BACKGROUND
human papillomaviruses  are non-enveloped tumour dna viruses about  <dig> nm in diameter with a double-stranded circular genome of approximately  <dig> kb  <cit> .
hpvs are the cause of approximately 5% of all cancers  <cit> .
high risk hpvs, of which hpv- <dig> is the most prevalent, are responsible not only for causing cervical cancer in women  <cit>  but also anogenital, head and neck tumours in men and women  <cit> .
the major structural protein of hpv is the l <dig> capsid protein that can spontaneously self-assemble in vivo and in vitro to form capsomers and virus-like particles  that are highly immunogenic  <cit> .
l <dig> can form two types of vlps, an icosahedral lattice with a t =  <dig> symmetry, composed of  <dig> pentamers which is morphologically identical to the native virions and a smaller t =  <dig> particle composed of  <dig> l <dig> pentamers  <cit> .
the structure of the hpv <dig> t =  <dig> particle has been solved by crystallography.
the l <dig> protein has a jellyroll β sandwich structure formed by conserved sequences with additional hypervariable loops protruding towards the outer surface of the capsid  <cit> .
the three c-terminal helices  <dig>   <dig>  and  <dig>  of l <dig> are surface exposed and their inter-capsomeric reactions influence vlp formation.
the deletion of the full h <dig> has no impact on capsomer assembly, but prevents the formation of either t =  <dig> or t =  <dig> vlps  <cit> .
recently prophylactic vaccines against hpv- <dig> have appeared on the market.
these are composed of l1-based vlps made in insect or yeast cells.
spontaneous assembly of hpv- <dig> l <dig> protein into vlps has also been achieved in plants  <cit> .
plant-produced vlps were immunogenic when administered to animals  <cit> , further justifying continuing research into the use of plants for producing heterologous proteins for vaccine use.
the hpv- <dig> l <dig> protein can assemble into vlps in non-plant expression systems not only in its native form, but also in chimeric forms  <cit> ; expressing the p1a peptide derived from the p <dig> tumour-associated antigen  <cit> , an epitope of human immunodeficiency virus type  <dig>  <cit> , a short epitope of the hepatitis b core antigen  <cit> , a neutralization epitope of the l <dig> minor capsid protein of hpv- <dig> and - <dig>  <cit> , and a neutralizing epitope of human respiratory syncytial virus   <cit> .
chimeric vlps have also been produced in transgenic and transplastomic plants  <cit> .
serological characterization of hpv- <dig> l <dig> particles has been carried out using monoclonal antibodies  that recognize either linear or conformational surface-exposed epitopes.
amino acid  residues  <dig>   <dig> and  <dig> of l <dig> are crucial for recognition by the conformation-specific and neutralizing mabs h16:v <dig> and h16:e <dig>  <cit> .
mabs h16:7e, h16:9a, and h16:u <dig> recognize conformational epitopes which are not yet fully mapped   <cit> .
linear epitopes are recognized by mabs h16:i <dig> , h16:j <dig> , and h16:d <dig>   <cit> .
these mabs are fundamental to investigate the use of hpv- <dig> l <dig> as carrier, since they provide information of the intactness and the stability of l1-based structures.
for the purpose of expressing an hpv- <dig> l1-based scaffold in plants that can be easily engineered to carry foreign epitopes, we constructed a synthetic l1Δc <dig> gene.
to investigate the impact of the presence of a foreign epitope on vlp assembly and conformation, we designed four different synthetic genes encoding the l <dig> protein where two different l <dig> regions were substituted by either a 23-aa or a 8-aa epitope from the ectodomain of the m <dig> protein  of influenza a virus.
the chimeric l <dig> proteins generated were tested for their ability to form t =  <dig> or t =  <dig> particles, and characterized by their reactivity towards a panel of linear and conformation-specific anti hpv- <dig> l <dig> mabs.
