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Journal
of General Virology (2001), 82, 1737–1747. Printed in Great Britain
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Naked RNA immunization with replicons derived from
poliovirus and Semliki Forest virus genomes for the generation
of a cytotoxic T cell response against the influenza A virus
nucleoprotein
Marco Vignuzzi, Sylvie Gerbaud, Sylvie van der Werf and Nicolas Escriou
Unite! de Ge! ne! tique Mole! culaire des Virus Respiratoires, URA 1966 CNRS, Institut Pasteur, 25 rue du Dr Roux, F-75724 Paris cedex 15,
France
The potential of RNA-based vaccines was evaluated for the generation of a protective immune
response in the mouse model of influenza type A virus infection using the internal nucleoprotein
(NP) as antigen. This antigen is of particular interest, since it has the potential to elicit protective
cytotoxic T lymphocytes (CTL) against heterologous strains of influenza A virus. In view of the short
half-life of RNA, self-replicating RNAs or replicons of the positive-stranded genomes of Semliki
Forest virus (SFV) and poliovirus were engineered to synthesize the influenza A virus NP in place
of their structural proteins. NP expression was demonstrated by immunoprecipitation after
transfection of cells with RNA from the SFV (rSFV-NP) and poliovirus (r∆P1-E-NP) genome-derived
replicons transcribed in vitro. C57BL/6 mice were injected intramuscularly with these synthetic
RNAs in naked form. Both replicons, rSFV-NP and r∆P1-E-NP, induced antibodies against the
influenza virus NP, but only mice immunized with the rSFV-NP replicon developed a CTL response
against the immunodominant H-2Db epitope NP366. Finally, the protective potential of the CTL
response induced by immunization of mice with rSFV-NP RNA was demonstrated by the reduction
of virus load in the lungs after challenge infection with mouse-adapted influenza A/PR/8/34 virus
and was comparable to the protective potential of the response induced by plasmid DNA
immunization. These results demonstrate that naked RNA immunization with self-replicating
molecules can effectively induce both humoral and cellular immune responses and constitutes an
alternative strategy to DNA immunization.
Introduction
The ongoing antigenic variability of influenza virus surface
antigens remains a problem for the production of current
commercial vaccines, which principally protect the host by
inducing neutralizing antibodies to these antigens and are
unable to induce strong mucosal or cellular immune responses.
In particular, the cytotoxic T lymphocyte (CTL) response,
which is directed against the nucleoprotein (NP) (Kees &
Krammer, 1984 ; Parker & Gould, 1996 ; Yewdell et al., 1985)
and other relatively conserved internal viral proteins implicated
in virus replication and expressed early in infected cells, is
essentially absent. In humans, the importance of this CTL
response is not yet clearly defined, but studies suggest that it
plays at least a co-operative role in virus clearance. In the
Author for correspondence : Nicolas Escriou.
Fax j33 1 40 61 32 41. e-mail escriou!pasteur.fr
0001-7634 # 2001 SGM
absence of pre-existing strain-specific neutralizing antibody,
protection against influenza virus infection in humans mediated
predominantly by CTLs has been inferred (McMichael et al.,
1983) and in immunosuppressed patients, cases of persistent
influenza virus infections have been reported (McMinn et al.,
1999 ; Rocha et al., 1991). Moreover, the importance of a CTL
response is supported by the existence of CTL escape mutants
(Voeten et al., 2000), indicating that during an epidemic season,
a CTL response in humans is operating and applying selective
pressure on circulating influenza virus strains. The role of the
CTL response is more clearly demonstrated in the mouse
model. For instance, it has been shown that reduction of virus
load in the lungs and histological recovery correlate with
specific CD8+ CTL responses (Allan et al., 1990 ; Topham &
Doherty, 1998) and that adoptive transfer of CTL clones
activated in vitro can provide protection against influenza virus
in mice (Graham & Braciale, 1997 ; Taylor & Askonas, 1986).
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M. Vignuzzi and others
Efforts to develop approaches that would fortify the
immune response by inducing CTLs against the influenza virus
NP, using systems that express whole NP, have received
particular attention. Immunization of mice with influenza virus
NP expressed by recombinant vaccinia virus (Endo et al., 1991 ;
Lawson et al., 1994 ; Stitz et al., 1990), fowlpox virus (Webster
et al., 1991), Semliki Forest virus (SFV) (Zhou et al., 1995) or
Sindbis virus (SIN) (Tsuji et al., 1998) has been shown to induce
immune responses that, in some cases, confer at least partial
protection.
An alternative to these approaches, gene immunization, is
based on the inoculation of DNA expression vectors that
contain gene sequences encoding a foreign protein. Immunization with naked DNA vectors encoding the influenza
virus NP has been shown to induce antibodies, cellular
responses and protection against both homologous and crossstrain challenge infection by influenza A virus variants (Bot et
al., 1996 ; Ulmer et al., 1993, 1998). The advantages of DNA
immunization include ease of production, purification and
administration of the vaccine and a long-lasting immunity.
This long-lived immunity lasts for more than 1 year in the
mouse model and is probably due to the long-term persistence
and expression of the injected DNA (Wolff et al., 1992). For
this very reason, some questions remain from a clinical
standpoint as to the potential risk of DNA sequence integration
into the host genome, although preliminary studies in animals
have not shown integration events to lead to insertional
mutagenesis (Nichols et al., 1995).
