Patent Application: US-201113885388-A

Abstract:
described is a vaccine against respiratory syncytial virus . more specifically , described is a recombinant subunit vaccine comprising the ectodomain of the rsv - encoded small hydrophobic protein . the ectodomain of sh is referred to as she . the ectodomain is typically presented as an oligomer , or a pentamer . further described are antibodies , raised against the ectodomain or specific for the ectodomain , and their use for protecting a subject against rsv infection and / or for treatment of an infected subject .

Description:
a plasmid containing the coding sequence of flag - compcc - she ( fig1 , panel b ) was ordered at genscript ( seq id no : 31 ). the flag - compcc - she coding sequence was ligated as a ndei / noti fragment in a ndei / noti opened plt32h bacterial expression vector ( mertens et al ., 1995 ). total rna of rsv a2 - infected hep - 2 cells was prepared using the high pure rna tissue kit ( roche , mannheim ) according to the manufacturer &# 39 ; s instructions . after cdna synthesis , the rsv a2 sh coding sequence was amplified using the following forward and reverse primers ( 5 ′ ataagaaagcggccgctatggaaaatacatccataacaatag3 ′ ( seq id no : 36 ); 5 ′ gaagatctctatgtgttgactcgagctcttggtaactcaaa3 ′ ( seq id no : 37 )). the pcr product was digested with noti and bglii and ligated in a noti / bglii opened pcaggs - ptb - etag expression vector ( cornelis et al ., 2005 ). the resulting vector plt32 - flag - compcc - she was deposited under the budapest treaty at bccm ( bccm / lmbp : technologiepark 927 , 9052 zwijnaarde , belgium ) under deposit number lmbp 6817 on 8 nov . 2010 . the construction of the pcaggs - luc expression vector was described earlier ( schepens et al ., 2005 ; referred as pcaggs - hif - rluc ). the coding sequence of mhbc , as described earlier by jegerlehner et al ., as part of the “ abi ” plasmid , was ordered at geneart ( seq id no : 32 ) ( de filette et al ., 2005 ; jegerlehner et al ., 2002 ). this coding sequence was cloned as a ndei / noti fragment in a ndei / noti opened plt32h bacterial expression vector . to construct plt32 she - tgcn4 , the she coding sequence was fused to the tgcn4 - flag coding sequence by fusion per . the she fragment for fusion per was amplified using the primers : 5 ′ ggaattccatatgaacaagttatgtgagtacaacg3 ′ ( seq id no : 38 ) and 5 ′ gatttgttttaaacctcctgtatttactcgtgcccgaggcaa3 ′ ( seq id no : 39 ) and a template plasmid that was ordered at geneart ( seq id no : 33 ) and that contains the coding sequence of the rsv a2 sh ectodomain ( nklceynvfhnktfelprarvnt ) ( seq id no : 40 ). the gcn4 fragment for fusion pcr was amplified using the primers 5 ′ cccaagcttctaacattgagattcccgagattgaga3 ′ ( seq id no : 41 ) and 5 ′ tattaaccctcactaaagggaagg3 ′ ( seq id no : 42 ) and a template plasmid that contains the tgcn4 coding sequence , c - terminally fused to the coding sequence of three successive flag - tag sequences ( seq id no : 34 ; de filette et al ., 2008 ). the two pcr fragments were fused using the primers : 5 ′ ggaattccatatgaacaagttatgtgagtacaacg3 ′ ( seq id no : 43 ) and 5 ′ tattaaccctcactaaagggaagg3 ′ ( seq id no : 44 ). this fusion pcr product was cloned as a ndei / hindiii fragment in a ndei / hindiii opened plt32h bacterial expression vector . the resulting plt32 she - tgcn4 - flag was deposited under the budapest treaty at bccm ( bccm / lmbp : technologiepark 927 , 9052 zwijnaarde , belgium ) under deposit number lmbp 6818 on 8 nov . 2010 . the construction of the plt32 m2e - tgcn4 expression vector was described earlier ( de filette et al ., 2008 ). a plasmid containing the coding sequence of the lpp ( 5 ) tryptophan - zipper fused to the coding sequence of the sh ectodomain separated by the coding sequence of a glygly linker was ordered at genscript . this coding sequence was amplified using the following forward and reverse primers ( 5 ′ gcgaaatgggatcagtggagcagc - 3 ′ ( seq id no : 53 ); 5 ′ aatataggatccctaggtcgcccagttatcccagcg - 3 ′ ( seq id no : 54 )), phosphorylated and digested with bamhi . the plh36 - hisdevd - lpp - she was constructed by a three - point ligation using the described pcr fragment , bamhi / psti - digested plt32 plasmid fragment and ecorv / psti - digested plh36 fragment . the sequence of the constructed plh36 - hisdevd - lpp ( 5 ) - she plasmid is displayed in seq id no : 49 . expression and purification of she - tgcn4 , m2e - tgcn4 , flag - compcc - she , mhbc and lpp ( 5 ) - she a 30 - ml preculture of plt32she - tgcn4 - transformed e . coli was grown at 28 ° c . in luria broth and used to inoculate 1 liter of fresh medium . at an a600 of 0 . 6 - 0 . 8 , the cells were treated with 1 mm isopropyl 1 - thio - β - d - galactopyranoside , incubated for another four hours , and then collected by centrifugation ( 6000 × g , 20 minutes , 4 ° c .). the bacterial pellet was resuspended in 20 ml tris - hcl buffer ( 50 mm tris - hcl , 50 mm nacl and 1 mm edta ), ph 8 , and sonicated . bacterial debris was pelleted by centrifugation ( 20 , 000 × g , one hour , 4 ° c .). the supernatant was applied to a deae sepharose column pre - equilibrated with tris - hcl buffer containing 50 mm nacl ( buffer a ). after washing , the bound proteins were eluted by a two - step gradient going from 0 - 40 % buffer b ( 50 mm tris - hcl , 1 m nacl ) and 40 - 100 % buffer b . fractions containing she - tgcn4 were pooled , adjusted to 25 % ammonium sulfate saturation , and applied to a phenyl - sepharose column pre - equilibrated with 25 % ammonium sulfate , 50 mm tris - hcl , ph 8 . bound proteins were eluted with a two - step gradient . the two - step elution was performed with 0 - 40 % and 40 - 100 % 50 mm tris - hcl buffer , ph 8 ( buffer a ). the fractions containing she - tgcn4 were loaded on a superdex 75 column . gel filtration was performed in phosphate - buffered saline ( pbs ), and the fractions containing she - tgcn4 were pooled and stored at − 70 ° c . expression and purification of flag - compcc - she was identical to she - tgcn4 apart from the use of a q sepharose column for anion exchange chromatography instead of a deae sepharose column . the expression and purification of m2e - tgcn4 was described before ( de filette et al ., 2008 ). expression and purification of mhbc was identical to she - tgcn4 apart from the use of a sephacryl s400 column for gel filtration chromatography instead of superdex 75 column . a 30 - ml preculture of plh36 - hisdevd - lpp ( 5 ) - she - transformed e . coli cells was grown at 28 ° c . in luria broth with ampicillin and used to inoculate 3 liters of fresh medium . at an a 600 of 0 . 