BACKGROUND
influenza is a widely distributed viral infection of humans and animals.
the high variability of the surface viral proteins, hemagglutinin and neuraminidase, results in the appearance of a new epidemic strain every one to two years  <cit> ; this requires the production of new vaccines at the same frequency.
a promising solution to this problem is the development of recombinant vaccines that can be rapidly produced in standard expression systems.
one of the most promising conserved antigens of influenza virus is the extracellular domain of transmembrane protein m <dig>  m2e  <cit> .
the length of m2e is only  <dig> amino acid residues .
its sequence is virtually unchanged in all human isolates since  <dig> , and in strains of avian influenza a it differs in only a few amino acids.
by itself m2e possesses low immunogenicity  <cit>  but this can be increased by delivery in a multimeric form or in combination with complex adjuvants or delivery systems .
toll-like receptors  have been shown to play a critical role in controlling the adaptive immune response, by properly arming dendritic and other antigen-presenting cells, triggering important costimulatory and regulatory mechanisms, and promoting antigen presentation  <cit> .
it has been previously shown that genetically fusing an antigen of interest to bacterial flagellin, the ligand for tlr <dig>  significantly increases the immunogenicity and protective capacity of the antigen  <cit> .
notably flagellin is particularly active as mucosal adjuvant, opening the possibility of non-invasive intranasal administration of vaccines.
the ability of flagellin to be both the platform and the adjuvant for different vaccines was demonstrated in various models of infection including influenza .
recombinant proteins, including those for medical purposes, can be produced in a variety of expression systems, including bacteria, yeast, plants, and mammalian cells.
the advantages of using plants for protein production include the low final cost and inherent safety of products due to the absence of pathogens common for plants and animals.
recombinant proteins in plants may be obtained by stable genetic transformation  or through transient expression .
creation of lines of stably transformed plants and, subsequently, production of a protein from such plants requires considerable time and cost.
the level of expression of target proteins by transgenic plants is usually relatively low, resulting in a high cost of products due to the difficulty of their purification  <cit> .
the use of self-replicating viral vectors or efficient non-replicating transient expression vectors is an alternative approach providing sufficient amounts of the desired protein   <cit>  to conduct immunological studies.
to create such vectors, sequences from tobacco mosaic virus , cowpea mosaic virus  or potato virus x  have frequently been used.
the genomes of these viruses are small and consist of positive-strand rna.
the vectors are introduced into plant cells by infiltration of the plant leaf tissue with agrobacterium suspensions carrying the vector within the t-dna portion of a binary plasmid resulting in transfer of vector dna into the recipient plant cells during the agroinfection  <cit> .
such expression systems have been used for production of a variety of pharmaceutical proteins, including antibodies and candidate vaccines  <cit> .
the latter have included influenza virus hemagglutinin  and hbc virus-like particles carrying m2e peptide  <cit> .
in this work four tandem copies of m2e sequences based on human and avian influenza virus isolates were genetically fused to salmonella typhimurium flagellin and produced in nicotiana benthamiana plants using transient expression using either a replicating pvx vector  <cit>  or the non-replicating cpmv-ht system  <cit> .
intranasal immunization of mice with the plant-produced hybrid protein induced the desired immune response and conferred protective immunity against a lethal influenza virus challenge.
RESULTS
viral vectors for expression of the hybrid protein consisting of flagellin linked to four copies of influenza m2e peptide in n. benthamiana plants
the highly conserved extracellular domain of the influenza virus m <dig> protein, m2e, is a promising target for the development of recombinant “universal” vaccines against influenza  <cit> .
however, m2e is poorly immunogenic and needs to be presented in a multimeric form or linked to a carrier molecule such as virus-like particles  <cit> .
here we use flagellin of salmonella typhimurium as such carrier and simultaneously as an adjuvant.
in order to make a broader range candidate vaccine, a hybrid protein flg-4m containing two copies of human consensus m2e sequence and two copies of the m2e peptide of avian influenza virus strain a/chicken/kurgan/05/ <dig> fused to the c-terminus of s. typhimurium fljb  was designed.
in order to facilitate folding of hybrid protein, copies of m2e peptides were separated from each other by flexible glycine-rich linker .
the empty flagellin  was used as a control.
both proteins, flg-4m and flg, were designed with n-terminal 6-histidine tag to facilitate their purification.fig.
