Evaluation of the influenza A replicon for transient expression of recombinant proteins in mammalian cells.

Abstract

Recombinant protein expression in mammalian cells has become a very important technique over the last twenty years. It is mainly used for production of complex proteins for biopharmaceutical applications. Transient recombinant protein expression is a possible strategy to produce high quality material for preclinical trials within days. Viral replicon based expression systems have been established over the years and are ideal for transient protein expression. In this study we describe the evaluation of an influenza A replicon for the expression of recombinant proteins. We investigated transfection and expression levels in HEK-293 cells with EGFP and firefly luciferase as reporter proteins. Furthermore, we studied the influence of different influenza non-coding regions and temperature optima for protein expression as well. Additionally, we exploited the viral replication machinery for the expression of an antiviral protein, the human monoclonal anti-HIV-gp41 antibody 3D6. Finally we could demonstrate that the expression of a single secreted protein, an antibody light chain, by the influenza replicon, resulted in fivefold higher expression levels compared to the usually used CMV promoter based expression. We emphasize that the influenza A replicon system is feasible for high level expression of complex proteins in mammalian cells.

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Evaluation of the Influenza A Replicon for Transient
Expression of Recombinant Proteins in Mammalian Cells
Florian Krammer, Jens Pontiller, Christopher Tauer, Dieter Palmberger, Andreas Maccani, Martina
Baumann, Reingard Grabherr*
Department of Biotechnology, Vienna Institute of BioTechnology, University of Natural Resources and Applied Life Sciences, Vienna, Austria
Abstract
Recombinant protein expression in mammalian cells has become a very important technique over the last twenty years. It is
mainly used for production of complex proteins for biopharmaceutical applications. Transient recombinant protein
expression is a possible strategy to produce high quality material for preclinical trials within days. Viral replicon based
expression systems have been established over the years and are ideal for transient protein expression. In this study we
describe the evaluation of an influenza A replicon for the expression of recombinant proteins. We investigated transfection
and expression levels in HEK-293 cells with EGFP and firefly luciferase as reporter proteins. Furthermore, we studied the
influence of different influenza non-coding regions and temperature optima for protein expression as well. Additionally, we
exploited the viral replication machinery for the expression of an antiviral protein, the human monoclonal anti-HIV-gp41
antibody 3D6. Finally we could demonstrate that the expression of a single secreted protein, an antibody light chain, by the
influenza replicon, resulted in fivefold higher expression levels compared to the usually used CMV promoter based
expression. We emphasize that the influenza A replicon system is feasible for high level expression of complex proteins in
mammalian cells.
Citation: Krammer F, Pontiller J, Tauer C, Palmberger D, Maccani A, et al. (2010) Evaluation of the Influenza A Replicon for Transient Expression of Recombinant
Proteins in Mammalian Cells. PLoS ONE 5(10): e13265. doi:10.1371/journal.pone.0013265
Editor: Man-Seong Park, Hallym University, Republic of Korea
Received July 5, 2010; Accepted September 14, 2010; Published October 11, 2010
Copyright: ? 2010 Krammer et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was funded by the FWF (Austrian Science Fund, http://www.fwf.ac.at/) project P17361-B13. The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: Reingard.grabherr@boku.ac.at
Introduction
Expression of therapeutic proteins in mammalian cells is a
growing field in biotechnology. Although yields can be optimized
by the choice of cell line, expression vector, and adequate process
design, there still is a need for new expression systems as well as
promoter elements [1,2]. A great number of viral replicons and
attenuated or replication deficient viruses for heterologues gene
expression derived from RNA viruses have been developed during
the last twenty years. Most of them were designed to be used as
vehicles for gene transfer and recombinant protein expression in
mammalian cells or as vaccines. These include the+sense single
stranded (+ss) RNA virus members of the Togaviridae like Semliki
Forest virus, equine encephalitis virus, rubella virus or Sindbis
virus and members of the Flaviviridae like Kunjin virus as well as –
sense single stranded (2ss) RNA virus members of the Rhabdovir-
idae, Paramyxoviridae and Orthomyxoviridae [3,4,5,6,7,8]. The system
which has shown the greatest potential for recombinant gene
expression is beyond doubt the Semliki Forest virus replicon. A
review from 1992 which reports the progress on alphavirus
replicons for recombinant gene expression also suggests to use an
influenza A replicon for the same purpose [9]. Over the years,
influenza virus reverse genetics techniques developed rapidly [10]
and culminated in the invention of bidirectional plasmid based
systems [11]. Although these systems are far away from being easy
to handle they provide a perfect tool to evaluate the influenza A
replicon for the expression of recombinant proteins. To date, a
small number of proteins, namely the chloramphenicol transferase
(CAT), the green fluorescent protein (GFP), and the firefly
luciferase have been expressed from influenza replicons for
matters of transfection control for influenza virus reverse genetics
and better understanding of the influenza replication machinery
[12,13,14]. Additionally, GFP, a mycobacterial epitope and
interleukin-2 have been expressed by an viable active influenza
A virus from an altered NS genomic segment [8,15,16]. Here we
describe the expression of the human anti-HIV-gp41 antibody
3D6 [17] by the replicon of the influenza A virus strain A/
Hiroshima/52/2005 (H3N2). Additionally, we expressed the light
chain individually, evaluated the effect of using different non-
coding regions (NCRs) on the expression level and investigated the
influence of temperature on the influenza replicon activity.
