Method of obtaining a vaccine for therapeutically and prophylactically treating infections of respiratory tracts of humans

<td rowspan="2">POLAND REPUBLIC CHINA</td><td>PATENT DESCRIPTION</td><td> 145565</td>

<td rowspan="3">Additional patent to the patent in</td><td rowspan="3"></td>

<td></td>

<td></td>

<td></td><td>Reported: 08, 30 (P. 235193)</td><td></td>

<td></td><td>Priority: Θ4 08 31 Swiss</td><td>Int. Cl.⁴ A61K 39/108</td>

<td></td><td></td><td>A61K 35/22</td>

<td>OFFICE PATENT</td><td>The application was announced: 86 07 13</td><td></td>

<td>PRL</td><td>Patent description published: 89 09 30</td><td></td>

Creators in ^ mlazku: LLubinko Stojko / ic, Rocdaila Fhvic, Vera S] psojevic Patent holder: Solco Bsel AG, fczylea (Sziwaccaia)

HOW TO MAKE THE VACCINE FOR CURATIVE AND PREVENTIVE TREATMENT OF URINARY ROAD INFECTIONS IN HUMANS

There is a way of raising the vaccine for curative and prophylactic treatment of urinary tract infections in humans ·

Vaccines against various diseases in humans or animals have been known and used for a long time · For example, according to Swiss Patent No. 158 980, a separate mixed vaccine for a sick person can be boiled in such a way that ascending at a given outbreak is taken germs, e.g. from tonsils, from the vagina, from sputum or feces, grow them without melting and deactivate. According to French Patent No. 2 034 743, antigens of various origins can be made insoluble in wcd by treating the appropriate liquid in^theseąg taninirnem gHoowm · The resulting product visualizes delayed absorption capacity, so its effect is prolonged.

Vaccine prepared on the basis of enterop: aLtogenic strains of the genus Escherichia coli according to European Patent No. 28 172, according to United States Patent No. 4,338,298 or ^ according to UListed Drugs (30, 1978 /), 123 Getrax K38 can be used for passive immunization of fresh-born slaughter animals. The vaccine is administered intramuscularly or subcutaneously shortly before the mutce of the animal and thus protects calves, piglets, etc. after birth before diarrhea caused by coliforms.

Rroponowint also for humans vaccines based on Cli. According to imported German Patent 1 931 193, microorganisms are collected from the sputum of persons suffering from respiratory tract infection, killed these microorganisms and processed into an aerosol vaccine. It is given by inhalation and can be used as an anti-inflammatory medicine. breathing. This vaccine contains, in addition to many other types and strains of ballery. also Escherichia coli.

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Another known vaccine, known from French Patent No. 2,397,839, consists of killed or weakened embryos of a certain number of genus W ballteria, including 11 different strains of Escherichia coli. It is born orally and is intended to immunize the gastrointestinal tract, and in particular to eliminate this cataract or to protect against catarrhal disease. This vaccine contains besides. including methionine, iron salts, vitamins, lactic bacteria and more.

To date, however, no vaccine strictly for urinary tract squeezing and based in particular on Escherichia coli has been developed.

The reason for the lack of such a vaccine is a very large number of serological types of techerichia coli. Manually, these bacteria have a very complex antigenic structure, with 3 major antigenic groups. So far, about 160 antigen types 0, more than 60 K types and more than 60 H types have been identified, resulting in approximately 10,000 serologically different Escherichia coli strains. With such a number of strains, the development of a vaccine effective against urinary tract infections in general on the basis of (possible) to some extent the number of 'Escherichia coli strains' seems impossible in advance.

For such a vaccine to be considered generally effective against urinary tract infections, it should actually provide immunity against cystitis, prostatitis, pelvic inflammation and nephritis, regardless of the bacteriological origin of the disease. However, it is clear that the wider the scope of the vaccine's immunizing activity, the greater is the potential for such effects to spread to other systems specific to the body of the person receiving the vaccine.

As is known, Cli bacteria infiltrate fimbriae (Italian, fiber), which cause adhesion of these intestinal bacteria to the intestinal mucosa. Without the interaction of fimbria, Cli bacteria would be flushed from the intestine, which would indicate a serious violation of the physiological balance between Cli bacteria and other bacteria in the intestine. therefore, it was equally feared that the Cli-based vaccine would cause antibodies to form in the body that would be able to react with normal fimbria-causing germ systems, including coliform coliforms, which would cause serious intestinal flora abnormalities mentioned above .