RESULTS
gene synthesis, cloning and protein structure modelling
a synthetic l1Δc <dig> gene lacking the 3'-terminal domain reported to code for a nuclear localization signal  <cit>  was designed, that contained unique restriction sites located within nucleotide regions encoding the structural helices and β-sheets .
restriction sites at the 5'- and 3'-terminus were introduced for appropriate cloning into the non-replicating plant expression vector peaq-ht   <cit> .
additionally, this gene sequence was optimized with human codon usage, as it was previously shown that this increased hpv- <dig> l <dig> yield in plants  <cit> .
the l1Δc <dig> gene sequence was used as a backbone to generate four chimeric variants  containing two different m2e influenza epitopes: m2e2- <dig> or m2e2- <dig>  that replaced l <dig> sequences predicted to form either h <dig> , or the coil region connecting h <dig> and β sheet j  .
l1Δc <dig> and chimeric genes were cloned in peaq-ht, and after sequencing control, all constructs were transformed into agrobacterium tumefaciens.
protein structure predictions revealed that all chimeras folded in a way that resembled the crystallographic structure of the native l <dig> protein  <cit> , but we observed that chimeras with the shorter version of the m2e epitope replacing either h <dig> or the coil region were more similar than those carrying the longer m2e epitope version .
expression of l1Δc <dig> and chimeric constructs
to determine whether l1Δc <dig> and its chimeric variants could be transiently expressed in nicotiana benthamiana, we analysed tissue extracts from agroinfiltrated leaves by western blot using the md2h <dig> mab  which is specific for the hpv16-l <dig> protein.
l1Δc <dig> and chimeric gene variants expressed an hpv- <dig> l <dig> monomer of the expected size of  <dig> kda, with the exception of chim2e2-24_h <dig> that migrated slightly slower and showed the strongest signal .
no evident proteolysis was observed with l1Δc <dig> or with chimeric l <dig> protein extracts.
expression of l1Δc <dig> and chimeras was confirmed with camvir- <dig>  another anti-hpv <dig> l <dig> mab .
testing for the presence of influenza epitopes using the anti-m <dig> 14c <dig> mab  resulted in the recognition of the longer epitope  in either h <dig> and the coil position, while this antibody was unable to detect constructs with the shorter epitope  .
thus we concluded that all synthetic chimeric constructs were successfully expressed and that two of them expressed also influenza epitopes.
further step was to determine if chimeras were able to preserve an appropriate conformation necessary to form assembled protein structures.
characterization of l1Δc <dig> and chimeric constructs by capture-elisa
all the linear and conformation-dependent mabs reacted with l1Δc <dig> and chimeric constructs from agroinfiltrated leaf extracts when analysed by capture elisa .
the highest l <dig> value was obtained six days post-agroinfiltration in a time course analysis of ten days .
binding of conformation-specific mabs with all l1-based chimeric protein constructs indicated that they aggregated in higher order structures, such as capsomers, or vlps in either t =  <dig> or t =  <dig> form.
recognition of chimeras by these conformation-specific mabs confirmed not only their expression in plants, but also proved that their self-assembly ability  was not abolished by the substitution of h <dig> or coil surface-exposed l <dig> regions with the foreign influenza epitopes.
among all chimeras, chim2e2-24_h <dig> showed the highest binding affinity with the conformation-dependent neutralizing mabs .
the estimated yield of l1Δc <dig> and chimeric proteins accumulated in agroinfiltrated leaves was measured six days post-agroinfiltration .
l <dig> quantification was calculated by capture-elisa using the h16:j <dig> mab which recognizes a linear epitope in both monomeric and assembled structures and the h16:v <dig> conformation-specific mab which binds only to assembled structures.
the highest l <dig> yield was obtained for chim2e2-24_h <dig> with both mabs , followed by chim2e2-24_c .
chim2e2-24_h <dig> accumulated also higher yield then the l1Δc <dig> backbone protein, suggesting that the substituted amino-acid sequence had a positive impact on protein stability.
on the other hand, the two chimeras bearing a shorter version of the m2e epitope accumulated at lower amount compared to both l1Δc <dig> and chimeras having longer substitution.
the greater difference observed between h16:j <dig> and h16:v <dig> in the case of l1Δc <dig> and chim2e2-24_h <dig> suggested that monomeric l <dig> protein was also accumulating, beside the assembled species.
electron microscopy of purified l1Δc <dig> and chimeric constructs
to investigate the structure of vlps and/or capsomers formed by l1Δc <dig> and chimeras in plants, we carried out cs2so <dig> density gradient and further analysed the fractions obtained by electron microscopy.
a begomovirus purification protocol  <cit>  adopted for purifying vlps of other papillomaviruses  was successfully employed to purify hpv- <dig> l <dig> particles.
in l1Δc <dig> preparations, spherical t =  <dig> vlps of expected size of  <dig> nm were formed; in addition small vlps with probable t =  <dig> symmetry of about  <dig> nm and capsomers ranging from 8- <dig> nm in diameter were also detected.
