most vaccines against infectious agents have a similar goal , that of priming the immune system so that the specific pathogen is destroyed before it can multiply and cause disease . historically , this priming has been achieved using vaccines based on whole viruses or bacteria that have been killed or on liveattenuated strains . although these vaccines have been proved to be highly effective against some pathogens , these approaches have not yielded efficacious vaccines against several other major targets . in addition , there are also some safety concerns associated with the use of attenuated strains and killed pathogens . for these reasons , there has been increasing interest in the development of subunit vaccines that are predominantly based on antigenic surface proteins or polysaccharides . recombinant dna technology has facilitated the development of subunit vaccines , such as hepatitis b , 
  and has led to a pangenomic approach to vaccine development . recent events , however , have demonstrated the need for the development and implementation of alternative technologies to overcome limitations in capacity , speed and costs of vaccine production . in recent years , there has been increasing interest in using plants as production systems for therapeutics , vaccines , and antibodies , 
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  with several vaccine candidates completing phase i clinical trials . , 
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  plants afford several advantages as production systems , including , safety from contamination with animal pathogens , high production capacity , and relatively low capital investment . to date , the following strategies have been used to produce recombinant proteins , including vaccine antigens , in plants : nuclear transformation , 
  chloroplast transformation , 
  and infection with plant rna viral vectors . 
  when tested in animal models , several plantproduced antigens have induced cellular and humoral immune responses conferring protection against challenge . 

  in spite of these encouraging reports , there are disadvantages to each of these plantbased approaches . nuclear transformants often show low levels of expression , and are subject to long lead times . chloroplast transformation , while giving high levels of target expression , 
  is limited to few plant species , has limited potential for posttranslational modification ( e.g. no glycosylation ) , and is also subject to long lead times . as with chloroplast transformation , engineered plant viruses allow for high levels of recombinant protein expression . plant viruses have the added advantages of short lead times , rapid bulkup capacity , and the potential to fuse peptides to the viral coat protein ( cp ) , so facilitating production and enhancing immunogenicity . 
  however , the potential instability of the plant viral expression vector is a concern , and may limit the range of targets . to address some of the limitations of the above plantbased approaches , we have developed an expression system based on a launch vector , pbid4 ( figure  1 ) , which consists of two key components , an agrobacterium binary plasmid , pbi121 
  and a plant virus expression vector . agrobacterium binary plasmids , the standard tools for generating stable transgenic plants 
  are based on the tumourinducing plasmid of agrobacterium tumefaciens . target sequences to be expressed , along with regulatory elements , are incorporated between the left border ( lb ) and right border ( rb ) of the binary plasmid . upon introduction of agrobacteria into plant tissue , multiple singlestranded dna ( ssdna ) copies of sequence between lb and rb are generated and released . it is this feature of the binary vector , to generate multiple ssdna copies , which we use to launch our viral vector into plant tissue . the viral vector we have used is based on tobacco mosaic virus ( tmv ) . tmv is the most extensively studied plant virus , and has been most widely used for the expression of foreign proteins in plants . the singlestranded positivesense rna genome of tmv encodes the viral replicase , the celltocell movement protein , and the cp that encapsidates the viral rna and mediates viral spread throughout the entire plant . in pbid4 , the cp sequence is replaced with the target gene sequence to be expressed . in the absence of cp , the resultant viral vector is a naked selfreplicating rna that is less subject to recombination than cpcontaining vectors , and that can not effectively spread and survive in the environment . the vector contains the 35s promoter of cauliflower mosaic virus ( a dna plant virus ) that drives initial transcription of the recombinant viral genome following introduction into plants , and the nos terminator , the transcriptional terminator of agrobacterium nopaline synthase . the tobacco mosaic virus genome contains genes for virus replication ( 126/183k ) and celltocell movement ( mp ) , and the cloned target gene , inserted into a unique cloning site and under the transcriptional control of the coat protein subgenomic mrna promoter ( arrow ) . left and right border sequences ( lb and rb ) of the agrobacterium binary plasmid delimit the region of the launch vector that is transferred into plant cells following infiltration of plants with the recombinant agrobacterium . the launch vector is delivered by agroinfiltration , during which , bacteria are artificially forced into the intercellular spaces of plant tissues , either by injection or vacuum infiltration . following infiltration , multiple ssdna copies of the viral vector sequences are launched into plant cells in a matter of minutes . the introduced viral sequences , along with the cloned targets , are then amplified through replication . translation of these recombinant viral mrnas typically results in the accumulation of 100  mg quantities of target protein per kg of plant tissue in less than a week . we have expressed a wide range of proteins in plants using the launch vector system . figure  3(a  c ) depict the expression of the green fluorescent protein in nicotiana benthamiana plants following the introduction of agrobacteriumcontaining recombinant launch vector by injection ( figure  3a , b ) or vacuum infiltration ( figure  3c ) . vacuum infiltration has the particular advantage over injection as a mode of introducing the launch vector into all exposed plant tissues , therefore eliminating the need for a virus capable of spreading through the entire plant , and allowing for the use of a cpdeficient viral vector . highlevel expression of target proteins can be achieved when the launch vector is used ( figure  3d ) . accumulation of an engineered thermostable protein lickm ( described below ) of up to 05  g / kg fresh leaf tissue has been achieved within 3  days postinfiltration . this expression level is c. 50fold higher than has been achieved with this target cloned into a standard binary plasmid . gleba et al . 

