KR970001707B1 - Pseudomonas infection preventing vaccines using attenuated pseudomonas cfcpa 50243 - Google Patents

Abstract

A attenuated pseudomonasaeruginosa isolate especially CFCPA 50234 is disclosed for formulating vaccine curing or preventing pseudomonas contagion. Pseudomonasaeruginosa isolate is extracted from the infected patient and classified into 7 classes by Fisher-Devilin immunotype; only type 5 germ is selected, repeatedly dosed to experimental animals and acclimatized to yield attenuated pseudomonasaeruginosa isolate CFCPA 50234 (KCCM 10033) which is not toxic in itself and attenuated for the cell wall protein to yield only antigen. The acclimation to yield the attenuated pseudomonasaeruginosa isolate is repeated, commonly 3-7 times, until it shows the desired value of LD50. The desired LD50 value is more than 2.0 x 107 ,desirably 3.0 x 107 cells. CFCPA 50234 can be used for preparing pseudomonasaeruginosa vaccine by nurturing the germs in various sized fermenters having an optimum environment; yielding many isolates; treating the isolates by a series of processes; yielding cell wall protein; and finally treating them by ordinary vaccine preparation methods.

Description

A vaccine against Pseudomonas aeruginosa infection using attenuated Pseudomonas aeruginosa CFCPA 50243

The present invention relates to the use of novel Pseudomonas aeruginosa strains, in particular the attenuated Pseudomonas aeruginosa CFCPA 50243, for the preparation of a vaccine for the treatment and prevention of Pseudomonas aeruginosa infections.

Pseudomonas aeruginosa is a bacillus with a length of 1.5 to 3.0 µm and a width of 0.5 µm. It has one flagellar, gram-negative motility, and is widely distributed in soil, water, sewage and human intestines. . Microbiol. 4, 1069-1075, 1990. Pseudomonas aeruginosa is a pathogenic bacterium that causes refractory infections such as sepsis, systemic infection, chronic airway infection, and pancreatic cystic fibrosis. In particular, sepsis caused by Pseudomonas aeruginosa is a disease caused by the invasion of microorganisms or secretion of toxic constituents in the blood of patients whose resistance has been reduced by surgery, laceration and trauma. It is known to cause death. In addition, Pseudomonas aeruginosa has recently been detected in urinary tract infections and has attracted more attention. Therefore, there is an urgent need for the development of a medicament capable of effectively preventing or treating refractory purulent diseases such as sepsis and urinary tract infection caused by Pseudomonas aeruginosa. However, Pseudomonas aeruginosa is not only resistant to commercial antibiotics except for some antibiotics, but preventive and therapeutic agents have not been developed that have a good effect until now, so the mortality rate due to Pseudomonas aeruginosa infection is increasing.

The only treatment for P. aeruginosa infections that has been developed and put to practical use is to neutralize toxins by making antitoxins for P. aeruginosa. However, antitoxin is a therapeutic agent that can be used only for patients who have already advanced sepsis, and has a disadvantage that it can not be used universally for general patients because it has no prophylactic function and is very expensive. In addition, the biggest drawback is that the rate of cure that can be treated through the administration of this antitoxin is relatively low, and side effects are also great.

Therefore, in recent years, a method of using a common antigen has been actively studied as one method of solving the disadvantages of the antitoxin use method. Currently, research on common antigens aimed at preventing or treating Pseudomonas aeruginosa infections can be divided into two categories. One method is to isolate and purify common antigens from Pseudomonas aeruginosa having different immune forms and use them as prophylactic vaccines against Pseudomonas aeruginosa infection [Japan, J. Exp. Med. 45, 355-359, 1975], and another method is to extract genes encoding this antigen by recombinant genetic engineering method which is rapidly developed in recent years, recombine and express in a suitable vector, and then mass-produce common antigens. It is an attempt to use it as an antigen. Although the development of prophylactic vaccines using common antigens is a very effective approach, it is pointed out that there are many problems to be solved in recent years as research progresses. The most decisive disadvantage is that only one common antigen can prevent all the Pseudomonas aeruginosas with various immune forms, so the effect is extremely low. This low effect suggests that in addition to the common antigens identified to date, some unknown major antigens must be present together to prevent effective prevention.

Another method is a mouse monoclonal antibody against Pseudomonas aeruginosa using cell fusion techniques [J. Inf. Dis. 152, 1290-1299, 1985] or human monoclonal antibody [FEMS Microbiol. Immunol. 64, 263-268, 1990], an antibody-producing cell line capable of neutralizing Pseudomonas aeruginosa best among each antibody is selected, and an attempt is made to develop a low-cost therapeutic agent through mass production using this as a production strain. However, this method is not only used as a prophylactic vaccine for the treatment of Pseudomonas aeruginosa infection through antibody production, but also by one type of monoclonal antibody to dozens of Pseudomonas aeruginosas known to be serologically and immunologically. Because it is not possible to treat all of the infectious diseases caused by the disadvantage that can not effectively treat all infected patients. In order to overcome these disadvantages, cross-reactivity is investigated to investigate the simultaneous administration of several antibodies and blood samples from Pseudomonas aeruginosa infected patients to identify serological or immunological forms of Pseudomonas aeruginosa. Although methods for administering monoclonal antibodies have been suggested. This is also time consuming and is therefore not suitable for the treatment of struggling patients.