purified preparations of chimeric proteins showed that chim2e2-24_c, chim2e2-9_c, as well as chim2e2-24_h <dig> successfully formed capsomers and vlps of similar structure.
on the contrary, the chimeric construct with the m2e2- <dig> substitution in h <dig> position  formed only capsomers .
electron microscopy analysis confirmed the results of the capture-elisa obtained with conformation-specific mabs indicating the self-assembly of chimeric l <dig> protein in higher order protein structures .
discussion
the current study supports the possibility of using plants as a method for the expression and folding of proteins of human viruses and raises the prospect of plants competing with other systems for the production of low-cost and effective vaccines.
in this work, we demonstrated that: 1) the synthetic hpv- <dig> l <dig> gene can be successfully expressed in a transient manner in n. benthamiana plants using a non replicating peaq-ht expression vector; 2) the hpv- <dig> l <dig> carrier bearing the m2e influenza epitope can be efficiently produced in plants; 3) m2e influenza epitope presentation in either h <dig> or h4-β-j coil positions is not detrimental for the formation of capsomers and does not disrupt the recognition by conformation-dependent mabs; 4) m2e epitopes in coil position allow vlp formation; 5) m2e epitopes in h <dig> position enable vlp assembly only when a longer sequence of amino acids is used.
the hpv- <dig> l <dig> gene and chimeras were expressed in plants using the small binary vector peaq-ht containing modified 5'- and 3'-untranslated regions from cowpea mosaic virus  rna- <dig>  avoiding the double cloning procedures necessary with standard binary vectors with a concomitant saving in time.
the successful use of replicating viral vectors for the expression of heterologous proteins in plants has been reported on several occasions  <cit> .
however, the size and complexity of viral vector systems, as well as the genetic stability of the constructs during viral genome replication and possible biocontainment issues can negatively affect their widespread use, justifying the selection of non-replicating peaq-ht plasmid as a vector of choice for the current studies.
the hpv- <dig> l <dig> protein carrier containing influenza epitopes was expressed in plants, but the recognition of m2e influenza sequences was achieved only in chimeras with the longer version of the epitope.
it is yet to be investigated if the lack of reaction with constructs carrying the shorter version of the m2e influenza epitope is due to the use of an inappropriate mab or to epitope inaccessibility on the carrier surface.
so far, there is only one report of the expression of a chimeric hpv16-l <dig> protein in transgenic plants, where the chimera was made of l <dig> fused to e6/e <dig> epitopes of the same virus  <cit> .
here we achieved for the first time the transient expression in plants of chimeric hpv16-l <dig> bearing an epitope of a heterologous virus, with a substitution rather than a fusion strategy.
several reports have demonstrated the usefulness of the m2e as a means of inducing neutralizing antibodies against influenza  <cit> .
the n-terminus of m2e, containing the highly conserved slltevet epitope, common to both m <dig> and m <dig> proteins, was sufficient to induce antibody production with an inhibitory effect against this pathogen  <cit> .
two different versions of this epitope were selected for the construction of hpv-chimeras.
the major concern in introducing heterologous epitopes into the l <dig> platform is to ensure their surface-exposure while preserving the conformation of the l <dig> oligomer.
hpv- <dig> pseudovirion infection is abolished by the neutralizing mabs v <dig> and e <dig> which recognize surface exposed conformational epitopes of l <dig>  <cit> ; these epitopes are considered of crucial importance for immunogenicity.
all chimeric proteins tested in this work reacted positively with a panel of linear and conformation-specific mabs.
furthermore, the positive reaction obtained with h16:v <dig> and h16:e <dig> indicated that both epitopes inserted in either l <dig> positions preserved these l <dig> immunogenic epitopes.
mab h16:d <dig> has affinity for the denatured vlp antigen  <cit> , and the weak binding of l1Δc <dig> and the chimeric proteins to this mab demonstrates that the assembled products are stable.
electron microscopy results showed that the l <dig> protein purified by cs2so <dig> density gradient centrifugation assembled into recognizable vlps.
maclean et al.