6 - 0 . 8 , the cells were treated with 1 mm isopropyl 1 - thio - β - d - galactopyranoside , incubated for another four hours , and then collected by centrifugation ( 6000 × g , 20 minutes , 4 ° c .). the bacterial pellet was resuspended in 300 ml buffer containing 20 mm nah 2 po 4 / na 2 hpo 4 , 300 mm nacl and 5 mm imidazole , ph 7 . 5 and sonicated . bacterial debris was pelleted by centrifugation ( 20 , 000 × g , one hour , 4 ° c .). the supernatant was loaded on a nickel - sepharose column pre - equilibrated with buffer containing 5 mm imidazole . after washing , the bound proteins were eluted by a step - wise ( 50 mm , 100 mm , 200 mm and 400 mm ) imidazole gradient . fractions containing lpp ( 5 ) - she were pooled , desalted and further purified on a q - sepharose column . the sample was applied to a deae sepharose column pre - equilibrated with tris - hcl buffer containing 50 mm nacl ( buffer a ). after washing , the bound proteins were eluted by a two - step gradient going from 0 - 40 % buffer b ( 50 mm tris - hcl , 1 m nacl ) and 40 - 100 % buffer . the fractions containing lpp ( 5 ) - she were loaded on a superdex 75 column . gel filtration was performed in phosphate - buffered saline ( pbs ) and the fractions containing lpp ( 5 ) - she . a detoxified mutant of heat - labile e . coli enterotoxin , ltr192g , was used for intranasal ( i . n .) administration ; this preparation was generously provided by dr . j . clements ( department of microbiology and immunology , tulane university medical center , new orleans , la ., usa ) ( norton et al ., 2010 ). she ( cc4s ), a chemically synthesized , hplc - purified she peptide in which the naturally occurring cysteine was replaced by a serine and to which a cysteine was added at the n - terminus was ordered at pepscan ( pepscan , lelystad ). the she ( cc4s ) peptide was via its n - terminal cysteine residue fused to a lysine in the immunodominant loop of mhbc on the surface of hbc vlps by chemical linkage using the heterobifuctional sulfo - mbs ( pierce ), according to the manufacturer &# 39 ; s instructions . in short , 400 μg mhbc , dissolved in 200 μl pbs , was incubated with sulfo - mbs ( at a final concentration of 1 mg / ml ) for one hour . after removal of unbound sulfo - mbs molecules by size exclusion chromatography , sulfo - mbs - linked mhbc vlps were diluted in 2 ml h 2 o , subsequently , 100 μl she ( cc4s ) peptide ( dissolved in 100 ml pbs ) was added and incubated for one hour at room temperature to allow cross - linking of the peptide to the mhbc vlps . finally , free she ( cc4s ) peptide was removed by size exclusion chromatography . the purity and cross - linking efficacy was tested via sds - page followed by coomassie staining . hep - 2 cells ( atcc , ccl - 23 ), vero cells ( atcc , ccl - 81 ), hek293t cells ( a gift from dr . m . hall ) and a549 cells ( atcc , ccl - 185 ) were grown in dmem medium supplemented with 10 % heat - inactivated fetal calf serum ( fcs ), 1 % penicillin , 1 % streptomycin , 2 mm l - glutamine , non - essential amino acids ( invitrogen , carlsbad , calif . ), and 1 mm sodium pyruvate . specific pathogen - free , female balb / c mice were obtained from charles river ( charles river wiga , sulzfeld , germany ). the animals were housed in a temperature - controlled environment with 12 - hour light / dark cycles ; food and water were delivered ad libitum . mice were immunized at 8 weeks of age after one week adaptation in the animal room . the animal facility operates under the flemish government license number la1400091 . all experiments were done under conditions specified by law ( european directive and belgian royal decree of nov . 14 , 1993 ) and authorized by the institutional ethical committee on experimental animals . rsv a2 , an a subtype of rsv ( atcc , rockville ), was propagated by infecting monolayers of vero cells , with 0 . 1 moi in the presence of growth medium containing 1 % fcs . five to seven days after infection , the cells and growth medium were collected , pooled and clarified by centrifugation ( 450 × g ). to concentrate the virus , the clarified supernatant was incubated for four hours at 4 ° c . in the presence of 10 % polyethylene glycol ( peg6000 ). after centrifugation ( 30 minutes at 3000 × g ), the pellet was resuspended in hank &# 39 ; s balanced salt solution ( hbss ), containing 20 % sucrose , aliquoted and stored at − 80 ° c . for intranasal immunization or infection , the mice were slightly anesthetized by isofluorane . the final volume used for administration of vaccine + adjuvant or virus was 50 μl ( 25 μl per nostril ). vaccines + adjuvant were formulated in pbs , whereas the viral inoculum was formulated in hbss . three or four days post - challenge , the mice were sacrificed . the mouse lungs were removed aseptically and homogenized with a heidolph rzr 2020 homogenizer for 30 seconds in 1 ml hbss containing 10 % sucrose . lung homogenates were subsequently cleared by centrifugation at 4 ° c . and used for virus titration on hep - 2 cells . monolayers of hep - 2 cells were infected with 50 μl of serial 1 : 3 dilutions of the lung homogenates in a 96 - well plate in serum - free opti - mem ® medium ( invitrogen ) supplemented with penicillin and streptomycin . four hours later , the cells were washed twice with dmem medium containing 2 % fcs and incubated for five days at 37 ° c . in 50 μl overlay medium ( completed dmem medium containing 1 % fcs , 0 . 5 % agarose ). the cells were fixed by adding 50 μl of a 4 % paraformaldehyde solution on top of the agarose overlay . after overnight fixation at 4 ° c ., the overlay medium and paraformaldehyde solution were removed , the cells were washed twice with pbs and blocked with pbs containing 1 % bsa ( pbs / bsa ). subsequently , polyclonal goat anti - rsv serum ( ab1128 , chemicon international ) was added ( 1 / 4000 ). after washing three times with pbs / bsa , the cells were incubated with hrp - conjugated anti - goat igg antibodies ( sc2020 , santa cruz ) for 30 minutes . non - binding antibodies were removed by washing four times with pbs / bsa containing 0 . 