<dig> structure of the expression constructs used in this study.
a amino acid sequence of flg-4m protein.
the sequences of human “consensus” m2e peptides, m2eh, are italicized; the sequences of m2e peptide of avian influenza virus strain a/chicken/kurgan/05/ <dig>  m2ek, are shown in bold; the linker sequences are underlined.
flagellin sequence is shaded in grey.
b structures of viral vectors pa7248amv-flg-4m and peaq-flg-4m.
the tdna regions of the binary vectors are shown.
rdrp, rna dependent rna polymerase of pvx; sgp <dig>  subgenomic promoter of the pvx protein 25 k; amv, the leader sequence of rna  <dig> of the alfalfa mosaic virus; 6his,  <dig> histidine tag; flg, flagellin of s. typhimurium; 4m2e, four copies of m2e peptide ; 35s, promoter; 35s-t, terminator of the cauliflower mosaic virus rna; nost, terminator of nopaline synthase gene; lb and rb are the left and right borders of tdna; p <dig>  gene of silencing suppressor p <dig> from the tomato bushy stunt virus; 5’ and 3’, the 5’-utr and the 3’-utr from pvx  and cpmv
two plant virus-based transient expression systems were used for production of recombinant flg-4m protein in n. benthamiana.
the first vector, pa7248amv-gfp  <cit> , is a replicating system based on the pvx genome.
this vector includes the 5’-utr of the pvx genome, the polymerase gene, the first promoter of subgenomic rna, the translation enhancer represented by the sequence of the 5’-untranslated region of rna  <dig> of the alfalfa mosaic virus , the gfp gene flanked by unique restriction sites asci and smai, the last  <dig> nucleotides of the coat protein gene, and the 3’-untranslated region of the pvx genome.
this construction is nserted between the 35s promoter and 35s terminator and cloned in the binary vector pbin <dig>  <cit> .
to create the viral vector allowing production of the flg-4m protein, a sequence of the hybrid gene was cloned in pa7248amv-gfp at the asci and smai sites to replace gfp, which resulted in construction of a recombinant vector pa7248amv-flg-4m .
recombinant vector pa7248amv-flg for expression of flg in plants was constructed in a similar way.
the second expression system, peaq, is based on cpmv.
this is a non-replicating system which uses production of a highly translatable mrna to achieve high level expression.
the recombinant vector peaq-flg-4m contains 5’-utr and 3’-utr from cpmv rna- <dig> upstream and downstream, respectively, of the hybrid gene flg-4m .
expression of the hybrid protein flg-4m in n. benthamiana plants
to express the protein flg-4m, vectors were inserted into a. tumefaciens strain gv <dig>  which was used for agroinfiltration of leaves of n. benthamiana plants.
agroinfiltration zones for two vectors were located within one leaf.
protein samples were isolated in  <dig>   <dig>   <dig>   <dig>  and 7 days after infection.
for every day three leaves were taken for analysis.
protein samples were analyzed using sds-page and western-blotting .
maximal expression was observed at fourth day.
summarizing the expression results for  <dig> leaves we found out that peaq-flg-4m construct provides expression level of about 30 % in comparison to pa7248amv-flg-4m.fig.
<dig> efficiency of pvx- and cpmv-based vectors.
coomassie brilliant blue stained gel  and western blot  of proteins isolated form n. benthamiana plants and separated by sds-page.
m, − molecular weight marker ;  <dig> and  <dig>  − total soluble proteins isolated at fourth day after agroinfiltration from leaves infiltrated with vector pa7248amv-flg-4m and peaq-flg-4m, respectively;  <dig>  − total soluble proteins isolated from empty leaves.
antibodies against m2e were used in western blotting
given the higher levels of expression obtained, we used vector pa7248amv-flg-4m for the scaled-up production of protein flg-4m in n. benthamiana plants.
the total soluble protein was isolated from leaves at 4th day after agroinfiltration and analyzed by sds-page.
the data presented in fig.
3a show that flg-4m protein is highly expressed and accounted for about 30 % of the soluble protein fraction .
the recombinant protein was specifically revealed in western blot analysis with the monoclonal antibodies specific to m2e .
the recombinant fusion protein carrying an n-terminal 6-histidine tag was purified to a purity of greater than 90 %  using standard affinity chromatography on ni-nta resin and used for animal experiments.
the recombinant flg protein was expressed in n. benthamiana using vector pa7248amv-flg and purified in a similar way.fig.
<dig> expression and purification of flg-4m protein.
coomassie brilliant blue stained gel  and western blot  of proteins isolated form n. benthamiana plants and separated by sds-page.
m, − molecular weight marker ;  <dig>  − total soluble proteins isolated from empty leaves;  <dig>  − total soluble proteins isolated at fourth day after agroinfiltration from leaves infiltrated with vector pa7248amv-flg-4m;  <dig>  − purified flg-4m protein.
antibodies against m2e were used in western blotting
antigenicity of plant-produced protein flg-4m
the display of the m2e peptide in an accessible manner when it is fused with flagellin in the flg-4m protein was examined by elisa.
to this end plates were coated with serial dilutions of flg-4m protein and empty flg, then probed with the m2e-specific and the 6-his tag-specific monoclonal antibodies .
while both flg-4m and flg reacted with the anti-his antibodies, only flg-4m reacted with the m2e-specific antibodies.
these results confirm the presence of m2e in the purified flg-4m protein and its accessibility for antibodies.fig.