<cit>  transiently expressed the l <dig> protein in the cytoplasm and observed a size-range of the vlps from 40- <dig> nm.
our data are consistent with this work, and we found vlps ranging from 30- <dig> nm in the same plant host.
chimeric particles with m2e substitutions in the coil region predominantly assembled into t <dig> = vlps.
the substitution of  <dig> aa of the l <dig> coil region with either  <dig> or  <dig> aa of the influenza m2e region was not detrimental for vlp formation, thus confirming that this region is not involved in the inter-pentameric reactions necessary to create a vlp.
the chimeric polypeptides having shorter substitution in h <dig>  could still assemble into capsomers, but the formation of t =  <dig> or t =  <dig> vlps was inhibited, as expected on the basis of the published crystal structure of hpv- <dig> l <dig>  <cit> .
interestingly, the substitution of the same h <dig> with a longer m2e sequence  allowed the assembly into t =  <dig> and t =  <dig> vlps, along with capsomer formation, indicating that the incorporation of a longer version of the m2e epitope partially restored the function of the deleted h <dig> region.
this suggests that the length of an epitope, as well as its amino acid sequence is important for efficient vlp formation.
furthermore, when h <dig> was substituted by a rsv epitope  <cit>  or by a l <dig> epitope of hpv- <dig> and - <dig>  <cit> , only capsomers were found assembled in preparations from insect cells systems.
the detection of l <dig> superstructures for chim2e2-24_h <dig> in plants suggests that the plant cell or the purification strategy could additionally play important role in hpv vlp formation/stability.
the yields reported in this work are in the range from  <dig> to  <dig> mg of total l <dig> protein per kg of fresh weight for chim2e2-24_c and chim2e2-24_h <dig> , thus fulfilling the often-requested quantity for commercially viable production of antibodies  <cit> .
previous works reported yields of l <dig> protein in n. benthamiana plants ranging from  <dig> μg per kg of plant material with a transient expression system of the native gene  <cit> , to  <dig> mg per kg of plant material in the case of a chloroplast-targeted l <dig> protein transiently expressed from a human codon-optimized gene, with a 5-fold decrease when the nls was removed   <cit> .
our yields of  <dig> mg for l1Δc <dig> and  <dig> mg for chim2e2-24_h <dig> are in accordance with the highest yields reported so far for plant-transiently expressed hpv- <dig> l1Δc <dig>  this approves the use of l <dig> gene with the human codon preferences and demonstrates the suitability of the peaq-ht vector for hpv- <dig> l <dig> transient expression in plants.
further enhancement of l <dig> yield could be obtained by appropriately decreasing the gc content, and by deleting regions prone to gene silencing, to reduce mrna secondary structure.
three of the four chimeric proteins tested assembled into vlps and capsomers in plants, and one of them produced only capsomers.
vlp assembly in the three chimeras emphasized their potential usefulness in future immunogenicity studies; in fact, to achieve comparable antibody responses at least  <dig> to  <dig> times more l <dig> are required in the form of capsomeres than in the form of vlps  <cit> .
out of three vlp-forming chimeras, chim2e2-24_h <dig> and chim2e2-24_c reacted also with anti-influenza mab, suggesting the availability of this epitope on vlp surface.
considering that these two chimeras had the highest l <dig> yield as well, we propose them as candidate multivalent vaccines for further immunological studies.
our work demonstrated that chimeric hpv- <dig> l <dig> proteins can be transiently expressed in plants in only one week, with a significant-time reduction compared to other plant-expression systems , thus offering new possibilities for rapid testing of new chimeras made in plants.
the use of a synthetic l <dig> gene designed to include unique restriction sites opens further perspectives for inserting other heterologous epitopes in different structural positions.
other interesting surface-exposed regions are the d-e and f-g <dig> loops for which the designed synthetic gene can be easily manipulated to present single or multiple foreign epitopes.
this synthetic gene could also be used as a backbone for exchanging longer portions of other hpv- <dig> or related papillomavirus genes in order to synthesize novel vaccines with therapeutic and/or preventive use, or vaccines with multiple features effective against more than one papillomavirus.
CONCLUSIONS
the hpv- <dig> l <dig> protein was successfully expressed by the cpmv-derived plant transient expression vector peaq-ht.
chimeric constructs bearing a heterologous influenza epitope were also successfully transiently expressed in plants as judged by their reaction with anti-hpv <dig> l <dig> specific mabs.
the characterization of chimeras by electron microscopy showed appropriate assembly of vlps, with the exception of one chimera, which formed only capsomers.
chimeras were recognized by assembly-dependent conformation-specific mabs, confirming particle stability.
the substitution of the l <dig> h <dig> region with foreign epitopes of suitable size had a positive impact on l <dig> self-assembly into vlps, compared to previous results showing that the deletion of the same h <dig> region abolished vlp assembly.
interestingly, this substitution had a positive effect on l <dig> yield, too.
moreover, we designed a synthetic gene allowing an easy manipulation of l <dig> structural positions suitable for inserting exogenous sequences, thus opening new perspectives for a rapid and easy construction of different-virus chimeras.