01 % triton ® x - 100 and once with pbs . finally , the plaques were visualized by the use of trueblue peroxidase substrate ( kpl , gaithersburg ). the plaques of different dilutions were counted and , for each dilution , the number of pfu per lung ( 1 ml ) was calculated as : number of plaques present in the dilution × the dilution × 20 (= 1000 μl total supernatant volume / 50 μl of the volume of supernatant used to infect the first well of the dilution series ). the number of pfu / lung was then calculated as the average number of pfu / lung calculated for the different dilutions . as each supernatant of the homogenized lungs was tested in duplicate , the final number of pfu / lung was calculated as the average of these duplicates . to determine the lung rsv load by qrt - pcr , lung homogenates were prepared and clarified as described above . total rna from these lung homogenates was prepared by the use of the high pure rna tissue kit ( roche , mannheim ) according to the manufacturer &# 39 ; s instructions . cdna was prepared by the use of hexamer primers and the transcriptor first strand cdna synthesis kit ( roche , mannheim ). the relative levels of genomic rsv m cdna were determined by the use of qrt - pcr using primers specific for the genomic rna of the rsv a2 m - gene ( 5 ′ tcacgaaggctccacataca3 ′ ( seq id no : 45 ) and 5 ′ gcagggtcatcgtctttttc3 ′ ( seq id no : 46 )) and a nucleotide probe (# 150 universal probe library , roche ) labeled with fluorescein ( fam ) at the 5 ′- end and with a dark quencher dye near the − 3 ′ end . the relative amount of gadph mrna was determined by qrt - pcr using primers specific for mouse gadph ( 5 ′ tgaagcaggcatctgaggg3 ′ ( seq id no : 47 ) and 5 ′ cgaaggtggaagagtgggag3 ′ ( seq id no : 48 ) and lightcycler ® 480 sybr ® green i master mix ( roche ). the relative amount of genomic rsv rna per lung homogenate was calculated as the ratio between the relative amount of rsv m - gene rna and the relative amount of mouse gadph mrna . two weeks after each immunization , blood samples were collected from the lateral tail vein . the final bleeding was performed by cardiac puncture of animals anesthetized with avertin . blood was allowed to clot for 30 minutes at 37 ° c ., and serum was obtained by taking the supernatant from two subsequent centrifugations . serum antibody titers were determined by elisa using pooled sera from the group . to determine m2e or she - specific antibody titers , microtiter plates ( type ii f96 maxisorp , nunc ) were coated with , respectively , 50 μl of a 2 μg / ml m2e - peptide solution or 2 μg / ml she - peptide solution in 50 mm sodium bicarbonate buffer , ph 9 . 7 , and incubated overnight at 37 ° c . after washing , the plates were blocked for one hour with 200 μl of 1 % bsa in pbs . after a one - hour incubation , the plates were washed again . a series of 1 / 3 dilutions of the different serum samples , starting with a 1 / 100 dilution , were loaded on the peptide - coated plates . the bound antibodies were detected with a peroxidase - labeled antibody directed against mouse isotypes igg1 or igg2a ( southern biotechnology associates , inc ., birmingham , ala ., usa ) and diluted 1 / 6000 in pbs + 1 % bsa + 0 . 05 % tween ® 20 . after washing , the microtiter plates were incubated for five minutes with tmb substrate ( tetramethylbenzidine , sigma - aldrich ). the reaction was stopped by addition of an equal volume 1 m h3po4 and the absorbance at 450 nm was measured . endpoint titers are defined as the highest dilution producing an o . d . value twice that of background ( pre - immune serum ). hek293t cells were transfected with the indicated expression vectors . twenty - four hours later , the cells were detached using enzyme - free dissociation buffer ( invitrogen , carslbad , calif . ), washed once with pbs and incubated for one hour in pbs containing 1 % bsa ( pbs / bsa ). subsequently , the cells were incubated with the indicated serum or antibodies at the indicated concentrations . one hour later , the cells were washed three times with pbs / bsa and incubated with the anti - mouse igg alexa 633 secondary antibodies for 30 minutes . after washing the cells four times with pbs / bsa and once with pbs , the cells were analyzed using a becton dickinson lsr ii flow cytometer . single gfp - expressing cells were selected based on the peak surface of the sideward scatter signal , the peak surface and peak height of the forward scatter signal and the peak surface of the green fluorescence signal . finally , of these gfp - positive single cells , the alexa 633 fluorescence signal was measured . vero cells were either mock infected or infected with 0 . 5 moi of rsv a2 in the presence of serum - free medium . four hours later , the free virus was washed away and the cells were incubated in growth medium containing 1 % fcs . sixteen hours later , the cells were washed once with pbs and fixed with 2 % paraformaldehyde for 20 minutes . subsequently , the cells were washed twice with pbs and permeabilized with 0 . 2 % triton ® x - 100 detergent for five minutes . after washing once with pbs , the cells were blocked in pbs / bsa . one hour later , she - specific 3g8 monoclonal antibody or isotype control antibody was added at a final concentration of 5 μg / ml . after washing the cells twice with pbs / bsa , polyclonal anti - rsv goat serum was added . one hour later , the cells were washed three times with pbs / bsa . the binding of the indicated antibodies to the cells was analyzed by the use of anti - mouse and anti - goat igg antibodies labeled with , respectively , alexa 488 and alexa 568 fluorescent dyes . confocal images of the stained cells were recorded with a zeiss confocal microscope . stable hybridomas cells producing she - specific monoclonal antibodies ( mab ) were generated by hybridoma technology ( kohler and milstein 1975 ). briefly , she - specific hybridomas were derived from individual mice that were immunized i . p . three times at three - week intervals with 10 μg of she - tgcn4 vaccine adjuvanted with alhydrogel ® ( brenntag biosector ). three days before fusion , mice were boosted an additional time with the same formulation and splenocytes were isolated then fused to sp2 / 0 - ag14 myeloma cells in the presence of peg 1500 ( roche diagnostics gmbh , germany ). fused cells were grown in rpmi 1640 medium supplemented with 10 % fetal bovine serum , 10 % bm condimed h1 ( roche diagnostics gmbh , germany ), 2 mm l - glutamine , and 24 μm beta - mercaptoethanol and 1 × hat supplement ( invitrogen , carlsbad , calif .). hybrids secreting she - specific antibodies were identified by she peptide elisa screening and monoclonal antibodies producing hybrids were obtained after two rounds of sub - cloning by limiting dilution procedure . monoclonal antibodies were purified on a protein a - sepharose column ( electrical engineering biosciences ). the resulting hybridomas were deposited under the budapest treaty at bccm ( bccm / lmbp : technologiepark 927 , 9052 zwijnaarde , belgium ) under deposit numbers lmbp 7795cb for 3g8 on 8 nov . 