasma was
spiked with high titre (1011 IU/ml) human parvovirus
B19
•Spiked freeze-dried product was treated for 24 and 72
hours at 80ºC
N I BSC
Inactivation bybydry
heat
treatment
Inactivation
dry
heat
treatment
•Bioproducts Laboratory, UK
•Although
quantification
showed
that the
•Factor
VIII B19
(8Y)DNA
was spiked
with high
titre human
unspiked factor
parvovirus
B19 VIII contained 104 IU/ml, no infectious
particles
could be detected
thetreated
infectivity
•Spiked
freeze-dried
productby
was
for 24assay
and 72
hours at 80ºC
• •There was a 2.5 log10 reduction in the number of B19
infectious units after 24 hours at 80ºC, compared to the
untreated control
•After 72 hours of treatment, no infectious units could be
detected by the infectivity assay
N IBSC
Inactivation bybydry
heat critical
treatmentfluids
Inactivation
super
•Bioproducts
Laboratory,
UK
• Aphios Corporation,
USA
•Factor VIII (8Y) was spiked with high titre human
parvovirus B19
•SuperFluids™ are normally gases which, when
•Spiked
freeze-dried
product
was
treated
for
24
and
72
compressed, show enhanced penetration and expansion
hours
at 80ºC
properties.
•
• SuperFluids™ penetrate the virus particles and inflate
them until the decompression causes their expansion.
The overfilled particles rupture at their weakest point.
N IBSC
Inactivation bybydry
heat critical
treatmentfluids
Inactivation
super
•Bioproducts Laboratory, UK
•Serum was spiked with high titre human parvovirus
•Factor
VIII
(8Y)
was
spiked
with
high
titre
human
B19
parvovirus B19
•Three supercritical fluids were tested: Freon-22, Freon
•Spiked
freeze-dried
-23 and
N2O/CO2 product was treated for 24 and 72
hours at 80ºC
• Pressure was 206 bars and temperature was either 25
• or 50°C
•Although the DNA titre was unchanged by the
treatment (1010 IU/ml), treatment with N2O/CO2 at 206
bars and 50°C resulted in more than 5 log10 reduction
of the number of infectious units (undetectable by our
infectivity assay).
Inactivation
heat treatment
byby
thedry
INACTINE™
system
N IBSC Inactivation
•Bioproducts Laboratory, UK
• VI technologies or Vitex, USA
•Factor VIII (8Y) was spiked with high titre human
parvovirus B19
• INACTINE™ system includes small molecules that, due
•Spiked freeze-dried product was treated for 24 and 72
to
their
size
and
stability
in
blood,
are
able
to
penetrate
hours at 80ºC
through the protective walls of resistant pathogens.
•
• These molecules are triggered only when they bind with
their target, the DNA or RNA of the pathogen. The loss of
DNA or RNA replication is a fatal event for viruses or
bacteria whereas the red blood cells do not need DNA or
RNA to function.
Inactivation
heat treatment
byby
thedry
INACTINE™
system
N IBSC Inactivation
•Bioproducts
Laboratory,inUK
• Red cells resuspended
medium AS1 were spiked with
•Factor
VIII
(8Y) parvovirus
was spikedB19
with high titre human
high titre
human
parvovirus B19
•Spiked
freeze-dried
treated
forhours
24 and
• Samples
were takenproduct
after 3,was
6, 18
and 22
of 72
hours
at 80ºC
treatment
•
• B19 DNA titre decreased with time (7 log10 reduction)
• The number of infectious units in the treated sample was
reduced from 105 to undetectable level after only 3 hours
of INACTINEä treatment
B19 inactivation by INACTINE
350000
300000
"infectious units"/per
250000
200000
150000
100000
50000
0
0
5
10
15
Time (hours)
20
25
N IBSC
Inactivationby
bythe
dryHelinxâ
heat treatment
Inactivation
technology
•Bioproducts Laboratory, UK
• Cerus Corporation
•Factor VIII (8Y) was spiked with high titre human
• Helinx® B19
compounds are able to cross cell walls or viral
parvovirus
membranes and bind to and crosslink nucleic acids (DNA
•Spiked freeze-dried product was treated for 24 and 72
and RNA), thereby preventing their replication
hours at 80ºC
• The psoralen compound S-59 requires UV light for
•
activity
•Although the reduction in B19 DNA titre was only 1
log10, the number of infectious units per ml was reduced
by 4.5 log10.
N IBSC
Removalby
bydry
nanofiltration
Inactivation
heat treatment
•Bioproducts
UK
• Asahi KaseiLaboratory,
Pharma, Japan
•Factor
VIII (8Y)solution
was spiked
with
high
titretitre
human
• 0.5% albumin
spiked
with
high
human
parvovirus
parvovirus B19
B19
•Spiked
freeze-dried
product
was treated
for 24 andPCR
72
• B19 DNA
was titrated
by real-time
LightCycler
hours at 80ºC
• Prefiltration through 35N PlanovaÒ filter removed 1
•log of human parvovirus B19
10
• Then filtration through 15N or 20N PlanovaÒ filters
removed up to 6 log10 reduction in the B19 DNA titre
• Results correlated with study on haemoglobin solutions
by Abe et al., 2000. Artif Cells Blood Substit Immobil
Biotechnol 28(5): 375-383.