KR100421453B1 - Vaccine for the prophylaxis of the disease caused by Gram negative bacteria and process for the preparation thereof - Google Patents

Abstract

The present invention includes attenuated O-lipopolysaccharide-deficient modified lipopolysaccharides and outer membrane proteins of Gram-negative bacteria, which have a lipo-proteosome structure. And E. coli CFCEC-97415 strains which produce modified vaccines for the prevention of diseases caused by the disease, in particular sepsis, and preparation thereof, and attenuated, O-liposaccharides, which are excellent in safety. And the antigenicity is greatly enhanced, showing excellent defense against various Gram-negative bacteria.

Description

Vaccine for the prophylaxis of the disease caused by Gram negative bacteria and process for the preparation

The present invention relates to a vaccine for the prevention of diseases caused by various Gram negative bacteria in the human body. More specifically, the present invention includes lipoproteins containing attenuated O-lipopolysaccharide-deficient modified lipopolysaccharides and outer membrane proteins of Gram-negative bacteria (hereinafter also referred to as "OMP"). Prophylactic vaccines for gram-negative bacteria, in particular sepsis, having a proteosome structure, in particular sepsis and methods for their preparation, and E producing attenuated, modified lipopolysaccharides lacking O-liposaccharides coli CFCEC-97415 strain.

Sepsis is a bacterial acute infection caused by a variety of causes, a horrible disease in which the pathogen itself or a bacterial toxin damages blood or tissues, causes severe toxins and lowers blood pressure, resulting in death. . Causing sepsis include major gram-negative bacteria E. coli (Escherichia coli), Pseudomonas aeruginosa (Pseudomonas aeruginosa), capsular Klebsiella (Klebsiella pneumoniae), Proteus (Proteus), Serratia marcescens (Serratia), Acinetobacter (Acinetobacter), Enterobacter (Enterobacter), etc. There is this. These bacteria cause sepsis by infecting patients with reduced immune function, including burn patients, car accidents involving severe wounds, incisions from surgical procedures, and cancer patients, and recently, sepsis caused by urethral infections. Is being reported.

In case of sepsis, the infection of bacteria itself is dangerous, but the endotoxins they secrete are so powerful that they are dangerous even if they exist in the order of tens of pg per ml of blood, and 10-20 ng of endotoxins destroy 1 million leukocytes. Has been reported. In addition, endotoxin shock by endotoxins poses a direct threat to the life of a curable patient, which is caused by the normal inflammatory response of various immune cells and the hypersensitivity reactions involving cytokines such as IL-10, IL-12, and INF. Happens.

This sepsis is the 13th leading cause of direct death in the United States, with more than 400,000 deaths per year, of which more than 100,000 die (MMWR 39, 31-34, 1990). Therefore, there is an urgent need for the development of a medicament capable of effectively preventing or treating sepsis caused by Gram-negative bacteria. Various antibiotics have been developed in response to such demands, but resistance bacteria have been continuously developed, the possibility of opportunistic infection has increased along with social development, and preventive vaccines and treatments with satisfactory effects have not been developed until now, which can prevent sepsis. There is great interest in vaccines.

Until now, efforts have been made to develop and use antibodies to the causative agent of sepsis or its toxin. For example, the endotoxin protective antibody Centoxin (Centoxin, USA, failed in Phase III clinical trials in 1993), T-88 (Chiron, USA, decommissioned in 1994), E5 (Xoma, USA in clinical trials in 1995) Monoclonal antibodies have been developed, but failed in clinical applications. Since then, with a different approach, Xoma is developing a bactericidal permeability increasing protein (BPI) under the name Neuplex. Applicant has isolated and purified the extracellular membrane protein of Pseudomonas aeruginosa which has the highest infection probability and high mortality among the causative agents of sepsis to develop Pseudomonas aeruginosa vaccine which shows the protective effect against various Fisher types, and completed the current clinical phase II and its effectiveness It is a proven state.

