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Gene Therapy (1999) 6, 873–881
1999 Stockton Press All rights reserved 0969-7128/99 $12.00
http://www.stockton-press.co.uk/gt
Photodynamic treatment of adenoviral vectors with
visible light: an easy and convenient method for viral
inactivation
FHE Schagen, ACE Moor, SC Cheong, SJ Cramer, H van Ormondt, AJ van der Eb,
TMAR Dubbelman and RC Hoeben
Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands
Recombinant adenovirus vectors are popular tools for sensitizer in phosphate-buffered saline (PBS), giving
⬎
7
gene transfer and gene therapy. However biosafety con- log
10
inactivation of the adenovirus. DNA isolated from MB-
straints require that all handling of the vectors and vector- and light-treated virions was inefficient as a template for
containing samples is restricted to dedicated containment transcription. Furthermore, Southern blot analysis revealed
laboratories, unless they had undergone a validated virus- fragmentation of the viral DNA. Based on its preference
inactivation procedure, which decontaminates the samples for DNA, MB is suited for adenovirus inactivation in blood
from any active virus. In this study we evaluated the feasi- plasma. Spiking experiments in which AdCMVLuc was
bility of photodynamic treatment (PDT) with visible light to added to plasma samples demonstrated a reduction (
⬎
4
inactivate recombinant adenovirus vectors in biological log
10
-fold) of reporter gene expression to almost back-
samples, with minimum associated effects on other biologi- ground levels. In contrast to MB, photodynamic treatment
cal activities. Several photosensitizers were tested for their with RB, UP or AlPcS
4
did not lead to DNA damage.
capacity to inactivate a model human adenovirus vector, Although alterations of the viral capsid could not be
AdCMVLuc, upon illumination. Four photosensitizers detected, the binding pattern of the particles to target cells
(methylene blue (MB), rose bengal (RB), uroporphyrin (UP) was significantly changed. Taken together, our data
and aluminum phthalocynine tetrasulphonate (AlPcS
4
)) demonstrate that PDT is an efficient, convenient and useful
could inactivate the adenovirus, as measured by method for the inactivation of adenovirus vectors in
expression of the luciferase reporter gene and by plaque biological samples.
assay. Of these, MB demonstrated to be the most effective
Keywords:
photo-inactivation; photodynamic treatment; blood plasma
Introduction
In the past decade, recombinant adenovirus vectors have
become very popular tools for in vivo gene transfer into
animals and more recently, for gene therapy purposes in
patients. Many vaccination and gene therapy studies
have borne out the relative safety of adenoviral vectors
for human application.
1–3
However, biosafety constraints
require that all handling of vectors and vector-containing
samples or materials is restricted to dedicated contain-
ment laboratories, unless the material has been exempted
and permitted to be released from the biosafety contain-
ment. Such a release is possible only if one has met two
requirements: the material is produced under GMP con-
ditions and the vector lot to be released is characterized
extensively, viz free of replication-competent adeno-
viruses.
Obviously, this cannot be achieved for all vector virus
lots used in a research setting. Thus, adequate and vali-
dated adenovirus inactivation procedures are needed in
Correspondence: RC Hoeben, Applied Virology Group, Department of
Molecular Cell Biology, Wassenaarseweg 72, 2333 AL Leiden, The
Netherlands
Received 12 August 1998; accepted 21 December 1998
order to decontaminate biological samples fully to allow
transfer to non-containment research facilities. In this
study, we have evaluated the use of photodynamic treat-
ment with photosensitizers which can be excited by vis-
ible white light, for the inactivation of adenoviruses in
general and in the context of plasma with the mainte-
nance of biological activities in particular.
So far, photodynamic inactivation has been proven to
be a powerful method for inactivating enveloped viruses,
such as murine retroviral vectors, human immunodefi-
ciency viruses (HIV-1 and -2), hepatitis-B and -C and ves-
icular stomatitis virus (VSV).
4–8
The procedures were also
effective in the presence of blood or blood plasma. In this
study, we demonstrate that also a non-enveloped virus,
the adenovirus, can be effectively inactivated with PDT.
One of the agents tested, MB, can be used specifically to
degrade the viral DNA. Our data show that PDT can be
an efficient, convenient and useful technique for the inac-
tivation of adenovirus vectors in biological samples.
