Patent Application: US-201715465312-A

Abstract:
the disclosure relates to a glycoconjugate vaccine conferring protection against francisella tularensis infections and a method to manufacture a glycoconjugate antigen .

Description:
escherichia coli strains were grown in lb at 37 ° c ., 180 r . p . m . antibiotics were used at the following concentrations ; tetracycline 20 μg / ml , ampicillin 100 μg / ml , spectinomycin 80 μg / ml , chloramphenicol 30 μg / ml . the host strain for initial cloning experiments was e . coli xl - 1 , subsequent strains used for glycoconjugate production were e . coli dh5α and clm24 ( table 1 ). for efficacy studies , mice were challenged with f . tularensis subsp . holarctica strain hn63 . the bacterium was cultured on blood cysteine glucose agar plates ( supplemented with 10 ml of 10 % ( wt / vol ) histidine per litre ) at 37 ° c . for 18 hours . cloning , sequencing and expression of the f . tularensis o antigen coding region dna was prepared from the f . tularensis subsp . tularensis strain schus4 by phenol extraction as described by karlsson et al . ( 2000 ). the o - antigen coding region was amplified using the primers ftfragment2rev ( 5 ′- ggatcattaatagctaaatgtagtgctg - 3 ′; seq id 10 ) and oant1ftfwd ( 5 ′- ttttgaattctacaggctgtcaatggagaatg - 3 ′; seq id 11 ) using the following cycling conditions : 94 ° c ., 15 sec , 55 ° c ., 15 sec , 68 ° c ., 20 min ; 35 cycles using accuprime taqhifi ( invitrogen u . k .). this was cloned into the ta cloning vector pgem - t easy to generate the vector pgab1 . the plasmid pgab1 was digested with ecori and the insert was subcloned into the vector plafr to generate the construct pgab2 . immunofluorescence imaging of e . coli cells carrying f . tularensis o antigen coding region immunofluorescence was carried out as previously described [ 17 ] with the modification that the igg2a mouse monoclonal antibody fb11 was used to detect f . tularensis o antigen ( 1 μl / ml in 5 % ( v / v ) fcs / pbs ). e . coli clm24 carrying the vectors pgab2 , pgvxn114 and pgvxn150 was grown for 16 h in 200 ml lb broth at 37 ° c ., 180 r . p . m . this was used to inoculate 1 . 8 l of lb broth and further incubated at 110 r . p . m . 37 ° c . to an od . 600 nm reading of 0 . 4 to 0 . 6 . l - arabinose was then added to a final concentration of 0 . 2 % and iptg to a final concentration of 1 mm to induce expression of exoa and cjpglb respectively ; following 5 hours of incubation , 0 . 2 % l - arabinose was added again and the culture left to incubate o / n . cells were harvested by centrifugation at 6 , 000 r . p . m . for 30 m , and pelleted cells were incubated at room temperature for 30 m in a lysis solution composed of 10 × bugbuster protein extraction reagent ( novagen ) diluted to 1 × in 50 mm nah2po 4 , 300 mm nacl , 10 mm imidazole , ph 8 . 0 supplemented with 0 . 1 % tween , 1 mg / ml lysozyme and 1 μl / ml benzonase nuclease ( novagen ). cell debris was removed by centrifugation at 10 , 000 r . p . m . for 30 m , the supernatant was collected and 1 ml ni - nta agarose ( qiagen ) was added to the supernatant . the slurry - lysate was incubated for 1 h at 4 ° c . with shaking then loaded into 10 ml polypropylene columns ( thermo scientific ). his tagged exoa was purified according to manufacturer &# 39 ; s instructions ( qia expressionist , qiagen ) with the addition of 20 % glycerol and 5 % glucose to the elution buffer . protein yields were estimated using a bicinchonic acid assay kit according to manufacturer &# 39 ; s instructions ( pierce ® biotechnology bca protein assay kit , u . s . a .). for large - scale protein purification , material was isolated using ge healthcare his trap columns and an akta purifier with an imidazole gradient of 30 - 500 mm . the collected fraction containing exoa glycosylated with f . tularensis o - antigen was further purified using a resource q anionic exchange column ( ge healthcare ) with a nacl gradient from 0 to 500 mm in 20 mm trishcl ph 8 . 