It has been surprisingly found, however, that from a small number of strains of cherichia coli and a low number of certain other types of balleria that have been isolated from the urine of people suffering from an urinary tract infection, it is possible to produce a vaccine that is effective against such specific infections and is generally effective and not practically no harmful effect on the intestinal flora. .

The new vaccine is prepared as a suspension in a sterile, isotonic solution by the method consisting of weeftug of the invention in that the following uropathogenic strains of bacteria, namely Escherichia coli DSM 3229-DSM 3234, Kebsiella pneumoniae DSM 3235, R ~ otous mirabilis DSM 3238, RMemus morganii 3237 and Streptococcus faeralis DSM 3236, which are known in the urine of people suffering from urinary tract infection, each is grown separately on a suitable medium and after the end of the culture, each of the biological materials produced is separated and inactivated by heating the suspension to a temperature of 55-6 ° C for about 1 hour, or by treatment with formaldehyde or by irradiation with radiation , then the obtained and prepared bacteria from individual strains are mixed together in such an amount and diluted with sterile isotonic solution, that in 1 m there are about 50-500 millionW of ZarockcW in each strain and inflicts color with aluminum phosphate to obtain a concentration of 1.5-10 mg ALPO ^ per 1 ml suspension.

The process according to the invention is carried out as described below.

First, medium mocr samples are taken from persons suffering from urinary tract infection, they are transplanted onto tocCnncey agar, they are cultured on agar plates at a temperature^{e 37}° C in ^cof the first^6-18 ^gthen, separate the resulting colonies and identify them by determining their biochemical and biological properties.

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Identification of Streptococcus faecalis (enterococci).

The characteristic nice colonies are colored by the Gram method and subject them to the following tests:

phytophysis (in the presence of warmed blood) ♦ hemolysis -! β increase at 45 ° C + increase at pH = 9.6 + directly in a 6.5% Na CL solution ♦ increase in 40% bile

The following criteria are used to identify strains E, coli, Rroteus and KebsielLa:

E. coli

Proteus aorganli

Rroteus mirabilis

Kebsiella pneumoniac

Ability to move actively

Growth in the environment KCN ^ yrate as a carbon source Carbohydrate gas from glucose Kws from Lactose

Karst from sucrose, a fissile from mitosis

Kws from mmnt

Acid from trechalose rkus from xylose and gelatin ddrolysis

Indole

Urease tyS with TSJ (3-ferric iron agar) +

This ♦

♦ +

T (dj '(d) ♦

+ ♦

♦ +

♦ +

+ ♦

Lizy ndo hy dr o rbolksy la for

The colonies identified in this way are allowed to grow on the plates^kac^h agar at 37 ° C for 24 hours, ^pabout which the suspension is made in physooogical table salt solution, tests their purity using Gram staining and exposes to dryness. The individual strains obtained are characterized and deposited with determinations as set out in the tables below.

Carbohydrate fermentation (+ means acid)

Escherichia coli strain

<td>Carbohydrate</td><td>455 UB</td><td>525 UB</td><td>560 UB</td><td>616 UB</td><td>654 UB</td><td>719 UB</td>

<td>lactose</td><td> +</td><td> +</td><td> ♦</td><td> +</td><td> ♦</td><td> +</td>

<td>saccharose</td><td> 0</td><td> 0</td><td> 0</td><td> 4-</td><td> +</td><td> +</td>

<td>mannitol</td><td> +</td><td></td><td> +</td><td> +</td><td> +</td><td> +</td>

<td>maltose</td><td> +</td><td> +</td><td> + -</td><td> +</td><td> +</td><td> + ·</td>

<td>mlibioza</td><td> +</td><td> ♦</td><td> +</td><td></td><td></td><td> +</td>

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cd array

<td> ——----</td><td> --------</td><td></td><td>--ς ---------</td><td></td><td>G ------ ------</td><td> :::2::::::::::</td>

<td>raffinose</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td>rhamnose</td><td> +</td><td></td><td> 4-</td><td> +</td><td> 4-</td><td> 4-</td>

<td>trehalose</td><td> ♦</td><td> +</td><td> 4-</td><td> +</td><td> +</td><td> +</td>

<td>salicin</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 4-</td>

<td>ribose</td><td> ♦</td><td> +</td><td> 4·</td><td> 4·</td><td> +</td><td> 4-</td>