Beside this decontamination purpose, PDT with MB can
also be used to study the effect of invading adenoviral
particles on recipient cells, since the photodynamically
inactivated adenoviruses are internalized, but have lost
the ability to express viral genes.
Photodynamic inactivation of adenovirus
FHE Schagen
et al
874
Results
Viral inactivation
Photoinactivation of human adenoviruses was studied
using AdCMVLuc, a recombinant adenovirus that carries
the firefly luciferase gene as a reporter. Initially, six
photosensitizers were tested: methylene blue (MB),
protoporphyrin (PP), rose bengal (RB), uroporphyrin
(UP), aluminum phthalocyanine (AlPc) and aluminum
phthalocyanine tetrasulfonate (AlPcS
4
). The effectiveness
was assessed by exposing HepG2 cells to the treated
virus samples and measuring the expression of the
reporter gene. From this series of photosensitizers, MB,
RB, UP and AlPcS
4
tremendously reduced the reporter
gene expression, without affecting the viability of the
cells (data not shown). To optimize the exposure con-
ditions, various sensitizer concentrations and illumi-
nation periods were tested at a light intensity of
106 mW/cm
2
. As shown in Figure 1, all four photosensiti-
zers were able to inactivate AdCMVLuc upon illumi-
nation, while no effect was seen when the samples were
protected from light. The degree of inactivation was
Figure 1 Luciferase activity as a measure of AdCMVLuc infectivity after PDT with MB, RB, UP or AlPcS
4
. HepG2 cells were infected with photoinactiv-
ated AdCMVLuc virus, lysed after 2 days and luciferase activity was measured. Open symbols represent samples exposed to light, filled symbols depict
samples shielded from light. Infectivity of AdCMVLuc after treatment with (a) 2.7
m
MB (circles), 1.3
m
MB (squares), 0.7
m
MB (triangles) and
0.3
m
MB (diamonds); (b) 20
m
RB (circles), 10
m
RB (squares), 5
m
RB (triangles) and 2
m
RB (diamonds); (c) 50
m
UP (circles), 20
m
UP (squares), 10
m
UP (triangles) and 2
m
UP (diamonds); (d) 50
m
AlPcS
4
(circles), 20
m
AlPcS
4
(squares), 10
m
AlPcS
4
(triangles) and
1
m
AlPcS
4
(diamonds). The background activity in the luciferase assay was 200 light units.
clearly dependent on the photosensitizer used, its con-
centration and on the applied illumination. A concen-
tration of 10
8
p.f.u./ml AdCMVLuc, giving a luciferase
activity of 5 × 10
7
light units was completely inactivated
upon a 1-min illumination in 2.7 m MB (Figure 1a). The
same holds for 1.3 m MB, although here a 10-min illumi-
nation period was necessary for complete inactivation.
With either 10 m or 20 m of the photosensitizer RB, a
⬎5 log
10
decrease in luciferase activity was reached only
after an illumination period of 20 min (see Figure 1b).
Figure 1c depicts the results obtained with UP: a 50 m
concentration and 30-min illumination were needed for
complete inactivation. Although AlPcS
4
can significantly
inactivate AdCMVLuc, even upon treatment with 50 m
ALPcS
4
and a 30-min illumination inactivation is
incomplete.
To verify that the luciferase data reflect the viral titer,
plaque assays were performed in parallel (Table 1). The
results of the plaque assays were in good agreement with
the results of the luciferase assay. Inactivation was com-
plete, when 10
8
p.f.u./ml AdCMVLuc were treated with
MB, RB or UP, whereas 50 m AlPcS
4
and 30-min illumi-
Photodynamic inactivation of adenovirus
FHE Schagen
et al
875
Table 1 AdCMVLuc titers after photodynamic treatment
Photosensitizer Concentration Illumination AdCMVLuc titer
(
m
) period (min) (p.f.u./ml)
MB 1.3 0 4 × 10
7
5ND
0.7 5 7 × 10
2
10 ND
RB 20 0 4 × 10
7
51× 10
2
10 10 ND
10 1 × 10
3
20 ND
UP 50 0 4 × 10
7
10 6 × 10
2
30 ND
AlPcS
4
50 0 5 × 10
7
20 5 × 10
5
30 3 × 10
3
ND, not detectable (⬍10
2
).
nation were insufficient for inactivating 10
8
p.f.u./ml
completely. In the unilluminated control samples, the
virus titer decreased no more than two-fold with each of
the photosensitizers.