However, since sepsis is caused by a single or multiple infection of various Gram-negative bacteria, a more fundamental preventive vaccine uses a common antigen that can protect against various causative bacteria. Antigen components expected to be available as common antigens include extracellular membrane proteins and lipopolysaccharides, which are common components of all Gram-negative bacteria. The extracellular membrane of Gram-negative bacteria consists of extracellular membrane proteins and lipopolysaccharides that span the structure of the outer membrane proteins. However, the extracellular membrane protein cannot be used as a common antigen because its components or structures are different for each species and genus, and lipopolysaccharide is not suitable as a common antigen by itself because its components and forms are different for each strain. That is, the lipopolysaccharide is composed of O-liposaccharide, lipid (A) and three parts of the core. Among them, the outermost O-liposaccharide is species-specific and thus cannot be used as a common antigen. Therefore, it is possible to develop sepsis vaccines by Gram-negative bacteria using lipid A or core sites, which are structures common to Gram-negative bacteria, as common antigens. However, lipopolysaccharides are toxic in humans as endotoxins and must be attenuated by treatment with alkalis such as NaOH or anhydrous hydrazine ( Infect. Immun. 66, 1891-1897, 1988). Attenuated lipopolysaccharides, however, have the disadvantage of low antigenicity that does not induce sufficient amounts of antibodies to defend. Due to this low antigenicity, the protein is used as a carrier to enhance antigenicity, and the protein used here is tetanous toxoid ( Infect. Immun. 64, 4047-4053, 1996), cholera. Toxin (cholera toxin ( Infect. Immun. 60, 3201-3208, 1992), high molecular weight protein ( Infect. Immun. 64, 4047-4053, 1996), GBOMP ( Neisseria mengingitidis group B outer menbrane protein) ( J. infect. Dis. 160, 1064-1067, 1989).

As described above, in order to use the core region of the lipopolysaccharide as a common antigen, E. coli J5 (Rc chemotype) or Salmonella minnesota R595 mutant ( Infect ) which produces a modified lipopolysaccharide lacking O-liposaccharide Immun. 59, 4491-4496, 1991). Cross et al. ( J. Endotox. Res. 3, 57, 1994) found that the blood of volunteers vaccinated with the boiled E. coli J5 strain had a protective effect against sepsis. Contrary results were obtained with no better results than normal plasma. This conflicting result is due to the fact that the cells themselves were used as antigens and the experiments did not clearly demonstrate whether antibodies against common antigens were produced. AK Bhattacharjee et al. ( J. Infect. Dis. 170, 622-629, 1994) reported that lipopolysaccharide-specific antibodies purified by affinity chromatography using purified lipopolysaccharide as antigens induce sepsis due to Gram-negative bacteria. It has been demonstrated that neutropenic rats have a protective effect. However, lipopolysaccharides have low antigenicity and did not produce sufficient amounts of antibodies to defend against when used alone as antigens. As a result, the above documents only demonstrate the usability of the lipopolysaccharide as a common antigen, and provide no alternative to the method of enhancing antigenicity.

Similarly, vaccines using polysaccharides have problems with their antigenicity, such as Pedavax and Actihib, which prevent meningitis caused by gram-negative bacteria. In addition, pneumo vaccines (4-valent, 9-valent, 23-valent) also use polysaccharides as antigen components. Vaccines that use these polysaccharides as antigens do not have any problem in producing sufficient amounts of antibodies for defense in those over 2 years of age. It does not induce cell-mediated immunity. Thus, since 1980, attempts have been made to solve this problem by developing vaccines coupled with proteins. Proteins used for coupling include tetanus toxin, cholera toxin, high molecular weight protein, GBOMP and the like as described above. In particular, GBOMP is effective against Haemophilus influenzae (Haemophilus influenzae) And Streptococcus pneumoniae (Streptococcus pneumoniaeIt is used as a coupling agent of a prophylactic vaccine (E. coliJ5 DLPS-GBOMP). As such, the reason for using GBOMP is that lipopolysaccharide and extracellular membrane proteins are similar to the extracellular membrane, and GBOMP is Neisseria meningitidis.Neisseria meningitidis)end Because it is used as an antigen component of causing bacterial meningitis, a boosting effect is also expected.