The prior art has also proposed a method for separating cellular proteins from Pseudomonas aeruginosa and using them as antigen vaccines. [Vaccine, Vol. 7, Experimental studies on the protective efficacy of a Pseudomonas aeruginosa vaccine 1989]. However, the method presented in this document used four general strains of NN 170041, NN 170015, NN 868 and NN 170046, which were not attenuated, as well as the toxicity of the strain itself. In the process of preparing protein vaccines from these strains, cell breakage occurs and impurities such as heterologous nucleic acids or toxic polymers such as lipopolysaccharides (LPS), which are present in the cytoplasm, are mixed in the vaccine components. There is a disadvantage to pay attention.

Accordingly, the present inventors have found a wide range of methods to find excellent immunogenic (ie antigenic) action by effectively inducing the formation of antibodies against Pseudomonas aeruginosa when administered to a human body without the risk of inherent toxicity of Pseudomonas aeruginosa itself. One study was conducted. As a result, the Pseudomonas aeruginosa isolated from Pseudomonas aeruginosa-infected patients were classified according to Fisher's immunity, and each of them was attenuated by repeated purifying. It was confirmed that the obtained and completed the present invention.

Thus, the present invention isolates these isolated microorganisms from novel safe Pseudomonas aeruginosa strains, particularly patients infected with Pseudomonas aeruginosa, which have been attenuated so that their cell wall proteins are only antigenic, without the toxicity of Pseudomonas aeruginosa itself. After dividing into seven types according to the Fisher-Devlin Immunotype, only the microorganisms corresponding to the Fisher Immunotype 5 were selected and repeatedly injected into the experimental animals to be purified to select the most attenuated strains. CFCPA 50243 strain which is a safe attenuated Pseudomonas aeruginosa strain obtained.

In addition, the cell wall protein purified purely from the attenuated safe Pseudomonas aeruginosa CFCPA 50243 as described above according to the present invention can be used as a vaccine for preventing Pseudomonas aeruginosa infection by itself. Alternatively, antibody immunoglobulins induced in animals by such cell wall proteins can be used as a therapeutic agent for Pseudomonas aeruginosa infection. Accordingly, the present invention relates to the use of attenuated Pseudomonas aeruginosa CFCPA 50243 for the preparation of a Pseudomonas aeruginosa infection treatment and a vaccine for preventing Pseudomonas aeruginosa infection.

Fischer-Deblin immune forms of Pseudomonas aeruginosa are classified into seven types from 1 to 7, and among the seven Pseudomonas aeruginosas, a strain of type 5 was selected as the attenuated strain.

The safe, attenuated Pseudomonas aeruginosa strain according to the present invention is obtained by purely separating the Pseudomonas aeruginosa, which corresponds to Type 5 of the Fisher's immune form, from the blood of a patient determined to be infected with Pseudomonas aeruginosa and attenuating iteratively. The safe attenuated Pseudomonas aeruginosa strain thus obtained is designated as strain CFCPA 50243.

The attenuated Pseudomonas aeruginosa strain CFCPA 50243 obtained by this repetitive purification process is almost identical to the parent strain before attenuation in several aspects, including morphological and bacteriological properties, but it does not show the pathogenicity of the parent strain, but rather Pseudomonas aeruginosa infection. It is classified as a novel strain because it shows new antigenic properties. Therefore, strain CFCPA 50243 was deposited on May 12, 1993 at the Korea Microbial Conservation Center, which is affiliated with the Korean spawn association, which is an international depositing institution, and was given a deposit number of KCCM 10033.

As described above, the purification process for obtaining the attenuated Pseudomonas aeruginosa strain is generally repeated until the strain shows the desired LD ₅₀ value. The number of repetitions varies depending on the engineer or the toxic state of the parent strain, but preferably 3 to 7 Repeat it several times. The LD ₅₀ value of the novel attenuated Pseudomonas aeruginosa strain thus obtained is at least 2.0 × 10 ⁷ cells, preferably at least 3.0 × 10 ⁷ cells.

The present invention also provides the use of the novel attenuated Pseudomonas aeruginosa CFCPA 50243 in the preparation of a Pseudomonas aeruginosa infectious agent and a vaccine for preventing P. aeruginosa infection.