DNA integrity
Adenoviruses are double-stranded DNA viruses coated
by an icosahedral protein capsid. Consequently, the pho-
tosensitizers can exert their inactivating effect at the viral
DNA or the protein capsid. To study the integrity of the
adenoviral genome, DNA was isolated from illuminated
virions and analyzed by transfection and Southern
blotting.
AdCMVLuc was treated with the various photosensiti-
zers during illumination periods sufficient to inactivate
the virus completely. Subsequently, the intactness of the
isolated viral DNA was evaluated by means of the
luciferase expression in 911 cells. As depicted in Figure 2,
MB-based photoinactivation abolished the luciferase
Figure 2 Luciferase activity upon transfection of DNAfrom photoinactiv-
ated AdCMVLuc. 911 Cells were transfected with 2.1 ng AdCMVLuc
DNA isolated from virus particles, which had been illuminated for 15 min
in the case of MB (1.3
m
), 20 min in the case of RB (20
m
) or 30 min
in the case of UP (50
m
) or AlPcS
4
(50
m
). Black bars represent the
unexposed samples, white bars the exposed ones.
expression completely. This is in contrast with the other
photosensitizers, which hardly affected, if at all, the
capacity of the DNA to serve as template for transcrip-
tion. Transfection with RB- or UP-treated unilluminated
DNA samples resulted even in a slightly lower luciferase
activity than their illuminated counterparts. The controls
without photosensitizer did not show any effect of light
exposure. Similar results were obtained in HepG2 cells
(data not shown).
The integrity of the extracted DNA from light-treated
samples was further studied by Southern blot analysis.
As shown in Figure 3, all DNA samples yielded a sharp
banding pattern as a result of the PstI digestion, except
the DNA isolated from virus photoinactivated with
1.3 m MB and 15-min illumination. This sample showed
a smear, indicative of random DNA fragmentation.
Electromicroscopic analysis
To study whether the phototreatment led to gross disrup-
tion of the virions, aliquots of 5.0 × 10
8
p.f.u. AdCMVLuc
were photoinactivated and the viral particles were vis-
ualized by electron microscopy. No major differences
were seen between the protein capsids or overall struc-
tures of illuminated or unilluminated virus particles or
between any of the four photosensitizers (see Figure 4).
Close inspection of MB and illuminated versus unillumi-
nated virions showed however a slight effect on the
organized structure in the capsid, although its regular
icosahedral form is completely preserved (Figure 4a and
b). Apart from complete virions, additional structures can
be seen in the AlPcS
4
-exposed samples (Figure 4c and d).
These structures, possibly capsid fragments, are present
in similar quantities in illuminated and unilluminated
samples and therefore, are presumably not a result of the
activated photosensitizers.
Binding pattern
To assess changes in the binding to and/or entry into the
cell, binding assays using
3
H-labelled AdCMVLuc were
performed. Upon addition of unilluminated radio-
labelled virus to HeLa cells, approximately 40% of the
Figure 3 Southern blot analysis of photodynamically inactivated
AdCMVLuc. PDT conditions were as indicated. Viral DNA was isolated,
digested with PstI and 1.0 ng was size-fractionated. Hybridization was
performed with PstI-fragmented pJM17. The Figure shown is representa-
tive of several blots.
Photodynamic inactivation of adenovirus
FHE Schagen
et al
876
Figure 4 Electron micrographs of adenovirus particles negatively stained with uranyl acetate. 5 × 10
8
p.f.u. AdCMVLuc treated with 2.7
m
MB and
15 min illumination (a), or treated with 50
m
AlPcS
4
and 30 min illumination (c). Control particles similarly treated but shielded from illumination
are for MB represented by (b) and for AlPcS
4
by (d). One unit of the scale bar is 114 nm in (a), (b) and 141 nm in (c) and (d).
radio-label was found bound to the cells, of which
approximately 75% was internalized (Figure 5). This
binding and especially the internalized fraction was low
(0–2%) in CHO cells, a cell line which lacks the primary
receptor and serves as negative control.
