Patent Application: US-72748607-A

Abstract:
an artificial invasin complex is prepared from purified or recombinantly prepared invasins and gram negative bacteria lipopolysaccharides . typically , ipab is mixed with ipac to form a ipab : ipac complex . this invasin protein complex is then mixed with the lipopolysaccharide to form an artificial invasin complex . additional bioactive molecules can be incorporated into the complex during manufacture . this artificial invasin complex is similar in function to native invaplex 24 or invaplex 50 . the artificial invasin complex has superior immunogenicity properties relative to the native complex and can be tailor made . its method of preparation lends itself to scale up . the artificial invasin complex can facilitate transport of biomolecules , therapeutics and antibiotics across cell membranes in a manner similar to native shigella invaplex .

Description:
biochemistry of artificial invaplex production and purification of recombinant ipab and ipac and native s . flexneri , s . sonnei and s . dysenteriae 1 lps the ipa proteins are highly conserved in all shigella spp [ 32 , 33 ]. recombinant e . coli expressing either ipab or ipac have been previously described . two strategies are available for purifying recombinant ipa proteins . histidine - tagged ( histag ) recombinant ipac is purified by affinity chromatography using nickel columns . the effect of the histidine residues on the ipa protein &# 39 ; s biological or immunological function or the ability of the proteins to form invaplex is not known but histag - ipac appears to maintain biological activity [ 30 ]. an alternative purification procedure has been used to produce recombinant ipab . this method takes advantage of ipab &# 39 ; s native affinity for the chaperone ipgc [ 34 , 35 ]. by co - expressing ipab together with the histag - chaperone ipgc in the same recombinant organism , it will be possible to purify the ipab / chaperone complex . after purification , the histag - ipac / ipab protein complex is denatured resulting in monomeric ipab protein and histag - ipgc which allows the histagged chaperone to be selectively removed on a nickel column while the free ipa protein runs through the column . the recombinant ipac was expressed in an e . coli background using an iptg inducible expression system ( pet plasmid ). specifically , the ipac gene was cloned into the pet15b plasmid ( novagen ) and expressed in the e . coli vector bl21 ( de3 ) plyss [ 30 ]. the recombinant ipac protein has several histidine residues on the amino terminal end that allows subsequent purification on a nickel column [ 30 ]. the ipac protein expressed in the recombinant is located in inclusion bodies and requires solubilization with urea . removing the urea often leads to insolubility if the ipac concentration is too high (& gt ; 1 mg / ml ). thus , ipac eluted from the nickel column was maintained in urea to maintain solubility . using small - scale cultures ( 3 l ) it has been possible to produce approximately 75 mg of purified ipac with a yield of approximately 8 mg / l . the product is greater than 90 % pure and is reactive with anti - ipac mab 2g2 [ 32 ] ( fig1 ). the purified protein is soluble and can be stored frozen . an example of purified ipac is in fig1 . the recombinant organism expressing ipab is in an e . coli bl21 ( de3 ) background . the ipgc gene was cloned into pet15b ; the ipab gene was cloned separately into the pacyc - duet vector . after iptg induction the histag ipgc / ipab complex is solubilized and purified on a nickel affinity column . the histag - ipgc / ipab complex is released with edta and prepared for second application to the nickel affinity column by removal of the edta and the addition of the non - ionic detergent opoe ( n - octyl - oligo - oxyethylene ) to a final concentration of 1 % v / v . the opoe will disrupt the histag - ipgc / ipab complex thereby allowing the free histag - ipgc protein to bind to the nickel ( ni - sepharose ) column and the untagged , free ipab to flow through the column for collection . fractions from the void volume were probed for ipab with anti ipab mab 2f1 . positive fractions were analyzed by sds - page ( stained ) to determine if contaminating histag - ipgc was present . if so , the material was treated again with opoe and reapplied to the nickel column to remove residual histag - ipgc . the resultant soluble ipab product has little to no histag - ipgc “ contamination ”, is over 90 % pure and reacts with anti ipab mab 2f1 [ 32 ] ( fig1 ). the yield of ipab per liter of starting culture was approximately 3 . 5 mg / l . purification of s . flexneri 2a , s . sonnei , or s . dysenteriae 1 lps s . flexneri 2a , s . sonnei , and s . dysenteriae 1 lps were produced by the westphal procedure [ 36 ] which involves a hot phenol / water extraction of the shigellae . virulent or attenuated strains of shigella can be used as source of lps as long as the smooth lps phenotype is expressed . in experiments described below , wild - type s . flexneri 2a ( strain 2457t ) and s . sonnei ( mosely ) were used . for s . dysenteriae 1 , the attenuated strain wrsd1 was used to minimize risk of infection to laboratory personnel . wrsd1 is a virg , stx knockout previously produced at wrair [ 37 ]. lps is extracted by the westphal procedure [ 36 ]. briefly , the bacterial cell pellets are suspended in hot ( 68 ° c .) distilled water ( 5 ml water for each gram of pellet ). an equal amount of phenol , heated to 68 ° c ., is added and the pellet solution is vigorously shaken for 15 minutes . the bottles are then cooled to approximately 10 ° c .