Based on the results of these previous studies, the present inventors conducted a continuous study to develop a method of attenuating the modified lipopolysaccharide lacking O-lipopolysaccharide to remove the risk and improving the low antigenicity. The result is a modified lipopolysaccharide that lacks O-lipopolysaccharide.E. coliFrom J5 strain New Attenuated StrainsE. coliWe developed CFCEC-97415 and found that a vaccine containing purified and attenuated lipopolysaccharide and gram-negative extracellular membrane proteins has a structure similar to that of the natural extracellular membrane, thereby promoting low antigenicity. . Also,E. coli, EspeciallyE. coliCFCEC-97415 orP. aeruginosaIt was found that by using the extracellular membrane protein of the antigenic enhancement effect can be obtained than the existing protein complement.

Accordingly, it is an object of the present invention to provide a vaccine and a method for producing a vaccine for preventing a disease caused by gram-negative bacteria, which is safe in human application and greatly enhanced antigenicity and shows excellent defense against various gram-negative bacteria. Another object of the present invention is to provide an E. coli CFCEC-97415 which produces a modified lipopolysaccharide that is attenuated and lacks O-lipopolysaccharide.

1 is attenuated lipoic polysaccharide (DLPS) - vaccine electron micrograph (× 15,000) of the outer membrane E. coli cells containing the protein (ECOMP); And

Figure 2 is an electrophoresis picture of lipopolysaccharide (LPS) and attenuated lipopolysaccharide (DLPS) isolated from E. coli J5 strain.

First, the present invention provides an antigen component comprising (i) an attenuated O-liposaccharide-deficient modified lipopolysaccharide; And (ii) a vaccine for preventing a disease caused by gram-negative bacteria, which contains a gram-negative bacterium extracellular membrane protein as a complement component for enhancing antigenicity.

Second, the present invention

(Iii) separating and purifying lipopolysaccharide from E. coli J5 strain or E. coli CFCEC 97415 strain;

(Ii) attenuating the lipopolysaccharide obtained in (iii);

(Iii) separating and purifying extracellular membrane proteins from Gram-negative bacteria;

(Iii) mixing the lipopolysaccharide obtained in (ii) and the extracellular membrane protein obtained in (iii):

It relates to a method for producing the vaccine comprising a.

Third, the present invention relates to an E. coli CFCEC 97415 strain (Accession No. KFCC-11276) which is attenuated and produces a modified lipopolysaccharide lacking O-liposaccharide.

Hereinafter, the present invention will be described in detail.

In one example of the present invention, a modified lipopolysaccharide lacking O-lipopolysaccharide isolated from E. coli J5 strain is used as an antigen component, which is attenuated by alkali treatment or the like, and then amplified with E. coli or P. aeruginosa . By mixing with extracellular membrane proteins of the same Gram-negative bacteria, lipopolysaccharide is inserted into the proteosome to form the same three-dimensional structure as the natural extracellular membrane, thereby improving low antigenicity. The vaccine of the present invention may further be used as a sepsis vaccine having a titer for all strains isolated directly from sepsis patients, further containing a pharmaceutically acceptable excipient such as alum-adjuvant, if necessary. have.

In addition, the present invention relates to an attenuated strain E. coli CFCEC-97415 which does not exhibit a lipopolysaccharide ladder having a molecular weight of 10,000 to 100,000 by repeatedly injecting and purifying E. coli J5 strain (ATCC 43745) into a laboratory animal. . In the present invention, the E. coli J5 strain (ATCC 43745) was injected and selected into Balb / C mice five times, and the strain having the lowest LD ₅₀ value was selected and named as E. coli CFCEC-97415. E. coli CFCEC-97415 showed no change in the morphological characteristics or the characteristics of the strain itself, and was confirmed by electrophoresis by separating lipopolysaccharides. It was confirmed to produce and was classified as a new strain. Accordingly, E. coli CFCEC-97415 was deposited on March 22, 2001 by the Korean spawn association, which was granted accession number KFCC-11276.