According to the present invention, the P. aeruginosa CFCPA 50243 attenuated according to the present invention is cultured in a fermenter of various sizes under optimal growth conditions to obtain a large amount of cells, and then the obtained cells are treated with organic solvents, extracted, and separated according to molecular weight size. After obtaining a cell wall protein by treatment according to a series of processes such as ultracentrifugation, and the like can be prepared according to the conventional method for producing a vaccine against Pseudomonas aeruginosa.

Hereinafter, the method for preparing the above-mentioned vaccine composition will be described in more detail.

In a first step, the attenuated Fisher type 5 Pseudomonas aeruginosa CFCPA 50243 according to the present invention is cultured in an appropriate size incubator. At this time, tryptic soy broth (tryptic soy broth) is used, or in particular, a special medium configured to be suitable for the culture of Pseudomonas aeruginosa as described above.

This special medium contains glucose (30g / l), peptone (15g / l), MgSO ₄ (0.5g / l), CaCO ₃ (5g / l), KH ₂ PO ₄ (1g / l), FeSO ₄ (5mg / l l), CuSO ₄ (5 mg / l) and ZnSO ₄ (5 mg / l). This special medium is specially designed by the present inventors to be suitable for cultivation of Pseudomonas aeruginosa strains. When these Pseudomonas aeruginosa strains are cultured in a special medium, it is more preferable to use a special medium because it is possible to obtain at least 30% higher dry cells on the basis of the weight of dry cells than when cultured in tryptic soy broth medium.

The optimal culture conditions in this cell culture step is the temperature 37 ℃, aeration amount 1vvm (volume / volume · minutes), the inoculation amount is 5 (v / v)% of the culture medium, the initial 2 hours the stirring speed to 100ppm And then incubated at 600 rpm for 10 to 20 hours, preferably 12 to 16 hours.

When the culture is completed, the cells precipitated from the culture medium in the second step is separated by a method such as centrifugation. In the third step, the isolated cells are treated with an organic solvent to inactivate microorganisms and remove cell wall lipid components. The organic solvent is preferably acetone, chloroform, ethanol, butanol, and the like, most preferably acetone.

Subsequently, in the fourth step, cell wall proteins are extracted five to six times while preventing the cells from being destroyed. To this end, the microbial cells are left in a solvent such as phosphate buffer, tris salt buffer, preferably phosphate buffer, or extracted using a mixer or homogenizer. The extraction process is preferably carried out by stirring at 4 to 100 to 2000rpm. In addition, care must be taken not to destroy the cells in the fourth step, in order to prevent the incorporation of toxic proteins in the cytoplasm into the desired cell wall proteins. Therefore, it is necessary to continuously check whether the cells are broken by measuring the intracellular marker enzyme lactate dehydrogenase or hexokinase during the extraction process.

Thus, the cell wall proteins of the attenuated Pseudomonas aeruginosa strain obtained as the supernatant are fractionated such that only proteins having a molecular weight in the range of 10,000 to 100,000 are obtained by primary and secondary ultrafiltration. At this time, it is very important that the molecular weight range of the cell wall protein is 10,000 to 100,000. If the molecular weight is less than 10,000, it is revealed by animal experiments. It does not exhibit the desired antigenic properties. The presence of rides (LPS) can cause toxicity.

Cell wall protein fractions in the molecular weight range of 10,000 to 100,000 were ultracentrifuge again to remove traces of toxic lipopolysaccharides that may remain, and then sterilized using a membrane filter to finally remove the attenuated Pseudomonas aeruginosa cell wall protein. To obtain.

The cell wall proteins from the attenuated Pseudomonas aeruginosa thus obtained are used as vaccines either alone or in a mixture in proportion to the corresponding cell wall proteins obtained by Pseudomonas aeruginosa of other Fischer immune forms.

When used as a vaccine against Pseudomonas aeruginosa infection, the dosage used depends on the sex, age, weight, and health condition of the subject of concern for infection with Pseudomonas aeruginosa, but in general, the dry weight of the cells is 0.1 to 0.5g (wet weight 0.5 to 2.5g) Cell wall protein, which is generally from 0.5 to 2.5 mg. The route of administration is preferably administered intramuscularly.

Cell wall proteins of attenuated Pseudomonas aeruginosa isolated purely according to the steps as described above may also be used in the preparation of a therapeutic for Pseudomonas aeruginosa infection. That is, the cell wall protein is used as an antigen to inoculate a test animal such as sheep, rabbits and goats to induce antibodies and formation, and then blood is collected from the test animal to separate serum. The isolated serum is treated by known methods to give the desired immunoglobulin in purified form. At this time, the method of purifying and separating the immunoglobulin from the serum is carried out using a method well known in the art (see: Practical Immunology, 3rd Ed., Pp 292-294). After mixing with distilled water and adsorbing to DEAE-cellulose, the supernatant is discarded and the immunoglobulin-adsorbed DEAE-cellulose is repeatedly washed several times with phosphate buffer to obtain purified immunoglobulin. The immunoglobulins thus obtained can be used alone. However, if necessary, immunoglobulin induced by immunizing an animal by mixing the cell wall protein obtained from the attenuated strain CFCPA 50243 of the present invention with the cell wall protein of Pseudomonas aeruginosa strains of other immune forms as a therapeutic agent for Pseudomonas aeruginosa infection Can be used.