± 5 ° c . the samples are centrifuged , the aqueous phase removed and stored at 4 ° c . extraction of the cell mass is performed a second time and all aqueous phases are pooled . the aqueous phase is dialyzed against distilled water for two days , and then centrifuged ( 8000 × g , 30 min ) to remove extraneous cellular debris . this supernatant is subjected to ultracentrifugation ( 90 , 000 × g ) for 2 hours and the pellet is saved . the pellet is rinsed with sterile distilled water , resuspended in sterile distilled water overnight at 4 ° c ., pooled and lyophilized . the final lyophilized product is weighed and then a small portion (& lt ; 10 mg ) is removed , dissolved in 1 ml of endotoxin - free water and characterized biochemically ( see below ). endotoxin content of the purified lps is performed by the chromogenic lal . e . coli endotoxin serves as a control reagent for this analysis . all results are reported in terms of international endotoxin units ( eu ). the purified lps is also analyzed by sds - page with silver stain to determine if the typical multiple band profile of smooth lps are present . fig1 shows a silver stained gel of the purified s . dysenteriae 1 lps demonstrating the typical multiple banding pattern of smooth lps . the final lps product has residual amounts of protein (& lt ; 5 %, determined by bradford assay ) and dna (& lt ; 5 %, determined by hoechst stain ) and is reactive with lps serotype specific antibodies ( anti - s . dysenteriae 1 lps mab mab753 ( chemicon international ) for s . dysenteriae 1 lps ; mab 2e8 for s . flexneri 2a lps ; mab755 ( chemicon ) for s . sonnei lps ) by western blot or elisa . quantitation of ipab and ipac content in invaplex ar by immunoassay the amount of ipab and ipac in invaplex ( artificial or native ) was determined using a modified elisa procedure . the elisa used purified recombinant ipab or ipac proteins to generate standard curves for determination of the quantity of the antigens in the invaplex preparations . immulon 1b elisa plates ( thermolab systems ) were coated overnight at 4 ° c . with either 50 μl of recombinant ipab , recombinant ipac , or invaplex ar . antigen was titrated ( in triplicate ) using 2 - fold serial dilutions in carbonate coating buffer ( 0 . 2 m carbonate , ph 9 . 8 ) with starting concentrations of 125 ng / ml ( ipab ), 200 ng / ml ( ipac ), and 10 ug / ml ( invaplex ). after washing and blocking with casein , affinity - purified monoclonal antibodies specific for ipab ( 2f1 ) or ipac ( 2g2 ) [ 32 ] were incubated with the antigen - coated plates for 2 hours . after washing , antigen - specific antibody was detected using anti - mouse immunoglobulin g ( igg ) conjugated with alkaline phosphatase ( kirkegaard & amp ; perry ). using the substrate para - nitrophenyl phosphate the optical density at 405 nm ( od 405 ) was measured using an elisa plate reader ( molecular devices , menlo park , calif .) after a 60 minute incubation with substrate . using the softmax pro 4 . 5 ( molecular devices ) program , a standard curve plotting od 405 versus concentration ( ng / ml ) was determined . the concentration of the unknown samples were then interpolated from the standard curve . invaplex ar is designed to have ipab and ipac concentrations and an ipac / ipab ratio that is similar to hp invaplex 24 . the ratio of the quantity of ipac to ipab was determined for the invaplex ar and compared to hp - invaplex 24 , the most pure form of invaplex . the ipac / ipab molar ratio in hp invaplex is approximately 8 . this was determined by densitometry analysis of sds - page gels and quantitative antibody based assays for ipab and ipac . in addition the lps content is expected to be at the same relative mass ratio ( approximately 0 . 5 to 0 . 6 mg of lps for every 1 mg of protein ) that is found in hp - invaplex . lps content in invaplex preparations was measured by determining the amount of 2 - keto - 3 - deoxyoctonate ( kdo ) in each preparation [ 43 ] or by using the limulus amoebocyte lysate assay ( cambrex inc .). method for formation of artificial invaplex . preparation of invaplex ar for s . flexneri 2a , s . sonnei and s . dysenteriae 1 the purified components were mixed at ratios similar to that found in highly purified native invaplex to form the artificial invaplex . analysis of the s . flexneri 2a hp invaplex indicated that the ipac / ipab molar ratio was approximately 8 . 0 and the lps to total protein ratio was approximately 0 . 56 mg lps / mg total protein . using these parameters as a guide for reconstituting invaplex from purified ipab , ipac and lps a series of experiments were conducted to create an invaplex ar . once formed , the artificial invaplex was purified by ion - exchange fplc . purified , soluble ipab and ipac , each in their respective final buffers were mixed together at an ipac / ipab molar ratio of 8 . after the ipab and ipac were mixed , the solution was slowly added to dry lps powder ( ratio of lps to total protein is 0 . 56 ). lps from any shigella species can be used ; for the described experiments s . flexneri 2a , s . sonnei or s . dysenteriae 1 lps was used . the mixture was incubated at 37 ° c . for approximately 2 hours with shaking . afterwards the protein / lps mixture was diluted to 20 mm tris - hcl , 0 . 10 m nacl and 1 . 2 m urea to reduce the nacl concentration prior to ion - exchange chromatography . the diluted mixture was then purified on a hitrap ™ q hp anion exchange column . preliminary assembly experiments accommodated the insolubility of ipac by maintaining both ipac and ipab proteins in a buffer containing ≧ 4m urea . once the ipac / ipab mixture was added to lps , solubility was no longer an issue which permitted the eventual removal of the urea . typically purified ipac by itself will precipitate upon dilution . in preliminary mixing experiments the 8 ipac / ipab molar ratio was maintained by adding 8 μm histagipac to 1 μm ipab in a final volume of 1 ml . both proteins were initially prepared in 20 mm tris - hcl , 0 . 