<dig> antigenicity of flg-4m protein.
two-fold dilutions of recombinant flg-4m or flg proteins  were coated on elisa plates which were then probed with monoclonal antibodies specific for m2e  or 6-his tag present both in flg-4m and flg
the m2e peptides naturally exposed on the surface of infected cells as tetramers tends to aggregate due to the presence of two cysteine residues per m2e copy that lead to disulfide bond formation and intra- and interprotein cross-linking.
in order to control the lack of aggregation of flg-4m protein we also performed western-blot analysis of purified protein under reducing and non-reducing conditions.
the recombinant protein migrated at expected position and was revealed by the monoclonal antibodies specific to m2e, suggesting the lack of aggregation .
no high-molecular weight aggregates or particulate structures were detected by the electron microscopy of purified protein samples.
immunogenicity and protectivity of plant-produced protein flg-4m
to characterize the immunogenicity and protective action, we performed two sets of animal experiments.
in the first case mice were immunized with the plant-produced preparation of flg-4m protein, in the second experiment an additional group of mice was immunised with plant-produced flagellin lacking m2e.
mice were vaccinated intranasally three times at two-week intervals with 10 μg protein without additional adjuvants.
blood samples were taken 2 weeks after the second and/or third immunizations.
serum was analysed by elisa to identify igg antibodies directed against m2e, using plates coated with synthetic peptides g- <dig> and g- <dig>  whose sequences corresponded to the human “consensus” m2e sequence and the m2e of influenza strain a/chicken/kurgan/05/ <dig>  respectively.
a strong immune response was activated after the second immunisation and further increased after the third .
induced antibodies efficiently bind to both synthetic m2e peptides g- <dig> and g- <dig>  which are present in the candidate vaccine, although the immune response towards g <dig>  the human m2e sequence, was stronger.fig.
<dig> titers of igg antibodies to synthetic m2e peptides g- <dig> and g- <dig> in sera of immunized mice.
the data obtained in two independent experiments after the second and the third immunizations are shown.
the results are expressed as the mean titer ± standard deviation for each group of five mice.
titers lower than  <dig> were detected for mice immunized with pbs or flg
in the first experiment the immunised and control mice were challenged with 10ld <dig> of mouse-adapted human influenza strain a/pr/8/ <dig>  and with 5ld <dig> of avian influenza strain a/chicken/kurgan/05/ <dig> .
as shown in fig.
6a, 75 % mice immunized with flg-4m, survived the lethal challenge of a/chicken/kurgan/05/ <dig> , while the rate of survival among the control mice was 14 %.
the challenge of immunized mice with 10ld <dig> of a/pr/8/ <dig>  led to a 50 % survival, while all control mice died upon infection .fig.
<dig> protective efficiency of the plant-produced flg-4m protein.
experiment 1: mice were challenged with 5ld <dig> of a/chicken/kurgan/05/ <dig>  or with 10ld <dig> of a/pr/8/ <dig> .
experiment 2: mice were challenged with 5ld <dig> of influenza strain a/pr/8/ <dig> .
survival  and weight  of immunized and control mice were monitored for 14 days post-challenge.
control, mice immunized with pbs
in the second experiment 5ld <dig> of influenza strain a/pr/8/ <dig>  was used for challenge.
figure 6c shows that mice immunised with flg-4m were completely protected from lethal virus challenge, while mice immunised with flg or pbs were not protected.
the morbidity of the disease was monitored by measuring the weight of the animals.
the data presented in fig.
6d reveal that immunisation did not eliminate morbidity but reduced it relative to the control.
discussion
the purpose of the present work was to develop a plant-produced candidate influenza vaccine based on m2e peptide linked to bacterial flagellin acting as an adjuvant for mucosal immunization.
it was previously demonstrated  <cit>  that immunization of mice with e. coli-produced flagellin linked to four tandem copies of human “consensus” m2e, provides protection against lethal challenge with human influenza a strain.
although the m2e sequence is well conserved in almost all influenza strains isolated from humans, the sequences of m2e peptides of newly emerging highly pathogenic strains of animal origin, such as avian influenza, differ from the human consensus, suggesting that a specific m2e-based vaccine against these strains would be required  <cit> .
combination of several m2e peptides of different origin in one vaccine protein could allow development of candidate vaccine efficient against the corresponding strains  <cit> .