In another embodiment of the present invention, lipopolysaccharide isolated from E. coli CFCEC-97415 is attenuated by alkaline treatment or the like, and then mixed with the extracellular membrane protein purified from E. coli or P. aeruginosa to obtain natural cells. Antigenicity is enhanced by forming lipo-proteosomes with the same three-dimensional structure as the outer membrane. If necessary, a pharmaceutically acceptable excipient such as alum-adjuvant may be added to provide a sepsis prevention vaccine having a protective effect against Gram-negative bacteria causing sepsis.

For example, the method for producing a vaccine according to the present invention will be described in detail below. In the present invention, in order to prepare a vaccine for preventing diseases caused by Gram-negative bacteria, in particular sepsis, E. coli CFCEC-97415 (rough colony), which first produces a modified lipopolysaccharide that is attenuated and lacks O-liposaccharide, is suitable. Political culture in the container. As a medium, tryptic soy broth or Luria broth, which is usually used, is used, and the temperature is 37 ° C., aeration volume of 1 minute vvm (volume / volume / minute), inoculation amount 3 v / v %, Incubate at 50 rpm (100 L jar) for about 12-16 hours.

When the culture is completed, cells are centrifuged from the culture medium or cells are obtained using a concentrator, and ethanol, acetone and ethyl ether are sequentially added to inactivate cells, and there is no O-liposaccharide to be used as an antigen component from the dry cells. Purified modified lipopolysaccharide. To purify lipopolysaccharide, lipopolysaccharide is extracted using the petroleum ether / chloroform / phenol method, which is the lowest known extraction method ( Clinical Infectious Dis . 20, S 149-153, 1995). The extracted lipopolysaccharide was treated twice with phenol and centrifuged to remove proteins, impurities and phenol, finally precipitated with ethyl ether and dried to separate pure lipopolysaccharide.

Subsequently, an attenuation process by alkali (eg, NaOH) treatment is performed. The lipopolysaccharide and NaOH are well mixed at 65 ° C. for 5 minutes. This process is continued for 1 hour and then reacted at the same temperature for 1.5 hours. After cooling for 60 min at 0 ° C., the pH is adjusted to 7.0 and leached with ethanol. Precipitated lipopolysaccharide is dissolved in distilled water and stored and analyzed by TNF-α analysis.

The attenuated lipopolysaccharide is mixed with an extracellular membrane protein to form a lipo-proteosome structure.E. coliorP. aeruginosafrom Extract.E. coliorP. aeruginosaIs grown on tryptic soy broth or luria broth and centrifuged to recover cells. The recovered cells are suspended in sodium acetate buffer and a solution containing 6% of the same amount of empigen as this volume is added to separate the extracellular membrane protein from the cells. Only the protein precipitated in 20-45% ethanol is centrifuged twice to obtain a fraction. Since the obtained protein contains lipopolysaccharides acting as endotoxins, their removal is essential to ensure stability as a vaccine. Therefore, 60% ammonium sulfate is added to precipitate only the protein, remove the lipopolysaccharide, and repeat this process three times. The final precipitate obtained here is composed of extracellular membrane protein, which is dissolved using 1% Empigen solution. The protein contains nucleic acid as another impurity, whose content is measured by₂₈₀/ A₂₆₀If the value is 1.16 (nucleic acid content 2% or less) or more, the nucleic acid is removed using DEAE-Sefadex resin.

The attenuated lipopolysaccharide obtained by the above method and the extracellular membrane protein are mixed at a constant ratio to prepare a vaccine, and the mixing ratio thereof is usually preferably 1: 1 to 1: 1.5. Since extracellular membrane proteins form proteasomes, the number and spacing of inserted lipopolysaccharides differ depending on the amount of lipopolysaccharides to be mixed. The most preferable structure is observed from 1: 1 to 1: 1.5 when observed with electron micrographs. This is because it has been confirmed (see Fig. 1).

In addition to the above two components, the vaccine according to the present invention may contain a pharmaceutically acceptable excipient, such as an ISCOM (immunostimulating complex), calcium hydroxide, calcium phosphate, aluminum hydroxide (alum hydroxide), etc. as necessary or for the purpose of enhancing antigenicity. Can be added.

