Patent Application: US-59461006-A

Abstract:
the current invention relates to vaccines that use baculovirus vectors to expose a host organism to an immunogen , thereby eliciting an immune response . a pseudo - typed baculovirus is used to display hemagglutinin on the cell membrane in order to increase host immunogenicity .

Description:
as mentioned above , highly pathogenic ai virus of the h5 subtype has imposed a tremendous threat to the global public health . for effective control of global ai pandemic , the best way to keep ai virus out of humans is to keep it out of birds ( normile , 2005 ). given the fact that baculovirus efficiently transduces mammalian cells and pseudotyped baculovirus is a promising vaccine vehicle , the primary objective of this invention is to develop pseudotyped baculovirus as a novel vaccine vehicle against ai virus infection . because ha is the primary immunogen eliciting immune responses , this application aims at constructing pseudotyped baculovirus with ha ( derived from h5 subtype ) displayed on the envelope , in a hope that the native conformation of ha is retained for elicitation of neutralizing antibodies . to ensure that ha is efficiently incorporated , two recombinant baculoviruses were constructed , bac - ha expressing ha fused with the cytoplasmic domain ( ctd ) derived from ha , and bac - ha64 expressing ha fused with the ctd derived from gp64 ( gp64 is the natural baculovirus envelope protein and its ctd may allow for more efficient incorporation of ha into baculovirus ). the full - length gene fragment encoding ha of ai virus ( a / chicken / taiwan / 1209 / 03 , h5n2 ) was cloned by polymerase chain reaction ( pcr ) into pfastbac - ha plasmid to express the recombinant ha protein in insect cells ( hu , 2006 ). in this study , the aim was to effectively display ha on the baculovirus envelope , therefore , an additional signal sequence derived from baculovirus envelope protein gp64 was genetically fused to the n - terminus of ha gene via a two - stage pcr approach ( hu , 2003a ). specifically , the wild - type gp64 gene was previously amplified by pcr from acmnpv dna and subcloned to pbac - ceh plasmid , thereby a stretch of 6 histidine residues ( his 6 tag ) was inserted between the signal sequence and the mature domain ( hu , 2003a ). five primers ( a , b , c , d and e as shown in table 1 ) were designed to construct the fusion genes using pbac - ceh and pfastbac - ha plasmids as the templates . primers a ( seq id no : 1 ) and b ( seq id no : 2 ) were used to amplify the gp64 signal sequence ( ss ) while adding a his 6 tag at the c - terminus . primers c ( seq id no : 3 ) and d ( seq id no : 4 ) ( or e [ seq id no : 5 ]) were used to amplify the mature domain ( md , including the ectodomain and transmembrane ( tm ) domain ) of ha , while adding a his 6 tag at the n - terminus and a cytoplasmic domain ( ctd ) at the c - terminus . owing to the significance of ctd on the recognition with nucleocapsid and subsequent protein incorporation , two versions of ctd , one derived from gp64 ( using primer d ) and the other derived from ha ( using primer e , not shown in fig1 ), were fused to c - terminus . the resultant pcr products were mixed and subject to the second pcr to amplify the whole fusion : ss - his 6 - md - ctd , using primers a and d ( or e ). the two - stage pcr procedure is illustrated in fig1 . fig1 shows the generation of the fusion gene ss - his 6 - md - gp64 ctd as an example . the gene fragments encoding gp64 ss - hi 6 ( left ) and his 6 - md - ctd ( right ) were amplified in the first pcr stage . the two fusion gene fragments were then fused in the second pcr stage . primers a and d were deigned to add restriction enzyme sites xho i and kpn i . table 1 : primers used in cloning experiments . primers a and b were used to amplify the gp64 signal sequence ( ss ) ( sequence in bold ) while adding a his 6 tag ( shaded sequences ). primers c and d were used to amplify the mature domain ( md , consisting of the ectodomain and transmembrane domain ) of ha , while adding a his 6 tag at the n - terminus and a cytoplasmic domain ( ctd , boxed region ) derived from gp64 at the c - terminus . primers c and e were employed to amplify the md and ctd of ha . the 5 ′ end of primers a and d ( and e ) were designed to harbor restriction enzyme sites