therefore in addition to human consensus m2e peptide we included m2e peptides of avian influenza virus into the hybrid protein based on bacterial flagellin.
although plants for a long time have been recognized as a promising host for expression of recombinant proteins, including vaccines, there are no universal approaches ensuring high level expression for any protein of interest.
here we used two plant viral-based expression system for production of flg-4m protein in n. benthamiana.
the first is replicating vector pa7248amv-gfp based on pvx genome, and the second vector peaq comprising fragments of cpmv genome is unable to replicate in plant cell.
in this instance the replicating pvx-based vector gave higher expression of the flg-4m protein than peaq.
the results obtained here are in contrast to those found when comparing the expression of the human papillomavirus 8 l <dig> protein using a replicating tmv-based vector and the peaq-ht system where the latter gave a 15-fold higher level  <cit> .
there are several possible explanations for this difference in expression levels between the two systems: it may reflect an intrinsic higher level of expression from the replicating versus the non-replicating system or it may relate to the strain of a. tumefaciens  used for infiltration.
the target protein was expressed in n. benthamiana cells at very high level up to 30 % of total soluble protein for pa7248amv-gfp .
it is much higher than 280 μg/g of fresh weight leaves reported for expression of the flagellin alone using non-replicating transient expression system  <cit> .
analysis of the plant-expressed flg-4m protein by transmission electron microscopy did not reveal the presence of aggregates.
this coupled with observation that the protein remained in the supernatant after ultracentrifugation suggests that the protein remains in an essentially unaggregated state.
intranasal immunization of mice with the plant-produced flg-4m protein induced an immune response against m2e and was responsible for development of protective immunity against the lethal influenza infection.
complete protection was observed in an experiment where immunized mice were challenged with 5ld <dig> of human influenza strain a/pr/8/ <dig>  notably the protection strongly depended on the presence of m2e in the hybrid protein, since immunisation with plant-produced empty flg confer no extra protection relative to the negative control.
in other experiments the protection was, however, not 100 % complete that could be related to low immunization dose  and, in case of experiment with 10ld <dig> of a/pr/8/ <dig> strain, by the use of too high infection dose, as evidenced by complete mortality in the control group.
the lack of complete protection of mice was observed by stepanova et al.
<cit>  in case of flg-4m protein produced in e. coli: 90 % survival upon challenge with 5ld <dig> of a/chicken/kurgan/05/ <dig> and 80 % survival upon challenge with 5ld <dig> of a/pr/8/ <dig> strain, although a higher dose of vaccine protein, 50 μg, was used for each of three immunisation.
three other examples of the plant-based expression of the m2e epitope have been reported.
ravin et al.
<cit> , using the pvx-based vector employed in this work, produced hbc virus-like particles carrying the m2e peptide with the yield about 1-2 % of total soluble protein; the plant-produced particles induced a protective immune response  against 1ld <dig> of homologous influenza a infection.
virus-like particles formed by l <dig> protein of human papillomavirus fused to m2e were produced in n. benthamiana with the peaq-ht expression system with the yield in the range from  <dig> to 120 μg/g of fresh weight  <cit> .
another approach is construction of recombinant tobacco mosaic virus carrying m2e epitope as a fusion with the coat protein; such viruses are capable of systemic infection of n. benthamiana plants and induced a protective immune response against challenge with 5ld <dig> of homologous influenza a virus  <cit> .
altogether these results indicated that production of m2e-based candidate influenza vaccines in plants is feasible.
the particular advantage of flagellin as a mucosal adjuvant suggests that expression of flagellin-m2e hybrid protein in plants by agroinfiltration with a recombinant viral vectors is a promising approach for production of recombinant vaccines against influenza.
CONCLUSIONS
in this study, we expressed the chimeric protein comprising flagellin of salmonella typhimurium fused to four tandem copies of the influenza m2e peptide in n. benthamiana plants.
the use of self-replicating viral vector based on pvx genome allowed to achieve very high level of expression up to 1 mg/g of fresh leaf tissue.
intranasal immunization of mice with purified vaccine protein induced high levels of m2e-specific serum antibodies and provided protection against lethal challenge with influenza virus.
this work shows that expression of fusion proteins based on flagellin and m2e in plants is feasible and could became a promising approach for manufacturing of “universal” recombinant vaccines against influenza.