2010 and lmbp 7796cb for 3d11 on 10 nov . 2010 , respectively . the sh protein is expressed at the surface of rsv virions and the plasma membrane of rsv - infected cells as a pentamer . the pentameric organization of sh is organized by the sh transmembrane domain , which oligomerizes as a coiled coil of five parallel alpha - helices . in order to present the c - terminal sh ectodomain ( she ) of rsv a as a pentamer that mimics its natural conformation , she was genetically fused to the short pentameric coiled coil domain of the rat cartilage oligomeric matrix protein ( compcc ), which is also composed of five parallel alpha - helices ( malashkevich et al ., 1996 ; fig1 ). a flag - tag was fused to the n - terminus of comp , rendering flag - compcc - she . flag - compcc - she was cloned in a plt - 32 ( mertens et al ., 1995 ) _expression vector , expressed in e . coli and purified . gel filtration analysis revealed that flag - compcc - she eluted as a 55 - 60 kda complex , indicating that the 11 kda flag - compcc - she proteins do indeed oligomerize into pentamers ( fig2 ). to test if vaccination with flag - compcc - she could evoke protection against rsv infection , we used a balb / c mouse rsv infection model . balb / c mice were immunized three times intranasally with 25 μg of flag - compcc - she in combination with 1 μg e . coli heat - labile enterotoxin ltr192g adjuvant . pbs and the influenza a m2 ectodomain fused to a tetrameric gnc4 scaffold ( m2e - tgnc4 ) ( de filette et al ., 2008 ) were used as negative controls . immunizations were performed every fortnight . a single rsv infection ( 5 × 10 5 pfu ) was used as positive control . between the first and the second week after each immunization , blood was collected to investigate the induction of she - specific igg antibodies . the presence of she - specific antibodies was first tested by she peptide elisa . m2e peptide elisa was used as negative control . fig3 demonstrates that she peptide - specific igg antibodies are induced and boosted after , respectively , the second and third immunization with flag - compcc - she . three successive flag - compcc - she / ltr192g immunizations resulted in high levels of igg2a she - specific antibodies but only low levels of igg1 she - specific antibodies , indicating a th1 - oriented / driven immune response . no she - specific igg antibodies could be detected in pbs or m2e - tgcn4 / ltr192g vaccinated mice ( fig3 , panels a , b and c ). as expected , no m2e - specific antibodies could be detected in the sera of flag - compcc - she / ltr192g or pbs vaccinated mice data . mice that were immunized with m2e - tgcn4 accumulated a high titer of m2e - specific igg2a antibodies , in accordance with previous results ( de filette et al ., 2008 ). next , we investigated if she - specific antibodies present in the flag - compcc - she immune serum could bind to cells expressing the rsv - sh protein at their surface by flow cytometry . hek - 293t cells were transfected with a gfp expression vector , in combination with either a sh expression vector ( pcaggs - etag - sh ) or a luciferase expression vector ( pcaggs - luc ) as negative control . twenty - four hours after transfection , the cells were detached , stained with different dilutions of flag - compcc - she or m2e - tgcn4 immune serum and analyzed by flow cytometry . fig4 illustrates that , in contrast to m2e - tgcn4 immune serum , serum from flag - compcc - she - vaccinated mice specifically binds sh protein expressed at the surface of living cells . to test if flag - compcc - she / ltr192g vaccination can elicit protection against rsv infection , the mice were challenged with 1 × 10 6 pfu rsv a2 nine weeks after the last immunization . four days after infection , the mice were sacrificed to determine the viral lung titer by plaque assay . fig5 illustrates that compared to pbs - and m2e - tgcn4 - vaccinated mice , vaccination with flag - compcc - she lowered rsv replication . no virus was detected in the mouse that was infected with living rsv before challenge . vaccination with formalin - inactivated virus or the rsv g protein can induce enhancement of disease upon infection , resulting in significant morbidity , by the induction of an unbalanced th2 immune response ( prince et al ., 1986 ). to test if flag - compcc - she vaccination might also induce enhancement of disease , we monitored the body weight before and after rsv challenge ( fig6 ). no weight loss was observed in any of the mouse groups after rsv challenge . this strongly suggests that flag - compcc - she vaccination does not result in enhancement of disease upon rsv infection . the hepatitis b virus core protein ( hbc ) virus - like particle ( vlp ) can present antigens as a dense array . in this way , hbc - vlps can induce a strong humoral immune response toward the presented antigen ( boisgerault et al ., 2002 ). therefore , as an alternative to presenting she as a pentamer , the sh ectodomain was presented in the immunodominant region loop of mhbc - vlps . hbc - she - vlps were obtained by chemical linkage of she peptides to mhbc , a mutant of hbc in which a lysine was introduced in the top of the hbc immunodominant region ( de filette et al ., 2005 ). to enable chemical linking , a cysteine residue was added to the n - terminus of she . in addition , the cysteine residue , present at position 4 of the she peptide , was replaced by a serine residue . this peptide was called she - cc4s . after purification of the mhbc - she - vlps , by size exclusion chromatography , the degree of cross - linking was examined by sds page . fig7 illustrates that approximately 50 % of the hbc proteins is chemically linked to a she - cc4s peptide . the slower migrating bands likely represent mhbc monomers to which two or three she ( cc4s ) peptides were linked . to test if shecc4s - linked mhbc proteins still assemble into vlps of the expected size ( 30 - 34 nm ), dynamic light scattering analyses was performed on the generated mhbc - she particles and the 1604 m2e - hbc vlp as fully functional