9,10
Photodynamic
treatment caused a dramatic change in the binding pat-
tern. Photoinactivation induced by RB, UP or AlPcS
4
,
resulting in complete inactivation of
3
H-labelled
AdCMVLuc (data not shown), reduced the internalized
fraction from about 30% of the total radioactivity to 0–5%.
At the same time the external-bound, but noninternalized
fraction increased from approximately 15% to 45% for UP
and AlPcS
4
, leaving the total amount of attached radiola-
bel almost unaffected. This may be a result of aspecific
binding due to an effect on the primary receptor–fiber
complex formation, or more likely, a result of modifi-
Photodynamic inactivation of adenovirus
FHE Schagen
et al
877
Figure 5 Binding pattern of adenovirus to HeLa or CHO cells after pho-
toinactivation by MB (1.3
m
), RB (20
m
), UP (50
m
) or AlPcS
4
(50
m
). An amount of 1.8 × 10
6
p.f.u.
3
H-AdCMVLuc was phototreated
with the illumination period (min) as indicated. Black/light gray bars rep-
resent the binding to HeLa cells with the black part representing the tryp-
sin-sensitive fraction and the light gray part representing the internalized,
trypsin-resistant fraction. Dark gray/white bars (right hand bar in each
group) represent the binding to CHO cells, with the dark gray part as
trypsin-sensitive fraction and the white part as trypsin-resistant fraction.
Binding fractions are depicted as percentages of total radioactivity in
3
H-
AdCMVLuc used.
cation of the pentonbase, which is involved in the
initiation of internalization. The increase in the external-
bound fraction was less evident for the RB sample.
After a 1-min illumination in the presence of MB, the
binding pattern of adenovirus to HeLa cells did not
change significantly. Only a slight reduction of the
internalized fraction is observed. This is fully consistent
with the hypothesis that damage induced by MB is
established at the DNA level.
Effectivity of photoinactivation in blood plasma
To study whether this phototreatment could be useful for
biological samples, 2 × 10
8
p.f.u./ml AdCMVLuc in
⭓90% (vol/vol) rat plasma and each of the four photo-
sensitizers were illuminated for 30 min with
106 mWatt/cm
2
white light (Figure 6). In the case of non-
Figure 6 Luciferase activity as a measure of the AdCMVLuc infectivity
after photoinactivation of 10
7
p.f.u. in a background of approximately 90%
rat blood plasma. Photoinactivated samples were used for infection of
HepG2 cells. White bars represent the samples exposed to 106 mW/cm
2
white light for a period of 30 min, black bars are treated similarly, but
were protected from illumination. The concentrations used are: 2.7
m
MB, 40
m
RB, 100
m
UP and 100
m
AlPcS
4
.
exposed samples and in the controls without photosensit-
izer, the infection with 10
7
p.f.u. resulted in a stable
luciferase activity of approximately 10
7
light units. In the
illuminated samples only MB caused a 4 log
10
reduction
in luciferase activity, whereas the others left the infec-
tivity almost unaffected: phototreatment with RB, UP or
AlPcS
4
yielded only a two- to three-fold reduction.
Side-effects of the photodynamic treatment
To study the effect of PDT on plasma proteins two para-
meters were evaluated. Firstly, luciferase was added to
rat plasma and its activity was measured to reflect the
integrity of the protein. Secondly, factor VIII levels in
human blood plasma were analyzed. As depicted in
Figure 7a, application of PDT with 30-min illumination
in 2.7 m MB resulted in a 14% decrease of luciferase
activity compared with illumination without photosensit-
izer. RB, UP and AlPcS
4
were much more pronounced in
their protein inactivation: 93%, 97% and 98%, respect-
ively. This is in accordance with the effect of the treat-
ment determined by measurement of the factor VIII lev-
els. After a 15-min illumination in 2.7 m MB we could
still detect 75% of the amount of factor VIII protein. Fac-
tor VIII was, as expected, much more susceptible to the
other photosensitizers. PDT with a 20-min illumination
in 40 m RB, a 30-min illumination in 50 m AlPcS
4
or
Figure 7 PDT induced side-effects evaluated by (a) luciferase activity or
(b) cell viability. (a) In the background of 70% blood plasma, extracts
from AdCMVLuc-infected cells were exposed to a 30-min illumination
without photosensitizer (control) or in presence of 2.7
m
MB, 40
m
RB, 100
m
UP or 100
m
AlPcS
4
. The luciferase activity is shown as
percentage of the control. (b)
3
H-thymidine incorporation of HepG2 cells,
incubated for 48 h in the indicated MB concentrations. White bars repes-
ent the MB samples, which were illuminated for a 30-min period before
they were added to the cells. Black bars represent samples, which were
protected from illumination.
