Vaccine for treating urinary tract infection

[Detailed Description of the Invention]

Many things are already proposed and humans or the vaccine against the various diseases of an animal is used from long before. For example, the bacteria which exist in a vagina, specific focus part, for example, tonsils, phlegm, or feces are brought together in the Switzerland patent No. 158,980, and the manufacturing method of the mixed vaccine for patients which cultivates this indiscriminately and inactivates it is indicated. By adding tannic acid aluminum to a suitable fluid extract, the antigen of the variety origin can be remarkably made into insoluble in water nature, and the France patent No. 2,054,745 says that absorption is delayed and an operation maintains output. European Patent No. 428,172, U.S. Pat. No. 4,538,298, or unlisted Drags (Unlisted Drugs), To the 60th volume and 123 pages (1978), it is indicated that the vaccine based on the enteric pathogen stock of a coliform bacillus kind and Gret back (G18tVax) K2S can use it for the passive immunity of the new animal for meat. The vaccine can prevent to a dam the inside of muscles, or the diarrhea which will take place by coliform bacillus after birth of a Offspring cow, a baby pig, etc. if it administers hypodermically at the suitable time before a delivery. The vaccine for humans based on a large 1! bacillus is also already proposed. In the German patent public presentation No. 1,951,195, a microorganism is extracted and annihilated from a tracheal gill infection patient's phlegm, and the method of preparing in the vaccine of aerosol Agent is indicated to it. This is prescribed for the patient by inhalation and means the immune therapy of a tracheal gill. this vaccine -- the bacteria of a coliform bacillus kind, and many others -- Seeds -- it ° calls and stock bacteria are included. the vaccine indicated to the France patent No. 2,397,839 -- a different four-sort stock of coliform bacillus -- many (the killed bacteria or the attenuated bacteria of bacteria d is included.) Oral Yo administration of this vaccine is carried out, and it is used for the immunity disposal of a gastrointestinal tract especially the medical treatment of gastroenteritis, and prevention. This vaccine contains methionine, iron salt, vitamin, Lactic acid rod @ (Lactobacillua) M, etc. In contrast, , (the vaccine mainly constituted considering coliform bacillus as a base for infection of a ureter has not been developed yet.) such infection -- one -- an antibiotic and a chemotherapeutic drug -- extensive (that development-of-symptoms frequency does not fall in spite of being used, but the high value is shown, and another side) a recurrence -- many (80% or more of rate of recurrence) (it is completely unexpected that there is no such nevertheless vaccine which is an important problem medically from moreover there being a tendency of chronic.) The reason which did not have a vaccine until now is considered because there are a huge number of human serum protein types in coliform bacillus. Especially these bacteria have very complicated antigenic structure. there are 6 m of main antigen groups -- present and O antigen type -- about 160 sorts and K store -- 60 or more sorts and H type -- 60 or more sorts are known and serologically different about 10 and 000 stocks exist in coliform bacillus with such combination. Thus, it seems that it was thought that it was impossible from the start to manufacture an effective vaccine to urinary tract infection generally from a number of coliform bacillus stocks which can be considered sensibly since there were many stocks. what guarantees immunity no matter it may twist the vaccine which is sufficient for asserting the general activity to all the urinary tract infection to what bacteria to cystitis, prostatitis, and a Kidney mouth disease VG Kidney glove nephritis -- it is not likely to be -- if it is Leave, there is nothing. However, it is also clear that a possibility that immunoreaction will attain to an immanency system becomes higher as the immune range becomes large. There is Fringe border (hair, Fibril) in coliform bacillus bacteria, and this enables adhesion in the intestinal mucous membrane of ° coliform bacillus in intestines as known well. If there is no Fringe border, coliform bacillus bacteria will begin to be passed from intestines, and it is possible that the physiological equilibrium in the intestines between coliform bacillus kind bacteria and other type bacteria is disturbed remarkably. Therefore, when the vaccine based on coliform bacillus is used, we are anxious about producing the far-reaching obstacle in a