In order to be effectively used as a vaccine for preventing sepsis, it must contain an antigenic component in an amount sufficient to produce an antibody so as to have a protective effect in the human body, which is dependent on the sex, age, weight, and state of health of the subject of concern. Depends. In general, the amount of the antigen component is determined by ELISA analysis of the results of fever test and antibody titer using mouse or rabbit. As a result of converting this value into weight ratio, it was found that administration of lipopolysaccharide and extracellular membrane protein in amounts of 10 to 100 µg, respectively. Routes of administration are generally intramuscular but may be subcutaneously administered in some cases. In principle, the vaccine of the present invention is inoculated three times like other bacterial vaccines.

Hereinafter, the present invention will be described in more detail with reference to the following examples, which are intended to illustrate the present invention but are not intended to limit the scope of the present invention in any way.

Example 1: E. coli Isolation and Identification of Attenuated Strains from J5 Strains

E. coli J5 ATCC 43745 strain was purchased from the American Type Culture Collection (ATCC). E. coli J5 strains produce modified lipopolysaccharides that lack O-liposaccharides but are not attenuated. Therefore, in order to prepare the E. coli J5 strain as a safe strain that can be used for vaccine preparation, the operation of repeatedly injecting mice to select purified strains, that is, strains having a high LD ₅₀ value, was repeated five times. At this time, the control strain E. coli J5 ATCC 43745 was used.

Ie, grown in pre-sterilized tryptic soy brothE. coliJ5 strains were diluted 1: 100,000-fold in phosphate buffer and 100 μl of them were plated in tryptic Soy Broth solid medium. After culturing at 37 ° C. for 12 hours or more, 5 of the colonies formed were inoculated into tryptic soy broth and grown in a 37 ° C. aerobic incubator. Cells obtained by centrifuging the logarithmic bacteria at 6,000 g for 10 minutes were suspended in physiological saline and centrifuged again under the same conditions and washed once. Fresh saline solution was then added to each bacteria at 5 × 10 ml.⁵, 2 × 10⁶, 8 × 10⁶, 3.2 × 10⁷, 1.28 × 10⁸, 5.2 × 10⁸And 2.1 × 10⁹Dilution was carried out as appropriate. Each dilution was administered intraperitoneally to 10 mice per group and observed for 3 days of survival from mice surviving the highest cell concentrations.E. coliTo Again aseptically separated. By repeating the above method four times for the isolated bacteria LD₅₀Value is 1.0 × 10⁸The above was made. Select one strainE. coliIt was named CFCEC-97415 (Accession No. KFCC-11276). This strain was attenuated approximately 100 times than the original strain, resulting in 1.0 × 10.⁶1.0 × 10⁸LD increased more than₅₀Has a value.

Example 2: Attenuated E. coli Physiological Characterization of CFCEC-97415 Strains

(1) The difference between the attenuated strain and the original strain was analyzed by polyacrylamide gel electrophoresis of lipopolysaccharide produced by each strain. After the cells were obtained, the cells were lysed with a cell lysis buffer and treated with proteinase K, a protease, to remove proteins, followed by electrophoresis on a polyacrylamide gel. As a result, it can be seen that the attenuated strain produces only lipopolysaccharides having a molecular weight of about 6,000 without the lipopolysaccharide ladder having a molecular weight of 10,000 or more appearing in the original strain (see FIG. 2: lane 1: size). Marker; lane 2: 5 μg of DLPS; lane 3: 10 μg of DLPS; lane 4: 20 μg of lane 4: lane 5: size marker; lane 6: LPS of 5 μg; lane 7: 10 μg of lane; lane 8: 20 μg of DLPS) .

(2) In order to investigate the change according to the growth temperature, the strain was inoculated with 100 ml of Luria broth adjusted to pH 7.0, and the growth curves at 25 ° C., 30 ° C., 37 ° C. and 42 ° C. were obtained at 600 nm. The absorbance was compared by measuring. In addition, in order to investigate the changes according to the use of different carbon sources, the strains were prepared using M9 medium (Na ₂ HPO ₄ · 7H ₂ O 12.8 g, KH ₂ PO ₄ 3 g, NaCl 0.5 g, respectively) containing different carbon sources. NH ₄ Cl 1.0 g, pH 7.0) was used to compare the use of carbon sources. As a result, it could be confirmed that the cause of attenuation was the lack of high molecular weight lipopolysaccharide.