Photodynamic inactivation of adenovirus
FHE Schagen
et al
878
50 m UP reduced the factor VIII levels to undetectable
levels (⬍1%; data not shown).
As MB is the peferred photosensitizer for adenovirus
inactivation in plasma samples, the effect of MB on the
viability of cells was assessed by measuring the incorpor-
ation of
3
H-thymidine. After a 48-h exposure to 2.7 m
MB of HepG2 cells, there was no effect detected on the
replication (Figure 7b), demonstrating that MB is not
affecting the viability of the cells.
Discussion
So far, photodynamic treatment has been used mainly for
inactivation of enveloped viruses. Although in some
studies inactivation of nonenveloped viruses has been
reported, the inactivation of nonenveloped viruses like
adenovirus, poliovirus, or parvovirus,
5,11–15
has never
been very efficient. In this study, we have analyzed sev-
eral sensitizers for their capacity to inactivate adenovirus
and determined their target site. White light plus low
concentrations of MB sufficed to inactivate completely
10
8
p.f.u./ml AdCMVLuc in PBS. RB and UP achieved
the same effect, but at higher concentrations, whereas
AlPcS
4
affected adenovirus to a lesser extent at all con-
ditions tested.
So far, other groups have never been able to show any
photoinactivating effect of AlPcS
4
on nonenveloped
viruses, such as encephalomyocarditis virus (EMCV) or
adenovirus.
6,7,12
These studies however, used less strin-
gent conditions and were performed on internalized
adenovirus and in the case of EMCV, in the presence of
human plasma.
7,12
This has possibly introduced too many
competitors for AlPcS
4
-induced viral damage, as con-
firmed by our AlPcS
4
data, which showed complete loss
of inactivation in a background of rat plasma. This is sup-
ported by the work of Malik et al,
16
who demonstrated
that protein damage (viz crosslinks) was responsible for
the loss of infectivity of HSV after PDT with several
phthalocyanines.
RB-induced inactivation of influenza and VSV was
reported to be correlated with their inability to fuse with
the host cell membrane.
17,18
After phototreatment, both
viruses showed cross-linking of viral membrane proteins,
which are involved in this fusion. Although the use of
uroporphyrin for virus inactivation has never been
reported, the photodynamic effects of porphyrins, includ-
ing uroporphyrin, on isolated cells, cell membranes, sub-
cellular organelles and on proteins of cytosol and plasma
have been investigated extensively.
19–21
All these studies
indicate that porphyrins induce photodynamic modifi-
cation of proteins.
In approximately 90% blood plasma the photosensitiz-
ers RB, UP and AlPcS
4
only cause a two- to three-fold
reduction in viral infectivity versus a more than 10
5
-fold
reduction in PBS. Apparently, the large amounts of
plasma protein quench the effect on the adenovirus, con-
firming the hypothesis of a protein basis of adenoviral
inactivation by these sensitizers.
The capacity of MB to photoinactivate enveloped
viruses is well documented and the molecular mech-
anism for this photoinactivation has been studied by sev-
eral groups.
5,22,23
It is generally accepted that MB strongly
associates with DNA and upon illumination, induces the
formation of 8-hydroxyguanine (8-OHG).
24–26
Single-
stranded nicks were observed as result of this 8-OHG for-
mation.