0 . this generated a typical yield of 2 - 3 mg / ml of glycoconjugate per 2 l of e . coli culture . the same techniques were used for the generation of the ‘ sham ’ c . jejuni heptasaccharide exoa glycoconjugate encoded by pacycpgl [ 18 ]. using the e . coli chromosomally inserted strain clm 24 cedapglb : escherichia coli strain clm24 with a chromosomally inserted copy of pgib were grown in luria - bertani ( lb ) broth at 37 ° c ., with shaking . antibiotics were used at the following concentrations : tetracycline 20 μg ml − 1 and ampicillin 100 μg ml − 1 . tetracycline was used to maintain the plasmid pgab2 coding for francisella tularensis o antigen and ampicillin was used to maintain the plasmid coding for the acceptor carrier protein . escherichia coli cells were grown for 16 h in 200 ml lb broth at 37 ° c ., with shaking . this was used to inoculate 1 . 8 l of lb broth and further incubated with shaking at 37 ° c . until an od600 reading of 0 . 4 - 0 . 6 was reached . at this point l - arabinose was added to a final concentration of 0 . 2 per cent and iptg to a final concentration of 1 mm to induce expression of the acceptor protein and pgib , respectively ; after another 5 h of incubation , 0 . 2 % l - arabinose was added again and the culture left to incubate overnight . cells were harvested by centrifugation at 5300 g for 30 min , and pelleted cells were incubated at room temperature for 30 min in a lysis solution composed of 10 × bugbuster protein extraction reagent ( novagen ) diluted to 1 × in 50 mm nah 2 po 4 , 300 mm nacl , 10 mm imidazole , ph 8 . 0 supplemented with 0 . 1 per cent tween , 1 mg ml − 1 lysozyme and 1 μl ml − 1 benzonase nuclease ( novagen ). cell debris was removed by centrifugation at 7840 g for 30 min , the supernatant was collected and 1 ml ni - nta agarose ( qiagen ) was added to the supernatant . the slurry - lysate was incubated for 1 h at 4 ° c . with shaking then loaded into 10 ml polypropylene columns ( thermo scientific ). his - tagged exoa was purified by the addition of an elution buffer according to manufacturer &# 39 ; s instructions ( qia expressionist , qiagen ) containing 250 mm imidazole with the addition of 20 per cent glycerol and 5 per cent glucose . alternatively cells were grown in lb agar plates containing tetracycline , ampicillin , iptg to a final concentration of 50 μm and l - arabinose to a final concentration of 0 . 2 % for 16 h at 37 ° c . cells were subsequently harvested by scraping and protein purified as indicated above . to verify transfer and presence of the f . tularensis o antigen , samples were analysed by western blotting . e . coli cells were grown o / n in 10 ml lb broth and diluted to an o . d . 600 nm of 1 . 0 . cells were centrifuged at 13 , 000 r . p . m . for 10 min , supernatant was removed and cells were resuspended in 100 μl laemmli buffer and lysed by boiling for 10 min before analysis by western blotting or silver staining . mouse anti f . tularensis o - antigen monoclonal antibody fb011 ( abcam u . k .) was used at a dilution of 1 : 1 , 000 , rabbit anti his monoclonal antibody was used to detect exoa at a dilution of 1 : 10 , 000 ( abcam u . k .). secondary antibodies used were goat anti mouse irdye680 and irdye800 conjugates used at 1 : 5000 dilutions . signal detection was undertaken using the odyssey ® li - cor detection system ( li_cor biosciences gmbh ). spleen supernatants were assessed using mouse inflammatory cytometric bead array kit ( cba - bd biosciences ) for il - 10 , il - 12p70 , ifn - γ , il - 6 , tnf - α , and mcp - 1 . samples were incubated with the combined capture bead cocktail , and incubated for 1 h at room temperature . following incubation , pe detection antibodies were added and incubated for a further 1 h . samples were then washed and resuspended in facs buffer . cytokine concentrations were measured via quantification of pe fluorescence of samples in reference to a standard curve . female balb / c mice were obtained from charles river laboratories ( kent , u . k .) at 6 - 8 weeks of age . the pilot study was done in groups of 10 mice immunised with either 0 . 