Example 3: E. coli Isolation of Extracellular Membrane Proteins from CFCEC-97415

The seed inoculation amount was 3% and incubated for about 6 hours in tryptic soy broth at 37 ° C., 400 rpm, 1 v / v / m. Logarithmic cells were centrifuged at 4,000 g for 10 minutes to obtain cells and then suspended at 1 g / ml in 1 M sodium acetate buffer. It stirred at room temperature for 15-20 second, and stirred and added the distilled water of 1.5 times of suspension amount to this. An equal amount of 1 M CaCl ₂ / MPZ 6% was then added and stirred at room temperature for 1 hour to release extracellular membrane proteins extracellularly. In order to leach the released extracellular membrane protein with ethanol, first, 20% (v / v) of ethanol was added and stirred for 30 minutes, followed by centrifugation at 10,000 g at 4 ° C. for 15 minutes to remove the protein leaching at 20% or less. Removed primarily. Ethanol was again added to the supernatant obtained so that the ethanol concentration was finally 45% and centrifuged under the same conditions as above to obtain a leachate. The obtained leachate was dissolved by adding 1 ml of Empigen solution at 1.5 ml per g of cells. To dissolve completely, the leach was suspended 5-10 times at 10-30 rpm using a motor-drivenhomogenizer. After sonication in a sonication bath at room temperature for 10 minutes, the mixture was centrifuged at 25,000 g for 20 minutes at 4 ° C. After centrifugation, the supernatant was collected, and the leachate was secondarily extracted in the same manner as above, and the third extraction was also performed if necessary.

Example 4: E. coli Purification of Extracellular Membrane Proteins from CFCEC-97415

The sample obtained in Example 3 contained other cell-derived impurities such as nucleic acids and lipopolysaccharides, and only the proteins required by ammonium sulfate to remove them were fractionated. First, 500 g of ammonium sulfate was added per 1 L of the solution of Example 3, and completely dissolved, followed by centrifugation at 4 ° C and 20,000 g for 20 minutes to discard the supernatant and recover only the leachate. 1% TEEN buffer solution (0.05M Tris, 0.01M disodium EDTA, 1% Empigen-BB, 0.15 M NaCl, pH 8.0) was added to the obtained leachate and completely suspended and sonicated for 5 minutes in an ultrasonic bath at room temperature. Treated to dissolve completely. 600 g of ammonium sulfate was added per liter of solution and centrifuged under the same conditions as above to recover the releach. If necessary, tertiary extraction was also performed on the supernatant obtained by the first and second centrifugation.

The leachate thus obtained was dissolved in 1% TEEN buffer solution, sonicated for 5 minutes in an ultrasonic bath at room temperature, and centrifuged at 28,000 g for 20 minutes to collect the supernatant. The same procedure as above was repeated three times to recover the supernatant.

The A ₂₈₀ / A ₂₆₀ value of this supernatant was measured by an absorbance system and used as an extracellular membrane protein only when the value was 1.13 or less, and when it was higher, the nucleic acid was removed using an anion exchange resin. Empigen, a surfactant component contained therein, was removed by dialysis.

Example 5: P. aeruginosa Purification of Extracellular Membrane Proteins from

As shown in Example 3, the inoculation amount was 3%, and incubated in Tryptic Soy Broth for about 6 hours at 37 ° C., 400 rpm, and 1 v / v / m, cells of log phase were obtained at 4,000 g. Washing once with 10 mM phosphate buffer solution, acetone in an amount corresponding to three times the weight of the cells was slowly added, suspended and left at 4 ° C. for 12 hours. The above procedure was repeated twice and the supernatant was completely removed. Then, suspended in 10 mM PBS buffer and centrifuged at 13,000 g to obtain a protein, the protein precipitated between 35-80% of ammonium sulfate was salted for 2 hours and then centrifuged at 13,000 g for 20 minutes. Since the molecular weight of the extracellular membrane protein is 20,000 to 100,000, the protein of 100,000 or more is removed by using a molecular weight 100,000-cut filter and the protein of molecular weight smaller than 10,000 is removed by using a 10,000-cut filter. Only extracellular membrane proteins were obtained.