24
The data presented here strongly indicate a
preference of MB for DNA, as it was the only sensitizer
tested that completely impaired the functional integrity
of the viral DNA after a 5-min exposure (Figure 2). Upon
extended illumination (15 min), Southern analysis
revealed DNA fragmentation. This suggests that forma-
tion of 8-OHG is sufficient for inactivation, which is in
agreement with the photoinactivation of M13, a non-
enveloped bacteriophage with a single-stranded circular
DNA genome.
22
Abe and Wagner observed a correlation
between the inactivating effect of MB and the number of
8-OHG piperidine-labile sites. Moreover, they described
that higher illumination doses introduced more piperi-
dine-labile sites, as shown by DNA smearing.
Although the inactivating capacity of MB diminished
in the presence of 90% rat plasma, PDT still induced a
⬎4 log
10
-fold reduction of the luciferase activity to almost
background levels. Despite the stringency of the con-
ditions, we were still able to detect 86% of luciferase
activity and 75% of blood clotting factor VIII in blood
plasma, as compared with untreated controls. Factor VIII
is known to be labile
27
and constitutes therefore an appro-
priate model to study the effects that MB and light
exposure has on biological samples, as confirmed by
Lambrecht et al.
5
This group showed that factor VIII is
even less affected at milder conditions. In this perspective
MB appears to be a promising agent for the decontami-
nation of blood or blood products, especially as this pho-
tosensitizer has been reported to induce no genotoxicity
in vivo
10
and did not show any effect on the viability of
HepG2s.
As has been described by Cotten et al
28
defective
virions can be used to facilitate the entry of ligand-coated
DNA particles into cells. In the latter study, inactivation
of adenovirus was based on UV exposure in the presence
of psoralens.
29
A 5-log
10
reduction in virus titer was gen-
erated with the use of 110 g/ml 8-methoxypsoralen (8-
MOP) and 280 g/ml 4′-aminomethyl-4,5′,8-trimethylp-
soralen (AMT), while DNA delivery capacity was main-
tained. Psoralen-derivatives are however, reported to be
mutagenic, which makes their removal mandatory and
their use laborious.
30
In contrast to psoralen, MB is not
reported to have any mutagenic or genotoxic effect. This
makes the use of MB together with the short white-light
illumination very easy and efficient. The plasmid-
delivering capacity of MB-inactivated adenovirus has not
been investigated and although the virions were shown
to be internalized, aspecific protein modification and con-
comitant loss of endosomolytic properties cannot be
excluded completely.
Materials and methods
Cell culture and virus production
The Ad5 E1-transformed human embryonic retina cell
line 911
31
and the hepatoma cell line HepG2 were grown
in Dulbecco’s modified Eagle medium (DMEM) sup-
plemented with 10% fetal calf serum (FCS), antibiotics
and 3 g/l glucose in a 5% CO
2
atmosphere at 37°C.
Incorporation of
3
H-thymidine in chromosomal DNA
was assessed on HepG2 cells, which were grown on a
six-well plate. At a 40% density, the medium was
replaced by 2 ml DMEM with 1.0% dialyzed FCS and
a MB concentrate. They were incubated at 37°C for 3 h,
Photodynamic inactivation of adenovirus
FHE Schagen
et al
879
whereafter 10 Ci
3
H-thymidine was added. After 48 h,
the medium was removed and the cells were washed
twice with PBS. Finally the cells were detached from the
dish by a trypsin treatment. The
3
H-thymidine content
of the resulting fractions was measured by liquid scintil-
lation counting.
The recombinant adenovirus AdCMVLuc (defective for
the E1 region), which was kindly provided by Dr J Herz
(University of Texas, Southwestern Medical Centre,
Dallas, TX, USA) contains the firefly luciferase gene
under control of the cytomegalovirus immediate–early
promoter.
32
This virus was propagated in the comp-
lementing cell line 911 and isolated as described by
Stratford-Perricaudet and Perricaudet.
33
Briefly, 48 h after
infection, detached cells were harvested and collected in
1 ml phosphate-buffered saline (PBS) containing 1%
horse serum (HS). This was followed by three rounds of
freeze-thawing, two steps of CsCl density-gradient puri-
fication and dialysis to remove the CsCl from the purified
virus suspension. The resulting AdCMVLuc stock was
stored as 50–100 l aliquots in 10% glycerol at −80°C.
