the present invention provides an oral preparation useful in vaccination against gram negative bacterial infection and method of its use . the same general preparation can also be used to treat gram negative bacterial infection by increasing the in vivo antibody response . the basic process for producing the oral preparation of the invention commences with growth and harvest of the bacteria , inactivation , preferably but not exclusively with formaldehyde , and lyophilization of the whole cells . this process appears to maintain the antigenic integrity of the bacteria . because of the feculent nature of the cells , the preferred diluent contains an aromatic oil , preferably a peppermint oil or a cherry - flavored oil , encapsulated in a non - phospholipid liposome ( novasome ®) to enhance the palatability of the vaccine . these flavored novasomes are composed of glycerol monostearate , soya sterols , soybean oil , cherry or peppermint oil , polysorbate 60 , oleic acid , and water for injection . details for preparation of lipid vesicles containing oil are disclosed in u . s . pat . no . 4 , 911 , 928 , the disclosure of which is incorporated herein by reference . however , other materials constituting the lipid vesicles , and other production methods , could be used so long as the flavor masking provision is met . an additional advantage of using the lipid vesicles described herein is that these vesicles appear to provide adjuvant activity in addition to their flavor masking capability . while such adjuvant activity is not necessary for practice of the invention , it may raise antibody titers , as described in u . s . ser . no . 08 / 201 , 346 , entitled &# 34 ; vaccines containing paucilamellar lipid vesicles as immunological adjuvants &# 34 ;, incorporated herein by reference . the following example shows a lyophilized , formalin - inactivated e . coli 0157 : h7 oral vaccine , reconstituted with cherry - flavored lipid vesicles in water for injection , which has been tested in animal models for both safety and immunogenicity . the vaccine is safe and immunogenic in mice and rats using a two - dose gavage regimen . this example illustrates the steps in preparing an oral vaccine of the invention , as well as tests for its safety . the e . coli 0157 : h7 vaccine described herein is a sterile , formalin - inactivated whole bacteria , lyophilized product in a single - use vial containing 300 mg bacterial protein in phosphate - buffered saline , ph 7 . 5 . the vaccine is stored at - 20 ° c . until reconstitution with 10 ml of the novasome - wfi water for invention ! diluent . the reconstituted vaccine should be administered within one hour . the novasome - wfi diluent is a paucilamellar , non - phospholipid liposome containing cherry - flavored oil to enhance the palatability of the e . coli 0157 : h7 vaccine . this novasome preparation is composed of glycerol monostearate ( 7 . 9 %), soya sterols ( 2 . 2 %), soybean oil ( 9 . 2 %), cherry oil ( 4 . 5 %), polysorbate 60 ( 2 . 1 %), oleic acid ( 0 . 1 %) and water for injection ( 74 %). after production of the novasome lipid vesicles , they are diluted with wfi in a ratio of novasomes : wfi of 1 : 32 ( v / v ). the final percentage of water in the novasome - wfi diluent is 99 . 2 %. the resultant novasome - wfi diluent is a sterile novasome suspension in water for injection in a single use vial . the novasome - wfi diluent is stored at room temperature . ten milliliters of the novasome - wfi diluent are utilized to reconstitute each bottle of the e . coli 0157 : h7 vaccine . ______________________________________glycerol monostearate 13 . 72 gpolysorbate 60 ( tween 60 ) 3 . 64 ggenerol 122 ( refined soya sterols ) 3 . 84 goleic acid 240 μlcherry oil 8 . 0 gsoybean oil 16 . 0 g______________________________________ all raw materials were added to a depyrogenated vessel which was then heated on a heating plate with agitation until all materials were melted . the lipid solution was then transferred to a sterile 50 ml conical centrifuge tube which was placed in an 85 ° c . circulating water bath for one hour . 3 . 7 ml sterile water for injection for each 5 ml of novasome preparation . for each preparation , the sterile water for injection was drawn up in a 5 cc syringe which was placed in an incubator and heated to 65 ° c . a syringe mixing machine using 5 cc syringes was set for a lipid / oil to diluent ratio of 1 . 3 : 3 . 7 . the speed controlling the force of pressure for mixing was adjusted to 70 . this instrument is described in u . s . pat . no . 4 , 895 , 452 , the disclosure of which is incorporated herein by reference . the heated lipid was drawn into a 5 cc luerlok syringe and connected to the diluent syringe by way of a two - way stainless steel connector . the syringes were then positioned into the teflon mixing block and placed on the mixing machine . the solutions in the syringes were allowed to mix by pushing the liquids back and forth from one syringe to the other under pressure for 10 strokes at room temperature and then rapidly cooled using co 2 for 90 strokes . several 5 cc novasome lipid vesicle preparations were combined into a single solution in a depyrogenated vessel . samples of the final pooled novasome preparation were taken for usp sterility testing , endotoxin testing , ph determination , stability testing , and sub - micron particle sizing . more particularly , usp sterility testing was performed on a 1 . 