Download Modified vaccinia virus Ankara multiplies in rat IEC

Journal of General Virology (2006), 87, 21–27
Short
Communication
DOI 10.1099/vir.0.81479-0
Modified vaccinia virus Ankara multiplies in rat
IEC-6 cells and limited production of mature
virions occurs in other mammalian cell lines
Malachy Ifeanyi Okeke,1 Øivind Nilssen2 and Terje Traavik1,3
1
Department of Microbiology and Virology, Faculty of Medicine, University of Tromsø, N-9037
Tromsø, Norway
Correspondence
Terje Traavik
2
[email protected]
Department of Medical Genetics, University Hospital of North Norway, N-9038 Tromsø,
Norway
3
GENOK-Norwegian Institute of Gene Ecology, Tromsø Science Park, N-9294 Tromsø, Norway
Received 31 August 2005
Accepted 28 September 2005
Recombinant viruses based on modified vaccinia virus Ankara (MVA) are vaccine candidates
against infectious diseases and cancers. Presently, multiplication of MVA has been demonstrated in
chicken embryo fibroblast and baby hamster kidney (BHK-21) cells only. The multiplication and
morphogenesis of a recombinant (MVA-HANP) and non-recombinant MVA strain in BHK-21
and 12 other mammalian cell lines have now been compared. Rat IEC-6 cells were fully permissive
to MVA infection. The virus yield in IEC-6 cells was similar to that obtained in BHK-21 cells at
low as well as high multiplicities of infection. Vero cells were semi-permissive to MVA infection.
Mature virions were produced in supposedly non-permissive cell lines. The multiplication and
morphogenesis of non-recombinant MVA and MVA-HANP were similar. These results are relevant
to the production and biosafety of MVA-vectored vaccines.
Modified vaccinia virus Ankara (MVA) is a highly attenuated vaccinia virus (VACV) strain derived from VACV
Ankara. MVA was developed by more than 570 serial passages in chicken embryo fibroblast (CEF) cultures during
which it incurred multiple DNA deletions (Meyer et al.,
1991). Genes that encode host-range and immune-evasion
factors are either deleted or fragmented in MVA (Antoine
et al., 1998; Blanchard et al., 1998). MVA multiplies efficiently in CEF and baby hamster kidney (BHK-21) cells. In
other mammalian cell lines incomplete morphogenesis
occurs (Caroll & Moss, 1997; Drexler et al., 1998). Recombinant and non-recombinant MVA have been reported
apathogenic in several in vivo models even when administered in high doses to immune deficient animals (Stittelaar
et al., 2001; Ramirez et al., 2003; Hanke et al., 2005). A recent
report indicated, however, that immunization of ferrets
with MVA-vectored vaccine against severe acute respiratory syndrome (SARS) is associated with enhanced hepatitis
(Weingart et al., 2004). Overall, the extreme attenuation
and un-impaired gene expression in non-permissive cells
recommend MVA as a promising vaccine vector. Presently,
several recombinant MVA constructs are being evaluated
for use as vaccine candidates against infectious diseases and
cancers (Sutter et al., 1994; Corona Gutierrez et al., 2002;
Drexler et al., 2004; Wyatt et al., 2004; Smith et al., 2005). In
the future, it is likely that MVA-vectored vaccines will be
Supplementary figures are available in JGV Online.
0008-1479 G 2006 SGM
licensed for treatment of human and animal diseases. Thus,
continued evaluation of MVA morphogenesis and multiplication in mammalian cells is essential to the production
of safe MVA-vectored vaccines. Assembly of MVA in the
only known permissive cell lines (CEF and BHK-21) is
similar to the assembly of other VACV strains (Sancho et al.,
2002; Gallego-Gomez et al., 2003). VACV assembly results
in the formation of four forms (Sodeik & Krinjse-Locker,
2002; Smith & Law, 2004). Details of the processes leading
to these morphological forms have been reported in a
number of articles (e.g. Tooze et al., 1993; Schmelz et al.,
1994; Hollinshead et al., 2001; Risco et al., 2002; Meiser et al.,
2003b; Carter et al., 2005).