The dosage varies widely depending on the age, weight, sex, health status of the patient infected with Pseudomonas aeruginosa and the severity of Pseudomonas aeruginosa infection, but generally 0.5 mg to 1000 mg per kg body weight per adult, preferably 5 mg to 100 mg intravenously. Administration.

The present invention is explained in more detail by the following examples, but the present invention is not limited in any way by these examples.

Example 1

Isolation and Identification of Fisher Immunotype 5 Pseudomonas Aeruginosa from Pseudomonas Aeruginosa Patients

260 different blood samples from patients with Pseudomonas aeruginosa infection were sterilely distributed for about 1 to 5 ml each, left at room temperature for 1 hour, and then stored in a refrigerator at 4 ° C. overnight, then stored at 4 ° C. at 1500 g. Centrifugation was performed for a minute to remove the solids and serum was obtained as supernatant. The serum obtained above was prepared in a tryptic soy broth culture plate prepared with 1.0-1.5% agar, which was prepared in advance, (1.15 g of Na ₂ HPO ₄ per ι, 0.2 g of KCl, 0.2 g of KH ₂ PO ₄ , After diluting each of them in a ratio of 1:10 with 8.766 g of NaCl), each was incubated for 12 hours or more in an aerobic incubator maintained at 37 ° C. The culture thus obtained was transferred to a new culture plate and microorganisms were separated in pure form using a known microbial pure separation method [Biology of Microorganisms].

The serological and immunological classifications of isolated Pseudomonas aeruginosa are known [Bergey's Manual] and are identified by the following analytical methods using commercially available assay kits [J. Gen. Microbiol. 130, 631-644, 1984]. 5 ml of Tryptic Soy Broth was placed in a test tube and inoculated with each bacterium, followed by incubation under aerobic conditions maintained at 37 ° C. to adjust the cell concentration so that the absorbance at 600 nm was maintained at about 2.0 or more. 1 ml of this culture solution was aseptically aliquoted and centrifuged at 6000 g for 10 minutes at 4 ° C. The upper culture medium was removed, and the same amount of sterile saline was added to the precipitated microorganisms and suspended. 15 μl of test serum and control serum (normal rabbit serum) were respectively added to the prepared 96-well microplates, and then 15 μl of the microbial suspension obtained above was added to each serum, mixed and agglutination was performed for 10 to 30 minutes. It was observed whether this happened. Herein, strains of wells in which a positive aggregation reaction occurs are easily distinguished since they have a serotype consistent with the serum added thereto.

Example 2

Screening of Bunter Attenuated Microorganisms with Isolated Pseudomonas aeruginosa

To attenuate Pseudomonas aeruginosa into a safe microorganism that can be used for vaccine production, microorganisms with type 5 immune forms were attenuated through repeated purification. The toxic Pseudomonas aeruginosa used as a control group was selected from GN 11189 (purchased from the Episome Institute in Japan), and GN11189 injected 2.0 × 10 ⁶ cells intravenously (or intraperitoneally) into a male ICR mouse weighing 20-25 g. A mortality rate of 100% is seen within three days.

Pseudomonas aeruginosa (LD ₅₀ 5.0 × 10 ⁶ ) having the selected type 4 immune form is liquid cultured in Tryptic Soy Broth under the same conditions as in Example 1, followed by centrifugation at 6000 g at 4 ° C. for 10 minutes. The obtained cell precipitate was suspended in 10 ml of physiological saline and recentrifuged under the same conditions as above, followed by 5 × 10 ⁵ , 5 × 10 ⁶ , 5 × 10 ⁷ and 5 × 10 ⁸ cells per ml with fresh physiological saline. Each cell was administered intravenously (or intraperitoneally) to 10 mice per group, with appropriate dilution. At this time, only GN 11189 strain 2.0 × 10 ⁶ cells were administered, and Pseudomonas aeruginosa was isolated aseptically from the ICR mice surviving at the highest cell concentration at 100% lethality within 3 days. The isolated Pseudomonas aeruginosa was plated on tryptic soy agar plate medium, and each microorganism was purely separated using the above-mentioned microbial pure separation method.

As described above, the first attenuated microorganism through the mouse was repeatedly administered 3 to 7 times in the same manner as above, and repeatedly treated so that at least LD ₅₀ was at least 2.0 × 10 ⁷ cells or more. The actual LD ₅₀ of the attenuated strain obtained here was measured at 3.0 × 10 ⁷ cells or more. The microbial strain thus safely attenuated was named CFCPA 50243 (Accession No .: KCCM 10033).