reference . fig8 illustrates that the size distribution of mhbc - she - cc4s overlaps with that of the 1604 m2e - hbc control , with a maximum at 30 nm , which corresponds with the reported size of hbc vlps ( clarke et al ., 1987 ). next to presenting the she peptide at the surface of mhbc vlps , she was also fused to tgcn4 , which is known to induce a strong humoral response toward fused peptides ( ref marina gcn4 ). she and a flag - tag were genetically linked to , respectively , the 5 ′- end and the − 3 ′ end of the tgcn4 coding sequence and cloned into a plt32 expression vector . after expression in e . coli , recombinant she - tgcn4 - flag was purified by anion exchange , hydrophobic interaction and gel filtration chromatography ( fig9 ). mhbc - she ( cc4s ) and she - tgcn4 vaccination induces she - specific antibodies and protection against rsv infections to test if vaccination with mhbc - she ( cc4s ) and she - tgcn4 can evoke protection against rsv infections , balb / c mice were vaccinated three times intranasally with 10 mhbc - she ( cc4s ) and she - tgcn4 in combination with 1 μg ltr192g adjuvant . pbs and empty mhbc , the latter in combination with 1 μg ltr192g , were used as negative controls . immunizations were performed every three weeks . a single rsv infection ( 5 × 10 5 pfu ) was used as positive control . between the second and the third week after each immunization , blood was collected to investigate the induction of she - specific igg antibodies . the presence of she - specific antibodies was tested by she peptide elisa . fig1 , panel a , demonstrates that she peptide - specific igg antibodies are induced and boosted after , respectively , the second and third immunization with mhbc - she ( cc4s ) and she - tgcn4 . three successive flag - compcc - she / ltr192g immunizations resulted in high levels of igg2a she - specific antibodies and somewhat lower levels of igg1 she - specific antibodies , indicating a th1 - oriented / driven immune response ( fig1 , panel b ). to test if vaccination with mhbc - she ( cc4s ) or she - tgcn4 can hamper rsv infection , the mice were challenged with 5 × 10 6 pfu rsv a2 three weeks after the last boost immunization . three days after challenge , the mice were sacrificed to determine the pulmonary rsv a2 levels by qpcr . fig1 shows that all mice that were vaccinated with mhbc - she ( cc4s ) or she - tgcn4 or mice that were infected beforehand with rsv , have lower pulmonary levels of genomic rsv rna than mice that were vaccinated with mhbc . these data confirm our previous observation that mucosal she - based vaccination can partially protect mice against rsv replication . remarkably , all mice that were vaccinated with an empty mhbc in combination with the ltr192g adjuvant , displayed lower levels of rsv than mice that were immunized with pbs without ltr192g adjuvant . this might be explained by the effect of ltr192g on the mouse innate immune system . the e . coli heat labile entertoxin has been shown to provide generic protection against lung viral infections , including rsv , via innate imprinting ( ref williams and hussel 2004 ). the effect of innate imprinting by ltr192g on lung viral replication appears to be transient as the impact of tlr192r on rsv replication is strongly reduced when viral infection occurs nine weeks after the last ltr192g administration . again , none of the mice showed significant body weight loss , indicating that vaccination with she when presented by vlps or tgcn4 is not inducing enhancement of disease upon challenge ( fig1 ). to investigate if she - specific antibodies that can interact with infected cells can provide protection against rsv infections , we developed rsv she - specific monoclonal antibodies based on she - tgcn4 immunized mice . one igg1 ( 3d11 ) and one igg2a ( 3g8 ) subtype hybridoma that produced antibodies that efficiently bound to she peptide in an elisa were selected , subcloned and used for antibody production . the 3d11 and 3g8 were purified via protein a affinity chromatography and tested for binding efficacy to she via an elisa . fig1 shows that 3d11 and 3g8 can bind to coated she peptide and are , respectively , of the igg1 and igg2a subtype . as antibodies can protect against viral infections via recognition and killing of infected cells by ( adcc ) or cdc , we investigated if the she - specific mabs 3d11 and 3g8 can recognize sh at the surface of cells . therefore , hek293t cells were transfected with an rsv sh expression vector or with a control firefly luciferase vector ( schepens et al ., 2005 ), both in combination with a gfp expression vector . twenty - four hours after transfection , live cells were stained with different concentrations of the she - specific monoclonal antibodies ( 3d11 and 3g8 ) or isotype matched influenza m2e - specific antibodies ( 14c2 igg1 and a ig2a m2e - specific mab ). polyclonal serum from flag - compcc - she - immunized mice was used as positive control . fig1 demonstrates that flag - compcc - she polyclonal serum , along with both 3d11 and 3g8 mabs , can readily bind to sh - expressing cells but not to control cells . in contrast , the igg1 and igg2a influenza m2e - specific antibodies could not bind to sh - expressing cells . these data clearly demonstrate that both 3d11 and 3g8 can recognize the ectodomain of sh expressed at the surface of cells . during infection , the rsv sh protein is mainly expressed at the er , golgi and cell membrane . in order to more directly investigate whether the rsv sh - specific antibodies can recognize infected cells via sh expressed at the surface of these cells , we performed immunostaining of rsv - infected and mock - infected cells . human a594 lung epithelial cells were either infected with 0 . 