To avoid these potential hazards, RNA has been proposed
as the expression vector ; however, development of this
approach faces new problems posed by the short intracellular
half-life of RNA and its degradation by ubiquitous RNases.
Initial attempts used mRNA to induce immune responses ; the
RNA was administered either intramuscularly (Conry et al.,
1995) by gold particle-coated gene gun delivery (Qiu et al.,
1996) or by liposome-encapsulated injection to protect the
RNA during administration (Martinon et al., 1993). To further
improve delivery of these molecules, encapsidated selfreplicating RNAs or replicons derived from the genomes of
positive-stranded RNA viruses have been developed. In such
systems, the structural protein sequences of the RNA genome
are substituted for heterologous sequences that express a
foreign protein, while the non-structural protein genes are
retained ; these replicons are capable of undergoing one round
of replication.
The genomes of the alphaviruses SFV and SIN have been
manipulated in this manner to allow the expression of foreign
proteins (Frolov et al., 1996). Packaging of such replicons
stabilizes the RNA molecules and injection of the resulting
virus-like particles induces an array of immune responses
against the given protein. Similarly, the capsid coding
sequences of poliovirus positive-sense RNA have been deleted
to permit the expression of foreign proteins (Choi et al., 1991 ;
Percy et al., 1992) and, when packaged into virus-like particles,
BHDI
this replicon can induce immune responses after injection into
mice transgenic for the poliovirus receptor (Moldoveanu et al.,
1995 ; Porter et al., 1997).
In this study, we evaluated the ability of recombinant
replicons to induce both humoral and cellular immune
responses when injected in the form of naked RNA, arguing
that packaging these vectors is unnecessary, since their
replicative nature alleviates the need for large quantities of
input RNA. In the case of recombinant SFV vectors encoding
the haemagglutinin and NP molecules of influenza A virus, the
injection of naked RNA has been found to induce specific
antibodies (Dalemans et al., 1995 ; Zhou et al., 1994). We
showed here that two recombinant replicons, one derived from
the SFV genome and the other from the poliovirus genome,
which both encode the internal influenza A virus NP protein in
place of the structural proteins, can elicit humoral responses
after injection of naked RNA. Moreover, injection of naked
rSFV-NP replicon RNA was found to induce a CTL response
that was specific for the immunodominant epitope of the
influenza virus NP and to reduce virus load in the lungs of
infected mice challenged with a mouse-adapted influenza virus
to the same extent as that seen using the DNA immunization
technique.
Methods
Cells and viruses. HeLa Young and BHK-21 cells were grown at
37 mC in complete Dulbecco’s modified Eagle’s medium (DMEM)
containing 1 mM sodium pyruvate, 4n5 mg\ml -glucose, 100 U\ml
penicillin and 100 µg\ml streptomycin and supplemented with 5 % heatinactivated foetal calf serum (FCS) (TechGen).
EL4 (mouse lymphoma, H-2b) and P815 (mouse mastocytoma, H-2d)
cells were maintained in complete RPMI 1640 medium containing
10 mM HEPES, 50 µM β-mercaptoethanol, 100 U\ml penicillin and
100 µg\ml streptomycin and supplemented with 10 % FCS.
Mouse-adapted influenza A\PR\8\34(ma) virus (H1N1) was derived
from serial passage of pulmonary homogenates of infected mice to naive
mice, as described previously (Oukka et al., 1996). Subsequent virus
stocks were produced by a single allantoic passage in 11-day-old
embryonated hen’s eggs, which did not affect virus pathogenicity for
mice.
Construction of the pCI-NP expression vector. Viral genomic
RNA was extracted from lung homogenates of influenza A\PR\8\34(ma)
virus-infected mice using 5 M guanidium isothiocyanate and phenol and
reverse-transcribed into cDNA. Next, the sequences encoding influenza
virus NP, including a SalI site before the initiation codon, were amplified
by PCR using Pwo DNA polymerase (Roche). The resulting DNA
fragment was cloned between the SalI and the Klenow-treated SacI sites
of plasmid pTG186 (Kieny et al., 1986). Based on the consensus sequence,
which can be obtained from the authors upon request, plasmid pTGNP82 was reconstructed. A silent mutation was introduced at codon 107
(E, GAG GAA), which destroyed an EcoRI site for the purpose of the
subsequent cloning steps. An additional mutation at codon 277 (Pro
Ser) was present in all NP sequences used in this study ; this mutation
does not directly affect the major histocompatibility complex (MHC)
class I-restricted immunodominant epitope of interest, NP366 (aa
366–374). The SalI–SmaI fragment of pTG-NP82 was then inserted
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Naked RNA vaccines against influenza A virus
between the SalI and SmaI sites of expression plasmid pCI (Promega) to
yield plasmid pCI-NP.
Construction of plasmids for the in vitro transcription of
recombinant replicons. pSFV-NP was constructed by the insertion of
the NP gene, derived from pTG-NP82, into the SmaI site of pSFV1,
which contains a subgenomic cDNA of SFV downstream from the SP6
RNA polymerase promoter (Liljestro$ m & Garoff, 1991).