Results
Transfection efficiency
The efficiency of transfecting mammalian cells can be
influenced by a number of factors like the choice of method,
condition of the cells, purity of DNA, plasmid size and number of
different ones to be delivered per cell. The influenza replicon that
we have chosen to evaluate is based on two plasmids (17035 bp
and 5116 bp) which encode viral RNA (vRNA) and proteins
which are necessary for the formation of the influenza replicon
(Figure 1). Plasmids which provide anti-sense RNA (analogous to
vRNA) of a reporter gene or gene of interest (Figure 1 and 2) were
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co-transfected. We compared this system to a single plasmid with
human cytomegalovirus immediate-early promoter (CMV) driven
expression in terms of transfection efficiency. As a reporter gene
for this particular experiment we chose EGFP and analyses were
performed by FACS. HEK-293 cells were transfected with
pEGFP-N1 (CMV) or pTripolis (bidirectional transcription of
influenza polymerases PB1, PB2 and PA), pBi-NP (bidirectional
transcription of influenza NP) and pMono-EGFP (Figure 1,
Table 1). The vector pMono-EGFP provides antisense transcrip-
tion of the EGFP reading frame driven by a human RNA
polymerase I dependent promoter fragment (below referred to as
RNA polymerase I promoter), the EGFP gene is flanked by 59 and
39 non-coding regions from the influenza NS genomic segment.
The percentage of EGFP expressing cells was determined over a
period of 101 hours (Figure 3 and 4). Eight hours post transfection
(h.p.t.) approximately 7.5% of the pEGFP-N1 transfected cells
were EGFP positive as compared to a very low percentage of
positive cells found in the populations transfected by the replicon
system. The percentage of EGFP expressing cells in the population
of pEGFP-N1 transfectants increased to 29.2% percent after 29
h.p.t. and peaked with 38.5% at 53 h.p.t. The cells transfected by
the replicon system showed a delayed peak of 19.4% positive cells
at 77 h.p.t. . In contrast to the pEGFP-N1 (CMV) driven control
the percentage of EGFP positive cells in the replicon transfected
fraction decreased very slowly after the peak was reached.
Another interesting phenomenon was that cells transfected with
the replicon system showed two distinct populations (Figure 5).
One population was EGFP negative while the other population
expressed high levels of EGFP. Almost no cells with low or
moderate EGFP expression were found in between these two
peaks. In contrast, the pEGFP-N1 transfected cells showed a broad
distribution spanning from completely negative to moderate and
high expressing cells.
Analysis of expression level by luciferase assay
Based on our results from the FACS experiments we speculated
that although the percentage of positive cells in populations
transfected with the replicon system was lower than in the pEGFP-
Figure 2. Schemata of bidirectional and monodirectional transcription cassettes. Bidirectional cassettes (A) were used for generation of
the influenza replicon. Monodirectional cassettes (B) were used to generate vRNA of reporters or genes of interest.
doi:10.1371/journal.pone.0013265.g002
Figure 1. Schematic drawings of plasmids used to generate the influenza replicon based expression system. pTripolis and pBi-NP
generate the influenza replicon (mRNA/protein and vRNA) whereas pMono plasmids with various genes of interest or reporter genes drive
transcription of vRNA.
doi:10.1371/journal.pone.0013265.g001
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Table 1. Plasmids and plasmid amounts used for various experiments.
Experiments
EGFP
CMV
EGFP
Replicon
Luciferase
CMV
Luciferase
Replicon
NCR NS
Luciferase
SV40
AB
CMV
1:1
AB
Replicon
1:1
AB
CMV
10:1
AB
Replicon
10:1
LC
CMV
LC
Replicon
Luciferase
Replicon
NCR M
Luciferase
Replicon
NCR PB1
negative
control
(HEK)
Plasmids
pEGFP-N1
9 mg
pcDNA-luc
9 mg
pGL3-control
9 mg
pRC-LC
4.5 mg
0.45 mg
9 mg
pRC-HC
4.5 mg
4.5 mg
pTripolis
3 mg
3 mg
3 mg
3 mg
3 mg
3 mg
3 mg
pBi-NP
3 mg
3 mg
3 mg
3 mg
3 mg
3 mg
3 mg
3mg
pMono-EGFP
3 mg
pMono-lucNS
3 mg
3mg
pMono-LC
3 mg
0.3 mg
3 mg
pMono-HC
3 mg
3 mg
pMono-lucM
3 mg
pMono-lucPB1
3 mg
pRL
1 mg
1 mg
1 mg
1 mg
1 mg
1mg
Total Amount
9 mg
9 mg
10 mg
10 mg
10 mg
9 mg
12 mg
4.95 mg
9.3 mg
9 mg
9mg
10 mg
10 mg
7mg
pEGFP-N1, pcDNA luc, pRC-LC and pRC-HC carry the CMV immediate-early promoter, pGL3-control and pRL carry SV40 promoter elements, pTripolis and pBi-NP drive bidirectional transcription (mRNA and vRNA) of the influenzapolymerases and the NP protein. pMono-EGFP, pMono-lucNS, pMono-LC, pMono-HC, pMono-lucM and pMono-lucPB1 drive monodirectional (vRNA) transcription of the gene of interest. AB stands for antibody, LC stands for light
chain experiments.
doi:10.1371/journal.pone.0013265.t001
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N1 transfected populations, however, the overall expression level
may still be higher due to a higher percentage of strongly
expressing cells. In order to quantify expression levels in more
detail we repeated the experiment using the firefly luciferase
instead of EGFP as the reporter. Non-transfected HEK-293 cells
were used as negative control in all luciferase experiments.