MVA assembly in mammalian cells, except BHK-21, seems
to be blocked at the immature virus stages (Caroll & Moss,
1997). However, this fact is based on the limited number of
mammalian cell lines studied so far. MVA-based vectors
and vaccines are currently being produced in CEF and
BHK-21 cell lines. The establishment and maintenance of
CEF cultures require experience in preparing primary tissue
cultures. CEF cultures survive few passages and weekly
de novo preparations are required (Drexler et al., 1998).
During serial passages, BHK-21 cultures rapidly deteriorate
on reaching confluence, and will hence be inadequate for
large-scale production purposes, especially in batch systems
that require high-density viable cells. Our aim was to
identify alternative cell lines that support efficient MVA
multiplication. Thus, we investigated the multiplication and
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21
M. I. Okeke, Ø. Nilssen and T. Traavik
morphogenesis of recombinant and non-recombinant MVA
in 13 mammalian cell lines.
The cell lines (Table 1) used in this study were purchased
from, and grown under conditions suggested by, ATCC. The
recombinant MVA (MVA-HANP) was kindly provided by
Dr Bernard Moss, National Institute of Health, USA. The
MVA-HANP genome contains the influenza virus (A/PR/8/
34) haemagglutinin (HA) and nucleoprotein (NP) cDNA
inserts (Sutter et al., 1994). MVAnr (non-recombinant
MVA; ATCC VR-1508) was purchased from ATCC. Antiinfluenza virus HA mouse monoclonal antibody, H28E23,
was also a gift from Dr Bernard Moss. Both MVA-HANP
and MVAnr-infected foci were visualized by immunostaining as described previously (Hansen et al., 2004;
Hornemann et al., 2003, respectively).
To determine whether other mammalian cell lines, besides
BHK-21, were permissive to MVA infection, different cell
lines were infected with MVA-HANP and MVAnr at an
m.o.i. of 0?05 IU per cell. After adsorption for 1 h, cell
monolayers were washed twice with PBS and incubated for
72 h post-infection (p.i.). Multiplication (fold increase in
virus titre) was determined by dividing virus yield at 72 h
p.i. by virus titre after adsorption. Permissiveness and cytopathic effect (CPE) were defined according to Caroll & Moss
(1997). Rat IEC-6 cells supported efficient multiplication
of MVA-HANP as well as MVAnr. IEC-6 and BHK-21
cells were permissive to MVA-HANP infection with a fold
increase in virus titre of 370 (2?56 log) and 477 (2?67 log),
respectively (Table 1). MVAnr multiplied more efficiently
than MVA-HANP in both cell lines. The multiplication of
MVAnr in IEC-6 and BHK-21 cells was 2218 (3?34 log) and
6068 (3?78 log), respectively (Table 1). Vero, NMULI and
A549 cells were semi-permissive to MVAnr, whereas only
Vero cells was semi-permissive to MVA-HANP (Table 1).
Other African green monkey kidney cell lines (CV-1 and BSC-1) have been reported previously to be semi-permissive to
MVA (Caroll & Moss, 1997). Host-range restriction could
result from inhibition of infectious virus formation or
spread (Caroll & Moss, 1997). To detect cell spread, cells
were infected with MVA-HANP and MVAnr at an m.o.i. of
0?01. At 24, 48 and 72 h p.i., the cells were immunostained.
The number of cells per infected focus at different time
points post-infection was used to quantify cell spread. IEC-6
cells supported efficient cell-to-cell spread of MVA-HANP
(Fig. 1a) and MVAnr (Fig. 1b). Similar results were obtained
with infected BHK-21 cells (data not shown). MVAnr had
enhanced cell spread and CPE compared with recombinant
MVA in IEC-6 cells (Fig. 1a and b) and BHK-21 cells (data
not shown). Limited cell spread was present in NMULI and
293 cells infected with MVA-HANP. Other non-permissive
cell lines formed exclusively single-cell immunostained
foci; an observation indicating lack of cell spread (data not
shown). Vero cells infected with MVAnr, but not MVAHANP, showed significant cell spread although not as pronounced as in IEC-6 or BHK-21 cells (data not shown). The
deletion or disruption of host-range genes may explain the
inability of MVAnr and MVA-HANP to multiply and spread
in most non-permissive cell lines. Except for MVA O18L (an
orthologue of VACV-Copenhagen), orthopoxvirus hostrange genes (Gillard et al., 1985; Perkus et al., 1990; Ali et al.,
1994; Beattie et al., 1996) are either deleted or disrupted in
MVA strains (Antoine et al., 1998).
