KR970001704B1 - Pseudomonas infection preventing vaccines using attenuated pseudomonas cfcpa 20215 - Google Patents

Abstract

A attenuated pseudomonasaeruginosa isolate especially CFCPA 20215 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 2 germ is selected, repeatedly dosed to experimental animals and acclimatized to yield attenuated pseudomonasaeruginosa isolate CFCPA 20215 (KCCM 10030) which is not toxic in itself and attenuated for the cell wall protein to yield only antigen. Type 2 isolate is selected as the objective isolate on the basis that it is detected from the blood of more than 17% of pseudomonas infected patients. 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 2.7 x 107 cells. CFCPA 20215 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

Prevention of Pseudomonas aeruginosa infection with attenuated Pseudomonas aeruginosa CFCPA 20215

The present invention relates to the use of novel attenuated Pseudomonas aeruginosa strains, in particular the attenuated Pseudomonas aeruginosa CFCPA 20215, for the preparation of vaccines for the treatment and prevention of Pseudomonas aeruginosa infections.

Pseudomonas aeruginosa is 1.5 to 3.0μm long and 0.5μm wide bacilli, which has one flagella, motility gram-negative bacteria, and is widely distributed in soil, water, sewage and human intestines [Mol .MIcrobiol. 4, 1069-1075, 1990. Pseudomonas aeruginosa is a 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 lead to death. In addition, Pseudomonas aeruginosa has recently been detected in urinary tract infections and has attracted more attention. There is an urgent need to develop a drug that can effectively prevent or treat refractory purulent diseases such as sepsis and urethral infection caused by Pseudomonas aeruginosa. However, Pseudomonas aeruginosa is not only resistant to antibiotic use, except for some antimicrobial agents, and the prevention and treatment of a good effect has not been developed to date, so the mortality rate due to Pseudomonas aeruginosa infection is increasing.

The only treatment for infectious diseases developed to date is the method of neutralizing toxins by making antitoxins against Pseudomonas 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 eliminating the disadvantages of the `antitoxin usage method. Currently, research on common antigens aimed at preventing or treating Pseudomonas aeruginosa infections can be broadly classified into two fields. One of them is to isolate and purify common antigens from Pseudomonas aeruginosa having different immune forms and use them as antigens for prophylactic vaccine against Pseudomonas aeruginosa infection [Japan, J.Exp. Med. 45, 355-359, 1975], and another method is to extract genes encoding this antigen by recombination in an appropriate vector by gene engineering method that is rapidly developing recently, and then mass production of pyrogen 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 Mierobiol. 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 to dozens of serological and immunologically known Pseudomonas aeruginosa produced by one type of monoclonal antibody. Because it is not possible to cure all infectious diseases, there is a point that it is impossible to 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 of administering monoclonal antibodies have been suggested, they are also time consuming and thus are inadequate for the treatment of struggling patients.

The prior art has also proposed a method of isolating cellular proteins from Pseudomonas aeruginosa strains and using them as antigen vaccines. See Vaccine, Vol. 7, Experimental studies on the protective dfficacy of a Pseudomonas aeruginosa vaccine 1989]. However, the method presented in this document used four common strains of NN 170041, NN 170015, NN 868, and NN 170046 that did not dire-solidize, so that the toxicity of the strain itself is not only a problem, but also a protein component vaccine from such strain. In the process of manufacturing the cell disruption occurs in the cytoplasm, heterologous nucleic acid or toxic polymer material such as lipopolysaccharide (LPS) is mixed in the vaccine components, there is a disadvantage that must be used with great care .

To develop low-cost therapeutics through mass production using production strains

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 P. aeruginosa upon administration of the human body, without the risk of inherent toxicity of P. aeruginosa itself. One study was conducted. As a result, the isolates of Pseudomonas aeruginosa isolated from Pseudomonas aeruginosa-infected patients were separated by amplification by Fischer immunity, and each of them was attenuated by repeated purifying to obtain new safe attenuated Pseudomonas aeruginosa strains whose cell wall proteins are useful for the production of a vaccine for preventing P. aeruginosa infection. It was confirmed that the present invention was completed.