Additionally we used a second negative control which was
transfected with pMono-lucNS and pBi-NP but not with the
pTripolis plasmid to verify that the vRNA alone is not translated.
The luciferase expression level was monitored over a period of
101 hours, including a pcDNA-luc (CMV) driven firefly luciferase
as positive control. Observed data resembled the previous
experiment to some degree, however the activity peak for the
pcDNA-luc control was detected at 29 h.p.t. in contrast to 53 h.p.t.
Figure 3. Transfection efficiency time course. HEK-293 cells either tranfected with pEGFP-N1 (CMV) or with the influenza replicon and pMono-
EGFP (Flu Replicon) were monitored over a period of 101 hours by FACS analysis. Data represents arithmetic mean values and standard deviation.
doi:10.1371/journal.pone.0013265.g003
Figure 4. EGFP expression. HEK-293 cells were either tranfected with pEGFP-N1 (CMV) or with the influenza replicon and pMono-EGFP (Flu
Replicon). Pictures were taken 56 and 98 hours post transfection. Non-transfected cells were used as negative control (Neg. Control).
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in the previous experiment (Figure 6). The replicon system showed
the same time course as observed in the FACS experiment
(Figure 6). However, the expression level of the replicon system
measured by luciferase activity was unexpectedly low, between
13% (53 h. p.t.) and 27% (77 h.p.t.) of the level of the pcDNA-luc
driven control.
We further decided to compare the influenza replicon system to
CMV and SV40 promoter (SV40) driven expression in HEK-293
cells. In order to be able to normalize firefly values to an internal
standard we co-transfected pRL DNA which codes for Renilla
luciferase driven by an SV40 promoter. Normalization was
applicable in all experiments except in time course experiments
described above because the Renilla expression is not stable over
time. HEK-293 cells were transfected with pTripolis, pBi-NP,
pMono-lucNS and pRL or pcDNA-luc and pRL or pGL3-control
(firefly luciferase under control of SV40 promoter) and pRL or
pBi-NP, pMono-lucNS and pRL (negative control) (Table 1). Cells
were harvested 48 hours post transfection and were subjected to
the luciferase assay. The expression value of the replicon system
(normalized value of 817.7) showed approximately one tenth of the
activity of the CMV driven construct (normalized value of 8479.9)
but approximately 100 fold activity of the SV40 driven construct
Figure 5. FACS analysis of cells transfected with either pEGFP-N1 (CMV) or the influenza replicon system and pMono-EGFP (Flu
Replicon) 77 hours post transfection. Cells transfected with the replicon system show two very distinct populations in the forward light scatter
(EGFP), one completely negative, the other highly positive. pEGFP-N1 driven EGFP expressions shows a broad distribution spanning from completely
negative to moderate and high expressing cells.
doi:10.1371/journal.pone.0013265.g005
Figure 6. Time course of firefly luciferase expression. HEK-293 cells either tranfected with pcDNA-luc (CMV) or with the influenza replicon and
pMono-lucNS (Flu Replicon) were monitored over a period of 101 hours by luciferase assay. Data represents arithmetic mean values and standard
deviation.
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(normalized value of 9.8). The negative control transfected with
pBi-NP and pMono-lucNS showed no activity at all (firefly
luciferase: 12865 RLU, Renilla luciferase: 466.8637.7 RLU,
normalized value of 0.3) (Figure 7).
The human RNA polymerase I promoter used to drive vRNA
transcription in our experiments is known to work in a very
restricted number of cell lines derived from primates. However, we
wanted to verify this restriction by comparing the activity of the
replicon system in the human HEK-293 cell line to activity in a
canine (MDCK) and in a rodent (CHO-K1) cell line. Cells were
transfected with pTripolis, pBi-NP, pMono-lucNS and pRL or
pcDNA-luc and pRL. As expected, luciferase activity was detected
in HEK-293 cells (firefly luciferase: 137040617119 RLU, Renilla
luciferase: 168.367.1 RLU) but not in the MDCK (firefly
luciferase: 89621 RLU, Renilla luciferase: 125.869.6 RLU) and
CHO-K1 (firefly luciferase: 9464.4 RLU, Renilla luciferase:
1349.36194.0 RLU) cells which showed values minimally higher
than the HEK-293 negative control (firefly luciferase: 12865
RLU, Renilla luciferase: 466.8637.7 RLU) or the untransfected
controls for MDCK (firefly luciferase: 7260 RLU) and CHO-K1
(firefly luciferase: 7767 RLU) (Figure 8).
Temperature dependence of the influenza A virus
replicon
As a next step, we decided to analyze the activity of the replicon
expression systems at different temperatures, namely 27uC, 37uC,
and 40uC. HEK-293 cells were transfected with pTripolis, pBi-NP,
pMono-lucNS and pRL or pcDNA-luc and pRL or pGL3-control
(firefly luciferase under control of SV40 promoter) and pRL. Cells
were allowed to settle at 37uC for 4 hours and were then
transferred to incubators heated to 27uC or 40uC or were further
incubated at 37uC. Cells were harvested 48 hours post transfection
and subjected to luciferase assay. Again, firefly luciferase values
were normalized to Renilla activity. Interestingly, the cells
transfected with the replicon system which were incubated at
27uC showed no activity at all. Activity at 40uC was reduced to
approximately 25% of the activity at the optimal temperature of
37uC indicating a strong temperature sensitivity of the influenza
viral polymerases (Figure 9).