5 m nacl , 6 m urea , ph 7 . 9 . the proteins were mixed in a glass test tube and incubated at 37 ° c . without shaking for 15 mins . dry lps ( 230 μg ) in a separate glass tube was also incubated at 37 ° c . without shaking for 15 mins . after the 15 min incubation , the ipab + ipac mixture was used to solubilize the pre - warmed lps by slowly adding the protein mixture down the side of the tube , followed by vortexing . no appreciable flocculation or precipitation was observed . the ipab / ipac / lps mixture was then incubated at 37 ° c . with shaking ( 200 rpm ) for 2 hrs . in preparation for ion exchange chromatography ( iec ), the mixture was diluted five - fold with pre - warmed 20 mm tris buffer , ph 9 . 0 to lower the salt concentration . no precipitation was observed as the mixture cooled to room temperature . for final purification , the diluted ipab / ipac / lps mixture was applied to an anion exchange column ( hitrap ™ q hp ) with buffer a consisting of 20 mm tris - hcl , ph 9 . 0 and buffer b consisting of 1 m nacl , 20 mm tris - hcl , ph 9 . 0 , and a step gradient of 0 % ( 5 column volumes ) to 24 % ( 10 column volumes ) to 50 % ( 6 column volumes ) to 100 % buffer b ( 5 column volumes ). one ml fractions were collected from a 1 ml hitrap ™ q hp column at an elution flow rate of 1 ml / min . fractions from each step were analyzed for the presence of ipab , ipac and lps by spotblot . the s . flexneri invaplex ar eluted in the 50 % b step ( fig2 ) which contained the greatest quantities of all three components ( ipab , ipac and lps ) as determined by western blots and silver stained gels ( fig2 a ). when applied to a size exclusion column the ipab / ipac / lps complex eluted at between the 1 mda and 669 kka standards which is the same size range as hp invaplex . formation of s . dysenteriae 1 artificial invaplex and larger scale production the above experiment was repeated but with a ten - fold increase in reactants to increase the invaplex ar yield . in addition s . dysenteriae 1 lps was used instead of s . flexneri lps . as such , 3 . 28 mg of ipac in 20 mm tris - hcl , 0 . 5 m nacl , 6 m urea , ph 7 . 9 , was mixed with 0 . 62 mg ipab in 20 mm tris - hcl , 0 . 5 m nacl , ph 7 . 9 , in a total volume of 5 mls . the protein mixture and a separate tube of lps ( 2 . 1 mg ) was pre - warmed to 37 ° c . the ipab + ipac mixture was added to the lps tube and vortexed to solubilize the lps . no precipitation was observed . the reaction mixture was incubated at 37 ° c . with shaking ( 200 rpm ) for 2 hours . next pre - warmed iec buffer a ( 20 mm tris - hcl , ph 9 . 0 ) was added to dilute the salt concentration 1 : 5 yielding a final volume of 25 mls . after cooling to room temperature , the diluted reaction mixture was applied to a 1 ml hitrap ™ q hp iec column and subjected to an invaplex purification step gradient ( see above ). the s . dysenteriae 1 invaplex ar eluted in the 50 % buffer b step . it contained ipab , ipac and s . dysenteriae 1 lps . ( fig2 c ). analysis of the 0 . 24 m and 1 . 0m nacl peaks did not detect significant quantities of ipab , ipac or lps . the yield of invaplex ar production was approximately 10 to 20 % of the amount of total protein used to initiate the assembly process . similar assembly experiments have been conducted for s . sonnei resulting in an invaplexar for s . sonnei ( see fig2 b ). a fifty - fold increase in reactants was used to produce more invaplex ar for advanced studies . using an 8 ipac / 1 ipab molar ratio , 16 . 4 mg of ipac ( in 20 mm tris - hcl , 0 . 5 m nacl , 6 m urea , ph 7 . 9 ) was mixed with 3 . 1 mg ipab ( in 20 mm tris - hcl , 0 . 5 m nacl , ph 7 . 9 ) in a total volume of 20 mls . the protein mixture and a separate tube of shigella flexneri 2a lps ( 11 . 4 mg ) were pre - warmed to 37 ° c . the ipab + ipac mixture was added to the lps tube and vortexed to solubilize the lps . the reaction mixture was incubated at 37 ° c . with shaking ( 200 rpm ) for 2 hours . next pre - warmed iec buffer a ( 20 mm tris - hcl , ph 9 . 0 ) was added to dilute the salt concentration 1 : 5 yielding a final volume of 100 mls . after cooling to room temperature , the diluted reaction mixture was applied to a 5 ml hitrap ™ q hp iec column and subjected to an invaplex purification step gradient ( see above ). the s . flexneri 2a invaplex ar eluted in the 50 % buffer b step . it contained ipab , ipac and s . flexneri 2a lps . analysis of the 0 . 24 m and 1 . 0m nacl peaks did not detect significant quantities of ipab , ipac or lps . the yield of invaplex ar production was approximately 9 % of the amount of total protein used to initiate the assembly process . analysis of invaplex ar preparations by electrophoresis and western blots for ipab and ipac the total protein composition of the invaplex ar preparations was determined by stained sds - page gels . western blots were used to determine the presence , size and relative concentrations of ipab and ipac . silver stained gels were used to assess the lps profile [ 18 ]. monoclonal antibodies specific for either the ipab or ipac proteins were used to probe the nitrocellulose blots [ 32 ]. after probing with alkaline phosphatase - conjugated protein a , blots are developed with asmx phosphate ( sigma ) and fast red tr salt ( sigma ). video images of the blots are displayed on a macintosh computer using a ccd video camera ( cohu ) and captured with nih image software ( version 1 . 62 ). densitometry analysis of the digital images were performed with gelpro image analysis software ( version 2 . 0 . 