Example 6: Production of Modified Lipopolysaccharide Lacking O-Lipopolysaccharide

Modified lipopolysaccharide lacking O-lipopolysaccharide, which is a component of the proteosome and used as an antigen component capable of recognizing several Gram-negative bacteria, was obtained by culturing E. coli CFCEC-97415 strain. The seed was inoculated at 3% and cultured in tryptic soy broth at 37 ° C., 20 rpm for 1 hour at 1 v / v / m for about 16 hours, and then cells were obtained at 4,000 g. Wash once with 10 mM phosphate buffer and acetone in the same amount as the cell weight was added slowly and suspended. The acetone was then removed by centrifugation and dried in a vaccum dessicator. To 200 g of dried cells, 600 ml of middle water was added and suspended and centrifuged at 7,000 g. Then, the suspension and centrifugation were repeated sequentially with ethanol and acetone, and then suspended in 200 ml of ethyl ether. Ethyl ether was removed and completely dried in the hood, and the lipopolysaccharide was purified as follows.

7.5 ml of extract mixture (90% phenol: chloroform: petroleum ether = 2: 5: 8) was added per 1 g of dry cells, and then suspended in a hood for 30 minutes and centrifuged at 4 ° C. and 4,000 rpm using a glass centrifuge tube. The supernatant was collected. The temperature of the rotary evaporator was adjusted to 40 ° C. and a round bottom flask was used to continue condensation until all components were volatilized at 100 rpm and disconnected when no longer condensed. The residual solution was transferred to a glass vessel and dissolved with minimal distilled water if the phenol condensed. When the solution was milky, centrifuged to remove phenol remaining in the hood, and 40 ml of ethyl ether was added again, followed by centrifugation at 4 ° C. and 4,000 rpm for 15 minutes to remove the supernatant ethyl ether. After drying completely in a vacuum dryer, the weight of dried lipopolysaccharide was measured. Distilled water was added to bring the final concentration of lipopolysaccharide to 4.0 mg / ml.

Example 7: Attenuation of Lipopolysaccharide

To the final concentration of 4.0 mg / ml lipopolysaccharide solution obtained in Example 6 was added 0.2 N NaOH in the same volume and shaken every 65 minutes for the first 60 minutes at 65 ℃, then left for 90 minutes and then 60 minutes at 0 ℃ Cooled. 1 N acetic acid was added to neutralize to pH 7.0. The attenuated lipopolysaccharide was leached with ethanol. The ethanol leaching process was repeated three times, and the finally obtained leachate was dissolved in distilled water corresponding to 20% of the original volume and used as an antigen component (see FIG. 2).

Example 8: Formation of Proteosomes

RefinedE. coliCFCEC-97415 orP. aeruginosaExtracellular membrane proteinsE. coliLipopolysaccharide purified from CFCEC-97415 was mixed to form a proteosome structure. At this time, the mixing ratio of each component was 1: 1 to 1: 1.5. Impurities such as organic solvents and surfactants contained in each component were removed by diafiltration with physiological saline by passing only a molecular weight of 3,000 or less using a Pellicon cassette system. As a result of observing the proteosome formed through dialysis with an electron microscope, it was confirmed that the lipopolysaccharide and the extracellular membrane protein had the same structure as the extracellular membrane (see FIG. 1).

Experimental Example 1: Antibody Value and Protective Capacity Test against Gram-negative Bacteria

The vaccine obtained in Example 8 was administered 3 μg intraperitoneally based on lipopolysaccharide to 6-week old ICR mice or Balb / C mice weighing 20-25 g and immunized three times weekly. Twenty five experimental animals were used for each group, and blood was collected from five animals at the time of pre-immunization and one week after immunization. The plasma obtained here was subjected to an enzyme-linked immunosorbent assay (ELISA). In this experiment, various Gram-negative bacteria themselves, lipopolysaccharides extracted from them, and extracellular membrane proteins were used, and antibody titers thereof were measured.