xho i and kpn i , respectively , for the enzyme digestion after pcr amplification . percentage identity of two nucleic acid sequences is calculated by first aligning the first sequence with the second sequence , inserting gaps in the first sequence where insertions have occurred in the second sequence , and inserting gaps in the second sequence where deletions have occurred in the second sequence . the aligned nucleotides are then compared between the first sequence and the second sequence from the first position to the final position . gaps , insertions , deletions or introns are not compared . the number of identical aligned nucleotides is then divided by the total number of aligned nucleotides to give the percent identity . percentage identity of two amino acid sequences is calculated by first aligning the first sequence with the second sequence , inserting gaps in the first sequence where insertions have occurred in the second sequence , and inserting gaps in the second sequence where deletions have occurred in the second sequence . the aligned amino acids are then compared between the first sequence and the second sequence from the first position to the final position . gaps , insertions , deletions are not compared . the number of identical aligned amino acids is then divided by the total number of aligned amino acids to give the percent identity . additionally , primers a and d ( or e ) were designed to add restriction enzyme sites kpn i and xho i which allowed for insertion of the fusion gene into kpni / xhoi site of mcs ii of pbac - ce plasmid , under the transcriptional control of p10 promoter . pbac - ce was constructed in our lab previously using pfastbac ™ dual ( invitrogen ) as the backbone ( hu , 2003b ) and harbored egfp ( encoding enhanced green fluorescent protein , egfp ) gene under the transcriptional control of cytomegalovirus immediate early ( cmv ) promoter . the expression of egfp served as a reporter for monitoring baculovirus transduction of mammalian cells . the resultant plasmids were designated pbac - ha and pbac - ha64 ( fig2 ). fig2 shows a schematic illustration of pbac - ha and pbac - ha64 plasmids . the fusion gene fragments generated by two - stage pcr as shown in fig1 were cloned into the kpn i / xho i site of mcs ii of pbac - ce plasmid , under the transcriptional control of p10 promoter . egfp expression was controlled by cmv promoter as a reporter . the recombinant baculoviruses were subsequently generated using bac - to - bac ® system ( invitrogen ) and designated bac - ha and bac - ha64 , respectively . bac - ha was designed to express his 6 - tagged rha ( recombinant ha fused with gp64 ss at the n - terminus and ha ctd at the c - terminus ). bac - ha64 was designed to express rha64 ( recombinant ha fused with gp64 ss at the n - terminus and gp64 ctd at the c - terminus ). ideally , during virus replication the gp64 signal sequence directs the translocation of the chimeric protein to the insect cell plasma membrane and is cleaved after protein anchoring thus exposing his 6 tag to the outer surface . the transmembrane domain enables the protein to anchor on the plasma membrane , while the ctd mediates ( 1 ) the recognition of budding baculovirus nucleocapsid with the membrane - bound protein and ( 2 ) the incorporation of the anchored protein into the virus envelope . the subsequent recombinant virus selection , plaque purification and amplification were performed using insect ( sf - 9 ) cell as the host as described earlier ( hu , 2003a ). the sf - 9 cells were maintained using tnm - fh medium supplemented with 10 % fetal bovine serum . the titers of recombinant baculoviruses were determined by endpoint dilution method ( hu , 2003a ) and are expressed as plaque forming units per milliliter ( pfu / ml ). confirmation of recombinant protein ( rha and rha64 ) expression in insect cells to confirm the expression of his 6 - tagged rha and rha64 in insect ( sf - 9 ) cells , the cells were respectively infected by bac - ce ( a control baculovirus expressing no ha [ hu , 2003b ]), bac - ha and bac - ha64 at a multiplicity of infection ( moi ) of 10 . the cells were harvested at 3 days post - infection ( dpi ) and lysed in phosphate buffered - saline ( pbs ). the lysates were subject to sds - page , followed by western blot analysis using anti - ha serum and anti - his 6 monoclonal antibody ( mab ) as the primary antibodies . as shown in fig3 , the cells were infected by bac - ce , bac - ha and bac - ha64 at moi 10 , harvested at 3 dpi and lysed in pbs . the lysates were subject to sds - page , followed by western blot analysis . sds - page was performed by mixing cell lysate with sample buffer ( 0 . 