Unité de Génétique Moléculaire des Virus Respiratoires, URA 1966 CNRS, Institut Pasteur, 25 rue du Dr Roux, F-75724 Paris cedex 15, France1
Author for correspondence: Nicolas Escriou. Fax +33 1 40 61 32 41. e-mail escriou{at}pasteur.fr
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Abstract |
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Introduction |
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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 cross-strain 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 self-replicating 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, 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.
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Methods |
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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 -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 hens 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 pTG-NP82 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 366374). The SalISmaI fragment of pTG-NP82 was then inserted 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 (Liljeström & 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 7463366 were replaced by a SacIXhoISalI 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 pP1-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 manufacturers 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 (1·5 U/µg DNA) (Promega) for 20 min at 37 °C, extracted with phenolchloroform, precipitated first in ammonium acetateisopropyl alcohol, then in sodium acetateisopropyl 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 acetateisopropyl alcohol and resuspended in dH2O.
Rabbit reticulocyte lysate in vitro translation.
RNA transcribed in vitro was translated using the in vitro Flexi Rabbit Reticulocyte Lysate system (Promega) supplemented with 0·8 mCi/ml [35S]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 °C, treated with 100 µg/ml RNase A in 10 mM EDTA for 15 min at 30 °C and finally analysed by 12% SDSPAGE 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, 8x106 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 then 150 V, 2100 µF, 99
) for BHK-21 cells in 0·4 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 post-transfection, 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 32P-labelled RNA probe complementary to nt 34174830 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/DNA-transfected cells were metabolically labelled with [35S]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 TrisHCl, pH 7·5, 150 mM NaCl, 1 mM EDTA, 1% NP40 and 0·5% protease inhibitor cocktail (Sigma). Cell extracts were then immunoprecipitated overnight at 4 °C in RIPA buffer (50 mM TrisHCl, 150 mM NaCl, 1 mM EDTA, 0·1% deoxycholate, 0·1% SDS, 0·5% NP40 and 0·5% Protease Inhibitor cocktail) in the presence of protein ASepharose 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 °C, were analysed by SDSPAGE 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 phenolchloroform and tested for the absence of endotoxin (<100 U/mg), as measured with the QCL-1000 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 0·5 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 2x106 cells/ml in complete RPMI 1640 supplemented with 10% FCS, non-essential amino acids, 1 mM sodium pyruvate and 2·5% concanavalin A. They were then re-stimulated for 7 days with 106 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 51Cr-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 51Cr-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 51Cr release was calculated as (experimental release-spontaneous release)/(maximal release-spontaneous release)x100.
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. (0·1 LD50) 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).
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Results |
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The recombinant RNAs, rP1-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 replicon-encoded 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 VP12A 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 NPVP1*2A fusion protein, was barely visible, suggesting that the addition of the NP sequences alleviated the spatial restriction seen in the previous case. Expression of the properly cleaved NPVP1* fusion protein was therefore revealed by the presence of a band with the expected molecular mass of 56 kDa (Fig. 2
).
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Induction of NP-specific antibodies after immunization with recombinant rSFV-NP and rP1-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 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 rSFV-NP 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 NP-specific 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.
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Discussion |
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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 non-human 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 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-I-dependent 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 non-coding 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-
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 mouse-adapted 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.
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Acknowledgments |
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Received 10 January 2001;
accepted 12 March 2001.