<td>amygdalina</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> +</td>

<td>galactose</td><td> 4-</td><td> +</td><td> 4-</td><td> 4-</td><td> 4-</td><td> 4-</td>

<td>sorbitol</td><td> +</td><td> ♦</td><td> 4-</td><td> 4-</td><td> 4&gt;</td><td> 4-</td>

<td>arabinose</td><td></td><td> 4-</td><td> +</td><td> +</td><td> 4-</td><td> 4*</td>

<td>glucose</td><td> +</td><td> +</td><td> 4-</td><td> . 4-</td><td> 4- '</td><td> 4-</td>

<td>Maruno's</td><td> +</td><td> 4-</td><td> 4-</td><td> 4-</td><td> 4-</td><td> 4-</td>

<td>fructose</td><td> +</td><td> 4-</td><td> +</td><td> 4-</td><td> 4-</td><td> 4-</td>

<td>Adonite (Rybit)</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td>inositol</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td>dulcitol</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td>cellobiose</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 4-</td>

<td>xylose</td><td> 4-</td><td> +,</td><td> +</td><td> 4-</td><td> +</td><td> 4-</td>

<td></td><td></td><td>W: a peculiarities</td><td>biochemical</td><td></td><td></td><td></td>

<td>Pr Uh.</td><td></td><td></td><td colspan="2">Escherichia strain</td><td>coli</td><td></td>

<td>FI hhca “</td><td>445 UB</td><td>525 UB</td><td>56o UB</td><td>616 UB</td><td>645 UB</td><td>719 UB</td>

<td>urea</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td>hemolysis on blood agar plates</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 4</td>

<td>hydrolysis gelatin</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td>citrate</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td>indole</td><td> 4·</td><td> 4-</td><td> 4-</td><td> +</td><td> +</td><td> 4-</td>

<td></td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td>ability to move actively</td><td> +</td><td> 4-</td><td> 4-</td><td> 4-</td><td> 4-</td><td> 4-</td>

In this table, the + sign indicates positive within 24 hours, and the 0 sign indicates negative after 72 hours.

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<td></td><td>Carbohydrate fermentation plants ^ (+</td><td>means acid)</td><td></td>

<td rowspan="2">Carbohydrate</td><td>Kebsiella</td><td>Frroeus</td><td>Proteus</td>

<td>pneumoniac</td><td>mirabilis</td><td>. mcogonii</td>

<td></td><td>KI 16 B</td><td>63 B</td><td>58 B</td>

<td>lactose</td><td> 0</td><td> 0</td><td> 0</td>

<td>saccharose</td><td> . +</td><td> 0</td><td> 0</td>

<td>mrmit</td><td> +</td><td> 0</td><td> 0</td>

<td>maltose</td><td> 4</td><td> 0</td><td> 0</td>

<td>mlibioza</td><td> 0</td><td> * 0</td><td> 0</td>

<td>raffinose</td><td> 0</td><td> 0</td><td> 0</td>

<td>rhamnose</td><td> 4</td><td> 0</td><td> 0</td>

<td>tretaloza</td><td> +</td><td> 4</td><td> 0</td>

<td>salicin</td><td> +</td><td> 0</td><td> 0</td>

<td>ribose</td><td> + -</td><td> 4</td><td> 4</td>

<td>amygdalina</td><td> 0</td><td> 0</td><td> 0</td>

<td>galactose</td><td> . 4</td><td> 4</td><td> 4</td>

<td>sorbitol</td><td> · 4</td><td> 0</td><td> 0</td>

<td>arabinose</td><td> +</td><td> 0</td><td> 0</td>

<td>glucose</td><td> 4</td><td> 4</td><td> 4</td>

<td>minnoza</td><td> 4</td><td> 0</td><td> 4</td>

<td>fructose</td><td> 4</td><td> 0</td><td> 4 ·</td>

<td>Adonite (Rybit)</td><td> 0</td><td> 0</td><td> 0</td>

<td> —————————————</td><td colspan="2"> — — —— ————— — — —</td><td> ’——————————————————</td>

<td>inositol</td><td> 0</td><td> 0</td><td> 0</td>

<td></td><td colspan="2"> ————————————————————————</td><td> ——————————————————</td>

<td>dulcitol</td><td> 0</td><td> 0</td><td> 0</td>

<td> — — —— ———</td><td> ————————————————</td><td></td><td> ——————— ———— — ——— — — —</td>