bacterial flora in intestines which reacts also to the coliform bacillus bacteria in all the systems in which a production Did do antibody usually has Fringe border, i.e., intestines, and was mentioned above inside of the body. however -- just because it was isolated from the urinary-tract-infection patient's urine -- many (from a number of coliform bacillus stocks which are not, and some stocks belonging to other bacteria kinds) Activity was specifically and generally shown in urinary tract infection, and, moreover, it was made clear to Things to save in spite of the above concern that the vaccine which does not have influence actually harmful to the bacterial flora in intestines could be manufactured. 8~14 kinds of urinary-tract-infection Pathogenesis bacteria stocks belonging to the kind of faecalis in which the present invention was isolated from (1) urinary-tract-infection patient's urine Inactivation bacteria obtained by cultivation of (however, 3/4 is a stock belonging to a coliform bacillus kind from the half of the stock used), 1.5~10■ provides the vaccine suspended in tension solution, such as sterilization, in four-sort Lt in each about 50 million stocks ~ 500 million pieces and (2) colloid aluminium phosphate (AJPO4), and 1M. 8~14 kinds of above-mentioned urinary-tract-infection Pathogenesis bacteria stocks with which the vaccine of the present invention was isolated from the urinary-tract-infection patient's urine (However, 3/4 is a stock belonging to a coliform bacillus kind from the half) It is on a suitable nutrition culture medium about each, It cultivates independently, the generated specific biological material is separated after the completion of cultivation, and it is inactivated by the publicly known method, The inactivation bacteria obtained from each Law are mixed mutually, and this mixture is diluted with sterilization isosmotic solution so that about 50 million inactivation bacteria [ from each Law ] ~ 500 million pieces may exist in 1 ml, It is manufactured by adding Colloy * Do-like aluminium phosphate until it becomes the concentration of AA!PO41 and 5~10 Da into 1 ml. Below, the present invention is explained still in detail. First, it was isolated from the urinary-tract-infection patient, and the urine sample (middle class urine) was inoculated on direct Matsukikonki- (Mac-Conkey) Agar. After inoculation, the colony which incubated by 0 67 degrees for 16~18 hours, and formed - Le Plate was isolated, and A was biochemical, and investigated biological character, and identified. Ny catalase by Durham dyeing of a special-feature small colony, and the following examinations (under existence of heating blood) +-hemolysis - Growth by growth 10-pH 9 and 6 in /beta-45-degreeC 10-6, 5%NaC! Growth in solution Detection of 10d of 10 to 40% growth ? LISA Tucaride antigen in the gallbladder and appropriation 2 of the following [ identification / of the stock belonging to 10 Dog intestine J1 Zolotheus J14 and (Proteus) Kumshuela sp. (Klebsiella) ]! I! : Semi was used. the colony identified by this method -- further -- it is made to grow by 37 degreeC on 1 Agar plate for 24 hours, and subsequently to a physiological saline is suspended, and purity was examined and it was made to freeze-dry by a Gram's method of staining The feature was described as follows about each obtained ridge, and it identified so that it might be seen in a table. Biochemical characteristic Growth by a nutrition culture medium Temperature resistance + : positivity 0: Negativity The morphological characteristics of an above-mentioned isolation stock can be summarized as follows. Dog jJIh The: It exercises by the flagellum projected without movement Durham negativity Bacillus and a sheath film from one mycelia cake 4~0.6X0.1~3.0 micrometers of skins by the flagellum projected from the Durham negativity Bacillus,X[ 1.1~1.5 ] 2.0~6.0 micrometers (raw bacillus), and a mycelia cake (they are not all). Pneumobacillus: The Durham negativity, aplanetic Bacillus, 0.6~1.5 micrometers x 0.6~6.0-micrometer nature About the coliform bacillus stock, the serological property was examined further and, for the reason, Many value OK serum A, B, and CX D and an agglutination test with E were first given on the slide. Di 7 piece -, 7 Bora Trease (Difc.) Directions fK of Laboratories, the Detroit (Detr, oit) XMLtrs person, and 1976], therefore a classification (and [ without 0 ] determination of H antigen) of a human serum protein type were performed. The following images were obtained by this identification. Ten strains which were described above and defined are as August 8, 1984 and CBS Fall 516.84 No.