5 m tris - hcl , ph 6 . 8 , 10 % glycerol , 5 % sds , 5 % β - mercaptoethanol , 0 . 25 % bromophenol blue ), boiling at 100 ° c . for 3 minutes , centrifuging for 1 minute , and loading onto a 10 % slab gel . after electrophoresis , the proteins on the gel were transferred onto a nitrocellulose membrane following standard procedures ( bio - rad laboratories ). two primary antibodies were used for western blot : chicken anti - ha polyclonal antibody and mouse anti - his 6 mab ( invitrogen ). the secondary antibodies were goat anti - checken and goat anti - mouse mab conjugated with alkaline phosphatase ( kirkegaard and perry laboratories , kpl , gaithersburg , md .). the color development was performed by immersing the membrane in bcip / nbt reagent ( sigma ). as shown in fig3 , the cells infected by bac - ce expressed no ha , whereas the cells infected by either bac - ha or bac - ha64 expressed a ≈ 69 kd protein that was detected by both anti - his 6 and anti - ha antibodies , thus confirming the expression of his 6 - tagged rha and rha64 . to confirm whether the his 6 - tagged rha and rha64 were properly translocated to the surface of plasma membrane , the cells were cultured on sterile cover slips , infected by bac - ce , bac - ha64 or bac - ha , and subject to immunofluorescence labeling / confocal microscopy visualization at 2 dpi ( fig4 ). as shown in fig4 , the sf - 9 cells were cultured on sterile cover slips ( placed in 6 - well plates ) and infected by bac - ha and bac - ha64 at moi 10 . two days later , the medium was removed , and the cells were fixed by methanol / acetone ( 1 : 1 ) for 5 min at − 20 ° c ., rinsed with 1 ml of pbs and then blocked with 2 % bovine serum albumin in pbs ( 1 ml ) for 30 min at 37 ° c . the cells were then incubated with the primary antibody ( mouse anti - his 6 mab , 1 : 300 dilution , amersham biosciences ) for 1 h at 37 ° c ., followed by pbs washes 3 times . the cells were then incubated with the secondary antibody ( fitc - conjugated anti - mouse mab , 1 : 50 dilution , kpl ) for 1 h at 37 ° c ., followed by 3 pbs washes . protein localizations were visualized by a confocal microscope ( tcs sp2 , leica , germany ). his 6 - tagged proteins emitted green fluorescence under the microscope . the merged photographs illustrate the colocalization of rha and rha64 with plasma membrane . both bac - ha and bac - ha64 expressed proteins that were detected by anti - his 6 and anti - ha antibodies . these proteins co - localized with the plasma membrane , indicating that both rha and rha64 were translocated and anchored on the plasma membrane . to examine whether the rha ( rha64 ) anchored on the plasma membrane was successfully displayed on the baculoviral envelope , bac - ha , bac - ha64 and bac - ce were purified by sucrose gradient ultracentrifugation and subject to immunogold electron microscopy using anti - his 6 antibody as the primary ab . as shown in fig5 , the recombinant viruses were produced by infecting insect cells ( sf - 9 ) at moi 0 . 1 and harvested at 4 days post - infection . the virus supernatant was individually purified by sucrose gradient ultracentrifugation ( o &# 39 ; reilly , 1992 ). briefly , 33 ml virus supernatant was loaded to each ultracentrifugation tube ( hitachi , 40pa ) and then underlaid with 3 ml sucrose solution ( 25 % ( wt / wt ), dissolved in phosphate buffered saline ). the virus was pelleted by ultracentrifuation at 90 , 000 × g ( cp100mx , hitachi ) for 1 . 5 hr , and the pellets were pooled and resuspended with 5 ml pbs . for further virus purification , the concentrated viruses ( 0 . 5 - 1 ml ) were loaded onto a sucrose gradient ( 10 ml ) consisting of four layers of 35 , 43 , 55 and 60 % ( wt / wt ) sucrose solution . after centrifugation at 90000 × g for 3 h , baculovirus that sedimented near 43 - 47 % sucrose was collected by syringe , diluted in pbs and ultracentrifuged ( 90 , 000 × g for 90 min ) again . the purified virus pellet was resuspended in pbs and then was subject to immunogold electron microscopy as follows . the carbon - coated copper grid ( pelco ) was floated on top of 10 μl purified baculovirus solution for 30 min . the grid was then floated on top of a drop of blocking solution ( pbs containing 1 % bsa and 25 mm glycine ph 6 . 