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 P. aeruginosa upon administration of the human body, without the risk of inherent toxicity of P. aeruginosa itself. One study was conducted. As a result, the isolates of Pseudomonas aeruginosa isolated from Pseudomonas aeruginosa-infected patients were separated by amplification by Fischer immunity, and each of them was attenuated by repeated purifying to obtain new safe attenuated Pseudomonas aeruginosa strains whose cell wall proteins are useful for the production of a vaccine for preventing P. aeruginosa infection. It was confirmed that the present invention was completed.

Therefore, the present invention isolates the new safe Pseudomonas aeruginosa, particularly Pseudomonas aeruginosa, from a patient infected with Pseudomonas aeruginosa, which has been attenuated so that its cell wall protein is only antigenic without the toxicity of Pseudomonas aeruginosa itself. After dividing into seven types according to the type (Fisher-Devilin Immunotype), only microorganisms corresponding to the Fisher-Devilin Immunotype 2 were selected and purified by repeatedly injecting them into the experimental animals to select the most attenuated strains. Safe attenuated Pseudomonas aeruginosa CFCPA 20215.

In addition, the cell wall protein purely isolated and purified from the safe attenuated Pseudomonas aeruginosa CFCPA 20215 according to the present invention can be used as a vaccine for preventing Pseudomonas aeruginosa infection by itself, or induced in an animal body by such a cell wall protein. Antibody immunoglobulin can be used as a therapeutic agent for Pseudomonas aeruginosa infection. Therefore, the present invention can be used as a therapeutic agent for Pseudomonas aeruginosa infection and vaccine for preventing P. aeruginosa infection, or the antibody immunoglobulin induced in the animal body by cell wall protein can be used as a treatment for P. aeruginosa infection. Accordingly, the present invention relates to the use of attenuated Pseudomonas aeruginosa CFCPA 20215 for the production of a vaccine for treating Pseudomonas aeruginosa infection and a vaccine for preventing P. aeruginosa infection.

The Fischer-Deblin immune forms of Pseudomonas aeruginosa are divided into seven types from 1 to 7, based on the fact that among these 7 Pseudomonas aeruginosa, type 2 Pseudomonas aeruginosa is most frequently detected from the blood of about 17% of infected patients. This strain was selected as the target strain.

The safe, attenuated Pseudomonas aeruginosa strain according to the present invention is obtained by purely separating Pseudomonas aeruginosa, which corresponds to Type 2 of the Fisher's immune form, from the blood of a patient determined to be infected with Pseudomonas aeruginosa and attenuating iteratively. The attenuated Pseudomonas aeruginosa strain thus obtained is named CFCPA 20215.

The attenuated Pseudomonas aeruginosa strain CFCPA 20215 obtained by this repeated purifying process is almost identical to the parent strain before attenuation in several aspects, including morphological and bacteriological properties. It is classified as a new strain because it shows new antigenic properties for infection. Therefore, strain CFCPA 20215 was deposited on May 12, 1993 at the Korea Microbial Conservation Center, an international depository institution, Korea Academic Spawn Association, and was given a deposit number of KCCM 10030.

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's engineer or the toxic state of the parent strain, but preferably 3 to 7 times. Repeat. The LD ₅₀ value of the novel attenuated Pseudomonas aeruginosa strain thus obtained is at least 2.0 × 10 ⁷ cells, preferably at least 2.7 × 10 ⁷ cells.

The present invention also provides the use of the novel Pseudomonas aeruginosa in the preparation of a Pseudomonas aeruginosa infection treatment agent and a vaccine for preventing Pseudomonas aeruginosa infection.

Pseudomonas aeruginosa CFCPA 20215 attenuated according to the present invention is cultured in a fermentor of various sizes under optimal growth conditions to obtain a large amount of cells, and then the obtained cells are treated with organic solvents and extracted, 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.

First, in step 1, the attenuated Fisher type 2 Pseudomonas aeruginosa CFCPA 20215 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 Clucose (30g / l), Peptone (15g / l), MgSO ₄ (0.5g / l), CaCO ₃ (5g / l), KH ₂ PO ₄ (1g / l), FeSO ₄ (5mg / 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, dry cells can be obtained at least 30% higher on the basis of the weight of the dry cells than when cultured in a tryptic soy broth medium.