<td>cellobiosa</td><td> 4</td><td> 0</td><td> 0</td>

<td>xylose</td><td> 4</td><td> 4</td><td> 0</td>

Rróba

Chemical properties

Kebsiella ftoteus pneuminiac mirabilis

KL 16b 65 B

Proteus morganii

B urea hemlysis on blood agar plates hydrolysis gelatin citrate indole active ability

In this table, the sign 4 means positive within 24 hours and the sign 0 means negative after 72 hours *

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Carbohydrate permeirt (+ means kwis)

Streptococcus faecalic carbohydrate

<td>lactose</td><td> ♦</td>

<td>saccharose</td><td> +</td>

<td>marnit</td><td> . +</td>

<td>mitosis</td><td> 4</td>

<td>melibiose</td><td> 0</td>

<td>raffinose</td><td> 0</td>

<td>rhamnose</td><td> + ·</td>

<td>trehalose</td><td> + · .</td>

<td>salicin</td><td> +</td>

<td>ribose</td><td> .+</td>

<td>awgńdlina</td><td> 4 </td>

<td>galactose</td><td> + </td>

<td>sorbitol</td><td> 4</td>

<td>and rab and no for</td><td> 0</td>

<td>glucose</td><td> +</td>

<td>mrrnoza</td><td> + ..</td>

<td>fructose</td><td>- «ί-</td>

<td>Adonite (Rybit)</td><td>Ο</td>

<td>inositol</td><td> 0</td>

<td>dulcite.</td><td> 0</td>

<td>cellobiose</td><td> +</td>

<td>xylose</td><td> 0</td>

<td colspan="2">Biochemical properties</td>

<td>Attempt</td><td>Streptococcus faecalis 676</td>

<td>urea</td><td> 0</td>

<td>type of hemoiosis</td><td>does not hemooize</td>

<td>gelatin hydrolysis</td><td> 0</td>

<td>reduction of laksum lotion</td><td> +</td>

<td>indole</td><td> 0</td>

<td>esculin</td><td> . +</td>

<td colspan="2">Growth on nutrient solution</td>

<td>4% bile</td><td> +</td>

<td>sodium chloride solution</td><td> +</td>

<td>pH value = 9.6</td><td> +</td>

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Temperature resistance 60 ° C for minutes, +

In this table, the plus sign means 'positive and the 0 sign' negative * for the morphological properties of the isolated and described strains can be summarized as follows:

Bschericha cola.

Gram - negative stamens, 1.1-1.5 x 2.0 - 5.0 pm, (live Oorma) motile thanks to flagellate whips. .

R * oteus mirabilis and P. morgan. .

Gram - negative stamens, 0.4 - 0.6 x 0.1 - 3.0 pm, uncoated, motile thanks to flagellate whips (not all).

Kebsiella pneumaniac. .

Gram - negative, immobile rods, 0.5 - 15 x 0.6 - 6.0 / um

Streptococcus Oaecalis.

Gam - negative ovate, 0.5-1.0 pm, immobile.

Escherichia coli strains have also been tested for their serological properties and, for this purpose, they were subjected to an agglutination test on a carrier using multivalent OK A, B, C, D and E sera. For this, serological values, i.e. antigen determination &lt;w 0, K and H, carried out by DiOco laboratories method. Dtroit St. Ser. Am. 1976. Ib identification gave the following strain

Ozraczenie

Serological type

<td>Echerichia salt</td><td rowspan="2">Ec 455 UB Ec 525 UB</td><td rowspan="2">0.6: K13: H 1 0.1: K 1: H 7</td>

<td>Echerichia coli</td>

<td>Echerichia coli</td><td>Ec 560 UB</td><td>0.4. : K 3: H 5</td>

<td>Echerichia coli</td><td>Ec 616 UB</td><td>0.75: ---: H 5</td>

<td>Echerichia coli</td><td>Ec 654 UB</td><td>character R</td>

<td>Echerichia coli</td><td>Ec 719 UB</td><td>you can't bet them</td>

hemonically

The strains described above were deposited on August 8, 1984 at OniΓaalbuftau voor Schimekulttures Csterstraat 1, 3740 AG fearm, Hlandia under the designations CBS 516.84 to CBS 525.84, and were transferred to Dutsche Samsung on December 5, 1984. von Mkroorganismen, Gisebachstrasse 8, 3400 GSingen, Rep. FOD. Nemiec, where she received input trash from DSM 3229 to DSM 3238.