To evaluate further the multiplication of MVA in IEC-6
and BHK-21 cells, the kinetics of virus multiplication were
Table 1. Screening of mammalian cell lines for productive infection of MVA-HANP and MVA
Virus multiplication (fold increase in virus titre) was determined by dividing virus yield at 72 h by virus titre after adsorption. The values
are the mean of two independent experiments titrated in duplicate. Letters in parentheses refer to permissiveness (P), semi-permissiveness
(SP) and non-permissiveness (NP).
Cell line
IEC-6
BHK-21
Caco-2
H411E
FHs74int
Hutu-80
Vero
RK-13
CHO-K1
A549
PK15
NMULI
293
22
ATCC code
CRL-1592
CCL-10
HTB-37
CRL-1548
CCL-241
HTB-40
CCL-81
CCL-37
CCL-61
CCL-185
CCL-33
CRL-1638
CRL-1573
Species
Rat
Hamster, syrian
Human
Rat
Human
Human
African green monkey
Rabbit
Hamster, chinese
Human
Pig
Mouse
Human
CPE
Multiplication
MVA-HANP
MVA
++
+++
2
+
2
2
+
+
2
2
+
2
2
+++
++++
2
+
2
2
++
+
2
+
+
++
2
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MVA-HANP
370
477
<1?0
<1?0
<1?0
<1?0
2?0
<1?0
<1?0
<1?0
<1?0
<1?0
<1?0
(P)
(P)
(NP)
(NP)
(NP)
(NP)
(SP)
(NP)
(NP)
(NP)
(NP)
(NP)
(NP)
MVA
2218
6068
<1?0
<1?0
<1?0
<1?0
10?0
<1?0
<1?0
1?8
<1?0
6?0
<1?0
(P)
(P)
(NP)
(NP)
(NP)
(NP)
(SP)
(NP)
(NP)
(SP)
(NP)
(SP)
(NP)
Journal of General Virology 87
Productive MVA infections
24 h p.i.
48 h p.i.
72 h p.i.
(a)
(b)
8
(d)
8
7
7
6
6
_
Virus titre (log10 IU ml 1)
_
Virus titre (log10 IU ml 1)
(c)
5
4
3
2
MVA-HANP/cell virus
1
MVA-HANP/medium virus
0
MVA/cell virus
0
12
24 36
48
60
4
MVA-HANP/cell virus
3
MVA-HANP-medium virus
2
MVA/cell virus
MVA/medium virus
1
0
MVA/medium virus
_1
5
_1
72 84
0
12
24
(f) 8
7
7
48
60
72
84
_
_
Virus titre (log10 IU ml 1)
(e) 8
Virus titre (log10 IU ml 1)
36
Time (h)
Time (h)
6
5
MVA-HANP/cell virus
4
MVA-HANP/medium virus
MVA/cell virus
3
6
5
MVA-HANP/cell virus
4
MVA-HANP/medium virus
MVA/cell virus
3
MVA/medium virus
MVA/medium virus
2
2
0
10
20
30
40
0
10
Time (h)
20
30
40
Time (h)
Fig. 1. Cell-to-cell spread and multiplication of recombinant and non-recombinant MVA in mammalian cells. Cell spread of
MVA-HANP (a) and MVA (b) in IEC-6 cells. The panels show representative fields at approximately6200 magnification.
Arrowheads point to foci containing many stained cells while arrows point to singly stained cells. Low multiplicity infection
(0?05 IU per cell) of IEC-6 (c) and BHK-21 (d) cells with MVA-HANP and MVA. Multiplication of MVA-HANP and MVA in
IEC-6 (e) and BHK-21 (f) cells at a high m.o.i. (5 IU per cell). Values are the mean of two independent experiments titrated in
duplicate.