In this cell culture step, the optimum culture conditions were 37 ° C., aeration rate 1 vvm (volume / volume. Minute), and the inoculation amount was 5 (v / v)% of the culture medium, and the stirring speed was 100 rpm at the initial 2 hours. After that, the mixture is incubated at 600 rpm for 10 to 20 hours, preferably 12 to 16 hours.

When the culture is completed, the precipitated cells in 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 standing 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 cells are broken by measuring the intracellular marker enzyme lactate dehydrogenase or hexokinase during the extraction process.

The cell wall proteins of the attenuated Pseudomonas aeruginosa strains thus obtained as supernatants are subjected to primary and secondary ultrafiltration and fractionated so that only proteins with a molecular weight ranging from 10,000 to 100,000 are obtained. At this time, it is very important that the molecular weight range of the cell wall protein is 10,000 to 100,000, which is revealed by animal experiments when the molecular weight is less than 10.000, and 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 residual toxic lipopolysaccharides, which were then sterilized using a membrane filter or the like 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 alone or as a mixture in a proportion with the corresponding cell wall proteins obtained by Pseudomonas aeruginosa of other Fischer immunoforms.

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 0.5g to 2.5). cell wall protein obtained from g), 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, inoculated to a test animal such as sheep and rabbit goats to induce antibody 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, for example, Practical Immunology, 3rd Ed., Pp 292-294. After mixing with DEAE-cellulose and adsorbing, the supernatant is discarded and the immunoglobulin-adsorbed DEAE-cellulose is repeatedly washed several times with phosphate buffer to obtain purified immunoglobulin. The immunoglobulin thus obtained can be used alone. However, if necessary, the cell wall protein obtained from the attenuated strain CFCPA 20215 of the present invention may be mixed with cell wall proteins of Pseudomonas aeruginosa strains of other immune forms in a proportion to use immunoglobulins derived from immunization of animals to treat P. aeruginosa infections. It may be.

The dosage varies widely depending on the age, body 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 embodiments.

Example 1

Isolation and Identification of Fisher Immunotype 2 from Patients with Pseudomonas Aeruginosa

Aseptically, 1 to 5 ml of each of 260 different blood samples from P. aeruginosa infections were collected aseptically, and left at room temperature for 1 hour, then stored in a refrigerator at 4 ° C. overnight, and then stored at 4 ° C. for 1500 minutes at 1500 g. The solids were removed by centrifugation and serum was obtained as supernatant. Serum obtained above was added to a tryptic soy broth culture plate to which 1.0-1.5% agar was prepared in advance. The phosphate buffer solution (1.15 g Na ₂ HPO ₄ per K, 0.2 g KCl, 0.2 g KH ₂ PO ₄ 0.2 g, NaCl 8.766 g) was diluted to a ratio of 1:10 and the resulting culture was transferred to a new culture plate and microorganisms were isolated in pure form using a known microbial pure separation method (Biology of Microorgani).

The serological and immunological classification of isolated Pseudomonas aeruginosa is known (Bergey's Manual) and identified by the following assay 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 was aseptically aliquoted, centrifuged at 6000 g at 4 ° C. for 10 minutes, the 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 added to the prepared 96-well microplates, and then 15 μl of the microorganism suspension obtained above was added to each serum, mixed, and agglutination was performed between 10 and 30 minutes. Was observed. 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

Selection of Attenuated Microorganisms from Isolated Pseudomonas Aeruginosa

To attenuate Pseudomonas aeruginosa into a safe microorganism that can be used for vaccine production, microorganisms with type 2 immune forms were attenuated through repeated purification. The toxic Pseudomonas aeruginosa used as a control group was selected from GN 11189 (purchased from Episome Institue, Japan), and GN 11189, when 2.0 × 10 cells were injected intravenously (or intraperitoneally) into male ICR mice weighing 20 to 25 g Shows 100% mortality within days.