~g 525.84 No., Arousal culture central office (Centraalbureau voorSchimmelcultura s) Male Chilsted lard (0osterstraat) and 1.374 Q AG, It Deposits to Baarn (Baarn) and the Netherlands], and is the German microorganism depository institution (Deutsche Sammlung von Mikroorganismen.) as December 5, 1984 and accession number [ DSM No. 6229 ] ~ No. 6268. It was moved to Grizeba 7 Ha, X Truth (Grisebachstrasse) 8.5400, and Ke 9 Tutedenen (Gdeltattingen) west r Germany]. When manufacturing a vaccine, the strain was separately cultivated on the solid or the fluid nutrition culture medium. It is preferred to use (Nutrient Agar Difco) nutrition Agar A/ - which has the following composition, for example as a solid nutrition culture medium. meat juices (Dirco beef extract) 34 sort / 1 bacto (Bacto)-peptone 59/1 salt 51 / 1 bacto (Bacto)-A -A / 15! l/1 -- this medium is sterilized for 15 minutes by 121 degreeC. Voice is about 7.2. In large-scale manufacture, a petri dish is used for laboratory manufacture using a roux (roux) bottle. By distributing the number d of inoculation materials over all the surfaces uniformly, inoculation is closed with a line 5o roux bottle cotton plug, turns the inoculation surface down, and saves a medium. An inoculation culture medium is incubated by 67 degreeC for 24 hours. That of bacteria washes out a part with requirements (based on the growth concentration of the culture in a container) of phosphate buffer solution / salt solution (PBS), agitating quietly so that the Agar surface may not be broken. A smear specimen is made from each bottle or a petri dish, and it dyes by the gram method, and throws away A cheese or the petri dish in which <> contamination which examines purity was accepted. One strain and the suspension from each batch are collected, and it filters through Extinction eta nylon gauze. - which performs inactivation as well as the cultivation itself for every strain. Inactivation can be carried out by heating to about 55~60 degreeC, formaldehyde fluid processing, or r line irradiation. Inactivation according to heating at the point of being easy is preferred. The culture obtained by cultivation is set, and during bathing, it heats for 1 hour, inactivates by 60 degreeC, and adds phenol after thermal inactivation (0.35% of the last concentration). Some suspension is taken, a sterility test and an identification test are done and the condensed main part is stored in a refrigerator. In three days, when impurities are not accepted by a sterility test, manufacture processing is continued. When growth of a certain microorganism is observed in the att guess during 14 days, and a sterile sample, these crops throw away a sterility test. About the content (inactivation suspension originating in one strain) of a preservation container, it is by cooling centrifuge [vine pole (5orvall) 4 CR and upward rotor 2,000, 3.000 It centrifuges by rpm for 1 hour, supernatant liquid is removed, and a bacteria sedimentation thing is suspended in 0.01% of thimerosal content salt solution. the degree of honey of a preservation concentrate -- a standard -- turbidity -- it uses and measures, a label is given to suspension, and it stores in [4-degreea< 2 2-degreeC. The concentration suspension of ten strains of coliform bacillus, a strange modification bacillus, the Morgan modification bacillus, a pneumobacillus, and excrement streptococci is mixed, and mixture 1Kt, EachX[ 2.5 ] 108 coliform bacillus 6 strain meter 1.5X10' Pieces Strange modification bacillus 1 strain 7.5X10' Pieces Morgan modification bacillus 1 strain 7.5X10' Individual pneumonia 1 strain 3X108-piece excrement streptococci 1 strain 5X107-piece total It is made for 2X10' Pieces / d to be included. it goes each preservation suspension but -- it mixes and the last concentration is obtained. a marks are diluted and attained by 0.01% thimerosal content 0.15 M phosphate buffer salt solution. After holding to room temperature for 24 hours, it saves at 4 degreeC (12degreeC). aluminium phosphate -- a pill -- since it adds -- concentration -- the part quantity (it must carry out.) Phosphoric acid aluminum Nitadel is prepared as follows. Since aluminum potassium sulfate and 12 HaO (854g) Luminicum solution become super saturation at room temperature, it holds to 37 degreeC. It fills underwater [ 24 sorts of ] with both solution simultaneously. Precipitation is centrifuged, a sedimentation thing is re-M Made muddy to water 16*!, and suspension is centrifuged again. Finally, a sedimentation thing is re-suspended, this suspension is made into 8.4 sorts of whole picture, and - is heated to 6.0 by 5NNaOH for 1 hour at iI1 ml!I Si