Influence of the influenza genomic non-coding regions
on the expression level
Recently, it was shown that the composition of the 59 and 39
non-coding regions (NCR) of influenza vRNA has strong influence
on influenza gene expression [18,19,20,21]. Therefore, we decided
to test NCRs from different genomic segments in the luciferase
assay. In the previously described experiments we used a construct
which contained the NCRs of the NS genomic segment of A/
Hiroshima/52/2005. The NS genomic segment codes for two
proteins, the non-structural protein 1 (NS1) which is not
incorporated into viral particles, and the nuclear export protein
(NEP). Additionally, we chose to fuse NCRs from the PB1
genomic segment and the M genomic segment to our reporter
gene. The two proteins encoded from the M genomic segments
(M1 and M2) are incorporated into the viral particles where M1 is
present in high abundance. The PB1 protein is part of the trimeric
polymerase complex of influenza A and eight copies of this protein
can be found per particle. In order to analyse the influence of the
different NCRs on the expression level we transfected HEK-293
cells with either pTripolis, pBi-NP, pMono-lucNS and pRL or
pTripolis, pBi-NP, pMono-lucPB1 and pRL or pTripolis, pBi-NP,
pMono-lucM and pRL (Table 1). Cells were harvested 48 h.p.t.
and subjected to a luciferase assay. Firefly luciferase values were
normalized to Renilla luciferase activity. As expected, the reporter
construct with NCR from the M genomic segment showed by far
the highest activity (normalized value of 3115.9) (Figure 10).
Interestingly, NCRs from the PB1 segment were able to drive the
second highest expression level (normalized value 2297.0) whereas
the construct with the NS NCRs showed just 25% of the activity of
the M NCR construct (normalized value of 814.7) (Figure 10).
Figure 7. Comparison of firefly luciferase expression driven by CMV, SV40 or by the influenza replicon. HEK-293 cells were either
tranfected with pcDNA-luc (CMV), pGL3-control (SV40) or with the influenza replicon and pMono-lucNS (Influenza Replicon) or with pMono-lucNS and
pBi-NP only (Neg. Contr.). Additionally, pRL, coding for Renilla luciferase, was cotransfected in all cases. Cells were harvested 48 hours post
transfection and subjected to luciferase assay. Firefly values have been normalized to Renilla values. Data represents arithmetic mean values and
standard deviation.
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Expression of a whole antibody by the influenza
replication machinery
In order to test the feasibility of the influenza A replicon system
to express secreted proteins, we made constructs encoding the
light and heavy chain of the human monoclonal anti-HIV-gp41
antibody 3D6. As positive control CMV promoter driven
expression plasmids for heavy (pRC-HC) and light chain (pRC-
LC) were used (Table 1). It is known that the heavy chain to light
chain ratio of 1:1 is in some cases suboptimal, therefore we
decided to apply heavy chain to light chain ratios of 1:1 and 10:1.
Figure 8. Activity of the influenza replicon in a human, a canine and a rodent cell line. HEK -293, MDCK or CHO-K1 cells were either
tranfected with pcDNA-luc (CMV) or with the influenza replicon and pMono-lucNS (Influenza Replicon). The HEK-293 negative control has been
transfected with pMono-lucNS and pBi-NP only. Additionally, pRL, coding for Renilla luciferase, was cotransfected in all cases. In case of MDCK and
CHO-K1, untransfected cells have been used as negative controls and can therefore not be normalized, absolute values can be found in Results
section. Cells were harvested 48 hours post transfection and subjected to luciferase assay. Data represents arithmetic mean values and standard
deviation.
doi:10.1371/journal.pone.0013265.g008
Figure 9. Temperature dependence of the influenza replicon. HEK-293 cells were either tranfected with pcDNA-luc (CMV), pGL3-control
(SV40) or with the influenza replicon and pMono-lucNS (Flu Replicon) or with pMono-lucNS and pBi-NP only (Neg. Contr.). Additionally, pRL, coding
for Renilla luciferase, was cotransfected in all cases. Four hours post transfection cells were transferred to 27uC or 40uC or remained at 37uC. Cells were
harvested 48 hours post transfection and were subjected to luciferase assay. Firefly values have been normalized to Renilla values. Data represents
arithmetic mean values and standard deviation.
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In the 10:1 expression series we basically used the same amount
of heavy chain coding plasmid as in the 1:1 expressions but
reduced the amount of light chain coding plasmid to a tenth of
the heavy chain amount. Briefly, HEK-293 cells were transfected
with pRC-HC and pRC-LC in ratios 1:1 or 10:1 or pTripolis,
pBi-NP, pMono-HC and pMono-LC (pMono-HC and pMono-
LC also in ratios 1:1 and 10:1) (Table 1). The antibody
concentration in the culture supernatant was analyzed at four
time points during a period of 101 hours by enzyme-linked
immunosorbent assay (ELISA) (Figure 11). Interestingly, expres-
sion level of the pRC driven 1:1 ratio samples (128.5 ng/ml) was
eight times higher than their influenza replicon driven counter-
part (20.9 ng/ml) at 101 h.p.t. (Figure 11). However, a
completely different expression profile could be observed for
the 10:1 ratio expressions. The pRC driven 10:1 expression
produced only 0.9 ng/ml whereas the replicon system produced
41.1 ng/ml in this setting which is approximately 45 times as
much antibody (Figure 11).