0 ml sample of the pooled novasome preparation . no bacterial growth was observed in the liquid medium . 4 , 850 ml of sterile water for injection was filtered through a 0 . 2 μm filtering unit into a depyrogenated vessel containing a stir bar . 150 ml of pooled novasome preparations was aseptically added to the diluent . the suspension was placed on a stir plate and allowed to mix for 24 hours . three 5 l bottles of novasome - wfi diluent were prepared . samples were taken from each 5 l novasome - wfi diluent bottle for usp sterility testing , endotoxin testing , and sub - micron particle sizing . a 10 μl sample of the vaccine was plated onto tsa which was incubated for 48 hours at 37 ° c . as a test for sterility of the larger batch of diluent . again , there was no growth observed on the solid or liquid medium . e . coli 0157 ; h7 vaccine manufacturing and control data preparation of e . coli 0157 ; h7 active substance the escherichia coli 0157 : h7 active material was derived from the escherichia coli strain 0175 : h7 ( atcc 43894 ). the seed was stored in 15 % glycerol trypticase soy broth at - 70 ° c . and identified prior to initiating the starter culture . culture identification was established by incubating the culture overnight on trypticase soy agar with 5 % sheep blood and macconkey &# 39 ; s agar . the culture showed a homogenous smooth colonial morphology and revealed only gram negative rods by gram stain . the api biochemical profile was consistent with e . coli 0157 : h7 . one colony from the tsa ii plate of the seed culture was used to inoculate each of two 15 ml tubes of trypticase soy broth ( bbl ). the cultures were grown on a rotor for 2 hours at 37 ° c ., after which a subculture was taken for identity check . 12 ml from the trypticase soy broth culture was used to inoculate a fernbach flask containing 1 l of lauria broth medium . the culture was grown on an incubator shaker at 150 rpm 37 ° c . for 3 . 5 hours after which a subculture was taken for identity check . the culture was incubated overnight on trypticase soy agar with 5 % sheep blood and macconkey &# 39 ; s agar . the culture showed a homogenous smooth colonial morphology and revealed only gram negative rods by gram stain . the api biochemical profile was consistent with e . coli 0157 : h7 . lauria broth medium ( ph 7 . 0 ) was used for the starter culture flask , as well as the fermentation process . the medium consisted of : the starter flask medium was sterilized in a steam autoclave for 30 minutes at a temperature setting of 121 ° c . the fermentation medium was sterilized in the fermentor at a temperature setting of 121 ° c . for 30 minutes . samples of both media were taken for sterility testing prior to initiating the starter culture or the fermentation . deionized water was added to a clean batch vessel . the ingredients for lauria broth medium were added with sufficient agitation for mixing and sufficient additional deionized water was then added to obtain 40 l . the solution was mixed , pumped into a 50 l b . braun fermento ( model biostat u - 50 ) and chased with sufficient deionized water to achieve a 50 l batch volume . the unit was sealed and the sterilization cycle started . 4 l of a 40 g / l dextrose solution was prepared and sterilized using a 0 . 2 μm membrane filter . the feed transfer line was aseptically connected to the fermentor and the dextrose feed was added . the incubation conditions ( ph 7 . 0 +/- 0 . 02 , temperature 37 ° c . +/- 1 ° c ., dissolved oxygen ( d . o .) .+/- 50 %, air flow 80 +/- 5 slpm , agitation d . o . cascade , 300 rpm / min .) were automatically maintained . the ph was maintained with an automatic controller utilizing 20 % glacial acetic acid and 4n sodium hydroxide . foaming was automatically controlled utilizing ppg 2000 in an addition vessel . the 1 l starter culture was used to inoculate the fermentor . the e . coli bacteria cells were allowed to ferment into log phase for 10 hours . samples were obtained hourly for optical density ( od ) measurement . when absorbance approached 1 . 0 , dilutions were performed . dextrose usage and foaming were monitored . subcultures were taken from the starter flask and at the end of the fermentation run for identity testing of the isolate . bacterial count ( cfu / ml ), ph , dextrose analysis and od were performed on the final product . cells were harvested using a heraeus varifuge ( model 20 rs ) continuous centrifuge for 5 . 25 hours . speed of the continuous rotor was controlled at 15 , 000 rpm and temperature was controlled to less than 25 ° c . supernatant samples were analyzed periodically during centrifugation for optimization of centrifugation . 