05 moi of rsv or mock infected . twenty - four hours after infection , the cells were fixed and stained with the she - specific mabs 3d11 or 3g8 in combination with polyclonal anti - rsv immune serum . fig1 illustrates that the she - specific mabs 3d11 and 3g8 can readily recognize sh at the cell membrane and near the nucleus ( likely corresponding to er and golgi ) of infected cells . this indicates that she mabs protect against rsv infection by recognizing rsv - infected cells . in this way , the herein - described she mabs 3d11 and 3g8 can be used as prophylactic or therapeutic treatment . to test if she - specific antibodies can reduce rsv replication in vivo , mice were passively immunized with she - specific monoclonal antibodies . she - specific 3g8 monoclonal antibodies , isotype control antibodies or pbs were intranasally administered to mice one day before and one day after rsv challenge . three days after rsv challenge , blood was collected to test for the presence of mabs in the serum of the treated mice . four days after rsv challenge , the mice were sacrificed to determine the viral titer in the lungs . peptide elisa demonstrated the presence of low concentrations of she - specific and isotype control antibodies in the serum of mice treated with the respective antibodies ( data not shown ). fig1 illustrates that mice that received she - specific monoclonal antibodies have reduced lung rsv titers as compared with mice that were treated with pbs or isotype control monoclonal antibodies . these data suggest that intranasal administration of she - specific antibodies can reduce rsv infection in mice . to test if she - based vaccines can also protect against rsv infections when this vaccine is administered via an alternative route with an alternative adjuvant and with a different carrier , the vaccine was tested intraperitoneally , with keyhole limpet hemocyanin ( klh ) as a carrier . maleimide - activated klh ( pierce ) was chemically linked to the peptide ( cgggs nklseynvfhnktfelprarvnt ( seq id no : 50 ); the sequence corresponding to the rsv a sh ectodomain ( she ) is underlined ) corresponding to the rsv a sh ectodomain . to promote directional chemical linking , a cysglyglyglyser ( seq id no : 55 ) linker was added to the n - terminus of the rsv a she peptide . in addition , the cysteine residue present in the natural rsv a she was substituted by a serine residue . chemical linkage was performed according to the manufacturer &# 39 ; s instructions ( pierce ). cross - linked klh - she proteins were isolated by size exclusion chromatography . to test if intraperitoneal ( i . p .) vaccination with a she - based vaccine can evoke protection against rsv infections , balb / c mice ( six mice per group ) were vaccinated three times intraperitoneally with 20 μg of klh - she or klh , each in combination with 50 μl of freund &# 39 ; s incomplete adjuvant ( millipore ). pbs vaccination without adjuvant was used as an additional negative control . between the second and third week after vaccination , blood was collected to determine the induction of she - specific igg antibodies . the presence of she - specific antibodies was determined and quantified by she peptide elisa . fig1 ( panels a and b ) demonstrate that three successive vaccinations with klh - she induces high levels of she - specific igg antibodies of both the igg1 and igg2a subtype . no she - specific igg antibodies could be detected in sera from pbs - or klh - vaccinated mice . in addition , flow cytometric analysis revealed that serum derived from mice that had been vaccinated intraperitoneally with klh - she can specifically bind to hek293t cells that express the rsv sh protein at their surface , whereas pre - immune serum did not . to test whether intraperitoneal klh - she vaccination can reduce rsv infection , the vaccinated mice were infected with 1 × 10 6 pfu of rsv a2 four weeks after the last vaccination . five days after challenge , the mice were sacrificed to determine the pulmonary rsv a2 titer by plaque assay . fig1 , panel d , illustrates that significantly less virus could be detected in the lungs of she - klh - vaccinated than in the lungs of klh - vaccinated mice ( p & gt ; 0 . 005 , mann - whitney u test ). the observation that among klh - she - vaccinated mice , higher titers of serum she - specific igg antibodies strongly correlated ( r 2 = 0 . 95 ) with lower levels of pulmonary rsv at day 5 post - infection , suggests that reduction of rsv replication by klh - she vaccination is mediated by she - specific antibodies ( fig1 , panel e ). the body weight of all mice was monitored at the day of infection and the day of sacrifice . fig1 , panel c , illustrates that mice that were vaccinated with klh - she gained significantly more weight than mice that were vaccinated with klh ( p & gt ; 0 . 005 , mann - whitney u test ). these data demonstrate that intraperitoneal vaccination with a she - based vaccine can reduce rsv replication without inducing morbidity . in addition , these data illustrate that next to mhbc , tgcn4 and compcc , klh can also be used as a protein carrier for she peptide - based vaccines . moreover , these data illustrate that next to titermax ®, also freunds &# 39 ; incomplete adjuvant can also be used as an appropriate adjuvant to induce she - specific immunity . to test if intranasal vaccination with klh - she can evoke protection against rsv infections , balb / c mice ( six mice per group ) were vaccinated three times intranasally with 20 μg of klh - she or klh , each in combination with 1 μg of ltr192g adjuvant . pbs vaccination without adjuvant was used as an additional negative control . between the second and third week after vaccination , blood was collected to investigate the induction of she - specific igg antibodies . the presence of she - specific antibodies was tested by she peptide elisa . fig1 ( panels a and b ) demonstrate that three successive vaccinations with klh - she induce she - specific igg antibodies of both the igg1 and igg2a subtype . no she - specific igg antibodies could be detected in sera from pbs - or klh - vaccinated mice . in addition , flow cytometric analysis revealed that serum derived from mice that were vaccinated intranasally with klh - she serum , but not pre - immune serum , can specifically bind to hek293t cells that express the rsv sh protein at their surface . to test whether intraperitoneal klh - she vaccination can reduce rsv infection , the vaccinated mice were infected with 1 × 10 6 pfu of rsv a2 nine weeks after the last vaccination . five days after challenge , the mice were sacrificed to collect bal ( broncho alveolar lavage ) fluid ( 3 ml ). the rsv a2 titer in the collected bal fluids was determined by plaque assay . fig1 , panel e , illustrates that significantly less virus could be detected in the lungs of klh - she - vaccinated mice than in the lungs of klh - vaccinated mice ( p & gt ; 0 . 