 influenza is a widely distributed viral infection of humans and animals. the high variability of the surface viral proteins, hemagglutinin and neuraminidase, results in the appearance of a new epidemic strain every one to two years; this requires the production of new vaccines at the same frequency. a promising solution to this problem is the development of recombinant vaccines that can be rapidly produced in standard expression systems. one of the most promising conserved antigens of influenza virus is the extracellular domain of transmembrane protein m m2e. the length of m2e is only amino acid residues. its sequence is virtually unchanged in all human isolates since, and in strains of avian influenza a it differs in only a few amino acids. by itself m2e possesses low immunogenicity but this can be increased by delivery in a multimeric form or in combination with complex adjuvants or delivery systems. toll-like receptors have been shown to play a critical role in controlling the adaptive immune response, by properly arming dendritic and other antigen-presenting cells, triggering important costimulatory and regulatory mechanisms, and promoting antigen presentation. it has been previously shown that genetically fusing an antigen of interest to bacterial flagellin, the ligand for tlr significantly increases the immunogenicity and protective capacity of the antigen. notably flagellin is particularly active as mucosal adjuvant, opening the possibility of non-invasive intranasal administration of vaccines. the ability of flagellin to be both the platform and the adjuvant for different vaccines was demonstrated in various models of infection including influenza. recombinant proteins, including those for medical purposes, can be produced in a variety of expression systems, including bacteria, yeast, plants, and mammalian cells. the advantages of using plants for protein production include the low final cost and inherent safety of products due to the absence of pathogens common for plants and animals. recombinant proteins in plants may be obtained by stable genetic transformation or through transient expression. creation of lines of stably transformed plants and, subsequently, production of a protein from such plants requires considerable time and cost. the level of expression of target proteins by transgenic plants is usually relatively low, resulting in a high cost of products due to the difficulty of their purification. the use of self-replicating viral vectors or efficient non-replicating transient expression vectors is an alternative approach providing sufficient amounts of the desired protein to conduct immunological studies. to create such vectors, sequences from tobacco mosaic virus, cowpea mosaic virus or potato virus x have frequently been used. the genomes of these viruses are small and consist of positive-strand rna. the vectors are introduced into plant cells by infiltration of the plant leaf tissue with agrobacterium suspensions carrying the vector within the t-dna portion of a binary plasmid resulting in transfer of vector dna into the recipient plant cells during the agroinfection. such expression systems have been used for production of a variety of pharmaceutical proteins, including antibodies and candidate vaccines. the latter have included influenza virus hemagglutinin and hbc virus-like particles carrying m2e peptide. in this work four tandem copies of m2e sequences based on human and avian influenza virus isolates were genetically fused to salmonella typhimurium flagellin and produced in nicotiana benthamiana plants using transient expression using either a replicating pvx vector or the non-replicating cpmv-ht system. intranasal immunization of mice with the plant-produced hybrid protein induced the desired immune response and conferred protective immunity against a lethal influenza virus challenge.  viral vectors for expression of the hybrid protein consisting of flagellin linked to four copies of influenza m2e peptide in n. benthamiana plants the highly conserved extracellular domain of the influenza virus m protein, m2e, is a promising target for the development of recombinant “universal” vaccines against influenza. however, m2e is poorly immunogenic and needs to be presented in a multimeric form or linked to a carrier molecule such as virus-like particles. here we use flagellin of salmonella typhimurium as such carrier and simultaneously as an adjuvant. in order to make a broader range candidate vaccine, a hybrid protein flg-4m containing two copies of human consensus m2e sequence and two copies of the m2e peptide of avian influenza virus strain a/chicken/kurgan/05/ fused to the c-terminus of s. typhimurium fljb was designed. in order to facilitate folding of hybrid protein, copies of m2e peptides were separated from each other by flexible glycine-rich linker. the empty flagellin was used as a control. both proteins, flg-4m and flg, were designed with n-terminal 6-histidine tag to facilitate their purification.fig. structure of the expression constructs used in this study. a amino acid sequence of flg-4m protein. the sequences of human “consensus” m2e peptides, m2eh, are italicized; the sequences of m2e peptide of avian influenza virus strain a/chicken/kurgan/05/ m2ek, are shown in bold; the linker sequences are underlined. flagellin sequence is shaded in grey. b structures of viral vectors pa7248amv-flg-4m and peaq-flg-4m. the tdna regions of the binary vectors are shown. rdrp, rna dependent rna polymerase of pvx; sgp subgenomic promoter of the pvx protein 25 k; amv, the leader sequence of rna of the alfalfa mosaic virus; 6his, histidine tag; flg, flagellin of s. typhimurium; 4m2e, four copies of m2e peptide; 35s, promoter; 35s-t, terminator