Figure 11. Expression of a the monoclonal anti-gp41 antibody 3D6. HEK-293 cells were either tranfected with pRC-LC and pRC-HC (CMV
HC:LC=1:1, CMV HC:LC=10:1) or with the influenza replicon and pMono-LC and pMono-HC (Flu Replicon HC:LC=1:1, Flu Replicon HC:LC=10:1).
Heavy chain to light chain ratios of 1:1 and 10:1 were tested. Antibody concentration in the culture supernatant was monitored for 101 hours by
ELISA. Data represents arithmetic mean values and standard deviation.
doi:10.1371/journal.pone.0013265.g011
Figure 10. Influence of different NCRs on the expression level. HEK-293 cells were tranfected with the influenza replicon and either pMono-
lucNS (NCR NS) or pMono-lucM (NCR M) or pMono-lucPB1 (NCR PB1) or with pMono-lucNS and pBi-NP only (Neg. Contr.). Additionally, pRL, coding for
Renilla luciferase, was cotransfected in all cases. Cells were harvested 48 hours post transfection and subjected to luciferase assay. Firefly values have
been normalized to Renilla values. Data represents arithmetic mean values and standard deviation.
doi:10.1371/journal.pone.0013265.g010
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Light chain expression
In order to eliminate the observed ratio effect and to be able to
determine single protein expression levels for a secreted protein we
chose to express the 3D6 light chain only. HEK-293 cells were
transfected with pRC-LC or the replicon system with pMono-LC.
The light chain concentration in the culture supernatant was
analyzed at four time points during a period of 101 hours by
ELISA. Surprisingly, in this experimental set-up, the replicon
driven expression showed higher expression levels than expression
driven by pRC-LC (Figure 11). At the end point of the
measurement period the culture supernatant of replicon transfect-
ed cells showed as much as 161.2 ng/ml light chain which is
approximately five times the amount that was achieved by the
pRC-LC control (Figure 12) (32.8 ng/ml light chain).
Discussion
In this study we investigated the feasibility of influenza A virus
replicons for transient expression of recombinant proteins in
mammalian cells. In a first experiment we compared the
transfection efficiencies of the plasmid based influenza replicon
system expressing EGFP and the expression vector pEGFP-N1
(CMV). The replicon based system showed a delayed expression
profile compared to the pEGFP-N1 driven expression, transfection
efficiency of the influenza replicon system was approximately 50%
lower (Figure 3). We hypothesize that there are two main reasons
for the reduced transfection efficiency of the replicon based
system. The CMV driven system is based on one plasmid whereas
the replicon system for EGFP expression is based on three
different plasmids, decreasing the statistical probability to have all
components present in the same cell. Additionally, pEGFP-N1 is
4733 bp in size whereas the plasmids used for the replicon system
are 3082, 5116 and 17035 bp in size. It is well known that the
transfection efficiency decreases dramatically with increased
plasmid size [22]. In addition, a certain degree of cytotoxicity of
the influenza replicon activity or its components and therefore a
negative influence on long-term expression cannot be ruled out. As
shown by FACS analysis, cells transfected with the replicon system
showed two distinct populations of cells (Figure 5). One population
remained totally negative whereas a second, quite large population
(19.4%) was highly EGFP positive. However, the overall
expression level was only between 13% and 27% of the levels
expressed by the CMV promoter (Figure 6). However, when
compared to the SV40 promoter, the influenza replicon system
showed approximately 100 fold higher activity (Figure 7).
We showed that the activity of the influenza replicon system is
tightly restricted to primate cells. Almost no activity was found in a
canine and a rodent cell line and full activity was detected in
human HEK-293 cells (Figure 8) and has also been shown
previously for Vero cells [23]. The RNA polymerase I promoter
used in this expression system for the initial transcription of vRNA
is derived from human non-transcribed spacer 59 rDNA regions.
RNA polymerase I promoters, which are used by the cell for
rRNA transcription, are known to be species specific [24].
However, by changing to a homologous RNA polymerase I
promoter the replicon system could be adapted to virtually any cell
line. Similar systems have been reported for canine and avian cells
[25,26].
Temperature is known to have a strong influence on influenza A
virus replication. For example, fever is involved in the defense of
the human body against influenza virus infections. On the other
hand, for recombinant protein expression lower temperatures have
some advantages such as lower energy consumption for heating in
large scale production, proper protein folding and less cell stress
[27]. We therefore, tested replicon activity at 27u, 37u and 40uC.
While pcDNA-luc (CMV) and pGL3-control (SV40) driven
expression was shown to be temperature independent, the
influenza replicon system turned out to be highly temperature
sensitive. Replicon based expression completely disappeared at
27uC. This finding becomes relevant for expression of influenza
polymerases and ribonucleoprotein complexes in insect cells,
which are usually grown at 27uC or below. The activity of the
replicon also decreased to about 25% at 40uC as compared to the
full activity at 37uC (Figure 9).
Initially, our reporter and gene of interest constructs for
generation of vRNA were flanked by 59 and 39 non-coding
regions of the NS genomic segment. In order to find out if
expression can be increased by the use of NCRs from other
Figure 12. Light chain expression. HEK-293 cells were either tranfected with pRC-LC (CMV) or with the influenza replicon and pMono-LC (Flu
Replicon). Light chain concentration in the culture supernatant was monitored for 101 hours by ELISA. Data represents arithmetic mean values and
standard deviation.