05 , mann - whitney u test ). the presence of she - specific iga and igg antibodies in the collected bal fluids was analyzed by she peptide elisa . this analysis revealed that in contrast to pbs - and klh - vaccinated mice , the bal fluids of mice vaccinated with klh - she contained both igg and iga she - specific antibodies ( fig1 , panels c and d ). the levels of igg she - specific antibodies present in the bal fluid of klh - she - vaccinated mice correlated with the levels of igg she - specific antibodies in the serum of the respective mice . the observation that among klh - she - vaccinated mice , higher titers of she - specific igg antibodies present in the bal fluid strongly correlate ( r 2 = 0 . 97 ) with lower levels of pulmonary rsv titers on day 5 post - infection , suggests that reduction of rsv replication by klh - she vaccination is mediated by she - specific antibodies ( fig1 , panel f ). these data demonstrate that intranasal vaccination with a she - based vaccine can reduce rsv replication without inducing morbidity . in addition , these data confirm that next to mhbc , tgcn4 and compcc , klh can also be used as a protein carrier for she peptide - based vaccines . passive transfer of klh - she immune serum protects against rsv infection in mice to further investigate if the reduction in rsv replication in mice that have been vaccinated with a she - based vaccine can be mediated by rsv she - specific antibodies , passive transfer experiments were performed . balb / c mice were vaccinated intraperitoneally with 20 μg of either klh - she or klh , both in combination with 75 μl of freund &# 39 ; s incomplete adjuvant . as an additional negative control , mice were vaccinated with pbs without adjuvant . she peptide elisa illustrated that the sera of all mice that had been vaccinated with klh - she contains high levels of she - specific igg antibodies . after final bleeding , the sera of the mice of each group were pooled and heat inactivated at 56 ° c . for 30 minutes . to test if klh - she sera can protect against rsv infections , 40 μl of klh or klh - she sera were administered to mice intranasally one day before ( day − 1 ) and one day after ( day 1 ) rsv challenge ( 2 × 10 5 pfu ) ( day 0 ). mice that were treated with pbs were included as additional controls . the weight of all mice was monitored daily ( fig1 , panel c ). five days post - infection , the mice were sacrificed to prepare lung homogenates . plaque assay analysis demonstrated that the lung homogenates of mice that had been treated with klh - she serum contained about 40 times less ( ratio of means of viral titers ) replicating virus than the lung homogenates originating from mice treated with klh serum ( fig1 , panel b ). the observation that the pulmonary rsv titer of mice that were treated with klh serum did not differ from the pulmonary rsv titer of mice that were treated with pbs , illustrates that administration of control serum does not impact pulmonary rsv replication in mice . although highly conserved within their subtype , the she amino acid sequences of rsv b viruses differs from that of the rsv a subtype viruses . therefore , to protect against rsv b viruses , a she - based vaccine most likely needs to include the rsv b she amino acid sequence . a rsv b she vaccine was constructed by chemically linking the consensus rsv b she peptide ( sheb : cgggsnklsehktfsnktleqgqmyqint ( seq id no : 51 ) to the mhbc virus - like particles . to promote chemical linking , a cysglyglyglyser ( seq id no : 55 ) linker was added to the n - terminus of the rsv b she peptide . in addition , the cysteine residue present in the natural rsv b she was substituted by a serine residue . the immunogen resulting from chemical linkage of the rsv b she peptide to mhbc was named mhbc - sheb . after purification of the mhbc - sheb vlps by size exclusion chromatography , the degree of cross - linking was analyzed by sds - page gel electrophoresis and coomassie staining . fig2 illustrates that more than half of the hbc monomers are cross - linked to at least one she peptide . immunization of mice with mhbc - sheb induces sheb - specific abs that bind to the surface of rsv b - infected cells to test whether mhbc - sheb vlps were immunogenic , one balb / c mouse was immunized three times subcutaneously with 20 μg of mhbc - sheb combined with 50 titermax ® ( sigma ). the three immunizations were performed with two - week intervals . bleedings were performed one day before each immunization and two weeks after the final immunization . to test whether mhbc - sheb immune serum can recognize rsv b sh proteins expressed on the surface of infected cells , vero cells were either mock infected or infected with a clinical isolate of rsv b virus ( kindly provided by dr . marc van ranst , university of leuven , leuven , belgium ). seventy - two hours after infection , the cells were fixed and either permeabilized using 0 . 2 % triton ® x - 100 or not permeabilized . the cells were then stained with either mhbc - sheb immune serum ( 1 / 100 dilution ) or control immune serum ( 1 / 100 dilution ) derived from balb / c mice that had been vaccinated with klh ( klh serum ) in combination with freund &# 39 ; s incomplete adjuvant . the samples were analyzed by immunofluorescent microscopy or flow cytometry . fig2 , panels a and b , illustrate that mhbc - sheb immune serum can bind to both permeabilized and non - permeabilized rsv b - infected cells but not to non - infected cells . in contrast , control immune serum did not bind to rsv b - infected cells . this demonstrates that vaccination of mice with mhbc - sheb induces serum antibodies that can recognize rsv b - infected cells , most likely by binding to the rsv b sh protein that is expressed at the surface of rsv b - infected cells . to test whether mhbc - sheb vaccination can protect mice from rsv b infection , two groups of six mice were immunized with mhbc or mhbc - sheb vlps , adjuvanted with 50 μl of freund &# 39 ; s incomplete adjuvant . as additional controls , six mice were vaccinated with pbs . vaccinations were performed intraperitoneally , three times with three - week intervals . bleedings were performed two weeks after each immunization . the induction of she - specific antibodies was determined by peptide elisa using shea or sheb as coating peptides . this analysis demonstrated that in all mice , three successive mhbc - sheb immunizations induced high titers of rsv b she - specific igg antibodies of both igg1 and igg2a subtype ( fig2 , panels a - c ). mhbc - sheb immune serum also bound to the shea peptide but to a much lower extent ( fig2 , panels a , b and d ). previous experiments in our and other laboratories have illustrated