Plasmids containing poliovirus cDNAs with P1 deletions and
substitutions were derived from plasmid pT7-PV1-52, which contains the
full-length poliovirus type I (Mahoney) infectious cDNA downstream of
the phage T7 promoter (Marc et al., 1989). Plasmid p∆P1-E contains a
subgenomic poliovirus cDNA in which nt 746–3366 were replaced by a
SacI–XhoI–SalI polylinker (GAGCTCGAGCTGTCGAC). In addition,
the SalI site of the pBR322 vector backbone of p∆P1-E was removed by
digestion with EagI\EcoN1, Klenow-filling and ligation.
Plasmid p∆P1-E-NP was constructed by the in-frame insertion of
sequences encoding the influenza A\PR\8\34(ma) virus NP, derived
from pTG-NP82 after digestion with NcoI\MfeI and treatment with the
Klenow fragment, into the Klenow-filled XhoI site of p∆P1-E.
In vitro transcription of plasmid DNA. The poliovirus and SFV
genome-derived plasmids were linearized with EcoRI and SpeI, respectively, and transcribed using the RiboMAX Large Scale RNA
Production systems (Promega) (T7 polymerase for the former, SP6
polymerase for the latter), according to the manufacturer’s instructions.
SFV transcripts were capped during transcription using 3 mM of cap
analogue (Epicentre Technologies). For in vivo studies, reaction mixtures
were treated with RQ1 DNase (1n5 U\µg DNA) (Promega) for 20 min at
37 mC, extracted with phenol–chloroform, precipitated first in ammonium
acetate–isopropyl alcohol, then in sodium acetate–isopropyl alcohol and
resuspended in endotoxin-free PBS (Life Sciences). For in vitro translation
studies, reaction mixtures were processed in the same way, but
precipitated once with sodium acetate–isopropyl alcohol and resuspended in dH O.
#
Rabbit reticulocyte lysate in vitro translation. RNA transcribed in vitro was translated using the in vitro Flexi Rabbit Reticulocyte
Lysate system (Promega) supplemented with 0n8 mCi\ml [$&S]methionine
(1000 Ci\mmol ; Amersham), 80 mM KCl and 20 % HeLa cell S10 extract
(a kind gift from Lisette Cohen, Institut Pasteur, Paris, France). Reaction
mixtures were incubated for 3 h at 30 mC, treated with 100 µg\ml RNase
A in 10 mM EDTA for 15 min at 30 mC and finally analysed by 12 %
SDS–PAGE and autoradiography on Kodak X-OMAT film.
RNA transfection. Transfection of RNA into HeLa or BHK-21 cells
was performed by electroporation using an Easyject plus electroporator
(Equibio). Briefly, 8i10' cells were trypsinized, washed twice, resuspended in 400 µl ice-cold PBS and electroporated in the presence of 16 µg
RNA or DNA using a single pulse (240 V, 900 µF, maximum resistance)
for HeLa cells or a double pulse (1200 V, 25 µF, 156 Ω then 150 V,
2100 µF, 99 Ω) for BHK-21 cells in 0n4 cm electrode gap cuvettes. Cells
were immediately transferred into DMEM supplemented with 2 % FCS
and distributed into four 35 mm diameter tissue culture dishes.
Analysis of RNA replication. At different time intervals posttransfection, cytoplasmic RNA was prepared using standard procedures
(Sambrook et al., 1989). After denaturation, RNA samples were spotted
onto a nylon membrane (Hybond-N, Amersham), hybridized with a $#Plabelled RNA probe complementary to nt 3417–4830 of poliovirus RNA,
essentially as described previously (Marc et al., 1989), and exposed on a
Storm phosphorimager (Molecular Dynamics).
Analysis of influenza virus NP expression in RNA-transfected cells. Influenza A\PR\8\34 virus-infected or RNA\DNAtransfected cells were metabolically labelled with [$&S]methionine
(50 µCi\ml) (1000 Ci\mmol ; Amersham) for 2 h at times of peak
expression. Next, cells were washed in PBS and lysed with 50 mM
Tris–HCl, pH 7n5, 150 mM NaCl, 1 mM EDTA, 1 % NP40 and 0n5 %
protease inhibitor cocktail (Sigma). Cell extracts were then immunoprecipitated overnight at 4 mC in RIPA buffer (50 mM Tris–HCl, 150 mM
NaCl, 1 mM EDTA, 0n1 % deoxycholate, 0n1 % SDS, 0n5 % NP40 and
0n5 % Protease Inhibitor cocktail) in the presence of protein A–Sepharose
beads (Amersham) with rabbit antibodies raised against influenza
A\PR\8\34 virus. The immunoprecipitates, washed in RIPA buffer and
eluted in Laemmli sample buffer at 65 mC, were analysed by SDS–PAGE
and visualized by autoradiography on Kodak X-OMAT film.
Immunizations. Male C57BL\6 mice (IFFA CREDO) 7 to 8 weeks
of age were injected intramuscularly with 100 µl PBS (50 µl in each tibialis
anterior muscle) containing 50 µg of plasmid DNA, 25 µg of poliovirus
replicon RNA or 10 µg of capped SFV replicon RNA. Booster injections
were administered at three or four week intervals. DNA used for injection
was prepared using the Nucleobond Megaprep kit, followed by
extraction steps with Triton X-114 and phenol–chloroform and tested for
the absence of endotoxin ( 100 U\mg), as measured with the QCL1000 Endotoxin kit (BioWhittaker). RNA preparations were analysed
before and after injection by agarose gel electrophoresis to verify the
absence of degradation.