To boil the vaccines, these strains are cultured individually on solid or liquid media, and preferably on solid media, e.g., nutrient agar (Ndrient® ar DiOco) having the following composition:

meat extract (DiOco ^ eO Ectract) 3 g / liter

Bucto - IRppon 5 g /; liter sodium chloride 5 g / liter&gt; Bacto - Ααζγ 15 g / liter

This medium is sterilized for 15 minutes at 121 ° C. It utilizes a pH value of about 7.2.

Large scale production uses Rcrnc cylinders and laboratory scale IRtrie plates. It is grafted to the material, which several milliliters are distributed evenly over the entire surface. Bitle Roux closes with cotton plugs and Wed.

145 565 a pit with a vaccinated surface is placed at the bottom. Inoculated cultures are cultured at 37 ° C for 24 hours. Bacterial herost is poured with the necessary amount, depending on the growth density of the culture in the dish, phosphoaao buffer with table salt solution (PBS) and lightly swaying, so as not to damage the surface of the agar, covers the entire pore * os-t,. *

One bottle is made from each cylinder or each Rftrie plate, stained using the Gram method and tested for purity. Cylinders or Fetrie plates that seem contaminated are discarded. Animals from one strain and one harvest are buried together through nylon gauze,

Inactivation, like culture, is carried out for a single strain. identical for this purpose, use maintained at a temperature of about 55-6 ° C for about 1 hour, or inactivated. with the help of formaldehyde or by irradiation with g-rays. it is inactivated in a free bath at 60 ° C for 1 hour, after which phenol (final concentration 0.35%) is added

The suspension is taken to check sterility and identification, after which the concentrated product stock is stored in the refrigerator. Since the sterility test after 3 days does not contain any contaminants, the vaccine development process continues, but the sterility test is carried out within 14 days and if it accumulates any organisms, the product is removed,

Inactivated suspensions in mgazinoy vessels, originating from one strain, are subjected to centrifugation in a cooled centrifuge at 3000 rpm / mimit for 1 hour (Sonrall 4 CR centrifuge, 2000 impeller), removing the remaining liquid and then from the bacterial sludge ^ The suspension is prepared in a solution of table salt containing 0.01% thiomereal. The density of the stock concentrates is determined by the turbidity and suspension methods, are described and stored at 4 ° C (♦ 2 ° C), preferably concentrated suspensions of 10 strains of E.coli, Ftoteus mirabilis, Rroteus mrganii, Kebsiella pneummiac and Streprococcus faecalls are mixed in such a way, that 1 milliliter contains:

^E.coli, ⁶ strains, from each 2.5 x together ^1,5 x ¹0 ^ embryos,

Rroteus mirab ^ ii, ¹ strain 7^,5 x 10? embryos ftOteus moo ^ h !, 1 strain. 7.5 x 10? zaΓ0^dkthat^,f

Kebsiella pieuminiac, 1 strain, 3 x 10 Zarock

Streptococcus faeoalii, 1 strain, 5 x 10 embryos together ² x ¹0® zarcdik &lt;W / l ml.

The final concentration is obtained by mixing together the individual, stored iine contents, the concentration is achieved by diluting with 0.15 m phosphate buffered saline containing 0.01% merthiolate (Tliomirsia), and then the product is stabilized at room temperature within 24 hours and then stored at 4 ° C (♦ 2 ° C),

Aluminum phosphate gel is added so that the concentration should be correspondingly higher. The aluminum phosphate gel is formed in such a way that 854 g of potassium aluminum sulfate, 12 H · 0 are dissolved in 6 liters of water and filtered, 685 g of trisodium phosphate,

Η? 0 dissolves in 6 liters of wsdy. The aluminum-containing solution is stored at 37 ° C, as it easily becomes supersaturated at room temperature. Both solutions are poured in evenly to 21 liters of water, centrifuged the precipitate, again mixed with 13 liters of water and centrifuged again,

The resulting precipitate is suspended, discharged to 8.1 liters, 5 m Na OH added until pH 6.0 and maintained in an autoclave at 121 ° C for 1 hour. This amount is sufficient to export 66 liters of vaccine, containing mg Al P0a or 0.66 mg AL mg in 1 milliliter, ** · ^ adsorbed final vaccine, after checking its pH, which should be 6.2 - 7.0, places in heat-resistant 1 m ampoules, with 0.5 ml of the vaccine as a single dose.