5 μg f . tularensis lps , 0 . 5 μg f . tularensis glycoconjugate , 0 . 5 μg f . tularensis glycoconjugate + sas , 0 . 5 μg ‘ sham ’ glycoconjugate + sas , 0 . 5 μg ‘ sham ’ glycoconjugate or sas only . one group of mice were left untreated as challenge efficacy controls . immunisations occurred on days 0 , 14 and 28 via intra - peritoneal ( ip ) route . mice were challenged 35 days post - immunisation with 100 cfu of f . tularensis strain hn63 by the ip route , delivered in 0 . 1 ml . subsequent experiments used the same schedule with 15 mice per group and doses of 10 μg of material per immunisation . four weeks following final vaccination 5 mice from each group were tail bled to obtain sera for antibody analysis and culled at day 3 post - infection with spleens harvested to analyse bacterial load and cytokine response . for the enumeration of bacteria , spleen samples were homogenized in 2 ml of pbs through 40 μm cell sieves ( bd biosciences ) and 100 μl aliquots were plated onto bcga plates . f . tularensis lps - specific igm and total igg levels were determined by elisa as previously described [ 19 ]. all work was performed under the regulations of the home office scientific procedures act ( 1986 ). statistical analyses were performed using the program pasw ( spss release 18 . 0 ). survival data was analysed by pair - wise log rank test stratified by experiment . cytokine and bacterial load data were analysed using univariate general linear models , using bonferroni &# 39 ; s post tests to further clarify significant differences . e . coli clm24 carrying the vectors pgab2 , pgvxn114 and pgvxn150 was grown for 16 h in 200 ml lb broth at 37 ° c ., 180 r . p . m . this was used to inoculate 1 . 8 l of lb broth and further incubated at 110 r . p . m . 37 ° c . until an o . d600 reading of 0 . 4 to 0 . 6 was reached . at this point l - arabinose was added to a final concentration of 0 . 2 % and iptg to a final concentration of 1 mm to induce expression of exoa and cjpg / b respectively ; after another 5 hours of incubation , 0 . 2 % l - arabinose was added again and the culture left to incubate o / n . cells were harvested by centrifugation at 6 , 000 r . p . m . for 30 m , and pelleted cells were incubated at room temperature for 30 m in a lysis solution composed of 10 × bugbuster protein extraction reagent ( novagen ) diluted to 1 × in 50 mm nah2po4 , 300 mm nacl , 10 mm imidazole , ph 8 . 0 supplemented with 0 . 1 % tween , 1 mg / ml lysozyme and 1 μl / ml benzonase nuclease ( novagen ). cell debris was removed by centrifugation at 10 , 000 r . p . m . for 30 m , the supernatant was collected and 1 ml ni - nta agarose ( qiagen ) was added to the supernatant . the slurry - lysate was incubated for 1 h at 4 ° c . with shaking then loaded into 10 ml polypropylene columns ( thermo scientific ). his tagged exoa was purified by the addition of an elution buffer according to manufacturer &# 39 ; s instructions ( qia expressionist , qiagen ) containing 250 mm imidazole with the addition of 20 % glycerol and 5 % glucose . protein yields were estimated using a bicinchonic acid assay kit according to manufacturer &# 39 ; s instructions ( pierce ® biotechnology bca protein assay kit , u . s . a .). for large - scale protein purification , material was isolated using ge healthcare his trap columns and an akta purifier with an imidazole gradient of 30 mm to 500 mm . the collected fraction containing exoa glycosylated with f . tularensis o - antigen was further purified using a resource q anionic exchange column ( ge healthcare ) with a nacl gradient from 0 to 500 mm in 20 mm trishcl ph 8 . 