  have reported a broadly similar approach , combining the features of agrobacterial and viral vectors to express several targets in plants . expression of target proteins in nicotiana benthamiana plants following infiltration with agrobacteriumcontaining pbid4 . ( a ) green fluorescent protein ( gfp ) expression 4  days postinfiltration achieved by injection . ( d ) expression of lickm at up to 05  g / kg fresh plant tissue . for the development of effective subunit vaccines , target antigens are often fused or conjugated to carrier proteins to enhance expression , stability , and/or immunogenicity . for example , to obtain effective vaccines against haemophilus influenza b , neisseria meningitidis and streptococcus pneumoniae target polysaccharides were covalently attached to carrier proteins . 
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  similarly , a variety of peptides have been fused to the receptorbinding subunits of cholera toxin and escherichia coli heatlabile enterotoxin . 
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  fusion of peptides and proteins to viral structural proteins has also been shown to enhance stability and immunogenicity , 
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  and to aid in target purification . 

  here this carrier molecule is based on a thermostable enzyme , 1,31,4glucanase ( lichenase ) , from clostridium thermocellum , 
  engineered versions of which have been used to express reporter genes in prokaryotic and eukaryotic systems . fulllength lichenase ( c. 35  kda ) consists of a signal peptide , a catalytic domain , a prothrrich box , and a docking domain ( figure  4a ) . for our purpose , we modified the molecule to replace the native signal peptide with the signal peptide from nicotiana tabacum pathogenesisrelated protein pr1a , 
  and deleted the prothrrich box and the docking domain . the catalytic domain has a loop structure dividing it into two regions , the nterminal region ( n : amino acids 3284 ) and the cterminal region ( c : amino acids 85246 ) . these two regions can be split at the loop structure and circularly permuted to make the molecule more receptive to insertions without affecting enzymatic activity . a multiple cloning site was introduced at the junction between these two regions , and a 6xhis tag and the sequence kdel were placed at the 3end of the permuted carrier to facilitate purification and retention in the endoplasmic reticulum , respectively . figure  4(b ) shows a schematic diagram of the modified lichenase ( lickm ; genbank accession number : dq776900 ) that has a molecular weight of c. 27  kda . target sequences can be expressed as n or c terminal fusions and/or as internal fusions , a feature that allows for the simultaneous expression of multiple targets .  the lichenase carrier molecule . , the vertical hatch corresponds to the nterminal region of the catalytic domain , and the dotted box corresponds to the cterminal region of the catalytic domain . lickm allows for fusions to molecules ranging in size from small peptides to fulllength proteins of up to 100  kda . we have engineered and successfully produced more than 30 target antigens from different pathogens , including respiratory syncytial virus , human immunodeficiency virus , plague , anthrax , influenza , and trypanosoma brucei as fusions with lickm . lickm maintains its enzymatic activity at high temperatures ( 65c ) , a property that also generally applies to fusions . this allows for easy and costeffective recovery of target proteins because a 10min heat treatment at 65c removes up to 50% of contaminating plant proteins ( figure  5a ) . the use of lickm as a carrier molecule contributes additional advantages , including the potential for enhanced expression ( figure  5b ) , and incorporation of multiple vaccine determinants ( figure  5c ) . ( a ) thermostability : equal amounts of tissue from uninoculated plants or plants expressing recombinant lickm or lickm fused to human papillomavirus ( hpv ) target protein were homogenized and equally split between two tubes . samples in lanes 2 , 4 , and 6 were incubated at 65c for 10  min , whereas samples in lanes 1 , 3 , and 5 were not heat treated . all samples were centrifuged to remove debris containing denatured proteins prior to analysis by sodium dodecylsulfate polyacrylamide gel electrophoresis ( sdspage ) followed by coomassie staining . lanes 1 and 2 show uninoculated plant extract , lanes 3 and 4 show extract