Antibody titre. Blood from mice was collected 1 week before
immunization and 3 weeks after each injection. Serial dilutions of pooled
serum samples were used to determine NP-specific antibody titres by
ELISA using 0n5 µg of detergent-disrupted influenza A\PR\8\34 virus
per well as antigen.
Cytotoxicity assay. Spleen cells were collected 3 weeks after the
last immunization and seeded into upright T75 flasks at 2i10' cells\ml
in complete RPMI 1640 supplemented with 10 % FCS, non-essential
amino acids, 1 mM sodium pyruvate and 2n5 % concanavalin A. They
were then re-stimulated for 7 days with 10' syngeneic spleen cells\ml,
which had been pulsed for 3 h with 10 µM NP366 peptide
(ASNENMETM, Neosystem), washed and irradiated (2500 rads). Cytotoxic activity of the re-stimulated effector cells was measured using a
standard &"Cr-release cytotoxicity assay, essentially as described previously (Escriou et al., 1995). EL4 and P815 target cells were pulsed or not
with NP366 peptide (10 µM) during &"Cr-labelling. Spontaneous and
maximal release of radioactivity were determined by incubating cells in
medium alone or in 1 % Triton X-100, respectively. The percentage
of specific &"Cr release was calculated as (experimental release
kspontaneous release)\(maximal releasekspontaneous release)i100.
Challenge infection of mice with influenza A/PR/8/34(ma)
virus. At 1 or 3 weeks after the third immunization, mice were lightly
anaesthetized with 100 mg\kg ketamine (Merial) and challenged intranasally with 100 p.f.u. (0n1 LD ) influenza A\PR\8\34(ma) virus in 40 µl
&!
PBS. Mice were sacrificed 7 days after challenge infection and lung
homogenates were prepared and titred for virus on MDCK cell
monolayers in a standard plaque assay. Statistical analyses were
performed on the log of the virus titres, measured for individual mice
using the Student t-test with the assumptions used for small samples
(normal distribution of the variable, same variance for the populations to
be compared).
Results
Production of recombinant replicons derived from the
SFV and poliovirus genomes
For the production of SFV genome-derived replicons, the
sequences encoding the mouse-adapted influenza A\PR\8\34
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M. Vignuzzi and others
Fig. 1. Schematic representation of plasmids encoding subgenomic recombinant replicons derived from the SFV or poliovirus
genomes. (a) pSFV-NP was constructed by the insertion of the influenza A/PR/8/34(ma) virus NP cDNA sequences (hatched
box) into the SmaI site of pSFV1 (Liljestro$ m & Garoff, 1991). The latter plasmid contains a subgenomic cDNA of the SFV
genome downstream from the SP6 RNA polymerase promoter. The NP sequences, which have been inserted in place of the
SFV structural genes, are therefore under the control of the 26S subgenomic RNA promoter (open arrow). (b) Nucleotides 746
to 3366 of the P1 region, which encodes the structural proteins, were deleted from pT7-PV1-52, which contains the complete
infectious cDNA of poliovirus type 1 Mahoney downstream of the T7 bacteriophage promoter. Deleted sequences were
replaced by a SacI–XhoI–SalI polylinker (not shown to scale), whereas sequences encoding the nine C-terminal amino acids of
VP1 comprising the optimal sequence for in cis cleavage by the 2A protease were retained, thus yielding plasmid p∆P1-E.
Plasmid p∆P1-E-NP was constructed by the insertion of sequences encoding the influenza A/PR/8/34(ma) virus NP (hatched
box) in-frame with the remainder of the poliovirus polyprotein sequences behind the poliovirus initiation codon. Extra amino
acid residues of the NP–VP1* fusion protein derived from the poliovirus sequences are shown on either side in single-letter
format. The Y–G cleavage site of proteinase 2Apro is indicated by an arrow.
virus NP were inserted into the polylinker of the SFV
expression vector pSFV1 (Liljestro$ m & Garoff, 1991) downstream of the 26S subgenomic promoter in place of sequences
encoding the SFV structural proteins, as described in Methods
(Fig. 1 a). SP6 RNA polymerase transcription of pSFV-NP
linearized with SpeI allowed the synthesis of capped positivesense recombinant RNAs capable of initiating a replication
cycle when transfected into cells (data not shown).
BHEA
For the production of poliovirus genome-derived replicons,
the plasmid vector p∆P1-E was first constructed, in which the
capsid protein-coding sequences of P1 were substituted with a
polylinker, such that sequences corresponding to an optimal
cleavage site for the 2A protease, consisting of the nine Cterminal amino acids of VP1 and the first amino acid of 2A (as
defined by Hellen et al., 1992), were maintained (Fig. 1 b). The
sequences coding for the influenza virus NP were then inserted
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Naked RNA vaccines against influenza A virus
Fig. 2. In vitro translation and processing of the recombinant poliovirus
polyprotein. Synthetic RNAs corresponding to the full-length poliovirus
genome rT7-PV1-52 (lane 1), the subgenomic replicon r∆P1-E (lane 2)
and the recombinant replicon r∆P1-E-NP (lane 3) were translated in vitro
in rabbit reticulocyte lysates supplemented with HeLa S10 cell extract
(see Methods). Translation products were analysed by SDS–PAGE and
visualized by autoradiography. Poliovirus protein precursors as well as
some of their major cleavage products are indicated on the left. Molecular
masses are indicated on the right next to the molecular mass marker (lane
4). The influenza virus NP encoded by the recombinant replicon is
indicated by an arrow (lane 3).
upstream of this reconstituted cleavage site and in-frame with
the rest of the sequences encoding the poliovirus polyprotein
to yield plasmid p∆P1-E-NP (Fig. 1 b).