0 . this generated a typical yield of 2 - 3 mg / ml of glycoconjugate per 2 l of e . coli culture . the same techniques were used for the generation of the ‘ sham ’ c . jejuni heptasaccharide exoa glycoconjugate . the plasmid coding for this heptasaccharide was pacycpgl carrying the entire cjpgl cluster from c . jejuni 81116 [ 1 ]. escherichia coli strain clm24 with a chromosomally inserted copy of pglb were grown in luria - bertani ( lb ) broth at 37 ° c ., with shaking . antibiotics were used at the following concentrations : tetracycline 20 μg ml - 1 and ampicillin 100 μg ml - 1 . tetracycline was used to maintain the plasmid pgab2 coding for francisella tularensis o antigen and ampicillin was used to maintain the plasmid coding for the acceptor carrier protein . escherichia coli cells were grown for 16 h in 200 ml lb broth at 37 ° c ., with shaking . this was used to inoculate 1 . 8 l of lb broth and further incubated with shaking at 37 ° c . until an od600 reading of 0 . 4 - 0 . 6 was reached . at this point l - arabinose was added to a final concentration of 0 . 2 per cent and iptg to a final concentration of 1 mm to induce expression of the acceptor protein and pglb , respectively ; after another 5 h of incubation , 0 . 2 per cent l - arabinose was added again and the culture left to incubate overnight . cells were harvested by centrifugation at 5300 g for 30 min , and pelleted cells were incubated at room temperature for 30 min in a lysis solution composed of 10 × bugbuster protein extraction reagent ( novagen ) diluted to 1 × in 50 mm nah2po4 , 300 mm nacl , 10 mm imidazole , ph 8 . 0 supplemented with 0 . 1 per cent tween , 1 mg ml - 1 lysozyme and 1 μl ml - 1 benzonase nuclease ( novagen ). cell debris was removed by centrifugation at 7840 g for 30 min , the supernatant was collected and 1 ml ni - nta agarose ( qiagen ) was added to the supernatant . the slurry - lysate was incubated for 1 h at 4 ° c . with shaking then loaded into 10 ml polypropylene columns ( thermo scientific ). his - tagged exoa was purified by the addition of an elution buffer according to manufacturer &# 39 ; s instructions ( qia expressionist , qiagen ) containing 250 mm imidazole with the addition of 20 per cent glycerol and 5 per cent glucose . expression of the f . tularensis schus4 o - antigen in e . coli dh5α cells the 20 kb f . tularensis schus4 o - antigen coding region was pcr amplified and cloned into pgem - t easy to generate the plasmid pgab1 . all bacterial strains and vectors used in this study are summarized in table 1 . to confirm o - antigen expression and transport to the outer cell surface of e . coli , pgab1 was transformed into dh5α cells and probed by immunofluorescence using mab fb11 , specific to the f . tularensis o - antigen . fig2 c demonstrates the expression of the o - antigen on the surface of e . coli dh5α cells , which is absent in the vector alone control ( fig2 d ). cjpgib can transfer f . tularensis o - antigen to the acceptor protein exotoxin a in order to generate a strong t - cell response and lasting immunity , a highly immunogenic protein is required as a carrier for the f . tularensis o - antigen . the selected carrier protein was an inactivated form of the p . aeruginosa exotoxin a variant l552v , ae553 ( exoa ) was selected [ 20 ]. the plasmid pgab2 containing the f . tularensis o - antigen expressed in the low copy vector plafr1 [ 21 ] was transformed into e . coli clm24 cells along with the plasmids pgvxn114 and pgvxn150 which contain cjpgib and exoa respectively . as negative glycosylation controls , clm24 cells were transformed with either pgvxn150 alone or with the combination of pgab2 , pgvxn150 and pgvxn115 , the latter coding for an inactive version of cjpgib [ 18 ]. following overnight induction of cjpglb and exoa expression with 1 mm iptg and 0 . 