Pseudomonas aeruginosa (LD ₅₀ 1.4 × 10 ⁶ ) having the selected type 2 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, and then 5 × 10 ⁵ , 5 × 10 ⁶ , 5 × 10 ⁷ and 5 × 10 ⁵ , Each cell was administered intravenously (or intraperitoneally) with 10 mice per group, diluted as appropriate. At this time, only GN 11189 strain 2.0 × 10 ⁶ cells were administered to the control group, and Pseudomonas aeruginosa was isolated aseptically from the ICR mice surviving at the highest cell concentration at the point of 100% mortality 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 microorganism 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 LD ₅₀ was repeatedly treated at least 2.0 × 10 ⁷ cells or more. The LD ₅₀ of the attenuated strain obtained here was measured to be 7.6 × 10 ⁷ cells or more. The microbial strain thus safely attenuated was named CFCPA20215 (Accession No .: KCCM 10030).

Example 3

Physiological Characteristics of Attenuated Pseudomonas aeruginosa

(1) The growth rate of the attenuated Pseudomonas aeruginosa CFCPA 20215 obtained in Example 2 according to pH change was measured. First, strain CFCPA 20215 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 concentration of the cells was measured by measuring absorbance at 600 nm using a spectrophotometer. As a result, strain CFCPA 20215 was found to be unable to grow under strong acidic conditions of pH 3.0 on the half side showing excellent growth rate under conditions of pH 5.0 or more.

(2) The growth rate of the attenuated Pseudomonas aeruginosa CFCPA 20215 according to temperature change was measured in the same manner as in (1). Strain CFCPA 20215 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 maintained at different temperatures 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 20215 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 20215 was inoculated into 50 ml of tricky soy broth, each stored at pH 7.0, incubated for 12 to 16 hours under aerobic conditions maintained at 200 rpm at 37 ° C, and then centrifuged at 6000g at 4 ° C for 10 minutes under aseptic conditions. The microorganisms were harvested and then suspended in 10 ml physiological saline. To 5 mg 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 different carbon sources, 5 μl of the microbial suspension obtained above was added and incubated for 12 hours. Then, the absorbance was measured at 600nm for each culture solution to investigate the presence of microorganisms.

As a result, strain CFCPA 20215 shows excellent growth rate in the presence of glucose or β-alanine and mannitol, but other carbon sources, such as xylose, galactose, sorbitol, inositol, maltose, sucrose, arabinose, etc. It was found that growth did not exist in existence. These carbon source utilization characteristics are substantially identical to those of Pseudomonas aeruginosa strains, type 2 of Fisher's immune form, which is the parent of CFCPA 20215 strains (Bergey's Manual). Something that shows some growth, but what is special is that it shows some growth in the presence of mannose.

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 properly 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, various organic acids such as acetic acid, pyruvic acid and lactic acid, and various organic and inorganic ammonium salts, peptone, yeast extract, casein hydrolysate and other nitrogen-containing nitrogen sources. Materials and the like can be used. As the inorganic compound, magnesium sulfate, calcium carbonate, iron sulfate, copper sulfate, sulfuric acid sulfate, potassium hydrogen phosphate and potassium dihydrogen phosphate may be used. Cultivation is preferably performed under aerobic conditions at 25 to 37 ° C., for example, plate culture, or liquid culture such as shaking culture or aeration stirring culture. The pH of the medium is preferably maintained at pH 5 to 9 during the incubation period. Preferably, 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. 2.5 l of the medium solution obtained by dissolving 30 g of trickic sobrie per 1 l of distilled water in a 5 L incubator was adjusted to pH 7.2 and sterilized before being introduced. The culture temperature was 37 ℃, the aeration amount is 1vvm, the inoculum was used as a condition of the attenuated Pseudomonas aeruginosa cells 5 (v / v)% obtained in Example 2 for the culture solution 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 liter of culture.

(2) Clucose (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 ( ₂ ) Strain CFCPA 20215 was cultured under the same culture conditions as in (1) except that a special Pseudomonas aeruginosa culture medium consisting of 5 mg / l), 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 Proteins from Attenuated Pseudomonas Aeruginosa

Cell wall proteins were partially purified from attenuated Pseudomonas aeruginosa CFCPA 20215 to prepare a vaccine against Pseudomonas aeruginosa.