Example 3

Physiological Characteristics of Attenuated Pseudomonas aeruginosa

(1) The growth rate of the attenuated Pseudomonas aeruginosa CFCPA 50243 obtained in Example 2 according to pH change was measured. First, strain CFCPA 50243 was inoculated into 50 ml of Tryptic Soy Broth maintained at pH 7.2 and pre-cultured for 12-16 hours while maintaining aerobic conditions at 200 ° C. at 37 ° C. The cells obtained by pre-incubation were inoculated in 500 ml of fresh tryptic soy broth maintained at pH 3.0, pH 5.0, pH 7.0, and pH 9.0, respectively, to a final concentration of 1 (v / v)%. Samples were taken at intervals of 2 hours while incubating under the same conditions, and the absorbance was measured at 600 nm using a spectrophotometer to determine the concentration of the cells. As a result, the strain CFCPA 50243 showed an excellent growth rate under the condition of pH 5.0 or more, while it was confirmed that the strain can not grow under the strong acid conditions of pH 3.0.

(2) The growth rate of the attenuated Pseudomonas aeruginosa CFCPA 50243 according to the temperature change was measured in the same manner as in (1). Strain CFCPA 50243 was inoculated in 50 ml of Tryptic Soy Broth maintained at pH 7.0 and pre-incubated for 12-16 hours while maintaining aerobic conditions at 37 ° C. at 200 rpm. The incubator was inoculated with 500 ml of Tryptic Soy Broth adjusted to pH 7.0 to a final concentration of 1 (v / v)%, and then the incubator maintained at 25 ° C, 30 ° C, 37 ° C, and 42 ° C. Each growth was measured by measuring the absorbance at 600 nm while incubating under the same conditions. As a result, it was confirmed that strain CFCPA 50243 shows excellent growth at 30 to 37 ℃ and relatively slow growth at 25 ℃ or 42 ℃.

(3) The following experiment is to measure the growth rate according to the carbon source of attenuated Pseudomonas aeruginosa. Strain CFCPA 50243 was inoculated into 50 ml of Tryptic Soy Broth each adjusted to pH 7.0 and incubated for 12 to 16 hours under aerobic conditions maintained at 200 rpm at 37 ° C., followed by centrifugation at 6000 g at 6000 ° C. for 10 minutes under aseptic conditions. The microorganisms were harvested and then suspended in 10 ml physiological saline. To 5 ml of M9 medium (Na ₂ HPO ₄ · 7H ₂ O 12.8g, KH ₂ PO ₄ 3g, NaCl 0.5g, NH ₄ Cl 1g) each containing a different carbon source, 50 μl of the obtained microbial suspension was added thereto for 12 hours. After culturing, the absorbance was measured at 600 nm for each culture to examine the growth of microorganisms.

As a result, strain CFCPA 50243 shows an excellent growth rate in the presence of glucose or β-alanine and mannitol, but other carbon sources such as xylose, sorbitol, inositol, maltose, sucrose, arabinose, mannose, etc. It was found that growth did not exist in existence. These carbon source utilization characteristics are substantially the same as the general growth characteristics of Pseudomonas aeruginosa strains corresponding to Type 5 of Fisher's immune form, the parent of the CFCPA 50243 strain (see Bergey's Manual).

As seen above, the attenuated Pseudomonas aeruginosa strain according to the present invention is produced by culturing in a conventional medium containing a carbon source, a nitrogen source, an inorganic compound, amino acids, vitamins and other nutrients under aerobic conditions while appropriately adjusting the temperature, pH, etc. Cell wall protein components obtained therefrom can be used for the production of Pseudomonas aeruginosa vaccine. The carbon source required for the cultivation may be various carbohydrates such as glucose, mannitol, and the like, and various organic acids such as acetic acid, pyruvic acid, and lactic acid, and nitrogen sources include various organic and inorganic ammonium salts, peptones, yeast extracts, casein hydrolysates, and other nitrogen-containing compounds. Materials and the like can be used. As the inorganic compound, magnesium sulfate, calcium carbonate, iron sulfate, copper sulfate, zinc sulfate and potassium dihydrogen phosphate, potassium dihydrogen phosphate and the like can be used. The culture is preferably carried out at 25 to 37 ° C. under aerobic conditions, for example, plate culture, or liquid culture such as shaking culture or aeration stirring. The pH of the medium is preferably maintained at pH 5 to 9 during the incubation period. Preferably, the cells obtained by centrifugation of the culture medium incubated for 12 to 16 hours are used as starting materials for vaccine production described below.