from plants expressing lickm , and lanes 5 and 6 show extract from plants expressing lickm fused to an antigen from hpv . arrows indicate positions of lickm ( lanes 3 and 4 ) , and lickmhpv antigen ( lanes 5 and 6 ) . ( b ) enhanced expression : domain 4 of protective antigen of bacillus anthracis expressed alone ( lane 3 ) or as a fusion to lickm ( lane 2 ) in escherichia coli using the pqe30 expression system . ( c ) multiple target expression : lickm carrying influenza a / vietnam/04 hemagglutinin stem domain as an internal fusion and a / vietnam/04 neuraminidase as a cterminal fusion ( lane 1 ) , lickm carrying influenza a / vietnam/04 hemagglutinin globular domain as an internal fusion and a / vietnam/04 neuraminidase as a cterminal fusion ( lane 2 ) . protein extracts were analyzed by sdspage followed by western blotting using polyclonal antilichenase antibody . in all panels , m corresponds to molecular weight marker , magic mark for western blots , and benchmark for coomassiestained gels . the influenza a virus presents one of the major infectious disease threats to human populations . the almost annual epidemics of influenza are associated with point mutations in the envelope antigens hemagglutinin ( ha ) and neuraminidase ( na ) , enabling the virus to evade immune responses induced by prior infections or vaccination . 
  of the greatest risks to public health is the emergence of strains of influenza a virus , with pandemic potential , that have new ha and na antigens as a result of recombination between strains . most of the world 's influenza vaccine supply is manufactured using embryonated eggs , which has insufficient capacity to immunize fully the world 's population against annual epidemics . the current potential for a pandemic , caused by emerging h5n1 strains , emphasizes the urgent need to develop new manufacturing platforms for influenza vaccines , including recombinant systems for subunit vaccines . ha is the immunodominant antigen that is believed to induce protective immunity , and is a key target for subunit vaccines . the efficacy of vaccination against influenza is predominantly determined by the amount of immunogenic ha that is present , 
  and the globular domain of ha contains the majority of the antigenic sites and neutralizing epitopes . na has been shown to contribute to immunity when delivered alongside ha , 
  and thus is a further potential candidate for subunit vaccine development . a recombinant habased vaccine candidate , produced in serumfree insect cell cultures , has been successfully tested in a phase ii clinical trial . however , despite promising data with subunit vaccine candidates , production capacity remains a major issue . some safety concerns and capacity limitations of current influenza vaccine production could be addressed using the launch vector / lichenase carrier system for plantbased production as described above . we have applied this system to express antigens from influenza type a / vietnam/04 ( h5n1 ) virus . we engineered the stem domain ( h5sd ) , amino acids 1758 followed by a triple glycine linker and amino acids 293535 , and the globular domain ( h5gd ) , amino acids 59292 , of influenza a / vietnam/04 virus ha ( genbank accession number : aax83397 ) as internal inframe fusions with lickm to give lickmh5sd and lickmh5gd , respectively , and inserted the fusions into pbid4 . we also inserted na ( n1 ) , amino acids 29449 of influenza a / vietnam/04 virus na ( genbank accession number : aat73327 ) into pbid4 , although not as a fusion with lickm . tissue samples were collected 47  days following infiltration , and each target protein was enriched to over 80% purity by affinity chromatography . the seroidentity of each target was determined by western blot and enzymelinked immunosorbent assay using reference polyclonal sheep sera . the plantproduced lickmh5sd and lickmh5gd were recognized by serum raised against ha purified from influenza a / vietnam/04 virus ( figure  6a , b ) , but not by serum raised against ha purified from influenza a / wyoming/3/03 virus ( figure  6b ) . the plantproduced na was recognized by serum raised against the reassorted virus nibrg17 ( antih7n1 ) ( figure  6c , d ) but not by serum raised against the reassorted virus nibrg18 ( antih7n2 ) ( figure  6d ) . furthermore , the plantproduced na showed na activity that was specifically inhibited by serum raised against nibrg17 , but not by serum raised against nibrg18 ( figure  6e ) . 