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23
M. I. Okeke, Ø. Nilssen and T. Traavik
studied at low as well as at high m.o.i. values. Cell monolayers in 25 cm2 tissue culture flasks were infected with
MVA-HANP and MVAnr at an m.o.i. of 0?05 IU per cell
(low) and 5 IU per cell (high), respectively. After adsorption
for 1 h at 37 uC, infected cells were washed twice with PBS
and incubated in appropriate medium supplemented with
2?5 % fetal bovine serum at 37 uC in a 5 % CO2 atmosphere.
At multiple time points post-infection, cells and medium
(culture supernatant) were harvested. Intracellular virions
were released by three cycles of freeze–thawing and brief
sonication. Virus titre in the cell lysate and culture medium
was determined by back titration onto BHK-21 cell monolayers. Infected cell-foci were visualized by immunostaining
after 24 h. The kinetics of MVA-HANP multiplication at
low and high m.o.i. in IEC-6 and BHK-21 cells were similar
(Fig. 1c–f). At both m.o.i. values, there were fast kinetics of
MVA-HANP in both IEC-6 and BHK-21 cells at early time
points post-infection. The initial faster replication kinetics
of MVAnr have also been reported by other investigators
(Caroll & Moss, 1997; Meiser et al., 2003a), and this phenomenon may be due to faster entry kinetics (Sancho et al.,
2002). At high m.o.i., medium virus titre of MVA-HANP in
BHK-21 but not IEC-6 cells was similar to the cell virus
titre (Fig. 1e and f). A reasonable explanation is that a high
amount of intracellular virus was liberated into the medium
following cell lysis, since extensive CPE occurred in BHK-21
cells infected with MVA-HANP at high m.o.i. Consistent
with the results in Table 1, MVAnr multiplied to higher
levels at low m.o.i. in both IEC-6 and BHK-21 cells rather
than MVA-HANP (Fig. 1c and d). The difference was more
pronounced in BHK-21 cells (Fig. 1d). At high m.o.i., the
kinetics of cell-associated and medium virus of MVAnr are
virtually identical to those of MVA-HANP in IEC-6 cells
(Fig. 1e). However, in BHK-21 cells, the cell virus titre of
MVAnr is approximately 1?0 log higher than that of MVAHANP across all time points post-infection (Fig. 1f). Taken
together these results demonstrate that the multiplication
of MVA-HANP and MVAnr in IEC-6 and BHK-21 cells is
comparable. Thus, IEC-6 cells can serve as an alternative to
BHK-21 and CEF cells for the production of MVA-based
vectors and vaccines. IEC-6 cells have features characteristic
of the intestinal epithelium in intact mammals (Quaroni
et al., 1979; Wood et al., 2003; Wang et al., 2003) and may
serve as a more authentic in vitro model for studying host
factors that modulate MVA host-range.
Our cell spread experiment suggested that mature viruses
might be present in some of the supposedly semi- or nonpermissive cell lines. We performed quantitative electron
microscopy (EM) with the aim of defining all viral forms
produced in the course of MVA infection. Cell monolayers
in six-well plates (Nunc) were infected with MVA-HANP
and MVAnr, respectively, at an m.o.i. of 5 IU per cell. After
adsorption for 1 h at 4 uC, the cells were washed three
times with PBS and incubated with fresh medium at 37 uC
in 5 % CO2 atmosphere for 6, 12, 24 and 48 h p.i. At
appropriate time points post-infection, infected cells were
fixed and processed for EM as described previously
24
(Mckelvey et al., 2002). Morphological forms of MVA
were counted in 50 section profiles of cells that were clearly
infected. Absolute and relative amounts of each viral form
were quantified for each of the 13 mammalian cell lines.
Complete morphogenesis of both MVA strains occurred in
IEC-6 cells. All four mature virion forms were produced
with MVA-HANP (Supplementary Fig. S1 available in JGV
Online, Table 2) as well as with MVAnr (Supplementary
Fig. S2). Although the morphogenetic structures present
in MVA-HANP-infected IEC-6 cells were the same as in
BHK-21 cells, there were differences in their abundance.