The recombinant RNAs, r∆P1-E and r∆P1-E-NP, derived
from in vitro transcription with T7 RNA polymerase of p∆P1-E
and p∆P1-E-NP-linearized DNAs were translated in vitro in
rabbit reticulocyte lysates. As shown in Fig. 2, the repliconencoded polyproteins were properly cleaved to express the
non-structural proteins necessary for RNA amplification (as
shown by the end products of cleavage), such as the 2A, 3CD,
2BC and 2C proteins. In particular, correct in cis cleavage of the
reconstituted VP1–2A site by the 2A protease was observed.
For the subgenomic replicon r∆P1-E, however, this cleaved
product migrated as a doublet, which could be explained as a
mixture of properly cleaved product and uncleaved VP1*–2A
fusion protein containing the 13 amino acid residues of the
reconstituted cleavage site and polylinker. It could be argued
that the very short stretch of amino acids before the cleavage
site was too short to permit 100 % cleavage. For the
recombinant replicon r∆P1-E-NP, such uncleaved product,
which would have appeared as a 72 kDa NP–VP1*–2A fusion
protein, was barely visible, suggesting that the addition of the
NP sequences alleviated the spatial restriction seen in the
Fig. 3. Replication of subgenomic poliovirus genome-derived replicons.
HeLa cells were mock-transfected (lane 4) or transfected by
electroporation with synthetic transcripts corresponding to the full-length
poliovirus genome rT7-PV1-52 (lane 1), the subgenomic replicon r∆P1-E
(lane 2) or the subgenomic recombinant replicon r∆P1-E-NP (lane 3). At
the indicated times post-transfection, cytoplasmic RNA was harvested,
slot-blotted onto a nylon membrane and hybridized to a 32P-labelled
riboprobe complementary to nt 3417–4830 of the poliovirus genome, as
described in Methods.
previous case. Expression of the properly cleaved NP–VP1*
fusion protein was therefore revealed by the presence of a band
with the expected molecular mass of 56 kDa (Fig. 2).
Replicative characteristics of poliovirus genomederived replicons r∆P1-E and r∆P1-E-NP
Since the influenza virus NP has been shown to associate
non-specifically with RNAs (Kingsbury et al., 1987 ; Yamanaka
et al., 1990), an interaction with the poliovirus RNA could
hypothetically affect overall replication efficiency. Therefore,
synthetic RNA transcripts of r∆P1-E and r∆P1-E-NP, as well as
parental rT7-PV1-52, were transfected into HeLa cells and
total cytoplasmic RNA was extracted at various times
post-transfection. Hybridization after slot blotting using a
radiolabelled riboprobe complementary to nt 3417–4830 of
poliovirus RNA revealed efficient replication of all RNAs
(Fig. 3). Similar results were obtained after transfection of L929
murine cells (data not shown). In our current studies, cells were
transfected by electroporation, which was more efficient than
the classic DEAE-dextran technique ( 90 % transfection
efficiency). Under these conditions, all three RNA species
induced cytopathic effects, regardless of the presence or
absence of capsid proteins, and resulted in the general
destruction of the cell monolayer (data not shown). Taken
together, these results illustrated that the insertion of sequences
encoding the influenza virus NP had no negative effects on
RNA replication.
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M. Vignuzzi and others
Fig. 4. Expression of the influenza virus NP by the recombinant replicons rSFV-NP and r∆P1-E-NP. Cells were transfected by
electroporation with replicon RNA or plasmid DNA, or infected with influenza virus at an m.o.i. of 10. At times of peak
expression, cells were metabolically labelled with [35S]methionine for 2 h. Cytoplasmic extracts were prepared and proteins
were immunoprecipitated with polyclonal antibodies raised against influenza A/PR/8/34 virus, analysed by SDS–PAGE and
visualized by autoradiography, as described in Methods. (a) Expression from SFV genome-derived replicons. Loaded samples
are as follows : 293 cells infected with influenza A/PR/8/34(ma) virus harvested at 20 h post-infection (lane 1), BHK-21 cells
transfected with recombinant replicon rSFV-NP (lane 2) or with replicon rSFV (lane 3) and harvested 18 h post-transfection,
and mock-transfected BHK-21 cells (lane 4). Molecular masses are shown on the left. (b) Expression from poliovirus genomederived replicons. Loaded samples are as follows : mock-transfected HeLa cells (lane 1), HeLa cells transfected with pCI (lane
2) or pCI-NP (lane 3) and harvested at 40 h post-transfection, HeLa cells transfected with replicons r∆P1-E (lane 4) or r∆P1E-NP (lane 5) and harvested at 12 h post-transfection. Molecular masses are shown on the right. The influenza virus NP is
indicated by filled arrows. Putative NP cleavage by-products are indicated by open arrows.