10 , media cybernetics , inc .). the total protein gel profile and the ipab and ipac content should resemble that found in hp - invaplex 24 or hp - invaplex 50 . size - exclusion chromatography was used to determine if the components of invaplex ar found in the ion - exchange peak are truly complexed . this is the same method used to produce hp - invaplex . a superose 6 hr 10 / 30 column ( amersham pharmacia ), calibrated with blue dextran 200 ( 2 mda ), thyroglobulin ( 669 kka ), and ovalbumin ( 43 kka ), was loaded with invaplex ar ( approximately 3 mg ). fractions ( 0 . 5 ml , flow rate 0 . 25 ml / min ) were collected and analyzed by immuno - spot blot for the presence of ipab , ipac and lps . all three invaplex ar components ( ipab , ipac and lps ) eluted in fractions between 2 mda and 669 kka indicating they are associated in a complex as they eluted at a size much greater than the individual components ( 43 kka for ipac ; 62 kka for ipab ). the sec size of invaplex ar is similar to the size where hp - invaplex elutes . evaluation of s . dysenteriae 1 invaplex ar in tissue culture model of internalization invaplex binds to and stimulates endocytosis in cultured mammalian cells . this property is dependent on the presence of the invasins ipab and ipac and utilizes host - cell acting during the endocytic process [ 38 ]. the invaplex internalization assay is an in vitro model used to measure the functional activity of invaplex preparations . the assay is modeled after the shigella plaque assay [ 39 ] and shigella uptake assays [ 40 , 41 ]. the assay involves incubating invaplex ar with bhk - 21 fibroblast cells ( 60 - 70 % confluent ) in chamber slides . after incubation for 5 , 15 , 30 , or 60 minutes the cells were washed 3 × with pbs and formalin - fixed . fixed cells were permeabilized with pbs supplemented with 0 . 1 % triton x - 100 and 0 . 1 % bsa ( wash buffer ) and probed with polyclonal mouse serum with specificity for lps , ipab , and ipac . bound polyclonal mouse antibodies were detected with gam - igg - oregon green 488 ( molecular probes ) followed by counterstaining with dapi to identify the nuclei . the invaplex treated cells were examined by fluorescent microscopy with digital image capturing . both native and artificial s . dysenteriae 1 invaplex were internalized by bhk cells with similar cytoplasmic staining pattern for both preparations indicating that the invaplex ar maintains the capacity to stimulate internalization by mammalian cells ( fig4 ). this indicates that the assembly of recombinant ipab and ipac with s . dysenteriae 1 lps into a complex isolated by ion - exchange fplc has resulted in a synthetic product which maintains the same biological activity as the native complex . the mouse lung - infection [ 31 ] and the guinea pig keratoconjunctivits models [ 44 ] were used to determine whether intranasal immunization with s . flexneri 2a invaplex ar stimulates a protective immune response that is effective against a homologous s . flexneri 2a challenge . separate mouse studies were used to determine whether immunization with s . sonnei invaplex ar could protect against a homologous s . sonnei challenge and against a heterologous challenge with s . flexneri 2a . additional studies were conducted to assess the adjuvant activity of invaplex ar using ovalbumin ( ova ) as an immunogen . in the adjuvanticity experiments , immune responses induced after immunization with ova alone were compared to immune responses induced with ova combined with either native invaplex , invaplex ar , or cholera toxin , an established , potent mucosal adjuvant . the guinea pig and mouse experiments were conducted at wrair under iacuc approved protocols . four separate animal experiments have been completed to evaluate the immunogenicity , adjuvanticity , and protective efficacy of artificial invaplex . common methods were used in each experiment and are outlined in the following paragraph . methods and materials specific to each study are indicated in separate sections . mice were immunized on days 0 , 14 and 28 . a total antigen volume of 25 ul was delivered in 5 to 6 small drops applied to the external nares with a micropipette . blood taken by tail bleed from all mice at days 0 , 28 and 42 and 2 weeks post - challenge ( day 63 ). on day 35 or 42 mice were euthanized followed by immediate collection of mucosal washes and removal spleen for in vitro proliferation and cytokine analysis s . flexneri 2a invaplex ar . three weeks after the final immunization mice ( 15 per group ) were challenged intranasally with a lethal dose ( 1 . 6 × 10 7 cfu ) of s . flexneri 2a ( 2457t ) or with s . sonnei ( mosely , 8 × 10 6 cfu ) as described for the mouse lung model [ 34 ]. prior to intranasal immunization or challenge , mice are anesthetized with a mixture of ketamine hydrochloride ( 40 mg / kg ) and xylazine ( 12 mg / kg ). challenged mice were monitored daily for two weeks for weight loss , fur ruffling and lethargy , with death used as an endpoint . surviving mice were bled 14 days post - challenge ( day 63 ). s . flexneri 2a invaplex ar immunogenicity and protection study i separate groups of female balb / c mice ( 10 - 15 mice / group ) were immunized intranasally on day 0 , 14 , and 28 with 5 ug of either the native s . flexneri 2a invaplex ( lot jwjx ) or 2 . 5 μg of artificial s . flexneri 2a invaplex ar ( lot kf - d3d4 ) vaccines . control animals were inoculated with saline . blood was collected on day 0 , 28 , 42 , and 63 . animals were challenged intranasally on day 49 . s . flexneri 2a invaplex ar immunogenicity and protection study ii separate groups of female balb / c mice ( 15 - 20 mice / group ) were immunized intranasally on day 0 , 14 , and 28 with 5 ug of either the native s . flexneri 2a invaplex ( lot jwjx ) or 2 . 