Cell-associated enveloped viruses (CEV) represented 41?3 %
(n=243) of mature viruses (IMV, intracellular mature virus;
IEV, intracellular enveloped virus; and CEV) produced in
IEC-6 as opposed to only 5?2 % (n=42) produced in BHK21 cells (Table 2). Conversely, in BHK-21 cells, a substantial
amount of IMV (70?5 %) and a low amount of CEV (5?2 %)
were produced (Table 2). Meiser et al. (2003a) reported that
50 % of mature viruses produced in MVA-infected CEF
were CEV. This is similar to what we obtained in IEC-6 cells.
Our results hence differ from those of Spehner et al. (2000),
which implied that enveloped viruses (IEV and CEV) were
the predominant mature viral forms in MVA-infected
BHK-21 cells. However, the difference may be a reflection
of different methodologies. Quantification by EM in which
50 cell sections were analysed may provide another picture
of relative proportion of enveloped particles rather than
quantification by CsCl gradient as reported by others. Our
EM data also demonstrated the heterogeneity in the block
of the MVA-HANP assembly in different cell lines (Table 2).
In addition to the normal morphogenetic structures, dense
particles (DPs) were produced in non-permissive and to a
lesser degree in permissive cell lines (Supplementary Fig. S1,
Table 2). There was a large accumulation of DPs in human
cell lines (Caco-2, A549 and 293) infected with MVAHANP. The DPs were slightly smaller in diameter and more
electron-dense than typical immature viruses (Supplementary Fig. S1). The DPs were naked or enveloped by single or
double membranes (Supplementary Fig. S1). Similar forms
of DPs have been observed in HeLa and CEF cells infected
with MVA (Meiser et al., 2003b; Caroll & Moss, 1997;
Gallego-Gomez et al., 2003). The enwrapment of DPs is
similar to the manner in which IMVs are enveloped to form
IEV, CEV and extracellular enveloped virus (EEV). This may
suggest that DPs encode signals for trans-Golgi network
wrapping, intracellular transport and a capacity to egress
from the cell. This is in contrast to earlier hypotheses,
suggesting that such signals were residing in the IMV only
(Krijnse-Locker et al., 2000). Although DPs have been
described as the transition between immature virus and
IMV (Caroll & Moss, 1997; Gallego-Gomez et al., 2003), it is
more likely that they are products of defective virion
morphogenesis. This is because DPs produced in 293 cells
failed to multiply when inoculated into permissive BHK-21
or IEC-6 cells (data not shown). Our result is consistent
with a previous report showing that DPs produced in
MVA-infected HeLa cells were non-infectious (Meiser
et al., 2003b). The morphogenesis of MVAnr in different
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Journal of General Virology 87
Productive MVA infections
Table 2. Quantification of viral forms produced in various mammalian cell lines infected for 24 h with MVA-HANP
Different viral forms were counted in 50 section profiles of cells that were clearly infected. Absolute and relative amounts of various viral
forms were calculated. The values in parentheses refer to the relative amount of viral forms as a percentage. Relative amount of each viral
form made at 24 h p.i. was calculated by dividing the number of each viral form in 50 cell sections by the total of all the viral forms
counted. C, Crescent; IV1, incomplete immature virus; IV2, complete immature virus; IV3, complete immature virus with DNA spot; DP,
dense particle (this includes forms that are naked or surrounded by single or double membranes); IMV, intracellular mature virus; IEV,
intracellular enveloped virus; and CEV, cell-associated enveloped virus.
Cell line
IEC6
BHK-21
Caco-2
H411E
FHs74int
Hutu-80
Vero
RK-13
CHO-K1
A549
PK15
NMULI
293
C
108
64
33
178
104
0
244
12
0
165
98
124
85
(7?4)
(4?2)
(5?9)
(16?7)
(15?8)
(0?0)
(23?1)
(19?1)
(0?0)
(14?2)
(12?7)
(9?0)
(11?3)
IV1
126
87
118
256
143
0
255
24
0
265
118
212
101
IV2
(8?6)
(5?7)
(20?9)
(24?0)
(21?7)
(0?0)
(24?2)
(38?1)
(0?0)
(23?0)
(15?3)
(15?3)
(13?5)
465
377
278
538
398
0
530
22
0
481
518
692
371
(31?8)
(24?8)
(49?3)
(50?4)
(60?4)
(0?0)
(50?3)
(34?9)
(0?0)
(41?7)
(67?2)
(50?0)
(49?5)
IV3
73
121
10
24
0
0
7
5
0
17
3
71
4
mammalian cell lines was similar to MVA-HANP except
that higher numbers of morphogenetic structures were
present for the former. In addition, mature virions were
easily detected in Vero cells infected with MVA, but not
MVA-HANP (Supplementary Fig. S3, Table 2).