After adding 2.5L of the special medium used in Example 4 (2) in a 5L fermenter, the culture was carried out for 12 to 16 hours while maintaining the same culture conditions as mentioned above. After completion of the incubator, the supernatant was removed by centrifugation of 2.5L of the culture medium at 4 ° C. at 6000 g for 20 minutes, and the precipitated cells were treated with 4 ° C. phosphate buffer (1.15 g Na ₂ HPO ₄ , 0.2 g KCl ₂ , KH _2). After suspension in 0.2 g of PO ₄ , 8.776 g of NaCl, pH 7.2) and centrifugation again, the Senpo was 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 12 or more. After removing the acetone from the precipitated cells, two volumes of fresh acetone were added again, agitated for 5 to 10 minutes, and centrifuged at 8000 g for 20 minutes at 4 ° C. 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 addition of the same volume of phosphate buffer, followed by secondary extraction under the same conditions as the above-described method 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 crushed was confirmed 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 separation gave a clear supernatant. 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 by protein quantification.

Example 6

Pure Protein Condition

The following experiment was performed to purely separate the crude cell wall protein adjusted to a concentration of 1 to 2 mg / ml to have only an active ingredient. Two to 2.5 liters of aqueous solution of crude cell wall proteins are first removed from the 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 molecular weights less than 100,000 to flow out of the filtrate. Proteins with molecular weights greater than 100,000 continue to circulate, separating molecules with molecular weights less than 100,000.

The initial volume was concentrated in an aqueous solution of protein concentrated to 10-20% in 20-25 L of sterile phosphate buffer (1.15 g Na ₂ HPO ₄ , 0.2 g KCl, 0.2 g KH ₂ PO ₄ , NaCl 8.766 g per 10 times the initial volume). By continuing to wash), more than 90% of protein with a molecular weight of 100,000 or less could be recovered. Proteins with a molecular weight of 100,000 or less separated by the filtrate were concentrated in the same system until a protein with a molecular weight of 10,000 to 100,000 was brought to a concentration of 1 mg / ml. 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 then the supernatant obtained was sterilized using a 0.2 μm filter to finally prevent vaccine composition against Pseudomonas aeruginosa infection. 250-260 mg were obtained.

Example 7

Cross-defense ability test by CFCPA 20215 cell wall protein antigen

The attenuated Pseudomonas aeruginosa CFCPA 20215 cell wall protein obtained by 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. One week after immunization, three mice received blood for ELISA (Enyzyme-linked immunosorbent assay). The remaining ICR mice were injected with wild type Pseudomonas aeruginosa, corresponding to immunoform type 2, in an amount corresponding to 10-50 times LD ₅₀ (1.4 × 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 20215 showed almost perfect protective effect against the corresponding Pseudomonas aeruginosa in its parent, Type 2 of the Fisher's immune form, and all of the results of ELISA against blood collected were positive. Appeared.

In addition, to examine whether the cell wall protein of CFCPA 20215 strain can act as a prophylactic antigen against infection caused by type 1,3,4,5,6,7 Pseudomonas aeruginosa strains in addition to the type 2 of the Fisher's immune form, The experiment was repeated using Pseudomonas aeruginosa strains corresponding to the type 1,3,4,5,6,7 of the Fischer immunoform. As a result, it was confirmed that the cell wall protein of the CFCPA 20215 strain exhibited excellent cross-protective ability against Pseudomonas aeruginosa strains corresponding to Fisher types 3 and 7.

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 stability of the strain itself described in Example 2, but also the stability of the cell wall protein itself for use as a 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. That is, the attenuated CFCPA 20215 strain was incubated in a 5 L fermentor using Tritic 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 protein source was intravenous injection, and the amount of protein used in the test group was 20 mg / kg and 100 mg / kg, and the control group was administered with 25 ml / kg of saline. As shown in Table 1, the cell wall protein of the CFCPA 20215 strain had no effect on the body weight of the experimental animals and showed normal growth, and no other symptoms were observed. There was also no specific change or symptom of each device.