250 g of cell paste was then aseptically transferred to each of 5 1 l nalgene centrifuge bottles for further processing . centrifuge bottles were weighed prior to addition of cell paste and the weight was recorded . six liters of 1 . 5 % formaldehyde solution were prepared using phosphate buffered saline ( pbs ) ( 146 mm na - 10mm po 4 ) diluent . samples of pbs were taken for usp sterility and endotoxin testing as well as ph determination . the 1 . 5 % formaldehyde pbs solution was added to each centrifuge bottle to achieve a weight of 1 , 035 g . the paste was resuspended by agitation after which centrifuge bottles were allowed to stir at 4 ° c . on a stir plate for 96 hours . samples from each centrifuge bottle were taken at 24 and 96 hours and plated on tsa ii plates for monitoring of viability . after 96 hours , the formaldehyde inactivated paste was centrifuged for 4 hours at 4000 rpm 4 ° c . in a beckman refrigerated centrifuge ( model j - 6b ). supernatant was discarded and sufficient pbs was added to each bottle to achieve the initial weight . paste was resuspended by agitation , after which bottles were centrifuged for 4 hours at 4000 rpm at 4 ° c . in a beckman refrigerated centrifuge ( model j - 6b ). the supernatant from this centrifugation was discarded and the wash process was repeated . samples of pbs were taken for usp sterility and endotoxin testing ( see ipc ) as well as ph determination . there was no growth observed in the liquid medium . an endotoxin assay was also performed using the limulus lysate assay ( cape cod associates ). the endotoxin level was at an acceptable level of & lt ; 0 . 03 eu / ml . as an additional check on the viability , a 10 μl sample of the product was taken at 24 and 96 hours after formalin inactivation and plated onto tsa ii which was incubated for 48 hours at 37 ° c . there was no growth observed on the solid medium . the formaldehyde inactivated bacterial paste was resuspended by agitation in pbs at a concentration of 1 g / 5ml of pbs . samples were taken from each centrifuge bottle of the final product for usp sterility testing . usp sterility testing was performed on a 1 . 0 ml sample of the resuspended formaldehyde inactivated paste . a 10 μl sample of the vaccine was also plated onto tsa which was incubated for 48 hours at 37 ° c . there was no growth observed on the solid or liquid medium . the formaldehyde inactivated vaccine was aseptically filled into 50 ml vials using a sepco filling machine with presterilized syringe and robing apparatus , with 10 ml of the paste placed in each vial . the product was frozen to - 40 ° c . on the lyophilizer shelf . the lyophilization chamber was evacuated to 100 μm and the product remained at - 20 ° c . for 16 hours . the temperature was then elevated to 0 ° c . for 7 hours , after which it was elevated to 25 ° c . for 14 hours . 40 samples were taken for usp sterility testing and 10 samples were analyzed for residual moisture assay . the vaccine passed both tests . the lyophilized vaccine was stored at - 20 ° c . mice were divided into 4 groups of 10 mice each . all the mice in a group received the e . coli 0157 : h7 vaccine reconstituted in cherry - flavored novasome - wfi diluent at doses of 7 . 50 , 1 . 875 , 0 . 9375 , and 0 . 1875 mg of protein , respectively . vaccine doses were given on day 0 and day 30 and were administered by garage . the animals were prebled 4 days before receiving their first vaccine dose , and were test - bled on days 14 , 28 , 44 , and 58 . two animals died during the course of the study : one in the 1 . 875 mg dose group died on day 30 , and one in the 0 . 9375 mg dose group died on day 5 . mouse sera were tested by elisa for the presence of antibody to e . coli 0157 : h7 lps . test sera were serially diluted in flat - bottomed elisa plates previously coated with e . coli 0157 : h7 lps and blocked . after two hours of incubation at room temperature , the plates were washed and incubated with enzyme - labeled goat anti - mouse igm or anti - mouse igg second antibody for 1 hour . after further washing , the plates were developed with a colorigenic substrate , and read using an automated elisa plate reader . anti - lps antibody titers were the reciprocal of the serum dilution producing an optical density ( od ) reading 3 × the od reading of the homologous prebleed . the igg subclass response was determined by using enzyme - labeled second antibodies specific for mouse igg 1 , igg 2a , igg 2b , and igg 3 . sera showing high pre - bleed elisa titers against e . coli 0157 : h7 lps were screened for antibody specificity by western blot . purified e . coli 0157 : h7 lps was separated by polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose sheets , which were then blocked , cut into strips , and dried . for screening , strips were incubated with a 1 : 100 dilution of the test serum for 2 - 4 hours , washed , incubated with an enzyme - labeled second antibody , rewashed , and developed with a precipitating colorigenic substrate . both the lps - specific igm and igg titers at day 58 were dose - dependent . the two higher doses of vaccine produced igm tilers on day 58 that were roughly equivalent ( fig1 ), as were the 58 - day igg titers at the same two doses ( fig2 ). in both cases , the tiers resulting from the lower doses of vaccine were correspondingly lower . the 58 - day igg titers were approximately 10 - fold higher than the igm titers . significant igm titers were obtained 14 days after the initial dose with the vaccine preparation at doses of 7 . 