Expression of influenza virus NP by recombinant SFV
and poliovirus genome-derived replicons
Nucleoprotein expression was analysed by immunoprecipitation (using antibodies against influenza A\PR\8\34
virus) of cytoplasmic extracts from cells transfected with SFV
or poliovirus genome-derived replicons or plasmid DNA, or
infected with influenza A\PR\8\34 virus, as described in
Methods. Cells were pulse-labelled with [$&S]methionine for
2 h prior to harvest at times of peak expression, as determined
in preliminary experiments. As shown in Fig. 4 (a), a protein
with an apparent molecular mass of 54 kDa was specifically
immunoprecipitated from extracts of cells transfected with
rSFV-NP (Fig. 4 a, lane 2) and co-migrated with the NP
produced by 293 cells infected with influenza A\PR\8\34
virus (Fig. 4 a, lane 1). No immunoreactive proteins could be
detected from the mock-transfected cells or from cells transfected with replicon RNA derived from the empty vector
pSFV1.
Similarly, transfection of HeLa cells with the recombinant
replicon r∆P1-E-NP (Fig. 4 b, lane 5) resulted in high levels of
NP-related protein expression. The majority of NP expressed
by the poliovirus genome-derived replicon migrated slightly
slower than the native form of NP expressed by pCI-NP DNABHEC
transfected cells (Fig. 4 b, lane 3). This difference in molecular
mass accounted for the additional amino acid residues of the
NP–VP1* fusion protein and was consistent with the fact that
proteolytic processing of the poliovirus polyprotein occurred
at the reconstituted VP1*–2A cleavage site. Interestingly, the
presence of bands of a smaller molecular mass (Fig. 4 b, lane 5)
suggested that the NP produced may have been cleaved, in
part, by one of the poliovirus proteases (2A or 3C).
Examination of the NP sequences for such cleavage sites (Y–G
for 2A protease or Q–G for 3C protease) confirmed this
possibility ; nevertheless, these potential cleavage by-products
remained a minority. Thus, both SFV and poliovirus genomederived recombinant replicons were shown to direct efficient
expression of the influenza virus NP in transfected cells.
Induction of NP-specific antibodies after immunization
with recombinant rSFV-NP and r∆P1-E-NP replicons
In order to establish the feasibility of using naked replicon
injection to elicit a heterospecific CTL response, we first
determined the conditions of RNA injection required to obtain
an antibody response comparable to that observed after one
injection of plasmid DNA, which in published literature was
shown to be sufficient to induce a CTL response (Ulmer et al.,
1993). To this end, naked RNA or DNA was administered
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Naked RNA vaccines against influenza A virus
Fig. 5. Anti-NP antibody response to immunization with naked RNA.
C57BL/6 mice were immunized two or three times at four week intervals
with 50 µg of plasmid DNA (pCI or pCI-NP), 10 µg of SFV genomederived replicon RNA (rSFV or rSFV-NP) or 25 µg of poliovirus genomederived replicon RNA (r∆P1-E or r∆P1-E-NP). Sera were collected before
(0) the first (1), second (2) or third (3) injection or after. Titres are
represented as the reciprocal of the highest dilution of pooled sera for a
given group of five or six mice giving an optical density measurement at
450 nm equal to three times that of background levels in a direct ELISA
test using purified split influenza A/PR/8/34 virions as antigen. Data from
one experiment representative of two are shown.
intramuscularly at monthly intervals (one, two or three times)
to C57BL\6 mice and the specific anti-NP antibody response
was examined by ELISA, as described in Methods.
As shown in Fig. 5, one injection of 10 µg of naked rSFVNP RNA induced serum antibodies against influenza virus NP,
although the specific ELISA titres were reproducibly lower
than those obtained after one injection of 50 µg of pCI-NP
DNA. At 3 weeks after a booster injection, a strong increase in
the anti-NP antibodies in the sera of rSFV-NP-injected mice
was observed, reaching a level comparable to that obtained
after two injections of pCI-NP DNA. Three injections of 25 µg
of naked r∆P1-E-NP RNA proved necessary to raise NPspecific antibodies to levels equal to or slightly higher than
those achieved by one injection of plasmid pCI-NP DNA.
Thus, these findings showed that poliovirus replicons were
immunogenic when injected as naked RNA, although less so
when compared to the SFV replicons.
Induction of an NP-specific CTL response after
injection of recombinant SFV replicon as naked RNA
In order to evaluate whether recombinant rSFV-NP and
r∆P1-E-NP injected as naked RNA were able to induce specific
CTLs directed against the dominant H-2Db-restricted epitope
NP366, C57BL\6 mice were injected intramuscularly either
once or twice with 10 µg of rSFV-NP RNA, three times with
25 µg of r∆P1-E-NP RNA or once with 50 µg of pCI-NP DNA
as a positive control. This immunization schedule was defined
according to the previously determined anti-NP antibody
response and based on the observation that one injection of
plasmid DNA was sufficient to induce a detectable NP-specific
Fig. 6. Induction of NP-specific CTL response by naked RNA immunization.