Example 9

Antigenicity of Cell Wall Proteins

In the same manner as in Example 8, the cell wall protein obtained by purification from attenuated Pseudomonas aeruginosa CFCPA 20215 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 and immunized. One week after the injection, a certain amount of blood was collected from each group of rats, and serum was separated to determine whether antibodies were produced using the ELISA method as described below [J. 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 and reacted for 2 hours at room temperature or 12 to 14 hours at 4 ° C. After attaching the protein to the plant, 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 from the mice immunized with the protein antigen was serially diluted with PBS, added 100 μl, and reacted at 37 ° C. for 2 hours. Serum of mice not immunized was used as the antibody of the control group. After the reaction, washed 5 to 6 times with phosphate buffer solution, 100 μl of a peroxidase-bound secondary antibody (Rabbit-anti mouse Ig-Peroxidase Conjugate) was added to the rabbit antibody against the mouse antibody, and then 1 hour at room temperature. After the reaction, the mixture was washed vigorously at least seven times with the same phosphate buffer wash. To the citrate-phosphate buffer (0.1 M Citrate-Phosphate buffer, pH 5.0), ortho-phenylenediamine dihydrochloride was added thereto in a concentration of 0.3 to 0.4 mg / ml, and then 50 μl was added as a substrate. After the reaction, 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 the analysis using the ELISA, it can be seen that the test group exhibited an 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, 9.5-1 ml of cell wall protein solution (containing 100-200 μg of cell wall protein) obtained from the CFCPA 20215 strain was inoculated 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 (Na ₂ HPO ₄ · H ₂ O 13.8 g / l, 0.2 g of KCl, 14.2 g / l Na ₂ HPO ₄ , pH 8.0) was washed three times with 200 ml 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 20215 strain obtained in Example 10 was confirmed by the following method. In the following, 10 mice were used in each group.

By pihyeo immune type 1, 2 and 3 in mice, 4, 5, 6 and 7 from 1.0 to 3 × 10 ⁶ of each of the pathogenic Pseudomonas aeruginosa strains each cell intraperitoneally by injection and cause Pseudomonas aeruginosa infection, mice after 2 hours to 4 hours The therapeutic effect was tested by intraperitoneal injection of immunoglobulin against the CFCPA 20215 strain in an amount of 1 to 10 mg / mouse. In the control group, 0.5 ml of saline for injection against immunoglobulin was injected intravenously.

As a result, injection of physiological saline only, but the control group showed a mortality rate of 50% or more within 48 hours, 100% at 72 hours, whereas 72 hours in the hepatitis group of the crowd fisher immunization type 2,3 and immunoglobulin Even after 80% of the surviving milk was shown. However, in the infected group of Fisher immunotypes 1, 2, 4 and 6, immunoglobulin against the CFCPA 20215 strain has been demonstrated to prolong survival.

Example 12

Formulation of Immunoglobulins

Purified Pseudomonas aeruginosa immunoglobulin against the attenuated Pseudomonas aeruginosa CFCPA 20215 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 solution as carriers were highly effective in liquid preparations, and manchitol, saccharose or lactose in freeze-dried preparations were highly effective. Indicated. 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 seen above, the attenuated Pseudomonas aeruginosa CFCPA 20215 (KKCM 10030) in the present invention is a highly safe microorganism in terms of toxicity and at the same time excellent in the safety and antibody production of cell wall proteins, and also the parent of the Fisher immune form of type 2 As well as Pseudomonas aeruginosa, Pseudomonas aeruginosas of type 3 and 7 exhibit excellent cross-protective ability and therapeutic effect, so 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.

Claims (3)

It is characterized in that it contains cell wall protein obtained by purely isolated and purified from the safe attenuated Pseudomonas aeruginosa CFCPA 20215 (KCCM 10030), which was purified, purified and attenuated Pseudomonas aeruginosa strains of the Fisher Immunotype 2 type. Pseudomonas aeruginosa infection prevention vaccine. The vaccine of claim 1, wherein the LD ₅₀ of the attenuated Pseudomonas aeruginosa CFCPA 20215 is at least 2.7 × 10 ⁷ cells. 2. The Pseudomonas aeruginosa infection prevention vaccine according to claim 1, further comprising a pharmaceutically acceptable excipient in addition to the cell wall protein.