3
H-thymidine-labeled AdCMVLuc was produced in
911 cells. A nearly confluent 911 monolayer in a 10-cm
dish was infected by AdCMVLuc in 1 ml 2% HS in PBS
at a multiplicity of infection (MOI) of 5. After 45 min at
room temperature, the inoculum was replaced by 10 ml
of fresh DMEM containing 1% dialyzed FCS. Seven hours
later, this was replaced again by 10 ml fresh DMEM now
containing 0.75% dialyzed FCS and 100 Ci
3
H-
thymidine. One day later the medium was enriched with
2% dialyzed FCS. The medium was collected 48 h after
infection, mixed with an equal volume of 20% PEG-6000
(BDH, Poole, UK) and incubated overnight at 4°C. The
PEG-6000
3
H-AdCMVLuc precipitate was sedimented by
centrifugation at 2800 g for 20 min at 4°C. The resulting
pellet was resolved in 1 × TD buffer (25 mm Tris, 137 mm
NaCl, 5 mm KCl, 0.73 mm Na
2
HPO
4
, 0.9 mm CaCl
2
,
0.5 mm MgCl
2
pH 7.45) and loaded on to a CsCl block-
gradient, consisting of a 2 ml heavy CsCl cushion
(1.45 g/cm
3
CsCl/10 mm Tris (pH 8.1), 1 mm EDTA;
refractive index (RI) 1.374) and a 3 ml light CsCl cushion
(1.20 g/cm
3
CsCl/10 mm Tris (pH 8.1), 1 mm EDTA; RI,
1.355). The centrifugation was performed in a SW-41
rotor at 130 000 g for 1 h at 17°C. The resulting virus band
was dialyzed and stored at −80°C in aliquots containing
10% glycerol.
AdCMVLuc infectivity assay
After PDT, the infectivity of AdCMVLuc was assessed
via the expression level of the luciferase reporter gene
and by determination of the titer. In the luciferase assay,
PDT samples containing photosensitizer and the equival-
ent of 10
7
p.f.u. AdCMVLuc, were adjusted to 200 l with
PBS and added to HepG2 cells in six-well plates. After a
20-min incubation at room temperature, the inoculum
was replaced by 2 ml fresh DMEM containing 2% HS.
Two days after infection, cells were lysed in 200 l lysis
buffer (25 mm Tris-phosphate, pH 7.8, 2 mm DTT, 2 mm
1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid, 10%
glycerol, 1% Triton X-100), the cell debris was sedimented
and supernatant was collected as whole lysate. Luciferase
activity was determined in 10 l of whole lysate by
addition of luciferase assay reagent (Promega, Madison,
WI, USA). After a 10-s incubation, the produced light was
measured in a luminometer (Lumat LB 9501, Berthold,
Bad Wildbad, Germany).
The adenovirus titers were determined in plaque
assays, as described by Fallaux et al.
31
Briefly, PDT
samples were serially diluted in 2 ml of DMEM contain-
ing 2% HS (heat-inactivated at 56°C for 30 min) and
added to confluent 911 cells in six-well plates. After 2 h
of incubation at 37°C, the inoculum was aspirated, and
the cells were overlaid by F15 minimum essential
medium (MEM) containing 12.3 mm MgCl
2
, 0.0025% l-
glutamine, 2% HS, 0.85% agarose (Sigma, St Louis, MO,
USA) and 20 mm HEPES pH 7.4. Plaques were scored
7–10 days after infection.
Photosensitizers
The photosensitizer MB was purchased from GT Baker
(Deventer, The Netherlands) and was stored as a 1.3-mm
stock solution at 4°C. RB was obtained from BDH (Poole,
UK) and AlPcS
4
and UP from Porphyrin Products
(Logan, UT, USA). The latter were stored as 1.0 mm stock
solutions at 4°C. All stocks were prepared in 50 mm
sodium-phosphate buffer (pH 7.4), filter-sterilized
(0.22 m filter) and stored in the dark.