Groups of four C57BL/6 mice were immunized at three week intervals with
the following vaccination protocols : one injection of 50 µg of pCI (#) or
pCI-NP ($) DNA, one injection of 10 µg of rSFV () or rSFV-NP (
)
RNA, two injections of 10 µg of rSFV (W) or rSFV-NP (X) RNA, or three
injections of 25 µg of r∆P1-E (=) or r∆P1-E-NP (>) RNA. Splenocytes
were collected 3 weeks after the last immunization and stimulated
in vitro for 1 week with irradiated syngeneic splenocytes loaded with
peptide NP366. These cells were used as effector cells in a classic
chromium-release assay against syngeneic EL4 target cells either loaded
with peptide NP366 (a) or not (b) (see Methods for details). The
percentage of specific lysis is shown at various effector : target ratios. Solid
lines indicate DNA or RNA encoding the influenza virus NP, hatched lines
indicate DNA or RNA not encoding the influenza virus NP. Data from one
experiment representative of three are shown.
CTL response at levels just below those of mice having
recovered from sub-lethal influenza A\PR\8\34(ma) virus
infection (data not shown). Splenocytes from immunized mice
were harvested 3 weeks after the last injection, stimulated in
vitro with peptide NP366 and tested for cytolytic activity 7
days later using a classic chromium-release assay, as described
in Methods. Spleen cell cultures initiated from mice injected
once with either rSFV-NP RNA or pCI-NP DNA (Fig. 6 a)
specifically lysed EL4 cells loaded with peptide NP366. A
much higher level of cytolytic activity was detected for spleen
cells of mice that had received a booster injection of rSFV-NP
RNA (Fig. 6 a). In all cases, no lysis was observed with
stimulated splenocytes from control naive mice or from mice
that were immunized with control vectors without the NP
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BHED
M. Vignuzzi and others
Fig. 7. Naked RNA immunization with
replicon rSFV-NP confers protection
against influenza virus challenge.
C57BL/6 mice (five or six per group)
were immunized three times at 3 week
intervals with either 10 µg of rSFV or
rSFV-NP replicon RNA or 50 µg of pCI or
pCI-NP plasmid DNA. A week after the
last injection, mice were challenged with
102 p.f.u. (0n1 LD50) of mouse-adapted
influenza A/PR/8/34 virus and virus titres
in the lungs were determined 7 days
post-challenge infection, as described in
Methods. Virus loads in mice injected
with each NP-encoding vector were
significantly lower than for mice injected
with the corresponding empty vector
(P 0n001 ; Student’s t-test). The mean
(open circles)pSD of each group is
shown. Data from one experiment
representative of six are shown.
sequences (Fig. 6, hatched lines), nor was any lysis detected on
syngeneic targets not charged with peptide (Fig. 6 b). Finally,
for all effector populations, lysis of allogenic P815 target cells
(H-2d) remained at background levels regardless of whether or
not they were incubated with peptide (data not shown),
indicating that the cytolytic activity was H-2-restricted and
was thus likely to derive from MHC-I-restricted CD8+ T cell
effector cells. In contrast, splenocytes from r∆P1-E-NP-injected
mice failed to lyse either of the EL4 targets, despite maintaining
the same immunization schedule (i.e. three injections of 25 µg
of RNA) that was found to induce NP-specific antibodies.
Three injections of 100 µg of r∆P1-E-NP RNA also failed to
induce a detectable CTL response (data not shown).
Protective immunity in vivo
Previous studies have found that CTLs directed against the
influenza virus NP and induced by naked DNA immunization
were protective against a challenge infection with influenza
virus (Bot et al., 1996 ; Ulmer et al., 1993). To determine
whether the CTL responses induced by naked RNA immunization with SFV genome-derived replicons could also
contribute to protection by reducing virus titre in the
lungs, C57BL\6 mice were injected three times with either
rSFV-NP RNA or pCI-NP DNA and challenged with a sublethal dose of the homologous influenza A\PR\8\34(ma) virus
(10# p.f.u.) 1 or 3 weeks thereafter. Virus load in the lungs was
evaluated 7 days after challenge by plaque assay on MDCK
cells. In all six studies, such as the one shown in Fig. 7,
immunization with either naked SFV RNA or naked DNA
encoding the influenza virus NP lowered virus lung titres by
one to two logs. The observed reduction of virus loads after
injection of each NP-coding molecule as compared to the
respective blank vector was found to be significant by the
Student t-test (P 0n001). It is worth noting that although
the drop in virus titre was moderate, which would correlate
BHEE
with the high virulence of our mouse-adapted virus strain
(LD was 10$ p.f.u. for C57BL\6 mice), the reduction in titre
&!
achieved with naked RNA immunization was as efficient as
that obtained with naked DNA immunization.
Discussion
The principal aim of this study was to explore and evaluate
the feasibility of adapting standard naked DNA immunization
protocols for naked RNA immunization using positivestranded RNA virus genomes that have structural proteincoding sequence deletions and are capable of a single round of
replication, as vaccine vectors against influenza virus.