Example 4

Culture of Attenuated Pseudomonas aeruginosa

(1) A 5 L incubator was used to obtain a large amount of Pseudomonas aeruginosa cells obtained according to Example 2. In a 5 L incubator, 2.5 배지 of the medium solution obtained by dissolving 30 g of tryptic soy broth per 1 증류 of distilled water was adjusted to pH 7.2 and sterilized. The culture temperature was 37 ℃, the aeration amount is 1vvm, the inoculum was used for the culture solution of the attenuated Pseudomonas aeruginosa cells 5 (v / v)% obtained in Example 2 and the stirring speed was maintained at 100rpm for the initial 2 hours After that, the cells were fixed at 600 rpm and incubated for 12 to 16 hours. The amount of cells obtained was on average about 8 g (dry weight) per culture ι.

(2) Glucose (30 g / l), Peptone (15 g / l), MgSO ₄ (0.5 g / l), CaCO ₃ (5 g / l), KH ₂ PO ₄ (1 g / l), FeSO ₄ (5 mg) / l), strain CFCPA 50243 was cultured under the same culture conditions as in (1), except that a special medium for Pseudomonas aeruginosa culture consisting of CuSO ₄ (5 mg / l) and ZnSO ₄ (5 mg / l) was used. By using this special medium, cells having a dry weight of about 10.4 to 10.5 g were obtained. Therefore, at least 30% higher dry weight cells could be obtained than in the case of using tryptic soy broth.

Example 5

Partial Purification of Cell Wall Protein from Attenuated Pseudomonas Aeruginosa

Cell wall proteins were partially purified from attenuated Pseudomonas aeruginosa CFCPA 50243 to prepare a vaccine against Pseudomonas aeruginosa.

2.5L of the special medium used in Example 4 (2) was added to a 5L fermenter, and then cultured for 12 to 16 hours while maintaining the same culture conditions as mentioned above. After the incubation was completed, the supernatant was removed by centrifugation of 2.5 L of the culture medium at 4 ° C. at 6000 g for 20 minutes, and the precipitated cells were phosphate buffered at 4 ° C. (1.15 g Na ₂ HPO ₄ per ι, 0.2 g KCl ₂ , and KH _2). After suspension in 0.2 g of PO ₄ , 8.766 g of NaCl, pH 7.2) and centrifugation again, the cells were washed with the same phosphate buffer. Three volumes of acetone were added to 130 g of the obtained cells, and left at 4 ° C. for at least 12 hours. After removing acetone from the precipitated cells, two volumes of fresh acetone were added again, stirred for 5 to 10 minutes, and centrifuged at 4 ° C. at 8000 g for 20 minutes. The acetone of the upper layer was removed, the same volume of acetone was added to the residue, the mixture was stirred in the same manner as described above, centrifuged, the supernatant was removed, and the precipitated cells were dried on a plate at room temperature to remove acetone. The dried microorganisms were suspended by adding 250 ml of the same phosphate buffer as above to a final concentration of 10 (w / v)%. The cell wall protein was extracted at a speed of 500 to 1500 rpm for 10 minutes using a homogenizer while kept at 4 ° C. The obtained suspension was centrifuged at 8000 to 10000 g for 30 minutes to obtain a supernatant, and then suspended in precipitated cells by adding the same volume of phosphate buffer, followed by secondary extraction under the same conditions as the extraction method above to obtain a supernatant.

Using the same conditions as the extraction method used above, the extraction was repeated 5 to 6 times while maintaining the cells in a state of being unbroken. At this time, whether the cells were broken or not was determined by measuring a specific protein that is a cytoplasmic marker, that is, lactate dehydrogenase or hexokinase. Each extracted supernatant, which was found to have extracted cell wall protein without the incorporation of intracellular proteins lactate dehydrogenase and hexokinase, was collected and stirred, and finally, financed for 30 minutes at 10,000 g at 4 ° C. Deep supernatant was obtained by deep separation. The protein solution thus obtained consisted of almost pure cell wall proteins only and was adjusted to maintain concentrations between 1 mg / ml and 2 mg / ml through protein quantification.

Example 6

Purification of Crude Protein

The following experiment was performed to purely separate crude cell wall proteins adjusted to a concentration of 1 to 2 mg / ml to have only active ingredients. Two to 2.5 liters of crude cell wall protein aqueous solution is first removed from a molecular weight 100,000 or more protein molecules using a molecular weight 100,000-cut membrane filter in a Pellicon Cassette System. This allows proteins with a molecular weight of 100,000 or less to exit the filtrate. Proteins with molecular weights greater than 100,000 continue to circulate, separating molecules with molecular weights less than 100,000.