5 μg of artificial s . flexneri 2a invaplex ar ( lot kr - c5 ) vaccines . control animals were inoculated with saline . blood was collected on day 0 , 28 , 42 , and 63 . lung and intestinal washes were collected on day 42 from 4 animals / group . remaining animals were challenged intranasally on day 49 . the ability of invaplex ar to function as a mucosal adjuvant was determined in mice using ovalbumin ( ova ) as a model protein antigen . balb / cbyj mice ( 5 mice / group ) were intranasally immunized on day 0 , 14 , and 28 with ova ( 5 μg ) alone , or ova ( 5 μg ) combined with either s . flexneri 2a invaplex ar ( lot kf - d3d4 ; 2 . 5 μg ), native s . flexneri 2a invaplex ( lot jwjx ; 5 μg ), or cholera toxin ( ct ; 5 μg ). blood was collected on day 0 , 28 , and 42 . control animals were inoculated with saline . lung and intestinal washes were collected on day 42 . serum endpoint titers on day 0 , 28 and 42 with specificity for invaplex 24 , invaplex 50 , lps , ova , ipab and ipac were determined by elisa . antigen - specific antibodies in mucosal washes were also assessed . the immunogencity and protective efficacy of s . sonnei invaplex ar was determined in mice using a homologous ( s . sonnei 53g ) and heterologous ( s . flexneri 2a 2457t ) challenge . balb / cbyj mice ( 10 - 15 mice / group ) were intranasally immunized on day 0 , 14 , and 28 with s . sonnei invaplex ar ( lot kj - d3d6 ; 2 . 5 μg ), native s . sonnei invaplex ( lot jojp ; 5 μg ), or native s . flexneri 2a invaplex ( lot jwjx ; 5 μg ). control animals were inoculated with saline . blood was collected on day 0 , 28 , and 42 . guinea pigs ( hartley strain , 6 to 10 per group ) were immunized intranasally with the artificial or native invaplex vaccines ( 25 ug / dose ). the dose volume ( 100 ul ) was split equally between each nostril . saline was used to immunize control animals . guinea pigs were immunized on days 0 , 14 , and 28 and bled from the lateral ear vein on days 0 , 28 , 42 , and 14 days after challenge . prior to intranasal immunization , guinea pigs were anesthetized with a mixture of ketamine ( 40 mg / kg ) and xylazine ( 4 mg / kg ). three weeks after the third immunization guinea pigs were challenged intraocularly with s . flexneri 2a ( strain 2457t ) and observed daily for 5 days to assess the degree of inflammation and keratoconjunctivitis as previously described [ 44 ]. elisa assays are used to measure antigen specific iga and igg antibodies in sera and mucosal secretions , including lung and intestinal lavages and stool extracts from immunized and / or challenged mice and guinea pigs [ 18 , 29 , 46 ]. antigens used in elisas , including purified s . flexneri 2a lps , s . sonnei lps , water - extracted shigella antigens , purified ipab and ipac proteins , and s . flexneri 2a native invaplex and ova were coated onto immunion ib 96 - well elisa plates ( thermolab systems ) overnight at 4 ° c . after blocking with 2 % casein ( in tris - saline buffer , ph 7 . 5 ) sera and mucosal washes were serially diluted in duplicate and incubated with the antigen - coated plates for 4 hours at 25 ° c . after washing with pbs containing 0 . 05 % tween 20 , antigen - specific antibody is probed with alkaline phosphatase conjugated anti - mouse igg or anti - mouse iga ( kirkegaard & amp ; perry ). alkaline phosphatase activity was detected with p - nitrophenyl phosphate ( 1 mg / ml ). after a 30 - min incubation in substrate , the optical density ( od 405 ) was measured using an elisa plate reader ( molecular devices , menlo park , calif .). endpoint titers were determined for each animal and was used to calculate geometric mean titers for each group at each time point . typically animals intranasally immunized with invaplex respond with a 4 to 8 - fold higher serum ( igg and iga ) and mucosal ( lung and intestine ) iga titers to shigella antigens as compared to preimmune samples or saline control animals . a capture enzyme immunoassay was used to determine total iga concentrations in mucosal samples . sample concentrations were determined from standard curves , using purified mouse iga assayed in parallel . specific mucosal iga activities were calculated by dividing the endpoint titer for each individual mucosal sample by the concentration of total iga within the same sample [ 47 ]. antigen stimulation of cultured lymphocytes from mice immunized with invaplex ar lymphocyte proliferation upon antigen stimulation was determined using splenocytes collected from invaplex - immunized mice and naive mice . mononuclear cells ( 2 × 10 5 per well ) were incubated with invaplex , ipab , or ipac or s . flexneri 2a lps preparations . simultaneously in separate microtiter wells , splenocytes were stimulated with concanavalin a to provide positive controls . negative controls included cells incubated with complete medium alone and cells from naive mice stimulated with antigen . after incubation with antigen for 4 to 7 days , cell proliferation was measured by a non - radioactive assessment of dehydrogenase activity using mts ( promega ) [ 48 ]. there is a strong correlation with increasing optical density readouts in this assay and the number of viable cells in a well . prior to measuring proliferation , cell culture supernatants were collected on days 3 and 5 for cytokine measurements ( see below ). stimulation indices ( si ) were calculated by dividing the mean optical density of antigen - stimulated cells by the mean optical density of medium - only stimulated cells . the si of cells from mice immunized with invaplex was compared to the si of cells from non - immunized mice . the immunogenicity of artificial s . flexneri 2a invaplex ( invaplex ar ), manufactured from individual purified components rather than the virulent organism ( native invaplex ) was evaluated in mice . groups of mice ( n = 6 - 10 ) were intranasally immunized on day 0 , 14 , and 28 with native s . flexneri 2a invaplex 24 ( 5 or 10 μg ), invaplex ar ( 2 . 