The genome of poliovirus is a good candidate for naked
RNA immunization because encapsidated subgenomic poliovirus replicons expressing viral, bacterial and tumour antigen
have already been described (Ansardi et al., 1994 ; Choi et al.,
1991 ; Porter et al., 1997). Furthermore, live recombinant
polioviruses expressing simian immunodeficiency virus proteins are being developed as prototypes for an AIDS vaccine
that elicits humoral and cellular immune responses in nonhuman primates (Crotty et al., 1999). We showed here that
when administered as naked RNA, a recombinant poliovirus
replicon, r∆P1-E-NP, engineered to express the influenza virus
NP was able to induce NP-specific antibodies in mice after
three injections, which suggested that the replicon did replicate
at least to some extent in vivo. This was further supported by
the fact that a neutralizing antibody response against the
poliovirus capsid proteins was elicited after injection with the
full-length poliovirus genome in the form of naked RNA into
C57BL\6 mice, which do not express the poliovirus receptor
(data not shown). However, no CTL response was detected
towards the immunodominant epitope of the influenza virus
NP in C57BL\6 mice injected with r∆P1-E-NP.
Firstly, this lack of CTL response could be due to the
possibility that the poliovirus genome replicates poorly in
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Naked RNA vaccines against influenza A virus
murine cells in vivo, particularly in myocytes, if these are
indeed the site of protein expression, after intramuscular naked
nucleic acid immunization (Corr et al., 1999). Secondly, there is
question as to whether or not the MHC-I-restricted NP366
peptide was properly presented by r∆P1-E-NP-transfected
cells. It was shown that the protein secretion pathway is altered
by poliovirus proteins 2B and 3A and recent evidence by
Kirkegaard and colleagues has shown that 3A protein
expression was responsible for strong inhibition of MHC-Idependent antigen presentation in poliovirus-infected cells
(Deitz et al., 2000 ; Doedens & Kirkegaard, 1995).
However, Mandl et al. (1998) have demonstrated that a
CTL response can be induced in mice transgenic for the
poliovirus receptor by a recombinant poliovirus containing
sequences that encode the H-2b-restricted CTL epitope of
chicken ovalbumin. In this situation, the genome of the virus
entered cells by infection rather than by uptake of injected
naked RNA and multiple cycles of replication and re-infection
of neighbouring cells were possible. Therefore, the inability to
detect a CTL response may be due to differences in the number
or type of cells receiving replicon RNA, levels of expression or
route of immunization. In vitro antigen presentation assays are
being developed to determine if NP peptide presentation does
indeed occur in cells replicating the r∆P1-E-NP recombinant
replicon.
Alternatively, the CTL response induced by the poliovirus
recombinant replicon might be skewed towards another
peptide, e.g. one of the poliovirus non-structural proteins that
could be hypothetically dominant to the NP peptide, in
C57BL\6 mice. Although sequences corresponding to the
H-2Db epitope-binding motif are found throughout the
proteins encoded by the poliovirus vector, there is no
information currently available on whether these are the
targets of a CTL response in H-2b mice. A means to clarify this
issue would be to examine the CTL response in H-2d mice, for
which the immunodominant epitope of the influenza virus NP
is known, since it is unlikely that the same phenomenon would
be repeated.
Finally, it is well accepted that the strong immunogenicity
of DNA vaccines is a consequence of long-lasting antigen
expression together with the adjuvant effect of short noncoding immunostimulatory sequences centred around unmethylated CpG motifs in the plasmid DNA backbone
itself (Sato et al., 1996). In the case of the highly effective naked
RNA immunization with SFV replicons, it has been suggested
that virus-like RNA replication could provide an adjuvant
effect by triggering a series of ‘ danger signals ’ which in turn
would activate the innate immune system of the host (Leitner
et al., 1999). It is tempting to speculate that poliovirus could
block, or at least inhibit, this process by an as yet unknown
mechanism, thus resulting in a poorly immunogenic replicon
that is able to avoid detection by the host immune system.
Injection of naked RNA from the recombinant rSFV-NP
replicon designed to express the influenza virus NP proved to
be effective at inducing anti-NP antibodies after two injections ;
antibody titres were at levels comparable to those obtained by
one injection of NP-encoding plasmid DNA. Induction of
antibodies specific for an influenza virus protein by this type of
vector when administered as naked RNA has already been
described (Dalemans et al., 1995 ; Zhou et al., 1994). Recently,
using LacZ as a model tumour antigen for cancer therapy, Ying
et al. (1999) have shown that injection of naked recombinant
SFV RNA can elicit an antibody response, activate CD8+ T
cells to release interferon-γ and protect from tumour challenge.
Here, we showed that the injection of naked recombinant
rSFV-NP RNA could induce, in addition to antibodies, CTLs
that target the NP366 epitope in a response that was found to
be comparable to that induced by plasmid DNA. Furthermore,
we showed that in a virus challenge model using a mouseadapted strain of influenza virus, RNA immunization worked
as well as DNA immunization in the degree of protection
conferred, as measured by reduction of virus load in the
lungs. It will be interesting to determine whether even greater
protection is conferred after challenge with less virulent strains
of mouse-adapted influenza virus and to evaluate the role of
anti-NP CTLs induced by naked RNA immunization against a
heterologous influenza virus of the same or a different subtype.
This work was supported in part by the Ministe' re de l’Education
Nationale, de la Recherche et de la Technologie (EA 302). We would like
to thank Ida Rijks for the production of influenza A\PR\8\34(ma) virus,
Annette Martin for helpful suggestions and discussions, Lisette Cohen
and Katherine Kean for valuable advice on in vitro translations and Nadia
Naffakh for critical reading of the paper.
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Received 10 January 2001 ; Accepted 12 March 2001
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