 in vitro characterization of plantproduced influenza antigens . ( a ) the plantproduced lickmh5sd ( lane 2 ) and lickmh5gd ( lane 3 ) analyzed by sodium dodecylsulfate polyacrylamide gel electrophoresis ( sdspage ) followed by western blotting using serum raised against purified hemagglutinin ( ha ) of influenza a / vietnam/04 virus . lane 1 shows influenza a / vietnam/04 virus , and m corresponds to molecular weight marker . ( b ) the plantproduced lickmh5sd and lickmh5gd analyzed by enzymelinked immunosorbent assay ( elisa ) using serum raised against purified ha of either influenza a / vietnam/04 virus ( antih5 pab ) or influenza a / wyoming/3/03 virus ( antih3 pab ) . ( c ) the plantproduced neuraminidase ( na ) ( n1 ) analyzed by sdspage followed by western blotting using serum raised against reassorted virus nibrg17 ( antih7n1 ) . ( d ) the plantproduced na ( n1 ) analyzed by elisa using serum raised against either reassorted virus nibrg17 ( antih7n1 pab ) or reassorted virus nibrg18 ( antih7n2 pab ) . ( e ) the na activity of plantproduced na ( n1 ) following preincubation with serum raised against either reassorted virus nibrg17 ( antih7n1 pab ) or reassorted virus nibrg18 ( antih7n2 pab ) . in all panels of ( a ) and ( c ) , m corresponds to magicmark molecular weight marker . in summary , expression of target influenza h5n1 antigens in plants using our launch vector system has successfully been achieved , and procedures have been established to enrich these proteins to at least 80% purity . in the case of ha , the target antigens were expressed as fusions to our engineered lichenase carrier molecule . these plantproduced targets have now been advanced into animal studies in mice and ferrets to assess their immunogenicity and protective efficacy . we have developed a plantbased expression system utilizing a  launch vector , pbid4 , to produce target vaccine antigens . the launch vector system combines the desirable features of agrobacterial and plant viral expression vectors to allow for consistent highlevel production of recombinant proteins under contained conditions . the launch vector described here retains the positive features of plant viral vectors , without the associated disadvantages of instability and potential environmental release . following inoculation , multiple copies of the vector are delivered to each recipient plant cell and then each copy is further amplified by viral replicases . in addition , as the launch vector can be introduced into all aerial plant tissue by vacuum infiltration , there is no requirement for cp to facilitate viral vector movement , and consequently , the expression vectors are more stable , and the virus can not survive in the environment . as with stable nuclear transgenic and plant viral expression systems , target antigens can be engineered into pbid4 and produced in bulk quantities in the time frame of a few months . furthermore , as largescale production depends on nontransgenic seeds , which can be prepared and stored in large quantities , the system has the potential for high capacity . these features make the launch vector system highly suitable for the production of vaccine targets , such as influenza antigens , that demand a short lead time and high capacity . as an additional optional feature for the expression of vaccine candidates , we have engineered a thermostable carrier molecule , lickm , to which target antigens can be fused . protein targets of up to 100  kda have been successfully fused to lickm , and furthermore , multiple targets can be fused to the same carrier molecule , allowing for the development of combination vaccines . , lickm simplifies the purification process , as heat treatment removes most contaminating plant proteins while leaving lickm fusions unaffected . the combination of the launch vector and lickm makes for a highly attractive system for vaccine antigen engineering and production .
historically , most vaccines have been based on killed or liveattenuated infectious agents . 
 although very successful at immunizing populations against disease , both approaches raise safety concerns and often have limited production capacity . 
 this has resulted in increased emphasis on the development of subunit vaccines . 
 several recombinant systems have been considered for subunit vaccine manufacture , including plants , which offer advantages both in cost and in scale of production . 
 we have developed a plant expression system utilizing a  launch vector , which combines the advantageous features of standard agrobacterial binary plasmids and plant viral vectors , to achieve highlevel target antigen expression in plants . as an additional feature , to aid in target expression , stability and purification , we have engineered a thermostable carrier molecule to which antigens are fused . 
 we have applied this launch vector / carrier system to engineer and express target antigens from various pathogens , including , influenza a / vietnam/04 ( h5n1 ) virus .