 human papillomaviruses are non-enveloped tumour dna viruses about nm in diameter with a double-stranded circular genome of approximately kb. hpvs are the cause of approximately 5% of all cancers. high risk hpvs, of which hpv- is the most prevalent, are responsible not only for causing cervical cancer in women but also anogenital, head and neck tumours in men and women. the major structural protein of hpv is the l capsid protein that can spontaneously self-assemble in vivo and in vitro to form capsomers and virus-like particles that are highly immunogenic. l can form two types of vlps, an icosahedral lattice with a t = symmetry, composed of pentamers which is morphologically identical to the native virions and a smaller t = particle composed of l pentamers. the structure of the hpv t = particle has been solved by crystallography. the l protein has a jellyroll β sandwich structure formed by conserved sequences with additional hypervariable loops protruding towards the outer surface of the capsid. the three c-terminal helices and of l are surface exposed and their inter-capsomeric reactions influence vlp formation. the deletion of the full h has no impact on capsomer assembly, but prevents the formation of either t = or t = vlps. recently prophylactic vaccines against hpv- have appeared on the market. these are composed of l1-based vlps made in insect or yeast cells. spontaneous assembly of hpv- l protein into vlps has also been achieved in plants. plant-produced vlps were immunogenic when administered to animals, further justifying continuing research into the use of plants for producing heterologous proteins for vaccine use. the hpv- l protein can assemble into vlps in non-plant expression systems not only in its native form, but also in chimeric forms; expressing the p1a peptide derived from the p tumour-associated antigen, an epitope of human immunodeficiency virus type, a short epitope of the hepatitis b core antigen, a neutralization epitope of the l minor capsid protein of hpv- and -, and a neutralizing epitope of human respiratory syncytial virus. chimeric vlps have also been produced in transgenic and transplastomic plants. serological characterization of hpv- l particles has been carried out using monoclonal antibodies that recognize either linear or conformational surface-exposed epitopes. amino acid residues and of l are crucial for recognition by the conformation-specific and neutralizing mabs h16:v and h16:e. mabs h16:7e, h16:9a, and h16:u recognize conformational epitopes which are not yet fully mapped. linear epitopes are recognized by mabs h16:i, h16:j, and h16:d. these mabs are fundamental to investigate the use of hpv- l as carrier, since they provide information of the intactness and the stability of l1-based structures. for the purpose of expressing an hpv- l1-based scaffold in plants that can be easily engineered to carry foreign epitopes, we constructed a synthetic l1Δc gene. to investigate the impact of the presence of a foreign epitope on vlp assembly and conformation, we designed four different synthetic genes encoding the l protein where two different l regions were substituted by either a 23-aa or a 8-aa epitope from the ectodomain of the m protein of influenza a virus. the chimeric l proteins generated were tested for their ability to form t = or t = particles, and characterized by their reactivity towards a panel of linear and conformation-specific anti hpv- l mabs.  gene synthesis, cloning and protein structure modelling a synthetic l1Δc gene lacking the 3'-terminal domain reported to code for a nuclear localization signal was designed, that contained unique restriction sites located within nucleotide regions encoding the structural helices and β-sheets. restriction sites at the 5'- and 3'-terminus were introduced for appropriate cloning into the non-replicating plant expression vector peaq-ht. additionally, this gene sequence was optimized with human codon usage, as it was previously shown that this increased hpv- l yield in plants. the l1Δc gene sequence was used as a backbone to generate four chimeric variants containing two different m2e influenza epitopes: m2e2- or m2e2- that replaced l sequences predicted to form either h, or the coil region connecting h and β sheet j. l1Δc and chimeric genes were cloned in peaq-ht, and after sequencing control, all constructs were transformed into agrobacterium tumefaciens. protein structure predictions revealed that all chimeras folded in a way that resembled the crystallographic structure of the native l protein, but we observed that chimeras with the shorter version of the m2e epitope replacing either h or the coil region were more similar than those carrying the longer m2e epitope version. expression of l1Δc and chimeric constructs to determine whether l1Δc and its chimeric variants could be transiently expressed in nicotiana benthamiana, we analysed tissue extracts from agroinfiltrated leaves by western blot using the md2h mab which is specific for the hpv16-l protein. l1Δc and chimeric gene variants expressed an hpv- l monomer of the expected size of kda, with the exception of chim2e2-24_h that migrated slightly slower and showed the strongest signal. no evident proteolysis was observed with l1Δc or with chimeric l protein extracts. expression of