5 μg ), purified ipab ( 2 . 5 μg ), purified ipac ( 2 . 5 μg ), lps ( 2 . 5 μg ), or saline . three weeks after the third immunization ( day 49 ), the mice were challenged with shigella flexneri 2a ( 2457t ). blood collected on day 0 , 28 , 42 , and 63 was analyzed by elisa for serum igg and iga responses to invaplex 50 , invaplex 24 , purified lps , ipab and ipac . fig5 and 6 outline the serum igg and iga endpoint titers determined in blood collected on day 42 ( two weeks after the third immunization ). saline - inoculated mice and preimmune sera from immunized mice did not have detectable levels of antigen - specific antibodies ( data not shown ). immunization with s . flexneri 2a invaplex ar induced serum igg and iga responses to invaplex 50 and invaplex 24 of comparable magnitude to those induced with native invaplex 24 ( fig5 ), and significantly higher ( p & lt ; 0 . 001 ) than those induced after inoculation with saline . a two - fold increase in the amount of native invaplex ( lot jwjx ) used for immunization did not result in an increase in the magnitude of the invaplex 50 , lps , or invaplex 24 - specific serum igg or iga responses measured on day 42 ( fig5 ). immunization with purified ipab resulted in a strong ipab - specific ( fig6 ) and invaplex 24 - specific response ( gmt & gt ; 5760 and 3800 , respectively ) and a moderate response to invaplex 50 ( gmt 950 ). immunization with purified lps did not induce a detectable serum igg or iga response to any of the antigens used in elisa in the majority ( 5 / 6 ) of mice in the vaccine group . similarly , immunization with purified ipac also did not induce a detectable immune response to any of the antigens assayed , including a ipac - specific elisa . interestingly , the mice immunized with invaplex ar had among the highest ipac - specific endpoint titers ( fig6 and 8 , significantly higher ( p & lt ; 0 . 001 ) than those induced after immunization with native invaplex . protection of mice from a lethal challenge of s . flexneri after intranasal immunization with invaplex ar the lethal lung model entails intranasal inoculation of mice with a lethal dose of shigellae which establish an infection of the lungs leading to severe weight loss , pneumonia and ultimately death over the observation period of two weeks . three weeks after intranasal immunization with s . flexneri 2a invaplex ar or native invaplex mice were challenged with a lethal dose of s . flexneri 2a ( strain 2457t ). in naive mice ( treated with saline ) 11 of 13 mice died with a mean maximum weight loss of 31 . 4 % of the pre - challenge weight ( see table 1 ). all mice immunized with s . flexneri 2a invaplex ar ( p & lt ; 0 . 001 ) or native invaplex ( p & lt ; 0 . 001 ) survived the lethal challenge with s . flexneri 2a . with respect to weight loss ( which is likely a more sensitive indicator of protective immunity ) mice immunized with invaplex ar lost less of their pre - challenge weight ( 21 . 3 %) as compared to 23 . 8 % to 26 . 0 % weight loss in the native invaplex - immunized mice . furthermore the day of weight rebound ( an indication of recovery from the challenge ) was day 7 for invaplex ar and day 13 ( native invaplex , 5 μg ) or day 7 ( native invaplex , 10 μg ). in addition the protective capacity of the individual components ( ipab , ipac and lps ) used to construct invaplex ar were also evaluated in the mouse lethal lung model ( see table 1 ). immunization with purified lps and purified ipac did not protect mice from death ( both p & gt ; 0 . 05 ) and although immunization with ipab protected 5 of 6 mice from a lethal challenge the mice never regained weight and remained symptomatic ( low weight , ruffled fur ) through the end of the observation period . the mean maximum weight loss after challenge was 29 . 9 % for lps , 31 . 0 % for ipab and 33 . 9 % for ipac all of which are very close to the naïve mice weight loss value of 31 . 4 %. the results of the challenge of invaplex ar mice with a lethal dose of s . flexneri 2a indicate that invaplex ar stimulates a level of protection that is comparable to or exceeds that of native invaplex . furthermore , it appears that the complex of ipab , ipac and lps is required in that individual components are incapable of stimulating a fully effective protective immune response . intestinal and lung washes collected on day 35 from mice immunized with s . flexneri 2a invaplex ar or native invaplex were assessed by elisa for antigen - specific iga levels . immunization with invaplex ar induced levels of lps and invaplex - specific intestinal iga that were comparable to levels induced by immunization with native invaplex ( fig7 ) and significantly higher levels ( p & lt ; 0 . 001 ) of ipab - specific iga . minimal ipac - specific intestinal iga was elicited after immunization with any of the invaplex vaccine preparations ( tables 7 and 8 ). intranasal immunization with invaplex ar elicited strong antibody responses in the lung , directed to lps , invaplex 50 , ipab and ipac ( fig8 and tables 7 and 8 ). minimal levels of lps - specific iga were induced in the lung after immunization with native invaplex with undetectable levels of antibodies specific for invaplex 50 , ipab and ipac . antigen - specific cellular proliferative response and secreted cytokine profiles after intranasal immunization with invaplex ar splenocytes collected from immunized mice on day 35 were stimulated in vitro with invaplex 24 , ipab , or ipac to assess induction of antigen - specific cell - mediated responses . proliferation of cells after incubation with antigen indicates prior exposure and immunological memory . concavalin a ( cona ), which non - specifically activates t cell proliferation , was used as a positive control to demonstrate viable cell levels . stimulation indices ( sis ) after stimulation with cona ranged from 13 . 