of the cauliflower mosaic virus rna; nost, terminator of nopaline synthase gene; lb and rb are the left and right borders of tdna; p gene of silencing suppressor p from the tomato bushy stunt virus; 5’ and 3’, the 5’-utr and the 3’-utr from pvx and cpmv two plant virus-based transient expression systems were used for production of recombinant flg-4m protein in n. benthamiana. the first vector, pa7248amv-gfp, is a replicating system based on the pvx genome. this vector includes the 5’-utr of the pvx genome, the polymerase gene, the first promoter of subgenomic rna, the translation enhancer represented by the sequence of the 5’-untranslated region of rna of the alfalfa mosaic virus, the gfp gene flanked by unique restriction sites asci and smai, the last nucleotides of the coat protein gene, and the 3’-untranslated region of the pvx genome. this construction is nserted between the 35s promoter and 35s terminator and cloned in the binary vector pbin. to create the viral vector allowing production of the flg-4m protein, a sequence of the hybrid gene was cloned in pa7248amv-gfp at the asci and smai sites to replace gfp, which resulted in construction of a recombinant vector pa7248amv-flg-4m. recombinant vector pa7248amv-flg for expression of flg in plants was constructed in a similar way. the second expression system, peaq, is based on cpmv. this is a non-replicating system which uses production of a highly translatable mrna to achieve high level expression. the recombinant vector peaq-flg-4m contains 5’-utr and 3’-utr from cpmv rna- upstream and downstream, respectively, of the hybrid gene flg-4m. expression of the hybrid protein flg-4m in n. benthamiana plants to express the protein flg-4m, vectors were inserted into a. tumefaciens strain gv which was used for agroinfiltration of leaves of n. benthamiana plants. agroinfiltration zones for two vectors were located within one leaf. protein samples were isolated in and 7 days after infection. for every day three leaves were taken for analysis. protein samples were analyzed using sds-page and western-blotting. maximal expression was observed at fourth day. summarizing the expression results for leaves we found out that peaq-flg-4m construct provides expression level of about 30 % in comparison to pa7248amv-flg-4m.fig. efficiency of pvx- and cpmv-based vectors. coomassie brilliant blue stained gel and western blot of proteins isolated form n. benthamiana plants and separated by sds-page. m, − molecular weight marker; and − total soluble proteins isolated at fourth day after agroinfiltration from leaves infiltrated with vector pa7248amv-flg-4m and peaq-flg-4m, respectively; − total soluble proteins isolated from empty leaves. antibodies against m2e were used in western blotting given the higher levels of expression obtained, we used vector pa7248amv-flg-4m for the scaled-up production of protein flg-4m in n. benthamiana plants. the total soluble protein was isolated from leaves at 4th day after agroinfiltration and analyzed by sds-page. the data presented in fig. 3a show that flg-4m protein is highly expressed and accounted for about 30 % of the soluble protein fraction. the recombinant protein was specifically revealed in western blot analysis with the monoclonal antibodies specific to m2e. the recombinant fusion protein carrying an n-terminal 6-histidine tag was purified to a purity of greater than 90 % using standard affinity chromatography on ni-nta resin and used for animal experiments. the recombinant flg protein was expressed in n. benthamiana using vector pa7248amv-flg and purified in a similar way.fig. expression and purification of flg-4m protein. coomassie brilliant blue stained gel and western blot of proteins isolated form n. benthamiana plants and separated by sds-page. m, − molecular weight marker; − total soluble proteins isolated from empty leaves; − total soluble proteins isolated at fourth day after agroinfiltration from leaves infiltrated with vector pa7248amv-flg-4m; − purified flg-4m protein. antibodies against m2e were used in western blotting antigenicity of plant-produced protein flg-4m the display of the m2e peptide in an accessible manner when it is fused with flagellin in the flg-4m protein was examined by elisa. to this end plates were coated with serial dilutions of flg-4m protein and empty flg, then probed with the m2e-specific and the 6-his tag-specific monoclonal antibodies. while both flg-4m and flg reacted with the anti-his antibodies, only flg-4m reacted with the m2e-specific antibodies. these results confirm the presence of m2e in the purified flg-4m protein and its accessibility for antibodies.fig. antigenicity of flg-4m protein. two-fold dilutions of recombinant flg-4m or flg proteins were coated on elisa plates which were then probed with monoclonal antibodies specific for m2e or 6-his tag present both in flg-4m and flg the m2e peptides naturally exposed on the surface of infected cells as tetramers tends to aggregate due to the presence of two cysteine residues per m2e copy that lead to disulfide bond formation and intra- and interprotein cross-linking. in order to control the lack of aggregation of flg-4m protein we also performed western-blot analysis of purified protein under reducing and non-reducing conditions. the recombinant protein migrated at expected position and was revealed by the monoclonal antibodies specific to m2e, suggesting the lack of aggregation. no high-molecular weight aggregates or particulate structures were detected by the electron microscopy of purified protein samples. immunogenicity and protectivity of plant-produced protein flg-4m to characterize the immunogenicity and protective action, we performed two sets of animal experiments. in the first