l1Δc and chimeras was confirmed with camvir- another anti-hpv l mab. testing for the presence of influenza epitopes using the anti-m 14c mab resulted in the recognition of the longer epitope in either h and the coil position, while this antibody was unable to detect constructs with the shorter epitope. thus we concluded that all synthetic chimeric constructs were successfully expressed and that two of them expressed also influenza epitopes. further step was to determine if chimeras were able to preserve an appropriate conformation necessary to form assembled protein structures. characterization of l1Δc and chimeric constructs by capture-elisa all the linear and conformation-dependent mabs reacted with l1Δc and chimeric constructs from agroinfiltrated leaf extracts when analysed by capture elisa. the highest l value was obtained six days post-agroinfiltration in a time course analysis of ten days. binding of conformation-specific mabs with all l1-based chimeric protein constructs indicated that they aggregated in higher order structures, such as capsomers, or vlps in either t = or t = form. recognition of chimeras by these conformation-specific mabs confirmed not only their expression in plants, but also proved that their self-assembly ability was not abolished by the substitution of h or coil surface-exposed l regions with the foreign influenza epitopes. among all chimeras, chim2e2-24_h showed the highest binding affinity with the conformation-dependent neutralizing mabs. the estimated yield of l1Δc and chimeric proteins accumulated in agroinfiltrated leaves was measured six days post-agroinfiltration. l quantification was calculated by capture-elisa using the h16:j mab which recognizes a linear epitope in both monomeric and assembled structures and the h16:v conformation-specific mab which binds only to assembled structures. the highest l yield was obtained for chim2e2-24_h with both mabs, followed by chim2e2-24_c. chim2e2-24_h accumulated also higher yield then the l1Δc backbone protein, suggesting that the substituted amino-acid sequence had a positive impact on protein stability. on the other hand, the two chimeras bearing a shorter version of the m2e epitope accumulated at lower amount compared to both l1Δc and chimeras having longer substitution. the greater difference observed between h16:j and h16:v in the case of l1Δc and chim2e2-24_h suggested that monomeric l protein was also accumulating, beside the assembled species. electron microscopy of purified l1Δc and chimeric constructs to investigate the structure of vlps and/or capsomers formed by l1Δc and chimeras in plants, we carried out cs2so density gradient and further analysed the fractions obtained by electron microscopy. a begomovirus purification protocol adopted for purifying vlps of other papillomaviruses was successfully employed to purify hpv- l particles. in l1Δc preparations, spherical t = vlps of expected size of nm were formed; in addition small vlps with probable t = symmetry of about nm and capsomers ranging from 8- nm in diameter were also detected. purified preparations of chimeric proteins showed that chim2e2-24_c, chim2e2-9_c, as well as chim2e2-24_h successfully formed capsomers and vlps of similar structure. on the contrary, the chimeric construct with the m2e2- substitution in h position formed only capsomers. electron microscopy analysis confirmed the results of the capture-elisa obtained with conformation-specific mabs indicating the self-assembly of chimeric l protein in higher order protein structures. discussion the current study supports the possibility of using plants as a method for the expression and folding of proteins of human viruses and raises the prospect of plants competing with other systems for the production of low-cost and effective vaccines. in this work, we demonstrated that: 1) the synthetic hpv- l gene can be successfully expressed in a transient manner in n. benthamiana plants using a non replicating peaq-ht expression vector; 2) the hpv- l carrier bearing the m2e influenza epitope can be efficiently produced in plants; 3) m2e influenza epitope presentation in either h or h4-β-j coil positions is not detrimental for the formation of capsomers and does not disrupt the recognition by conformation-dependent mabs; 4) m2e epitopes in coil position allow vlp formation; 5) m2e epitopes in h position enable vlp assembly only when a longer sequence of amino acids is used. the hpv- l gene and chimeras were expressed in plants using the small binary vector peaq-ht containing modified 5'- and 3'-untranslated regions from cowpea mosaic virus rna- avoiding the double cloning procedures necessary with standard binary vectors with a concomitant saving in time. the successful use of replicating viral vectors for the expression of heterologous proteins in plants has been reported on several occasions. however, the size and complexity of viral vector systems, as well as the genetic stability of the constructs during viral genome replication and possible biocontainment issues can negatively affect their widespread use, justifying the selection of non-replicating peaq-ht plasmid as a vector of choice for the current studies. the hpv- l protein carrier containing influenza epitopes was expressed in plants, but the recognition of m2e influenza