8 to 15 . 9 ( table 2 ). cells from saline inoculated animals did not proliferate after incubation with invaplex , ipab , or ipac . splenocytes from animals ( 4 / 4 ) immunized with invaplex ar proliferated after incubation with invaplex ( si = 10 . 2 ), ipab ( si = 8 . 7 ), and ipac ( si = 6 . 9 ) indicating immunological memory to these antigens was present . the ipab and ipac - specific proliferative responses in groups immunized with invaplex ar were significantly higher ( p & lt ; 0 . 01 ) than the proliferative responses in groups immunized with native invaplex . splenocytes from 4 / 4 animals immunized with native invaplex proliferated after incubation with invaplex ( si = 6 . 8 ). low to undetectable levels of proliferation occurred in splenocytes from mice immunized with native invaplex after ex vivo incubation with ipab ( 1 / 4 mice ) or ipac ( 0 / 4 mice ). confirmation of protection of mice from a lethal challenge of s . flexneri after intranasal immunization with invaplex ar using a different lot of invaplex ar the experiment describing protection with invaplex ar in the mouse lethal lung model was repeated with a different lot of s . flexneri 2a invaplex ar . in addition the evaluation of the purified components was repeated along with mice immunized with mixtures of two purified components ( ipab + ipac ; ipab + lps ; ipac + lps ). for this challenge , mice were inoculated intranasally with a slightly higher challenge dose ( 1 . 6 × 10 6 cfu ) of s . flexneri 2a ( strain 23457t ). in naive mice ( treated with saline ) 14 of 14 mice died with a mean maximum weight loss of 34 . 5 % of the pre - challenge weight ( see table 3 ). mice immunized with s . flexneri 2a invaplex ar ( 13 of 14 survived ; p & lt ; 0 . 001 ) survived the lethal challenge with s . flexneri whereas mice immunized with native invaplex had a much lower survival rate for the challenge dose used in this experiment ( see table 3 ). with respect to weight loss mice immunized with invaplex ar lost less of their pre - challenge weight ( 26 . 6 %) as compared to 31 . 6 % weight loss in the native invaplex - immunized mice and 34 . 5 % in the mice inoculated with saline . the protective capacity of the individual components ( ipab , ipac and lps ) or pairs of purified components used to construct invaplex ar confirmed previous results in that ipac and lps are not protective . purified ipab was not protective either . ( see table 3 ). mixtures of two of the purified components resulted in protection in the ipab + lps combination and the ipac + lps combination whereas the ipab + ipac combination was not fully protective . the results of the second experiment evaluating the protective capacity of invaplex ar clearly shows that invaplex ar stimulates a level of protection that exceeds that of native invaplex . furthermore , it appears that the individual components ( ipab , ipac or lps ) are incapable of stimulating a fully effective protective immune response . serum antibody responses directed to s . sonnei invaplex 50 , s . sonnei lps , ipab and ipac were determined by elisa . mice inoculated with saline ( negative control ) did not have detectable levels of antigen - specific serum igg or iga on day 42 ( table 4 ). similar s . sonnei invaplex 50 and lps - specific serum igg and iga endpoint titers were achieved after immunization with s . sonnei invaplex ar and native invaplex ( table 4 ). groups of mice immunized with s . sonnei invaplex ar had higher levels of ipab - specific serum igg ( gmt & gt ; 5760 , p & lt ; 0 . 001 ) as compared to groups of mice immunized with native s . sonnei invaplex ( gmt 546 ). animals immunized with invaplex ar had an anti - ipac serum igg mean titer of 1091 ( p & lt ; 0 . 001 ) whereas animals immunized with native invaplex or saline had undetectable levels of ipac - specific igg . the protective capacity of invaplexar was also evaluated for an invaplexar manufactured from purified s . sonnei lps and recombinant ipab and ipac using the mouse lethal lung model . it was compared to native s . sonnei invaplex . in addition the capacity of s . sonnei invaplex ar to protect against a heterologous s . flexneri challenge was also evaluated . in nave mice ( treated with saline ) 15 of 15 mice died with a mean maximum weight loss of 23 . 4 % of the pre - challenge weight ( see table 5 ). mice immunized with s . sonnei invaplex ar survived ( 15 of 15 survived ; p & lt ; 0 . 001 ) the lethal challenge with s . sonnei whereas mice immunized with native invaplex also exhibited solid protection ( 14 of 14 survived , p & lt ; 0 . 001 ) ( see table 5 ). with respect to weight loss , mice immunized with s . sonnei invaplex ar lost 7 . 7 % of their pre - challenge weight as compared to 9 . 2 % weight loss in the native s . sonnei invaplex - immunized mice and 23 . 4 % in the mice inoculated with saline . interestingly , the s . sonnei invaplex ar also provided significant protection ( 15 or 15 challenged mice survived , p & lt ; 0 . 