Photoinactivation
Photoinactivations were performed, unless indicated
otherwise, on 10
7
p.f.u. AdCMVLuc in 100 l PBS. All
illuminations were done at a constant temperature of
20°C using a slide projector with a 150 W Xenophot HLX
64640 lamp as light source. The dose rate of white-light
irradiation was 106 mW/cm
2
, as measured with a Gentec
(Saint Foy, CA, USA) TMP-310 photometer. Controls
were prepared and incubated identically, but were shi-
elded from light by wrapping them in aluminum foil.
Analysis of viral DNA
The DNA integrity of the photoinactivated virions was
tested by transfection of the viral DNA into HepG2 and
911 cells and by Southern blot analysis. After photoinac-
tivation of 3 × 10
8
p.f.u. AdCMVLuc, aliquots of the
exposed virus were mixed with 5 g carrier DNA and
400 g proteinase K, the volume adapted to 100 l with
PBS and incubated overnight at 48°C to degrade the viral
capsid. The DNA was further purified by phenol/
chloroform (1:1) extraction and ethanol precipitation and
2.1 ng was used to transfect HepG2 or 911 cells in six-
well plates. DNA transfections were performed with 0.5
mg/ml DEAE-dextran in 1 × TBS (25 mm Tris Cl, 137 mm
NaCl, 5 mm KCl, 0.7 mm CaCl
2
, 0.5 mm MgCl
2
, 0.6 mm
Na
2
HPO
4
; pH 7.4).
Other aliquots of the exposed virus were used for
Southern blot analysis. DNA was isolated essentially as
described above, with 1 g tRNA as carrier. The DNA
was subsequently digested with PstI, and 1 ng of each
sample was size-fractionated by electrophoresis on a 1.2%
agarose gel. The fractionated DNA was transferred on to
Hybond N+ (Amersham, UK) with 0.4 m NaOH/0.6 m
NaCl as the transfer solution. The resulting blot was
hybridized with the radio-labeled PstI-digested plasmid
pJM17 (containing the entire adenovirus genome) as
probe. Labelling was performed according to the ran-
dom-primer method using ␣-
32
P-dATP, random hexanu-
cleotides and Klenow DNA polymerase. Finally, the
Southern blot was exposed to Fuji-RX film at −80°C.
Photodynamic inactivation of adenovirus
FHE Schagen
et al
880
Electron microscopy
Aliquots of 5.0 × 10
8
p.f.u. AdCMVLuc were exposed to
light in 2.7 m MB for 0 and 15 min, in 20 m RB for 0
and 20 min and in either 50 m AlPcS
4
or 50 m UP for
0 and 30 min. All samples were fixed for 5 min in equal
volumes of 1% glutaraldehyde at room temperature.
Hereafter, virus samples were loaded on to carbon-
coated grids, negatively stained with 3–7% uranyl acetate
and analyzed with a Philips electron microscope
(Eindhoven, The Netherlands).
Binding characteristics of adenovirus
The general procedure of the virion-attachment assay
was outlined by Wohlfart et al.
34
60–70% confluent mono-
layers of either HeLa or CHO cells were preincubated
with 1% BSA for 10 min at 20°C.
35
Samples of 1.6 × 10
7
p.f.u./ml
3
H-thymidine-labeled virions were photoinac-
tivated, added to the monolayer at a MOI of 1–2 and
incubated for 120 min at 37°C. Subsequently, the inocu-
lum was aspirated and the cells were washed twice with
ice-cold 2% HS in PBS to remove the unbound virus.
Finally, the monolayers were incubated in the presence
of trypsin, until the cells detached from the dish. This cell
suspension was centrifuged at 150 g for 5 min and div-
ided into the cell pellet, representing the trypsin-insensi-
tive adenovirus fraction (the internalized virions) and the
supernatant, representing the trypsin-sensitive fraction
(the external bound, but non-internalized virions).
36
Radioactivity of all fractions was measured by liquid
scintillation counting and the percentage of attached or
internalized virus was calculated.
Acknowledgements
We are grateful to Ferry Spies and Hans van der Meulen
for the electron microscopy and Diana van den Wollen-
berg for excellent technical assistance. We also want to
thank Nico van Tilburg for the analysis of the plasma
samples. This investigation was financially supported by
the Dutch Organisation for Scientific Research (NWO),
project No. 901–01–096, and the Dutch ‘Praeventiefonds’,
grant No. 28–2178–1.
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