MVA is considered a safe vaccine vector because mature
virions are supposedly not produced in cells defined as
semi- or non-permissive. However, the results of our EM
analysis indicate that mature virions were produced in semiand non-permissive cell lines infected with MVA-HANP
(Table 2) and MVAnr (Supplementary Fig. S3). Mature
virions present in these cell lines were products of virion
morphogenesis and not leftover of input virus material.
There are at least three reasons for this conclusion. First,
mature virions were present within the cell cytoplasm and
on the cell surface (Supplementary Fig. S3). Leftover of
input virus material will only be on the cell surface. Second,
mature virions were not detected in these cell lines at early
time points post-infection (6 and 12 h p.i.) (data not
shown). Third, mature virions were found in the presence of
other products of on-going virion morphogenesis, including
viroplasm, immature viruses and DPs (Supplementary
Fig. S3). These structures are so fragile that they cannot
possibly be found intact in infecting virus material. To test
whether the mature virions were infectious, we infected
Vero, NMULI, A549, H411E and 293 cells with MVAHANP and MVAnr, respectively. Cell lysates from these cells
were used to infect BHK-21 and IEC-6 cells. In all cases more
than a 2 log increase was recorded (data not shown). A549
cells infected with MVAnr, but not MVA-HANP, resulted in
more than a 2 log increase in virus titre when passaged in
BHK-21 cells. Collectively, these results suggest that mature
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DP
(5?0)
(7?9)
(1?8)
(2?2)
(0?0)
(0?0)
(0?7)
(7?9)
(0?0)
(1?5)
(0?4)
(5?1)
(0?5)
101
70
118
41
12
0
18
0
0
173
34
161
189
(6?9)
(4?6)
(20?9)
(3?8)
(1?8)
(0?0)
(1?7)
(0?0)
(0?0)
(15?0)
(4?4)
(11?6)
(25?3)
IMV
245
565
4
31
2
0
0
0
0
29
0
118
0
(16?8)
(37?2)
(0?7)
(2?9)
(0?3)
(0?0)
(0?0)
(0?0)
(0?0)
(2?5)
(0?0)
(8?5)
(0?0)
IEV
100
194
3
0
0
0
0
0
0
13
0
6
0
(6?9)
(12?8)
(0?5)
(0?0)
(0?0)
(0?0)
(0?0)
(0?0)
(0?0)
(1?1)
(0?0)
(0?4)
(0?0)
CEV
243
42
0
0
0
0
0
0
0
11
0
1
0
(16?6)
(2?8)
(0?0)
(0?0)
(0?0)
(0?0)
(0?0)
(0?0)
(0?0)
(1?0)
(0?0)
(0?1)
(0?0)
virions produced in cell lines previously considered to be
semi- and non-permissive were infectious. These observations are relevant to the safety of MVA since mature viruses
produced in semi- and non-permissive cells can be a source
of infection of permissive cells or hosts. Although we have
shown that infectivity can be transferred from supposedly
semi- or non-permissive cells to permissive cells in vitro,
such an outcome may seem far fetched in vivo. This is
because MVA infection of some animal species has so far
only resulted in abortive infections (Stittelaar et al., 2001;
Ramirez et al., 2003; Hanke et al., 2005). In conclusion,
we have demonstrated that MVA multiplied efficiently in
IEC-6 cells, and that limited production of mature virions
occurred in cell lines so far considered to be semi- or nonpermissive. Further research is required to unravel the
molecular and cellular basis for these observations.
Acknowledgements
This work was supported by the Norwegian Research Council (project
no. 148535/V10) and the University of Tromsø, Norway. The authors
acknowledge with thanks the technical assistance of Randi Olsen,
Electron Microscopic Unit, University of Tromsø, Norway.
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