001 )) against a heterologous s . flexneri 2a challenge suggesting that the immune response to either ipab or ipac may contribute significantly to the protective immune response . the results of this experiment evaluating the protective capacity of s . sonnei invaplex ar clearly shows that invaplex ar from another shigella species stimulates a level of protection that is comparable to that of native invaplex . the guinea pig keratoconjunctivitis model is used to evaluate shigella vaccines and is often used as the precursor to human trials . the model involves infection of the guinea pig eyes with shigella which establish infection in the corneal epithelium . this outcome ( severe keratoconjunctivitis ) is a result of invasion of the corneal epitheilium by the shigellae and the subsequent inflammatory response by the host not unlike that observed in the human intestinal tract . in this experiment guinea pigs were immunized three time intranasally with either s . flexneri 2a invaplexar or native invaplex . a saline control group was also utilized . three weeks after the final immunization animals were challenged ocularly with s . flexneri 2a . both the invaplexar ( 90 % protection , p & lt ; 0 . 001 ) and native invaplex ( 100 % protection , p & lt ; 0 . 001 ) provided solid protection ( see table 6 ) indicating that the invaplex ar is comparable to the native invaplex . intranasal immunization with ova alone or ova combined with ct or pre - immune samples from immunized animals ( day 0 ) did not induce detectable levels of serum igg or iga with specificity to s . flexneri 2a invaplex 50 , s . flexneri 2a invaplex 24 ( fig9 ), s . flexneri 2a lps ( fig1 ), ipab or ipac ( fig1 ). immunization with ova combined with either invaplex ar or native invaplex induced similar invaplex 50 , invaplex 24 and lps - specific serum igg and iga endpoint titers ( fig9 and 10 . endpoint titers against purified ipab ( p & lt ; 0 . 05 ) and ipac ( p ≦ 0 . 001 ) were higher in mice immunized with ova + invaplex ar as compared to mice immunized with ova + native invaplex ( fig1 ). comparable levels of ova - specific serum igg and iga were induced in mice immunized with ova combined with invaplex ar , native invaplex , or ct on day 42 and were significantly higher ( p ≦ 0 . 001 ) than the responses induced in mice after immunization with ova alone ( fig1 ). antigen - specific antibodies in lung washes were also assessed by elisa to investigate the mucosal immune responses induced after immunization ( fig1 ). immunization with ova combined with invaplex ar induced similar levels of lps and invaplex 24 - specific igg and iga in lung washes as compared to responses after immunization with ova combined with native invaplex and higher than the levels induced after immunization with ova alone , or ova combined with ct . similar levels of ova - specific lung igg and iga were induced after immunization with ova combined with invaplex ar , native invaplex , or ct which were significantly ( p & lt ; 0 . 001 ) higher than those induced after immunization with ova alone . moderate levels of lps and invaplex - specific iga in intestinal washes ( data not shown ) were detected in washes from mice immunized with ova combined with invaplex ar or native invaplex and were undetectable in mice immunized with ova alone or ova combined with ct . ova - specific intestinal iga responses were below levels of detection in all samples assayed . a serum responses in the s . flexneri 2a invaplex ar mouse study i are bllod collected on day 42 . serum responses in the s . flexneri 2a invaplex ar adjuvanticity study are from blood collected on day 35 . b geomean ± 1 standard deviation from the mean ( n = 5 mice / group ) ( range of endpoint titers ) * significantly higher endpoint titers ( one way anova of log transformed endpoint titers ; p ≦ 0 . 001 ) as compared to endpoint titers of animals immunized with native invaplex . many therapeutic biochemicals , including antibiotics , are often ineffective against intracellular target because they are unable to cross mammalian cell membranes or because they require high extracellular concentrations to achieve therapeutic concentrations inside of mammalian cells . the artificial invaplex provides a mechanism to create a complex of invasins , lps and antibiotic by mixing the components during the assembly stage for creating artificial invaplex . once assembled , the native properties of invaplex allow it to transport the complexed antibiotic or therapeutic molecule into mammalian cells . purified , soluble ipab and ipac , each in their respective final buffers are mixed together at an ipac / ipab molar ratio of 8 . after the ipab and ipac are mixed , a solution of antibiotic , for example gentamicin at 5 to 100 μg / ml , is added to the mixture . next the ipab , ipac and antibiotic solution is slowly added to dry lps powder ( ratio of lps to total protein is 0 . 56 ). lps from any shigella species can be used ; for the described experiments s . flexneri 2a , s . sonnei or s . dysenteriae 1 lps was used . the mixture is incubated at 37 ° c . for approximately 2 hours with shaking . afterwards the protein / lps / antibiotic mixture is diluted to 20 mm tris - hcl , 0 . 10 m nacl and 1 . 2 m urea to reduce the nacl concentration prior to ion - exchange chromatography . for final purification , the diluted ipab / ipac / lps / antibiotic mixture is applied to an anion exchange column ( hitrap ™ q hp ) with buffer a consisting of 20 mm tris - hcl , ph 9 . 0 and buffer b consisting of 1 m nacl , 20 mm tris - hcl , ph 9 . 0 , and a step gradient of 0 % ( 5 column volumes ) to 24 % ( 10 column volumes ) to 50 % ( 6 column volumes ) to 100 % buffer b ( 5 column volumes ). one ml fractions were collected from a 1 ml hitrap ™ q hp column at an elution flow rate of 1 ml / min . fractions from each step were analyzed for the presence of ipab , ipac and lps by spotblot . the fractions containing ipab , ipac and lps are the artificial invaplex fractions . the effectiveness of the artificial invaplex - antibiotic complex will be evaluated in its ability to kill intracellular microorganisms such as shigellae growing in tissue culture cells . the artificial invaplex - 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