and 121 degreein auto crepe C. This Site is 1Jp04. It is sufficient quantity to manufacture vaccine 661 containing 3Da/d, or # 0.66 Da / d. capacity 1J which does not contain the pyrogen which carried out the mature rice-seedling after checking that the voice of the adsorbed last vaccine is in a 6.2~7. meeting! The ampul of 7 is filled up with every [ 1 time dosage 0.5 d ]. The vaccine needs to continue shaking during restoration. The total of the inactivation microorganism in the 1-time Fortress, i.e., 0.5d, is abbreviation I X 10' Pieces. <> which follows method of Europe office (2nd edition, 1st supplement 1980) K Follow, and toxicity in the sterility of a vaccine, follows the method of the Europe office in special toxicity by the standard (weight increase of a mouse) of WHO (World Health Organization) at the end, and it examines A vaccine can also be made into the freeze-drying drug design instead of an above-mentioned fluid drug design. The manufacturing method is as follows. In 0 dilution diluted to Yo 5 containing inactivation bacteria abbreviation 1X10'1liil of composition of a Shrink inactivation suspension obtained in the above-mentioned manufacture process as stated above in 0.5ILt, The inside of 0 containing thimerosal 0.01% and 85N salt solution, and human albumin Up and It was are Masaires (Haemaccel') to a plasma substitute. Beilin Gubaque(Behrlng-yerke AG) a and 3500 Marl Delk (Marburg) and the liquid containing West Germany]1% are used. Under a sterile condition, the vial of 2 A go is filled up with 1, 0.54 sort of these dilution suspension freezes it in abbreviation-45-degreeC, and it dries under a vacuum within freeze dehydration equipment. The drying temperature must not exceed 66 degreeC. All the processes of freeze-drying require about 24 hours. In order to introduce aluminium phosphate for water phosphoric acid aluminum Nitadel of 24 sorts of 0AJPO4 concentration 1 / ILI which seals a vial by rubber orange under a nitrogen air current after freeze-drying, and attaches an aluminum Nikumu cap before medication, Manufacture of 0 aluminium phosphate r Le simultaneously used as a solvent for remelting is as having described the above-mentioned manufacturing method. thus, the obtained phosphoric acid aluminum -- it dilutes with Nitadel salt solution, the last (about 12 times) concentration is set to 21R9-/IR1, and an ampul is filled up. The ampul of capacity 1- is filled up with 0.5 d. In order to examine the preservation life of a vaccine, capability to make an antibody form in a mouse was Race course(ed) twice. Namely, it measured as December 3, 1981 immediately after manufacture on December 7, 1983 two years after. Immunity disposal of a mouse: The vaccine was injected intraperitoneally in mouse (maleness, NMRI distant relative mating, weight 16~209) 6 group of each ten groups, and immunity disposal was performed in it. The interval of two weeks was set and injected twice (each time vaccine 0.5 M). Only phosphoric acid aluminum Nitadel (0, 5m) was injected into 20 mice used as contrast of of the same kind and the weight. Two weeks after the 2nd injection, blood was extracted abacterially and collected for every group. After clotting, it centrifuged, the serum of each pool was obtained, and freeze preservation was carried out by -22 degreeC to implementation of a serological examination. The of-the-same-kind bacteria stock of nine hammers contained in the vaccine was used for manufacture of an antigen. Strain Ec 654 was not condensed (R type) and was not able to be used as a condensation field. Nutrition culture-medium (Offspring Cush meat exudation culture-medium-Dirco) 9 d K suspension of the freeze-drying culture of a bacteria stock was carried out, and the culture was incubated by 67 degreeC for 24 hours. Secondary culture was used for manufacture of an antigen. Nutrition culture-medium [ 6004 sort / four sort ] (Child Si meat exudation culture medium) 1C seed culture medium was inoculated (each stock separately). It is 37degreeC about a culture and is 36-hour ink Eupertisi. Next, formalin was added so that it might become 0.1% of the last concentration. The culture was incubated for seven days by 67 degreeC. The examination of the nonexistence of a raw bacillus and sterility was done about each culture. The suspension of inactivation bacteria was centrifuged at 3.000 rpm with the cooling centrifuge (8orval) for 1 hour. a sedimentation thing is re-suspended to phosphate buffer solution / salt solution (p) i, and 7.2 -- the inside of 1' -- about [ bacteria ] -- 20 X 10' -- it was made to become the concentration of Pieces As a condensation field to a