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genomic segments, we prepared luciferase reporter constructs
flanked by 59 and 39 regions of the M and PB1 genomic segment.
By changing the flanking regions we were able to increase
expression 2.8-fold in case of the PB1 NCRs and 3.8-fold in case of
the M NCRs (Figure 10). In contrast to the M NCR, which is only
present on the reporter vRNA, the PB1 NCR is also present on the
PB1 vRNA derived from plasmid pTripolis. This has to be
considered since replicational competition between two vRNA
species with the same NCR cannot be ruled out. It is also notable,
that NCRs from the NS genomic segment, which encodes for non-
structural proteins, showed the lowest activity whereas NCRs from
the M genomic segment, coding for the most abundant structural
protein M1, showed the highest activity. The activity of the NCRs
derived from the PB1 genomic segment lied in between.
To test whether the replicon system is suitable as an expression
system for secreted proteins the anti-HIV-gp41 antibody 3D6 was
expressed applying different ratios of heavy and light chain gene
dosage. In the 1:1 ratio series the pRC (CMV) driven system
produced a six fold higher antibody concentration (128.5 ng/ml)
than the replicon system (Figure 11) (20.9 ng/ml, 101 h.p.t.).
Interestingly, in the 10:1 series (amount of light chain vector was
reduced to a tenth of the heavy chain vector concentration) we
found 41.1 ng/ml (101 h.p.t.) in the supernatant of the replicon
driven system which was approximately twice the amount found in
the 1:1 series (Figure 11). The CMV promoter system produced
only small amounts (0.9 ng/ml) of antibody in this experimental
setting (Figure 11). The optimal ratio between heavy chain and
light chain vector for the CMV driven constructs was found
somewhere in between 1:1 and 10:1, at least in CHO cells [28].
Both, heavy chain and light chain sequences used for vRNA
transcription were flanked by NCRs of the influenza NS genomic
segment. The vRNA comprising the heavy chain sequence is
approximately twice as long as the vRNA hosting the light chain.
Therefore, we speculate that there was has been a replicational
competition between both vRNA species. The shorter vRNA was
probably amplified at a higher rate and to a higher number. This
would explain why an initial excess of heavy chain vector was
beneficial for the expression of the antibody. This effect was
definitely not observed for pRC driven expression, where
reduction of light chain coding plasmid led to an almost complete
shut down of antibody expression. In order to eliminate this
competition effect and to be able to directly compare expression
levels of secreted proteins, we investigated the behavior when only
light chain was present. In this experimental set-up the replicon
system was able to drive expression far more efficiently than the
pRC control (Figure 12). Light chain concentrations in the
supernatant were fivefold higher for the replicon system
(161.2 ng/ml) than for the CMV driven expression 101 h.p.t.
(Figure 12). Interestingly, influenza replicon driven light chain
expression exceeded CMV driven light chain expression while for
EGFP and firefly luciferase expression the CMV promoter showed
higher activity than the replicon. This might be explained by the
fact that, in contrast to EGFP and firefly luciferase, the antibody
light chain is transported through the Golgi and secreted to the
supernatant. The folding and secretion pathway is the major bottle
neck in secreted protein production, strongly depending on the
individual protein and the metabolic burden by over-expression.
We speculate that influenza replicon transcription activity rates
may meet the optimal transcription rates for accurate folding and
secretion, while these pathways are overloaded by CMV promoter
activity. Measuring the actual transcription rates might help to
elucidate this phenomenon.
In conclusion, we demonstrated that expression efficiencies of
the influenza replicon were comparable with a widely used
transient CMV expression system. Luciferase assays were suitable
to evaluate temperature sensitivity of the replicon system, showing
complete shut-down of activity at 27uC. Additionally, we
demonstrated that the choice of flanking NCRs has strong
influence on the overall expression level, and have to be chosen
accordingly. We could further show that the influenza replicon is
feasible for high level expression of complex secreted proteins in
human cell lines. As has been shown for a human antibody light
chain, for some proteins the product yield can be increased
substantially above the levels of CMV promoter driven expression.
Materials and Methods
Cells and Viruses
The human cell line HEK-293 (ATCC # CRL-1573) was
cultured in RPMI 1640 medium (Biochrom, Berlin, Germany)
supplemented with 10% fetal calf serum (FCS) (PAA, Pasching,
Austria). The canine cell line MDCK (ATCC # CCL-34), the
african green monkey cell line Vero (ATCC # CCL-81), and the
Chinese hamster cell line CHO-K1 (ATCC # CCL-61) were
cultured in serumfree DMEM/Ham’s F12 medium (Biochrom,
Berlin, Germany).Influenza
(H3N2, Gene Bank Entries EU285583, EU283414, EU597800–
EU5978005) was grown on Vero cells in serum free DMEM/
Ham’s F12 supplemented with antibiotics and trypsin (Polymun,
Vienna, Austria).
strain A/Hiroshima/52/2005
Vectors and Vector Construction
Bidirectional transcription cassettes for influenza genes were
designed as described by Hoffmann and Neumann et al [11,29]. A
CMV promoter (derived from pCI, Promega, Madison, USA) in
sense direction drives mRNA transcription which is terminated by
a SV40 polyA signal. Minus-sense single strand (2ss) RNA
transcription is mediated by a human RNA polymerase I
dependent promoter fragment (RNA polymerase I promoter) in
antisense direction and terminated by a mouse RNA polymerase I
dependent terminator (Figure 2). Between RNA polymerase I
promoter and terminator two BsmBI restriction sites are located as
described by Neumann et al [30]. The bidirectional cassette was
synthesized by Geneart (Regensburg, Germany), amplified by
PCR with primers carrying BamHI and NotI restriction sites and
cloned into a modified pUC19 (NEB, Ipswitch, USA) vector
lacking the lacZ gene resulting in pBi (Figure 2). The core of the
transcription cassette consisting of the RNA polymerase promoter
and terminator was also amplified by PCR using primers carrying
BamHI and NotI restriction sites and cloned into a modified
pUC19 vector (NEB) lacking the lacZ gene resulting in pMono.