2 ) for 20 min and washed with pbs 3 × 5 min . following the washing step , the grid was transferred onto a drop ( 15 μl each ) of the anti - his 6 mab solution ( 1 : 100 diluted ) and incubated for 30 min at room temperature . after two pbs washes , the grid was then floated on the anti - mouse mab conjugated with 5 - nm gold particle ( 1 : 50 - dilution , sigma ) for 30 min at room temperature . after washing with pbs , the grid was negatively stained with 2 % phophotungstic acid ( pta , sigma ) for 2 min and air - dried at room temperature . the gris were examined under transmission electron microscope ( hitachi , h - 7500 ). fig5 reveals no gold particles on the surface of bac - ce , whereas gold particles were evidently observed on the surface of both bac - ha64 and bac - ha , indicating the incorporation and display of rha64 and rha on the viral envelope . the differential incorporation of rha64 and rha into the purified baculoviruses ( bac - ha64 and bac - ha ) was visualized by sds - page / western blot and quantified by scanning densitometry ( fig6 ) by which the amounts of his 6 - tagged ha and gp64 incorporated into the viruses were normalized based on equal amounts of vp39 ( the major capsid protein ). as shown in fig6 , the purified viruses ( bac - ce , bac - ha64 and bac - ha ) were subject to sds - page and western blot using anti - ha or anti - gp64 ab as the primary antibodies . the band intensities for ha and gp64 on the western blot , as well as the band intensities for vp39 on the sds - page , were scanned and analyzed by scion image shareware . compared to bac - ha , bac - ha64 incorporated ≈ 77 % more his 6 - tagged ha into the virus , but contained virtually equal amounts of gp64 . conversely , bac - ce contained no ha , but incorporated ≈ 24 % more gp64 than bac - ha . whether the choice of ctd influenced the ability of baculovirus to transduce mammalian cells was further examined . toward this end , four different mammalian cell lines ( beas - 2b , a549 , hela and vero e6 ) were transduced by bac - ha , bac - ha64 or the control virus bac - ce using the same virus dosage ( moi 35 ). the cells were harvested at 1 day post - transduction and analyzed for total fi by flow cytometry ( fig7 a ). among these 3 viruses , bac - ha64 resulted in significantly higher egfp expression levels in all 4 cell lines . compared with bac - ce , however , bac - ha led to similar egfp expression levels ( p & gt ; 0 . 05 ) in beas - 2b , a549 and vero e6 cells , and inferior egfp expression ( p & lt ; 0 . 05 ) in hela cells . the amounts of intracellular egfp genes within beas - 2b , a549 and hela cells were also quantified at 1 day post - transduction by q - pcr . for each cell line , the quantities of egfp genes carried by bac - ha and bac - ha64 were normalized against that carried by bac - ce ( fig7 b ). in comparison with bac - ce , the amounts of intracellular egfp delivered by bac - ha64 significantly excelled in all 3 cell lines ( p & lt ; 0 . 05 ). conversely , the amounts of intracellular egfp delivered by bac - ha were only higher in beas - 2b ( p & lt ; 0 . 05 ), but were lower in a549 and hela cells ( p & lt ; 0 . 05 ). these data indicate that bac - ha64 yielded improved transduction whereas . bac - ha did not . to examine whether the incorporated ha via different ctd affected the virus stability , viruses ( bac - ce , bac - ha64 and bac - ha ) of identical concentrations ( 5 × 10 7 pfu / ml ) were incubated at 37 ° c . and the remaining titers were monitored . as shown in fig8 , baculovirus supernatants ( bac - ce , bac - ha64 and bac - ha ) of identical concentrations ( 5 × 10 7 pfu / ml ) were incubated at 37 ° c . and the decay of virus transducing titer was monitored . briefly , the virus supernatants sampled at various times were diluted 2 - fold serially in tnm - fh medium in microfuge tubes . one hunderd microliter diluted virus solutions were directly used to transduce hela cells as described ( chan , 2006 ). at 24 hr post - transduction , the cells were trypsinized and