condensation temple, the suspension of bacteria abbreviation 2X10' Pieces / d was used. Agglutination test: Mouse serum was diluted with 1:2 dilution methods with the phosphate buffer physiology salt solution of silk 7.2. In any case, dilution serum 0.3d was mixed with the antigen of the capacity in the Curnow (1cahn) pipe. The pipe which did not add serum was used as antigen contrast. It was dressed after 18:00 time Switched to 57 degreeC for 24 hours at 4 degreeC, and the reaction was checked after It was. Condensation of bacteria made the maximum dilution detectable with the naked eye the potency of the serum. Negativity and a positive serum were also used for all the batches as contrast. The geometric average value of the cohesive force value of mouse serum was as follows. Colon Ul 455UB 1,015.5 905.1*Jyk bacillus 525 -- 905.1 1,015.5 Joy 560UB 1,015.5 905.1'j4 direct 616 UB 905.1 806.5 Size Ylf Ul 719.UE 1,280 1,280 pneumobacillus 16 B 201.6 201.6 strange modification 163 B 160.0 142.5 Morgan Transformation 58B 4 sort 3.1 4 sort 3.1 -- for at least two years, a vaccine holds activity completely and the result of these shows it in -\ and To, when saved at 4 degreeC. Especially adaptation of this vaccine is cystitis, prostatitis, bladder Kidney mouth disease, and a Kidney glove nephritis. Intramuscular injection of the vaccine of each 0.5 time is carried out to the patient who is not in acute letter H of generation of heat (contraindications) 3 times at intervals of 1~2 weeks. Disposal should be interrupted when a severe reaction is produced to inoculation of a vaccine. In one year, injection will be repeated as a booster by the <0.5m same dosage. A vaccine is inoculated into the patient of the urinary tract infection microbiologically checked by the owner symptoms nature from many hospitals until now at 383 examples, and it will be To continue about one-year or more observation. By inoculation, the improvement of the urinary tract resistance maintained for at least one year arose, and the result that the relapse of urinary tract infection was prevented was obtained. The result of a clinical test is summarized in the following table. The recurrence / the number of re-infection in a 1~12 power monthly observation period Q I H It may be said that tolerability is good. Critical side effects did not arise. This vaccine can be satisfactorily used also for a pregnant woman. This is proved from the experience about the pregnant woman of 100 or more examples. Finally, the main features and advantages of the present invention, and the present invention vaccine which was not described until now are pharmacologic, and it summarizes the result of a clinical approach. Quantitative composition of ten strains is based on 25% of Temple of 75% of the distribution frequency 2 coliform-bacillus stock (uniformly existence) of the disease germ of urinary tract infection, a strange modification bacillus, A / l / cancer modification bacillus, a pneumobacillus, and excrement streptococci. In the vaccine of the present invention, these urinary-tract-infection disease germ stocks containing the antigen in which a defense effect like a Fringe border antigen and O-antigen is shown, for example are inactivated. On the other hand, it is also shown clearly that these strains selected as an antigen show the intersection defense effect also to the disease germ of the different-species stock belonging to the same kind in a defense examination with a mouse. As shown by the mouse weight increase examination, toxicity fell [ the inactivation antigen contained in the vaccine of the present invention ] to the cooling storeroom clearly after at least 6 power month-long preservation. This vaccine induces an immune response to the humans who inoculated the inoculation animal (a mouse, rabbit) again, defends a mouse from death-dealing infection, and defends a rat from an experimental Kidney glove nephritis. in the quantitative and qualitative composition equivalent to the amount of antigens which induces a good immune response in addition -- a patient -- Ii -- any side effects do not arise as well as Serious oJ operation. This vaccine also induces the secretion to the immunoglobulin to the inside of urine to an inoculation patient. Simultaneously with To relax, this vaccine defends an at least 12 power monthly and a patient for urinary tract infection from re-infection. The vaccine of this @ Ming can carry out the product made from 'fA to a fluid drug design and a freeze-drying drug design. In a freeze-drying drug design, it is used as a re-Rebirth solvent before prescribing phosphoric acid aluminum Nitadel for the patient.