case mice were immunized with the plant-produced preparation of flg-4m protein, in the second experiment an additional group of mice was immunised with plant-produced flagellin lacking m2e. mice were vaccinated intranasally three times at two-week intervals with 10 μg protein without additional adjuvants. blood samples were taken 2 weeks after the second and/or third immunizations. serum was analysed by elisa to identify igg antibodies directed against m2e, using plates coated with synthetic peptides g- and g- whose sequences corresponded to the human “consensus” m2e sequence and the m2e of influenza strain a/chicken/kurgan/05/ respectively. a strong immune response was activated after the second immunisation and further increased after the third. induced antibodies efficiently bind to both synthetic m2e peptides g- and g- which are present in the candidate vaccine, although the immune response towards g the human m2e sequence, was stronger.fig. titers of igg antibodies to synthetic m2e peptides g- and g- in sera of immunized mice. the data obtained in two independent experiments after the second and the third immunizations are shown. the results are expressed as the mean titer ± standard deviation for each group of five mice. titers lower than were detected for mice immunized with pbs or flg in the first experiment the immunised and control mice were challenged with 10ld of mouse-adapted human influenza strain a/pr/8/ and with 5ld of avian influenza strain a/chicken/kurgan/05/. as shown in fig. 6a, 75 % mice immunized with flg-4m, survived the lethal challenge of a/chicken/kurgan/05/, while the rate of survival among the control mice was 14 %. the challenge of immunized mice with 10ld of a/pr/8/ led to a 50 % survival, while all control mice died upon infection.fig. protective efficiency of the plant-produced flg-4m protein. experiment 1: mice were challenged with 5ld of a/chicken/kurgan/05/ or with 10ld of a/pr/8/. experiment 2: mice were challenged with 5ld of influenza strain a/pr/8/. survival and weight of immunized and control mice were monitored for 14 days post-challenge. control, mice immunized with pbs in the second experiment 5ld of influenza strain a/pr/8/ was used for challenge. figure 6c shows that mice immunised with flg-4m were completely protected from lethal virus challenge, while mice immunised with flg or pbs were not protected. the morbidity of the disease was monitored by measuring the weight of the animals. the data presented in fig. 6d reveal that immunisation did not eliminate morbidity but reduced it relative to the control. discussion the purpose of the present work was to develop a plant-produced candidate influenza vaccine based on m2e peptide linked to bacterial flagellin acting as an adjuvant for mucosal immunization. it was previously demonstrated that immunization of mice with e. coli-produced flagellin linked to four tandem copies of human “consensus” m2e, provides protection against lethal challenge with human influenza a strain. although the m2e sequence is well conserved in almost all influenza strains isolated from humans, the sequences of m2e peptides of newly emerging highly pathogenic strains of animal origin, such as avian influenza, differ from the human consensus, suggesting that a specific m2e-based vaccine against these strains would be required. combination of several m2e peptides of different origin in one vaccine protein could allow development of candidate vaccine efficient against the corresponding strains. therefore in addition to human consensus m2e peptide we included m2e peptides of avian influenza virus into the hybrid protein based on bacterial flagellin. although plants for a long time have been recognized as a promising host for expression of recombinant proteins, including vaccines, there are no universal approaches ensuring high level expression for any protein of interest. here we used two plant viral-based expression system for production of flg-4m protein in n. benthamiana. the first is replicating vector pa7248amv-gfp based on pvx genome, and the second vector peaq comprising fragments of cpmv genome is unable to replicate in plant cell. in this instance the replicating pvx-based vector gave higher expression of the flg-4m protein than peaq. the results obtained here are in contrast to those found when comparing the expression of the human papillomavirus 8 l protein using a replicating tmv-based vector and the peaq-ht system where the latter gave a 15-fold higher level. there are several possible explanations for this difference in expression levels between the two systems: it may reflect an intrinsic higher level of expression from the replicating versus the non-replicating system or it may relate to the strain of a. tumefaciens used for infiltration. the target protein was expressed in n. benthamiana cells at very high level up to 30 % of total soluble protein for pa7248amv-gfp. it is much higher than 280 μg/g of fresh weight leaves reported for expression of the flagellin alone using non-replicating transient expression system. analysis of the plant-expressed flg-4m protein by transmission electron microscopy did not reveal the presence of aggregates. this coupled with observation that the protein remained in the supernatant after ultracentrifugation suggests that the protein remains in an essentially unaggregated state. intranasal immunization of mice with the plant-produced flg-4m protein induced an immune response against m2e and was responsible for development of protective immunity against the lethal influenza infection. complete protection was observed in an experiment where immunized mice were challenged with 5ld of human influenza strain a/pr/8/ notably the protection strongly depended on