sequences was achieved only in chimeras with the longer version of the epitope. it is yet to be investigated if the lack of reaction with constructs carrying the shorter version of the m2e influenza epitope is due to the use of an inappropriate mab or to epitope inaccessibility on the carrier surface. so far, there is only one report of the expression of a chimeric hpv16-l protein in transgenic plants, where the chimera was made of l fused to e6/e epitopes of the same virus. here we achieved for the first time the transient expression in plants of chimeric hpv16-l bearing an epitope of a heterologous virus, with a substitution rather than a fusion strategy. several reports have demonstrated the usefulness of the m2e as a means of inducing neutralizing antibodies against influenza. the n-terminus of m2e, containing the highly conserved slltevet epitope, common to both m and m proteins, was sufficient to induce antibody production with an inhibitory effect against this pathogen. two different versions of this epitope were selected for the construction of hpv-chimeras. the major concern in introducing heterologous epitopes into the l platform is to ensure their surface-exposure while preserving the conformation of the l oligomer. hpv- pseudovirion infection is abolished by the neutralizing mabs v and e which recognize surface exposed conformational epitopes of l; these epitopes are considered of crucial importance for immunogenicity. all chimeric proteins tested in this work reacted positively with a panel of linear and conformation-specific mabs. furthermore, the positive reaction obtained with h16:v and h16:e indicated that both epitopes inserted in either l positions preserved these l immunogenic epitopes. mab h16:d has affinity for the denatured vlp antigen, and the weak binding of l1Δc and the chimeric proteins to this mab demonstrates that the assembled products are stable. electron microscopy results showed that the l protein purified by cs2so density gradient centrifugation assembled into recognizable vlps. maclean et al. transiently expressed the l protein in the cytoplasm and observed a size-range of the vlps from 40- nm. our data are consistent with this work, and we found vlps ranging from 30- nm in the same plant host. chimeric particles with m2e substitutions in the coil region predominantly assembled into t = vlps. the substitution of aa of the l coil region with either or aa of the influenza m2e region was not detrimental for vlp formation, thus confirming that this region is not involved in the inter-pentameric reactions necessary to create a vlp. the chimeric polypeptides having shorter substitution in h could still assemble into capsomers, but the formation of t = or t = vlps was inhibited, as expected on the basis of the published crystal structure of hpv- l. interestingly, the substitution of the same h with a longer m2e sequence allowed the assembly into t = and t = vlps, along with capsomer formation, indicating that the incorporation of a longer version of the m2e epitope partially restored the function of the deleted h region. this suggests that the length of an epitope, as well as its amino acid sequence is important for efficient vlp formation. furthermore, when h was substituted by a rsv epitope or by a l epitope of hpv- and -, only capsomers were found assembled in preparations from insect cells systems. the detection of l superstructures for chim2e2-24_h in plants suggests that the plant cell or the purification strategy could additionally play important role in hpv vlp formation/stability. the yields reported in this work are in the range from to mg of total l protein per kg of fresh weight for chim2e2-24_c and chim2e2-24_h, thus fulfilling the often-requested quantity for commercially viable production of antibodies. previous works reported yields of l protein in n. benthamiana plants ranging from μg per kg of plant material with a transient expression system of the native gene, to mg per kg of plant material in the case of a chloroplast-targeted l protein transiently expressed from a human codon-optimized gene, with a 5-fold decrease when the nls was removed. our yields of mg for l1Δc and mg for chim2e2-24_h are in accordance with the highest yields reported so far for plant-transiently expressed hpv- l1Δc this approves the use of l gene with the human codon preferences and demonstrates the suitability of the peaq-ht vector for hpv- l transient expression in plants. further enhancement of l yield could be obtained by appropriately decreasing the gc content, and by deleting regions prone to gene silencing, to reduce mrna secondary structure. three of the four chimeric proteins tested assembled into vlps and capsomers in plants, and one of them produced only capsomers. vlp assembly in the three chimeras emphasized their potential usefulness in future immunogenicity studies; in fact, to achieve comparable antibody responses at least to times more l are required in the form of capsomeres than in the form of vlps. out of three vlp-forming chimeras, chim2e2-24_h and chim2e2-24_c reacted also with anti-influenza mab, suggesting the availability of this epitope on vlp surface. considering that these two chimeras had the highest l yield as well, we propose them as candidate multivalent vaccines for