that no or very little replicating virus can be rescued from rsv b - infected mice . nevertheless , we could observe that infections with clinical rsv β isolates induce pulmonary inflammation and weight loss in balb / c mice ( data not shown ). therefore , we tested whether mhbc - sheb vaccination could protect mice from rsv b - induced pulmonary inflammation . six days after intranasal challenge of mice with 2 × 10 6 pfu of an rsv b clinical isolate , broncho alveolar lavage ( bal ) was performed . mock - infected mice were used as negative control for analysis of bal cell infiltration . the bal fluid was analyzed for immune cell infiltration by flow cytometry as described in bogaert et al ., 2011 . fig2 , panels e and f , show that rsv b infection results in pulmonary infiltration of immune cells , especially cd8 + t lymphocytes , which are known to be responsible for rsv - induced morbidity in mice . however , compared to pbs - or mhbc - vaccinated mice , mhbc - sheb - vaccinated mice displayed significantly lower pulmonary cell infiltration . these data demonstrate that mhbc - sheb vaccination reduces rsv - related immune pathology . as an alternative protein scaffold to present she as a pentamer , we used the pentameric tryptophan - zipper described by liu et . al . ( lpp ( 5 ) ), which is derived from the e . coli lpp - 56 lipoprotein ( liu et al ., 2004 ). the coding sequence of the lpp ( 5 ) tryptophan - zipper was genetically fused to the she coding sequence and cloned into an e . coli expression vector ( plh36 ) containing a hexahistidine peptide and a caspase cleavage site as described by mertens et al ., 1995 . this expression plasmid was named plh36 - hisdevd - lpp - she ( seq id no : 49 ). expression from this plasmid renders the chimeric lpp ( 5 ) - she protein ( seq id no : 52 ) ( m hhhhhh pggs devd akwdqwssdwqtwnakwdqwsndwnawrsdwqawk ddwarwnqrwdnwatggnklceynvfhnktfelprarvnt ( seq id no : 52 ), his - tag sequence is underlined , linkers are in italic , devd caspase cleavage site is in italic + underlined , pentameric lpp tryptophan - zipper is in bold and the rsv a sh ectodomain is in bold + italic ). after induction of expression in e . coli , the lpp ( 5 ) - she protein was purified by subsequent nickel affinity , anion - exchange and gel filtration chromatography . fig2 demonstrates that the lpp ( 5 ) - she protein can be recognized by she - specific 3g8 monoclonal antibodies , both in a crude cell extract ( fig2 , panel a ) and as a purified protein ( fig2 , panel b ). in order to prove the efficacy of the vaccine in an independent animal model , cotton rats are used . cotton rats ( sigmondon hispidus ) are susceptible to rsv infection ( prince et al ., 1978 ). five groups of six cotton rats each are used . two groups of animals are immunized intraperitoneally ( i . p .) with 100 μg of klh ( vehicle control ) or 100 μg of klh - she ( i . e ., a chemical conjugate of she peptide derived from rsv - a with klh as a carrier ). klh and klh - she vaccine antigens are formulated with freund &# 39 ; s incomplete adjuvant and used to immunize cotton rats on days 0 , 21 , and 42 . a third group of animals is immunized intramuscularly with formalin - inactivated rsv ( fi - rsv ) in the presence of alum adjuvant . the latter group serves as a positive control for the induction of vaccine - enhanced disease that becomes apparent upon subsequent challenge with rsv . a fourth group is infected with 2 . 04 × 10 5 plaque forming units per cotton rat of rsv - tracy on day 0 and serves as positive control for protection against subsequent challenge . a fifth group of cotton rats remains untreated until the day of challenge and served as control for the challenge with rsv . the schedule of the vaccination is shown in fig2 . sera are collected before each immunization and on the day of challenge . on day 63 , cotton rats are challenged intranasally with 2 . 04 × 10 5 plaque forming units of rsv - tracy . the challenge virus is administered intranasally in a volume of 100 microliters while the animals are lightly anesthetized with isofluorane . on day 68 , serum is collected and all animals are sacrificed to collect lungs for virus titration and histopathological analysis . each lung is divided in two to perform histopathological analysis and virus titration . the left lungs are tied off and used for histopathological analysis . the lobes of the right lung are lavaged using 3 ml of iscove &# 39 ; s media with 15 % glycerin . the lavage fluid is stored on ice until titration . in addition , nasal lavages are prepared with 2 ml ( 1 ml for each nare ) in the same medium . the viral load in the lung and nasal lavages is determined by plaque assay on hep2 cells . cells are infected for 90 minutes with a serial dilution of the lavage samples . after removal of the inoculum , the cells are overlaid with 2 % methylcellulose in mem - containing antibiotics . after six days of incubation at 37 ° c . in a co 2 - incubator , plaques are counterstained with 0 . 1 % crystal violet / 10 % formalin solution and left at room temperature for 24 hours . for histopathological analysis , the left lung is perfused with 10 % neutral buffered formalin . fixed lung tissue is subsequently processed with a microtome to produce sections that are stained with hematoxilin and eosin and scored for the degree of histopathological lesions . serum samples are assayed for the presence of anti - she - and anti - rsv - neutralizing antibodies by peptide elisa and by a microneutralization assay . for peptide elisa , plates are coated overnight at 37 ° c . with 2 μg of she - peptide in 50 μl of 0 . 1 m carbonate buffer ph 9 . 6 . after coating , plates are blocked with 3 % ( w / v ) milk powder in pbs , followed by application of three - fold serial dilutions on cotton rat sera . retained she - specific cotton rat igg are detected using horseradish peroxidase conjugated secondary antibodies and tetramethylbenzidine substrate . the endpoint anti - she peptide igg titer in the samples is defined as the highest dilution for which the absorbance is at least twice as high as that of the pre - immune serum . neutralizing antibody titers are determined for rsv - a and - b in 96 - well microtiter plates with hep2 cells . serial dilutions of serum samples are mixed with a fixed amount of inoculum virus . the neutralizing antibody titer is defined as the serum dilution at which & gt ; 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