2 % l - arabinose ( w / v ) respectively , cells were lysed and his tagged exoa purified using nickel columns . elution fractions from each sample were separated by sds page and tested by immunoblotting with mabfb011 specific for f . tularensis lps . a band matching the expected size of exoa and an o - antigen ladder pattern could only be seen when a functional cjpgib was present ( fig3 , lanes 2 and 2b ). in the absence of a functional cjpgib there was no cross - reaction with mabfb11 ( fig3 , lanes 1 and 3 ). to demonstrate that the o - antigen was bound to the carrier protein , his tagged exoa f . tularensis o - antigen conjugate was purified and digested with proteinase k . the disappearance of the o - antigen ladder after proteinase k treatment but not in the untreated control confirmed that the o - antigen was anchored to exoa ( data not shown ). vaccination with the glycoconjugate provides significant protection against f . tularensis subsp . holarctica infection in mice in a pilot study we compared lps alone against the glycoconjugate vaccine and monitored antibody levels and murine survival . the sigma adjuvant system ® was selected for use in this study because it is based on monophosphoryl lipid a ( mpl ), a low toxicity derivative of lps that has been demonstrated to be a safe and effective immunostimulant [ 22 ]. in order to demonstrate the specificity of the glycoconjugate we used controls including mice with sas adjuvant alone , unvaccinated mice and mice vaccinated with a ‘ sham ’ glycoconjugate control ( c . jejuni heptasaccharide conjugated to exoa ). only mice vaccinated with 0 . 5 test glycoconjugate + sas ( p & lt ; 0 . 05 ) or 0 . 5 μg lps ( p & lt ; 0 . 001 ) demonstrated increased survival compared to the appropriate controls as determined by log rank test ( fig2 ). these candidates were selected for further assessment at higher doses and an additional group consisting of lps + sas was also added as a further control . protection was compared between mice immunised with either 10 μg glycoconjugate + sas , 10 μg lps or 10 μg lps + sas . all three vaccines were protective when compared to the unvaccinated mice ( p & lt ; 0 . 001 ), while the sas adjuvant alone did not elicit any protection ( p & gt ; 0 . 05 ) ( fig4 ). this experiment also indicated that lps + sas did not elicit the same level of protection as the glycoconjugate + sas combination ( p & lt ; 0 . 05 ) and thereafter lps + sas was deemed unnecessary for testing . the study was repeated in order to provide further bacterial organ load and immunological response data and no statistically significant difference was found between replicates . mice vaccinated with test glycoconjugate and challenged with f . tularensis subsp . holarctica have lower bacterial loads and pro - inflammatory cytokines 3 days post challenge three days post challenge 5 mice per group were sacrificed and bacterial loads in the spleens and inflammatory responses were evaluated ( fig5 ). fig5 shows the bacterial loads from vaccine with 10 μg of each candidate . mice that were immunised with the glycoconjugate + sas or lps both had significantly decreased bacterial loads in spleens ( p & lt ; 0 . 01 ) when compared to the sas and unvaccinated controls . mice vaccinated with glycoconjugate + sas had significantly less bacteria compared to those vaccinated with lps alone ( p & lt ; 0 . 05 ). inflammatory cytokine profiles between the different vaccine groups were also analysed ( fig6 ). reduced levels of inflammatory cytokines were seen in mice vaccinated with glycoconjugate + sas and lps alone ( p & lt ; 0 . 05 ), corresponding with decreased bacterial loads . there was no significant difference between cytokine profiles for both experiments ( p & gt ; 0 . 05 ). vaccination with the f . tularensis glycoconjugate induces a greater igg immune response the levels of lps - specific igg were assessed in mice 7 days prior to challenge for both experiments . increased lps - specific igg was observed in the glycoconjugate + sas vaccinated group when compared to animals vaccinated with lps only ( p & lt ; 0 . 001 ). although experiment 2 had higher levels of antibody ( p & lt ; 0 . 01 ), we observed no difference in pattern between experiments ( p & gt ; 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