further immunological studies. our work demonstrated that chimeric hpv- l proteins can be transiently expressed in plants in only one week, with a significant-time reduction compared to other plant-expression systems, thus offering new possibilities for rapid testing of new chimeras made in plants. the use of a synthetic l gene designed to include unique restriction sites opens further perspectives for inserting other heterologous epitopes in different structural positions. other interesting surface-exposed regions are the d-e and f-g loops for which the designed synthetic gene can be easily manipulated to present single or multiple foreign epitopes. this synthetic gene could also be used as a backbone for exchanging longer portions of other hpv- or related papillomavirus genes in order to synthesize novel vaccines with therapeutic and/or preventive use, or vaccines with multiple features effective against more than one papillomavirus.  the hpv- l protein was successfully expressed by the cpmv-derived plant transient expression vector peaq-ht. chimeric constructs bearing a heterologous influenza epitope were also successfully transiently expressed in plants as judged by their reaction with anti-hpv l specific mabs. the characterization of chimeras by electron microscopy showed appropriate assembly of vlps, with the exception of one chimera, which formed only capsomers. chimeras were recognized by assembly-dependent conformation-specific mabs, confirming particle stability. the substitution of the l h region with foreign epitopes of suitable size had a positive impact on l self-assembly into vlps, compared to previous results showing that the deletion of the same h region abolished vlp assembly. interestingly, this substitution had a positive effect on l yield, too. moreover, we designed a synthetic gene allowing an easy manipulation of l structural positions suitable for inserting exogenous sequences, thus opening new perspectives for a rapid and easy construction of different-virus chimeras. 
human papillomavirus l protein has the capacity to self-assemble into capsomers or virus-like particles that are highly immunogenic, allowing their use in vaccine production. in this study, we report for the first time the transient expression and the self-assembly of a chimeric hpv- l bearing the m2e influenza epitope in plants, representing also the first record of a successful expression of chimeric hpv- l carrying an epitope of a heterologous virus in plants. chimeras were recognized by a panel of linear and conformation-specific anti hpv- l mabs, and two of them also reacted with the anti-influenza mab. electron microscopy showed that chimeric proteins made in plants spontaneously assembled in higher order structures, such as vlps of t = or t = symmetry, or capsomers. a synthetic hpv- l gene optimized with human codon usage was used as a backbone gene to design four chimeric sequences containing either the m2e2- or the m2e2- epitope in two predicted surface-exposed l positions. we investigated the potential of hpv- l to act as a carrier of two foreign epitopes from influenza a virus: m2e2- ectodomain of the m protein, that is highly conserved among all influenza a isolates, or m2e2- a shorter version of m2e containing the n-terminal highly conserved epitope, that is common for both m and m influenza proteins. all chimeric constructs were transiently expressed in plants using the cowpea mosaic virus-derived expression vector, peaq-ht.
 human papillomavirus l protein has the capacity to self-assemble into capsomers or virus-like particles that are highly immunogenic, allowing their use in vaccine production. successful expression of hpv- l protein has been reported in plants, and plant-produced vlps have been shown to be immunogenic after administration to animals.  we investigated the potential of hpv- l to act as a carrier of two foreign epitopes from influenza a virus: m2e2- ectodomain of the m protein, that is highly conserved among all influenza a isolates, or m2e2- a shorter version of m2e containing the n-terminal highly conserved epitope, that is common for both m and m influenza proteins. a synthetic hpv- l gene optimized with human codon usage was used as a backbone gene to design four chimeric sequences containing either the m2e2- or the m2e2- epitope in two predicted surface-exposed l positions. all chimeric constructs were transiently expressed in plants using the cowpea mosaic virus-derived expression vector, peaq-ht. chimeras were recognized by a panel of linear and conformation-specific anti hpv- l mabs, and two of them also reacted with the anti-influenza mab. electron microscopy showed that chimeric proteins made in plants spontaneously assembled in higher order structures, such as vlps of t = or t = symmetry, or capsomers.  in this study, we report for the first time the transient expression and the self-assembly of a chimeric hpv- l bearing the m2e influenza epitope in plants, representing also the first record of a successful expression of chimeric hpv- l carrying an epitope of a heterologous virus in plants. this study further confirms the usefulness of human papillomavirus particles as carriers of exogenous epitopes and their potential relevance for the production in plants of monovalent or multivalent vaccines. 