Complementary DNA (cDNA) fragments of NP (EU597803.1),
PA (EU597802.1), PB1 (EU597801.1), PB2 (EU597800.1) from
A/Hiroshima/52/2005 were obtained by TRIZOLTMextraction
and reverse transcription of Vero infection supernatant and cloned
into pBi by the use of BsmBI restriction enzyme or by the In-
Fusion PCR Cloning System (Clontech, Mountain View, USA)
resulting in pBi-NP, pBi-PB1, pBi-PB2 and pBi-PA.
The pTripolis plasmid which hosts bidirectional expression
cassettes for all three influenza polymerases was cloned as follows:
pBi-PB2 was digested by NotI and BamHI and the bidirectional
transcription cassette was cloned in a NotI and BamHI digested
pTriEx1.1 plasmid (Merck, Darmstadt, Germany) resulting in
p3X-PB2. pBi-PB1 was digested by NotI and BamHI and the
bidirectional transcription cassette was cloned in a NotI and BamHI
digested pFastBac Dual plasmid (Invitrogen, Carlsbad, USA)
resulting in pFB-PB1. pBi-PA was digested by NheI and PciI and
the bidirectional transcription cassette was cloned in a NheI and
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Page 11

PciI digested pFastBac Dual (Invitrogen) resulting in pFB-PA.
Vector p3X-PB2 was then cut by PmlI and PacI restriction
endonucleases and the PB2 cassette was cloned into a PacI and
BstZ17I digested pFB-PB1 resulting in pFB-PB1-PB2. Vector pFB-
PA was finally cut with SphI and PacI restriction endonucleases and
the PA cassette was cloned into the SphI and PacI digested pFB-
PB1-PB2 resulting in pTripolis (Figure 1).
The plasmid pEGFP-N1 (Clontech) was used as control vector
in FACS experiments. The CMV driven control vector for the
luciferase experiment was constructed by BamHI and XbaI excision
of the firefly luciferase gene from pGL3-Control (Promega) and
cloning into a BglII and XbaI restricted pcDNA3 (Invitrogen)
resulting in pcDNA-luc. Additionally, the original pGL3 was used
as SV40 promoter control.
The plasmids pRC-HC (heavy chain) and pRC-LC (light chain)
encode heavy and light chain from the human monoclonal anti-
HIV-gp41 antibody 3D6 under the control of a CMV promoter as
described by Ru ¨ker et al [17]. The heavy chain nucleotide
sequence was altered on genetic level by site directed mutagenesis
to remove the BsmBI restriction site. The amino acid sequence was
not altered.
Heavy chain and light chain of 3D6, EGFP-N1 and firefly
luciferase were amplified by PCR with primers containing 59 and
39 non-coding regions of the NS genomic segment of A/
Hiroshima/52/2005 and cloned into pMono using BsmBI
restriction endonuclease resulting in pMono-HC, pMono-LC,
pMono-EGFP and pMono-lucNS. The firefly luciferase gene was
further amplified by PCR with primers containing 59 and 39 non-
coding regions of the PB1 or M genomic segment of A/
Hiroshima/52/2005 and cloned into pMono using BsmBI
restriction endonuclease resulting in pMono-lucPB1 and pMono-
lucM. Sequences were confirmed by Sanger sequencing.
The plasmid pRL-SV40 (Promega, Madison, USA) which
encodes the Renilla luciferase was used as internal control for
luciferase experiments. All plasmids used were prepared from over
night cultures of E. coli K12 JM109 (NEB) with a NucleoBond
Xtra Midi EF kit (Machery-Nagel, Du ¨ren, Germany). Primer
sequences are available on request.
Transfection
Transfections were carried out with an Amaxa Nucleofector I
device (Lonza, Basel Switzerland). The day before transfection,
cells were split 1:3 to obtain cells in log phase. Briefly, HEK-293
and MDCK cells were washed with PBS and treated with trypsin
(PAA) to detach them from flask surfaces. After detachment, cells
were diluted in PBS containing trypsin inhibitor (Polymun). CHO
cells were grown in suspension and were harvested directly. Cells
were counted and aliquots containing 46106cells were centrifuged
at 1000 g for 5 minutes. Supernatant was removed and 46106
cells per transfection were used for Nucleofection with a
Nucleofector Kit V as described by the manufacturer (HEK-293
program A23, MDCK program A24 and CHO program H14).
After transfection cells were resuspended in pre-warmed medium
containing 10% FCS and antibiotics. Cells transformed for
antibody or light chain expression were directly resuspended in
2.5 ml medium in 6-well plates whereas cells for other experiments
were resuspended in 10 ml medium and aliquots of 1 ml were
seeded in 12-well plates.