analyzed for the transduction efficiencies and mean fluorescence intensity by flow cytometry . the percentage of gfp - emitting cells was determined for each viral dilution . the transducing titers were calculated according to the following equation ( transfiguracion , 2003 ; logan , 2004 ) and are expressed as transducing units per milliliter ( tu / ml ): fig9 depicts that both bac - ha64 and bac - ce exhibited comparably higher tolerance to the incubation at 37 ° c ., as ≈ 88 % of transducing titer was retained after 12 h . however , the titer of bac - ha dropped to ≈ 69 % of the original after 12 h incubation , indicating the inferior stability of bac - ha . to examine whether the ha displayed on baculoviral envelope could serve as the immunogen eliciting anti - ha antibodies in vivo , 2 groups of mice , each comprising 8 female balb / c mice ( 6 weeks old , purchased from national experimental animal center ), were immunized intraperitoneally ( facciabene , 2004 ) with 1 . 5 × 10 8 pfu purified bac - ha and bac - ha64 , respectively . as negative controls , five mice were injected with purified bac - ce or pbs . for each mouse , the purified virus ( 250 μl ) was mixed with 250 μl complete freund &# 39 ; adjuvant for injection . two weeks after the primary injection , the mice received one booster shot using equal amount of the purified virus ( 1 . 5 × 10 8 pfu / mice ) with the incomplete freund &# 39 ; adjuvant . blood samples were taken from the tail vein on week 6 and heat inactivated . whether the serum antibodies were capable of inhibiting hemagglutination were determined by hemagglutination inhibition ( hi ) assay ( coligan , 2005 ). as shown in fig8 , bac - ce , bac - ha and bac - ha64 were purified by ultracentrifugation as described above . two groups of mice , each comprising 8 female balb / c mice ( 6 weeks old , purchased from national experimental animal center ), were immunized intraperitoneally ( facciabene , 2004 ) with 1 . 5 × 10 8 pfu of purified bac - ha and bac - ha64 , respectively . as negative conrols , 5 mice were injected with purified bac - ce or pbs . one of skill in the art would recognize that a variety of means for introducing the vaccine into the host exist , including subcutaneous injection , intravenous injection , intra - arterial injection , electroporation , and transdermal administration , among others . for each mouse , the purified virus ( 250 μl ) was mixed with 250 μl complete freund &# 39 ; s adjuvant for injection . two weeks after the primary injection , the mice received one booster shot using equal amount of the purified virus ( 1 . 5 × 10 8 pfu / mice ) with the incomplete freund &# 39 ; adjuvant . blood was taken from the tail vein on week 6 . the collected blood samples were left at room temperature for 1 h , centrifuged at 1000 × g for 5 min to remove cells , and incubated at 56 ° c . for 30 min to inactivate complement proteins . the sera were stored at − 80 ° c . until analysis . whether the serum antibodies were capable of inhibiting hemagglutinin were titered by hemagglutination inhibition ( hi ) assay ( coligan , 2005 ). the hemagglutination unit of the inactivated aiv were first measured according to the standard protocols ( coligan , 2005 ). as shown in fig9 , the negative control bac - ce and pbs induced no hi titers while both bac - ha and bac - ha64 induced hi titers . intriguingly , immunization with bac - ha64 elicited significantly higher hi titers compared to bac - ha . these data indicated that the incorporated ha successfully induced functional antibodies , and the amount of antibody was correlated with the amount of ha incorporated . although baculovirus displaying immunogen as a vaccine candidate has been proposed , this is the first report demonstrating that ha displaying baculovirus can be used as a vaccine to induce antibody responses to ai virus . normally other researchers pseudotype the baculovirus simply using the envelope protein ( e . g . vsv - g ) and the homologous ctd ( e . g . ctd derived from vsv - g ). however , here we demonstrate that , the way ha is displayed on the baculoviral envelope has a tremendous impact on the virus property and vaccine efficacy . the ctd derived form the baculovirus endogenous gp64 endows more efficient incorporation of ha protein ( compared to the ha ctd ) into the baculovirus , which in turn , results in the display of more ha on baculoviral envelope and confers higher immunogenicity . as a vaccine vehicle , baculovirus possesses the following advantages over other vaccine types : ( 1 ) baculovirus is non - pathogenic to humans , thus obviating bsl - 3 facilities for vaccine production . ( 2 ) baculovirus can be easily produced to high titers simply by infecting natural host insect cells , thus facilitating large - scale production . ( 3 ) recombinant baculovirus vaccine enables the differentiation between infected and vaccinated animals , because it does not induce the production of antibodies against the nucleoprotein antigen , which is common to all ai viruses . ( 4 ) baculovirus vaccine should avoid pre - existing immunity problem ( i . e . humans do not have anti - baculovirus antibodies that can immediately inactivate baculovirus . this platform can be extended to construct other pseudotyped baculoviruses . for example , we may replace egfp with neuraminidase ( na ) to construct a virus displaying ha and capable of expressing na in the animals . the ha displayed on baculovirus envelope supposedly retains the native conformation for eliciting the humoral immunity , and the na expressed in animals supposedly undergoes accurate post - translational modifications . since ai virus infection poses a tremendous threat to poultry industry and global public health , the application of recombinant baculovirus - based vaccine provides an alternative promising strategy to prevent avian flu epidemic and will contribute greatly to the development of anti - ai vaccine . as ai infection has turned into a tremendous threat to humans around the globe , the development of ai vaccine has become imminent . the approval of this novel vaccine platform can open a new avenue to vaccine development and provide an alternative option to existing vaccine technology . it is expected that this invention can result in a rapid technology transfer . the term “ conservatively modified variations ” of a particular nucleic acid sequence refers to those nucleic acids which encode identical or essentially identical amino acid sequences , or where the nucleic acid does not encode an amino acid sequence , to essentially identical sequences . because of the degeneracy of the genetic code , a large number of functionally identical nucleic acids encode any given polypeptide . for instance , the codons cgu , cgc , cga , cgg , aga , and agg all encode the amino acid arginine . thus , at every position where an arginine is specified by a codon , the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide . such nucleic acid variations are “ silent variations ,” which are one species of “ conservatively modified variations .” every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation . one of skill will recognize that each codon in a nucleic acid ( except aug , which is ordinarily the only codon for methionine ) can be modified to yield a functionally identical molecule by standard techniques . accordingly , each “ silent variation ” of a nucleic acid which encodes a polypeptide is implicit in each described sequence . furthermore , one of skill will recognize that individual substitutions , deletions or additions which alter , add or delete a single amino acid or a small percentage of amino acids ( typically less than 15 %, more typically less than 5 %, and even more typically less than 1 %) in an encoded sequence are “ conservatively modified variations ” where the alterations result in the substitution of an amino acid with a chemically similar amino acid . conservative amino acid substitutions providing functionally similar amino acids are well known in the art . every amino acid sequence herein also describes every possible variation containing one or more conservative amino acid substitution . the following six groups each contain amino acids that are generally considered conservative substitutions for one another : the following u . s . patent documents and publications are hereby incorporated by reference . abe , t , takahashi , h , hamazaki , h , miyano - kurosaki , n , matsuura , y , takaku , h . baculovirus induces an innate immune response and confers protection from lethal influenza virus infection in mice . j immunol 2003 ; 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