An aqueous protein solution concentrated to 10-20% of the original volume was added to 20-25L of sterile phosphate buffer (1.15 g of Na ₂ HPO ₄ , 0.2 g of KCl, 0.2 g of KH ₂ PO ₄ , NaCl 8.766 g, 10 times the initial volume). By continuing to wash), 90% or more of protein having a molecular weight of 100,000 or less was recovered. A protein with a molecular weight of 100,000 or less separated by the filtrate was removed in the same system using a molecular weight 10,000-cutting membrane filter to remove proteins and other components having a molecular weight of less than 10,000, while at a concentration of 1 mg / ml of protein with a molecular weight of 10,000 to 100,000. Concentrate until. In addition, the purity was confirmed by the 10-15% concentration gradient SDS-PAGE electrophoresis method for the protein solution, it was confirmed the presence of the cell wall protein in the range of the target molecular weight 10,000 to 100,000.

Finally, the supernatant was ultracentrifuged to remove lipopolysaccharide (LPS) or cell wall related fragments present in traces in the supernatant obtained above. The ultracentrifugation was carried out at 180,000 g to 200,000 g for 3 hours at 4 ° C. to remove the precipitate, and the obtained supernatant was then sterilized using a 0.2 μm filter to finally prevent vaccine composition against Pseudomonas aeruginosa infection. 250 to 260 mg were obtained.

Example 7

CFCPA 50243 Cross-Protection Test by Cell Wall Protein Antigen

The attenuated Pseudomonas aeruginosa CFCPA 50243 cell wall protein obtained by isolation and purification according to the method of Example 6 was intraperitoneally administered to 15 ICR male mice 6 weeks old at a dose of 0.2 mg / kg for 6 weeks. One week after immunization, blood was taken from three mice for an Enzyme-linked immunosorbent assay (ELISA). The remaining ICR mice were injected with wild-type Pseudomonas aeruginosa, corresponding to Fischer immunoform type 5, in an amount of 10-50 times the LD ₅₀ value (5.0 × 10 ⁶ ) of wild-type Pseudomonas aeruginosa. The protective effect of Pseudomonas aeruginosa was continuously investigated for up to one week. In this test, the cell wall protein of CFCPA 50243 showed almost complete protection against Pseudomonas aeruginosa, a type 5 of its parent, Fisher's immune form, and all of the results of ELISA against blood collected were positive. Appeared.

Example 8

Toxicity Test of Cell Wall Proteins

In order to be used as a component vaccine for the purpose of prevention against Pseudomonas aeruginosa infection, not only the safety of the strain itself described in Example 2 but also the safety of the cell wall protein itself to be used as the component vaccine should be ensured. In this example, the cell wall protein was purified and examined to determine whether it was safe in mice, experimental animals. In other words, the attenuated CFCPA 50243 strain was cultured in a 5L fermentor using tryptic soy broth as a culture medium, and then treated according to Examples 4, 5, and 6 to adjust the concentration of the extracted cell wall protein to 1 mg / ml. Afterwards, it was disinfected and used as a protein source for toxicity test.

In the toxicity test, ICR-based male mice having a body weight of 20 to 22 g were used. The injection route of the protein source was intravenous injection, and the amount of protein used in the test group was 20 mg / kg and 100 mg / kg, respectively, and 25 mLl / kg of saline was administered to the control group. As shown in Table 1, the cell wall proteins of the CFCPA 50243 strain had no effect on the body weight of the experimental animals, showed normal growth, and no other symptoms were observed. Also, no specific changes or symptoms of each organ were found.

Example 9

Antigenicity of Cell Wall Proteins

In the same manner as in Example 8, the cell wall protein obtained from the attenuated Pseudomonas aeruginosa CFCPA 50243 was used as an antigen and immunized with ICR mice, which are experimental animals, at a dose of 0.1 mg / kg or 0.2 mg / kg, One week after the immunization, a certain amount of blood was collected from the rats of each group, and serum was separated. Antigen production was measured using the ELISA method as follows. Clin. Microbiol. 15, 1054-1058, 1982].

First, 100 μl of the antigen adjusted to a concentration of 0.1 mg / ml in the coating buffer (0.05 M carbonate buffer, pH 9.6) was added to each well of a 96-microplate for 2 hours at room temperature or 12 to 14 hours at 4 ° C. After the reaction, the protein was attached to the plate, the aqueous solution was removed, and 200 μl of 1% bovine serum albumin (BSA) was added thereto to react at room temperature for 1 hour to block the protein-non-binding portion. At this time, the serum obtained from the mouse immunized with the protein antigen was added by dilution of 100 μl with PBS and then reacted at 37 ° C. for 2 hours. Serum of mice not immunized was used as the antibody of the control group. After completion of the reaction, washed 5 to 6 times with phosphate buffer again, 100 μl of a peroxidase-conjugated secondary antibody (Rabbit-anti mouse Ig-Peroxidase Conjugate) was added to the rabbit antibody against the antibody of mouse at room temperature. After reacting for a period of time, it was washed vigorously at least seven times with the same phosphate buffer wash. To the citrate-phosphate buffer (pH 5.0), ortho-phenylenediamine dihydrochloride was added thereto as a substrate, adjusted to a concentration of 0.3 to 0.4 mg / ml, and added in 50 µl, and shaded for 20 minutes. After the reaction was carried out, 50 µl of 1N-sulfuric acid was added to stop the reaction. Then, the absorbance of each reaction solution was measured at 490 nm using a spectrophotometer.