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Ensure that the vaccine is shaken when filling the ampoules. The total number of ziaaktwwwantch microorganisms &lt;W in 1 dose, that is, 0.5 ml, is peritoneum¹ 'x Ί0⁹.

The vaccine is ultimately tested for its Sterility, by the method given in Europaische HNirmahopoe (2nd Edition, Appendix 1970), regarding toxicity, according to the recipe of the World Health Organization (increase in weight of the mouse) and regarding abnormal toxicity, according to the Tiropean Pharmacopoeia .

According to the invention, the vaccine can be prepared not only in liquid form as described above, but also in lyophilized form. To this end, the seed suspension obtained is obtainedⁱⁿr described above, it is broken up like this^s in Ο,⁵ ml contained ¹ x 10⁹viral zantalectrical bacteria of the above composition. Dilution uses a 1% solution of human albumin or a plasma replacement agent called feenmacel (Bełuringwrte AG,

3500 Nrbturg, Germany) with O 'O1% thiomersal in 0.85 n sodium chloride solution. The 2 ml bites are filled under sterile conditions of 0.5 ml of the diluted suspension described in ^ J, frozen at about -45 ° C, and then dried under reduced pressure in a freeze-drying apparatus. The drying time should not be higher than 36 ° C. The entire lyophilization process lasts 24 hours and then the bottles are sealed under nitrogen with rubber stoppers with aluminum caps.

For the introduction of aluminum phosphate and at the same time as a solvent, prior to the administration of such a vaccine, an aqueous aluminum phosphate gel with an AL PO ^ concentration of 2 mg / ml is used. This gel is prepared as described above and is diluted with table salt solution (about two and a half fold) before entering into ampoules to give a final concentration of 2 mg / ml. 0&gt; 5 ml of vaccine is placed in 1 ml ampoules.

In order to test the stability of the vaccine by the method of the invention, its ability to produce antibodies in mice was determined twice, and titer was immediately after vaccine production, on December 3, 1981 and after 2 years, that is December 7, 1983.

Mouse mice. Five groups of 10 mice (male, strain NffilS, body weight 16-20 g) were immunized by giving them vaccine vaccines, injecting 2 doses of 0.5 ml vaccine 2 weeks apart. 20 mice from the same breeding group, with the same body weight, being the comparative group, were injected only with 0.5 ml of aluminum phosphate gel. Fb 2 weeks after the second dose of vaccine, blood was collected and collected in the group response. Fb coagulation was centrifuged the sera from each group and stored at -22 ° C for serological testing.

Antigens are agglutinous. Nine homologous bacterial strains contained in the vaccine were used to develop antigens. Strain EC 654 could not be agglutinated (form R), so it could not be used as a gluten, no gen. L. and. Lyophilization of the cultures of the strain was mixed with 9 ml medium (Difco calf extract), balancing the suspension and cultivating at 37 ° C in 24 hours. A large subculture was used to extract antigens. 600 ml of medium (flesh extract) was inoculated with a seed culture (each strain separately) and grown for 36 hours at ³⁷° C. Formalin was initially added up to toroidal concentration of 0, Ί%,^pat part^sm the culturing process was continued at 37 ° C for 7 days. The absence of live bacteria was then checked in each culture and sterility tested. The seeded bacterial suspension was centrifuged in a refrigerated centrifuge (Soorzal) for 1 hour at 3000 rpm and the pellet was re-suspended in a solution of table salt with fzsfoΓanoγym buffer (pH value "^7,2) this one^k, and^by uzts^rand^Æ s-setting approx. 20 x ¹0⁹ bacteria in ml. Down^pdo^s and^gl^att^taac^jand used about ² x ¹^ ® tekteri.i / ml as a^glutyno^gen.

Agglutination. Mouse sera are diluted at 1: 2 dilution with physiological saline, phosphate buffered and having a pH value of 7.2.

Ib 0.3 ml of diluted serum was mixed in fehn tubes with the same volume of antigen. One tube, not containing serum, served as an antigen test. Fb 18 hours at 37 ° C and a further 24 hours at 4 ° C the reaction was determined. The highest dilution at which one can be used was considered the titre of each serum

145 565 it was still possible to see visible agglutination of the balkeria. IN? all assays used both negative and positive sera for controls. The following geometric mean values ​​were found for agglutin mouse sera.