Cells dedicated to FACS analysis were transfected with 9 mg of
pEGFP-N1 or 3 mg of pTripolis, pBi-NP and pMono-EGFP (9 mg
total DNA) (Table 1). Untransfected HEK-293 cells served as
negative control.
Cells dedicated to luciferase assays were transfected with 9 mg
pcDNA-luc or 9 mg pGL3-control or 3 mg of pTripolis, pBi-NP
and pMono-lucNS (9 mg total DNA) or 3 mg of pTripolis, pBi-NP
and pMono-lucPB1 (9 mg total DNA) or 3 mg of pTripolis, pBi-NP
and pMono-lucM (9 mg total DNA) or 3 mg of pBi-NP and
pMono-lucNS (6 mg total DNA) as negative control. Additionally
1 mg of pRL was added to each of the transfections dedicated to
luciferase assays. In case of MDCK and CHO-K1 cells,
untransfected cells were used as negative control.
Cells dedicated to CMV driven antibody or light chain
expression were either transfected with 4.5 mg pRC-HC and
4.5 mg pRC-LC (9 mg total DNA) or 4.5mg of pRC-HC and
0.45 mg of pRC-LC (4.95 mg total DNA) or 9 mg of pRC-LC. Cells
dedicated to influenza replicon driven antibody or light chain
expression were transfected with 3 mg of pTripolis, pBi-NP,
pMono-HC and pMono-LC (12 mg total DNA) or 3 mg of
pTripolis, pBi-NP, pMono-HC and 0.3 mg of pMono-LC (9.3 mg
total DNA) or 3 mg of pTripolis, pBi-NP and pMono-LC (9 mg
total DNA) (Table 1). Untransfected HEK-293 cells served as
negative control.
Transfected cells dedicated to temperature experiments were
allowed to settle for 4 hours at 37uC and were then transferred
either to 40uC or 27uC incubators. All transfection experiments
were performed in duplicates.
FACS
FACS experiments were conducted to determine the proportion
of GFP expressing cells over a period of 101 hours. HEK-293 cells
were transfected, seeded into 12-well plates as described and
analyzed 8, 29, 53, 77 and 101 hours post transfection. Non-
transfected HEK-293 cells were used as negative control.
Supernatant was removed, cells were resuspended in 300 ml PBS
and 10 000 cells were analyzed using a BD FACS Canto II device.
FACS experiments were performed in technical duplicates
resulting in four measuring points taking the biological duplicates
into account. Data represents arithmetic mean values and
standard deviation.
Luciferase Assay
Briefly, the cell supernatant was removed and the cells were
resuspended in 300 ml PBS. Aliquots of 50 ml (approximately
6.66104cells) were taken from the cell suspension and were
transferred into black 96-well plates. Samples were analysed
regarding firefly and Renilla luciferase activity using the Dual Glo
Luciferase Assay System (Promega, Madison, USA) according to
the manufacturer’s recommendations. Readouts were performed
in a Tecan infinite M1000 reader (Tecan, Ma ¨nnedorf, Switzer-
land) using i-control 1.6 software (readout settings for both, firefly
and Renilla luciferase: integration time 10000, attenuation: none,
settle time 0). Luciferase assays were performed in technical
duplicates resulting in four measuring points taking the biological
duplicates into account. Firefly luciferase values were normalized
to Renilla luciferase activity. In case of luciferase time curve
experiments absolute firefly values were used due to the time
dependence of the Renilla expression. Data represents arithmetic
mean values and standard deviation.
Antibody and light chain quantification
HEK-293 cells were transfected with full antibody or light chain
only expressing constructs as described, seeded into 6-well plates
and samples from supernatant were taken 29, 53, 77 and
101 hours post transfection. The amount of 3D6 antibody or
3D6 light chain was determined by enzyme-linked immunosorbent
assay (ELISA). 96-well microtiter plates (Nunc-Immuno Plate
Maxisorp) were coated with anti-human k light chain antibody
(Sigma K3502, diluted 1:1000, goat origin) in case of light chain
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Page 12

detection or with anti-human IgG c-chain specific antibody
(Sigma I3382, diluted 1:1000, goat origin) in case of whole
antibody detection in coating buffer over night at 4uC. Plates were
washed with PBS containing 0.005% Tween 20 and incubated
with serial two-fold dilutions of 1:3 or 1:5 pre-diluted supernatant
samples (room temperature, 2 h). Afterwards plates were washed
three times and incubated with peroxidase conjugated anti-human
kappa light chain antibody (Sigma A7164, diluted 1:2000, goat
origin) in PBS containing 1% BSA and 0.005% Tween 20 (room
temperature, 1h). Unbound antibody was removed and plates
were washed. Samples were incubated with substrate (o-phenylene
diamine and H2O2), reaction was stopped using H2SO4and the
colorimetric change was measured at 492 nm. Samples were
quantified relative to a 3D6 antibody standard of known
concentration. In case of light chain detection, the same 3D6
antibody standard was used, a concentration of one third of the
whole antibody was assumed because the light chain represents
approximately one third of the mass of a whole antibody. ELISA
assays were performed in technical duplicates resulting in four
measuring points taking the biological duplicates into account.
Data represents arithmetic mean values and standard deviation.
Acknowledgments
We thank Martina Lo ¨schel for competent help with ELISA and Thomas
Sterovsky for MDCK cell supply.
Author Contributions
Conceived and designed the experiments: FK RG. Performed the
experiments: FK JP CT DP AM MB. Analyzed the data: FK JP CT DP
RG. Wrote the paper: FK RG.
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