As a result of analysis using the ELISA, it was found that the test group showed a high immunity of about 2 to 5 times higher than the control group by the administration of 0.1 or 0.2 mg / kg of antigen.

Example 10

Generation of immunoglobulin against Pseudomonas aeruginosa

In Example 5, 0.5-1 ml of cell wall protein solution (containing 100-200 µg of cell wall protein) obtained from the CFCPA 50243 strain was inoculated into the albino rabbit three times at 7 day intervals. Thereafter, rabbit blood was collected at regular intervals to separate serum. 100 ml of the separated rabbit serum was mixed with 300 ml of distilled water and added to 500 g (wet weight) of DEAE-cellulose at 4 ° C. After the mixture was sufficiently shaken at 4 ° C. for 1 hour, the mixture was left to stand to remove the supernatant, and the residue was washed several times with distilled water, followed by 0.01 M phosphate buffer (constitution: NaHPO · HO 13.8g / l, NaHPO14.2g / 1, pH 8.0) washed three times with 200 ml each to obtain 600 mg of immunoglobulin IgG with a purity of 96% or more.

Example 11

Therapeutic Effect of Immunoglobulin on Pseudomonas Aeruginosa Infection

The therapeutic effect of immunoglobulin against Pseudomonas aeruginosa infection against the CFCPA 50243 strain obtained in Example 10 was confirmed by the following method. In the following, 10 mice were used in each group.

The mice were challenged with 1.0 to 3.0 × 10 strains of each of the pathogenic Pseudomonas aeruginosa strains of Fisher Immunotypes 1, 2, 3, 4, 5, 6 and 7. Cells were intraperitoneally injected to cause Pseudomonas aeruginosa infection, and 2 to 4 hours later, mice were intraperitoneally injected with immunoglobulin for the CFCPA 50243 strain at an amount of 1 to 10 mg / mouse to examine the therapeutic effect. In the control group, 0.5 ml of saline for injection against immunoglobulin was injected intravenously.

As a result, the control group administered with only saline for injection showed a mortality rate of 50% or more within 48 hours, and 100% at 72 hours, whereas 80% after 72 hours in the group infected with the group of immuno-fluorine-inoculated group Fisher Immunotype 5 Survival rate was shown above. But. In the strain infected group of Fisher immunotypes 1, 2, 3, 4, 6 and 7, it has been demonstrated that immunoglobulin against CFCPA 50243 strain does not prolong survival.

Example 12

Formulation of Immunoglobulins

Purified Pseudomonas aeruginosa immunoglobulin against attenuated Pseudomonas aeruginosa CFCPA 50243 obtained in Example 10 was formulated using various pharmaceutically acceptable carriers so that 1 to 100 mg of antibody immunoglobulin per kg body weight could be administered. Formulated immunoglobulin was administered to mice inducing Pseudomonas aeruginosa infection to observe the difference in efficacy of immunoglobulin according to the type of carrier. As a result, immunoglobulin preparations using physiological saline or phosphate buffer as a carrier were highly effective in liquid preparations, and mannitol, saccharose or lactose in freeze-dried preparations were highly effective. It was. On the other hand, in the case of lyophilized formulations, human albumin is used in combination with the Pseudomonas aeruginosa immunoglobulin according to the present invention, but the effectiveness is lowered, and in the case of liquid formulations, the effect is decreased when aluminum hydroxide is used as a pharmaceutical carrier. The test results are summarized in Table 2 below.

As can be seen from the above, the attenuated Pseudomonas aeruginosa strain CFCPA50243 (KCCM10033) is a highly safe microorganism in terms of toxicity, and has excellent safety and antibody production of cell wall proteins, and is also a type of Pseudomonas aeruginosa, which is the parent of Fisher's immune forms. Since it shows excellent protective ability and therapeutic effect against, it can be seen that it is very excellent as a microorganism for the production of Pseudomonas aeruginosa preventive ingredient vaccine and Pseudomonas aeruginosa infectious agent treatment.

Claims (3)

It contains the cell wall protein obtained by purely separating and purifying the purified Pseudomonas aeruginosa strain of the Fisher Immunotype 5 from the purified, attenuated Pseudomonas aeruginosa strain CFCPA 50243 (KCCM 10033). Pseudomonas aeruginosa infection prevention vaccine. The vaccine of claim 1, wherein the LD ₅₀ of the attenuated Pseudomonas aeruginosa CFCPA 50243 is 3.0 × 10 ⁷ cells or more. 2. The Pseudomonas aeruginosa infection prevention vaccine according to claim 1, further comprising a pharmaceutically acceptable excipient in addition to the cell wall protein.