the presence of m2e in the hybrid protein, since immunisation with plant-produced empty flg confer no extra protection relative to the negative control. in other experiments the protection was, however, not 100 % complete that could be related to low immunization dose and, in case of experiment with 10ld of a/pr/8/ strain, by the use of too high infection dose, as evidenced by complete mortality in the control group. the lack of complete protection of mice was observed by stepanova et al. in case of flg-4m protein produced in e. coli: 90 % survival upon challenge with 5ld of a/chicken/kurgan/05/ and 80 % survival upon challenge with 5ld of a/pr/8/ strain, although a higher dose of vaccine protein, 50 μg, was used for each of three immunisation. three other examples of the plant-based expression of the m2e epitope have been reported. ravin et al., using the pvx-based vector employed in this work, produced hbc virus-like particles carrying the m2e peptide with the yield about 1-2 % of total soluble protein; the plant-produced particles induced a protective immune response against 1ld of homologous influenza a infection. virus-like particles formed by l protein of human papillomavirus fused to m2e were produced in n. benthamiana with the peaq-ht expression system with the yield in the range from to 120 μg/g of fresh weight. another approach is construction of recombinant tobacco mosaic virus carrying m2e epitope as a fusion with the coat protein; such viruses are capable of systemic infection of n. benthamiana plants and induced a protective immune response against challenge with 5ld of homologous influenza a virus. altogether these results indicated that production of m2e-based candidate influenza vaccines in plants is feasible. the particular advantage of flagellin as a mucosal adjuvant suggests that expression of flagellin-m2e hybrid protein in plants by agroinfiltration with a recombinant viral vectors is a promising approach for production of recombinant vaccines against influenza.  in this study, we expressed the chimeric protein comprising flagellin of salmonella typhimurium fused to four tandem copies of the influenza m2e peptide in n. benthamiana plants. the use of self-replicating viral vector based on pvx genome allowed to achieve very high level of expression up to 1 mg/g of fresh leaf tissue. intranasal immunization of mice with purified vaccine protein induced high levels of m2e-specific serum antibodies and provided protection against lethal challenge with influenza virus. this work shows that expression of fusion proteins based on flagellin and m2e in plants is feasible and could became a promising approach for manufacturing of “universal” recombinant vaccines against influenza. 
intranasal immunization of mice with purified flg-4m protein induced high levels of m2e-specific serum antibodies and provided protection against lethal challenge with influenza virus. the extracellular domain of matrix protein of influenza a virus is a promising target for the development of a universal vaccine against influenza because m2e sequences are highly conserved among human influenza a strains. the chimeric construct was expressed in nicotiana benthamiana plants using either the self-replicating potato virus x based vector, pa7248amv-gfp, or the cowpea mosaic virus -derived expression vector, peaq-ht. viral vectorpotato virus xm proteinflagellininfluenzavaccineissue-copyright-statement© the author 2015 in this study, we report for the first time the transient expression in plants of a recombinant protein flg-4m comprising flagellin of salmonella typhimurium fused to four tandem copies of the m2e peptide. this study confirms the usefulness of flagellin as a carrier of m2e and its relevance for the production of m2e-based candidate influenza vaccines in plants. the highest expression level up to 30 % of total soluble protein was achieved with the pvx-based expression system. it was previously shown that fusion of m2e to bacterial flagellin, the ligand for toll-like receptor and powerful mucosal adjuvant, significantly increases the immunogenicity and protective capacity of m2e.
 the extracellular domain of matrix protein of influenza a virus is a promising target for the development of a universal vaccine against influenza because m2e sequences are highly conserved among human influenza a strains. however, native m2e is poorly immunogenic, but its immunogenicity can be increased by delivery in combination with adjuvants or carrier particles. it was previously shown that fusion of m2e to bacterial flagellin, the ligand for toll-like receptor and powerful mucosal adjuvant, significantly increases the immunogenicity and protective capacity of m2e.  in this study, we report for the first time the transient expression in plants of a recombinant protein flg-4m comprising flagellin of salmonella typhimurium fused to four tandem copies of the m2e peptide. the chimeric construct was expressed in nicotiana benthamiana plants using either the self-replicating potato virus x based vector, pa7248amv-gfp, or the cowpea mosaic virus -derived expression vector, peaq-ht. the highest expression level up to 30 % of total soluble protein was achieved with the pvx-based expression system. intranasal immunization of mice with purified flg-4m protein induced high levels of m2e-specific serum antibodies and provided protection against lethal challenge with influenza virus.  this study confirms the usefulness of flagellin as a carrier of m2e and its relevance for the production of m2e-based candidate influenza vaccines in plants. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords viral vectorpotato virus xm proteinflagellininfluenzavaccineissue-copyright-statement© the author 2015 