50 mg ( fig3 ) and 1 . 875 mg ( fig4 ). igm displayed an anamnestic response after the second vaccine dose . igg titers , which were low 28 days after the initial dose , rose dramatically after the second dose , and remained high at day 58 ( 28 days after the second dose ; fig3 and 4 ). this example illustrates a second oral vaccine of the invention . like the e . coli preparation of example i , this vaccine is also prepared using sterile formalin - inactivated whole bacteria . in this case , the bacteria is shigella flexneri 2a . the vaccine , 419 mg bacterial protein in pbs at ph 7 . 15 , is stored at - 20 ° c . until reconstituted with 10 ml of the novasome wfi diluent . the diluent and the vaccine are prepared according to the procedures set forth in example i . mice were divided into four groups of 15 mice each . the mice in each group received the s . flexneri 2a vaccine reconstituted in cherry flavored novasome diluent at doses of 10 . 47 mg , 2 . 618 mg , 1 . 309 mg , and 0 . 2618 mg of protein , respectively . vaccine doses were given on day 0 and day 30 , and were administered by garage . the animals were bled 4 days before receiving their first vaccine dose , and were test - bled on days 14 , 28 , 44 and 58 . sera showing high pre - bleed elisa titers against s . flexneri lps were screened for antibody specificity by western blot . purified s . flexneri lps was separated by polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose sheets , which were then blocked , cut into strips , and dried . for screening , strips were incubated with a 1 : 100 dilution of the test serum for 2 - 4 hours , washed , incubated with an enzyme - labeled second antibody , rewashed , and developed with a precipitating colorigenic substrate . both the lps - specific igm and igg titers at day 58 were dose - dependent . igg and igm liters rose dramatically after the second dose , and remained high at day 58 ( 28 days after the second dose ; fig5 and 6 ). this example illustrates a third oral vaccine of the invention . like the e . coli preparation in example i , this vaccine is also prepared using sterile formalin - inactivated whole bacteria . in this case , the bacteria is salmonella enteriditis . the vaccine , 653 mg bacterial protein in pbs at ph 7 . 15 , is stored at - 20 ° c . until reconstituted with 10 ml of the novasome wfi diluent . the diluent and the vaccine are prepared according to the procedures set forth in example i . mice were divided into four groups of 15 mice each . the mice in each group received the salmonella enteriditis vaccine reconstituted in cherry flavored novasome diluent at doses of 15 . 89 mg , 3 , 973 mg , 1 . 986 mg , and 0 . 397 mg of protein respectively . vaccine doses were given on day 0 and day 30 , and were administered by garage . the animals were bled 3 days before receiving their first vaccine dose , and were test bled on days 14 , 28 , 44 , and 58 . sera showing high pre - bleed elisa titers against s . enteriditis lps were screened for antibody specificity by western blot . purified s . enteriditis lps was separated by polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose sheets , which were then blocked , cut into strips , and dried . for screening , strips were incubated with a 1 : 100 dilution of the test serum for 2 - 4 hours , washed , incubated with an enzyme - labeled second antibody , rewashed , and developed with a precipitating colorigenic substrate . both the lps - specific igm and igg tilers at day 58 were dose - dependent . igg and igm titers rose dramatically after the second dose , and remained high at day 58 ( 28 days after the second dose ; fig5 and 6 ). from the foregoing , it is apparent that the orally administered , inactivated whole bacteria vaccines of the present invention are safe and immunogenic . the foregoing description of the invention is meant to be only exemplary and is not intended to limit the scope of the invention . the invention is defined by the following claims .
the present invention concerns an oral preparation useful as an immunizing agent or vaccine against gram negative bacterial infection . this oral preparation can also be used as a treatment for those infected with gam negative bacteria . the preparations can be used against any gram negative bacterial infection , including escherichia coli , shigella flexneri 2a , and salmonella enteriditis .