Mano (or reverse)

Antigen

<td></td><td>rehearsal 1981</td><td>trial 19!</td>

<td>E. coli 455 UB</td><td> 1015,5</td><td> 905,1</td>

<td>E coli 525 UB</td><td> 905,1</td><td> 1015,5</td>

<td>E. coli 560 UB</td><td> 1015,5</td><td> 905,1</td>

<td>E. coli 616 UB</td><td> 905,1 ·</td><td> 800,3</td>

<td>E. coli 719 UB</td><td> 1280</td><td> 1280</td>

<td>Kebsiella</td><td> 201,6</td><td> 201,6</td>

<td>pneumoniac 16 B</td><td> 160,0</td><td> 142,5</td>

<td>Rroteus mooganii 58 B</td><td> 113,1</td><td> 113,1</td>

<td>Strepcoccus faecalis 676</td><td> 71,3</td><td> 71,3</td>

These results show that the vaccine stored at 4 ° C is fully effective for at least 2 years.

The use of the vaccine produced according to the invention is indicated especially in cases of cystitis, prostatitis, cystitis and renal pelvitis and pyelonephritis.

For patients who do not indicate acute fever (contraindication), this vaccine is given at intervals of 1 to 2 weeks, for a total of 3 intramuscular injections of 0.5 ml each. If a strong reaction occurs after injection, this treatment should be discontinued.

Rb after 1 year, the injection should be repeated to refresh, also using a 0.5 ml dose.

The vaccine produced by the method of the invention was injected in various hospitals to 383 patients with bacteriologically confirmed symptoms of urinary tract infections and these patients were further observed within 1 year. Wynk has found that administration of the vaccine results in an increase in urinary tract immunity for at least 1 year, providing far-reaching protection against recurrence of infection. the results of clinical trials are given below.

Number of relapses / reinfections during observation within 1-12 months.

Investigator

AND

The baby needs 1-2 months of service

VI

7-12 months

IA / BS

Li / SH

Ri / H, DE

Total vaccinated

Comparative attempt

<td> 62/62</td><td> 6/62</td><td></td>

<td> 100%</td><td> 9,3%</td><td></td>

<td> 118/118</td><td> 12/113</td><td> 2/115</td>

<td> 100%</td><td> 10,6%</td><td> 1,7%</td>

<td> 203/203</td><td> 15/183</td><td> 3/194</td>

<td> 100%</td><td> 8,2%</td><td> 1,5%</td>

<td> 383/383</td><td> 33/358</td><td> 5/309</td>

<td> 100%</td><td> 9,2%</td><td> 1,6%</td>

<td> 198/198</td><td> 26/148</td><td> 18/146</td>

<td> 100%</td><td> 17,6%</td><td> 12,3%</td>

145- 565

The vaccine produced according to the invention is well tolerated and no serious side effects have been reported. It can also be used without problems during pregnancy, as evidenced by over 100 such use cases.

Significant features of the vaccine produced according to the invention and established on the basis of tests. pharmacological and clinical benefits of this vaccine are listed below

The quantitative composition of 10 'different strains is compiled based on the' frequency of factors causing urinary tract infections, namely: 75% of the strains are E * coli (even occurring) strains, and 25% are strains of Fhoteus mirabilis, Rroteus morganii · KLebsiella pneumaniac and Streptococcus faecalis ·

Ib pathogenic strains - are inactivated so that they contain antigens that give protection, e.g. · filamentous antigens and 0 antigens · Strains selected As antigens tested in a mouse protection test, they also show cross-protection against heterologous strains of the same type of pathogen ·

As demonstrated by the mouse weight gain test, inactivated antigens contained in the vaccine^pionce produced p^pa person wetoug of the invention., after being stored in a cold room for at least 6 months, they become clearly less toxic · This vaccine, given to animals (mice, rabbits), as well as humans, causes them to increase immunity, protects mice against fatal infection and rats from artificially induced pyelonephritis · The quantitative and qualitative composition of the vaccine corresponds to the amount of antigen that causes good immunization and does not cause severe patients, and even no side effects · this vaccine when given to patients also causes secretion. immunoglohulin A in the urine of patients · It heals patients suffering from urinary tract infections, and at relapse protects at least 12 months against re-infection ·

The sfccepionka produced by the method of the invention may be liquid or lyophilized and in the latter case aluminum phosphate gel is used as the solvent for reconstitution prior to administration.