J5LPS: LPS isolated from E. coli J5

PA-LPS: LPS Isolated from Pseudomona aeruginosa

K-LPS: LPS Isolated from Klebsiella

En-LPS: LPS isolated from Enterobacter

S-LPS: LPS isolated from Serratia

As shown in Table 1, the antigen is derived from E. coli, but it can be seen that the antibody against the antigen-antibody reaction against other Gram-negative bacteria. Moreover, it turned out that it has high antibody titer not only about the bacterium itself but the lipopolysaccharide purified from the bacterium. When the lipopolysaccharide isolated from the parent strain E. coli J5 was used as the antigen, the highest antibody titer was expected, and the antibody titer was about 80-160 fold. From this, it was confirmed that a sufficient amount of antibody was produced to defend against Gram-negative bacteria. In addition, it was confirmed that the lipopolysaccharide of the E. coli J5 strain exhibited the desired result as a common antigen, as it had an antibody titer of about 100 times higher than the lipopolysaccharide obtained from the parent strain as well as the lipopolysaccharide of other Gram-negative bacteria. In addition, it was found that ECOMP and PAOMP play a sufficient role as protein complement, and that antigenicity was enhanced by generating about five times more antibodies than conventional GBOMP.

The effectiveness of the vaccine was examined by passive immunization, another evaluation method. As described above, a vaccine made in the form of lipo-proteosome, i.e., DLPS-ECOMP and DLPS-PVOMP, was intraperitoneally administered to 3 weeks of lipopolysaccharide on 3 weeks of 6 weeks old ICR mice with a body weight of 20-25 g Immunized twice. Whole blood was collected from mice 2 weeks after the final immunization. Passive immunization was performed to investigate the protective ability of pre-immune sera obtained before immunization and post-immune sera obtained after three immunizations. First, 50 mice per group were intraperitoneally administered with 0.2 ml of each serum, and after 2 hours, the Pseudomonas aeruginosa GN11189 strain, which had been attacked, was serially diluted into 5 groups of 1.0 × 10 ⁶ to 5 times in total, and 10 of them each into 1 group. By intraperitoneal administration by concentration. 1 week later, life and death were observed to determine the efficacy index (EI). Effective index was defined as the LD ₅₀ obtained from the test group in each test divided by the LD ₅₀ of the control group. In the control group, sterile saline was used instead of serum. The results are shown in Table 2.

As shown in Table 2, the EI value of the pre-immune serum was 1.3 to 3 and the EI value of the post-immune serum was 7 to 14, depending on the species. In general, when the vaccine has an EI value of 3 or more, it is determined that the immune effect is excellent. The vaccine of the present invention was determined to have excellent protection against Gram-negative bacteria. In addition, the EI value of the post-immune serum to the pre-immune serum was 4 to 6, which also showed a significant result.

Vaccine according to the present invention is not only excellent safety in human application but also greatly enhanced antigenic ability to protect against a variety of Gram-negative bacteria, very useful for the prevention of diseases caused by Gram-negative bacteria, especially sepsis.

Claims (9)

(Iii) an attenuated O-lipopolysaccharide-deficient modified lipopolysaccharide isolated from E. coli CFCEC 97415 as an antigen component; And (ii) an outer membrane protein purified from E. coli or P. aeruginosa as a complement component for enhancing antigenicity, and having a lipo-proteosome structure. , Preventive vaccine of diseases caused by gram-negative bacteria. delete The vaccine of claim 1, wherein the attenuated O-liposaccharide-deficient modified lipopolysaccharide is attenuated by alkali treatment. delete The vaccine of claim 1, wherein the weight ratio of component (i) to component (ii) is 1: 1 to 1: 1.5. The vaccine of claim 1, further comprising a pharmaceutically acceptable excipient. The vaccine according to any one of claims 1, 3, 5 or 6, wherein the disease caused by Gram-negative bacteria is sepsis. (Iii) separating and purifying lipopolysaccharide from E. coli CFCEC 97415 strain; (Ii) attenuating the lipopolysaccharide obtained in (iii); (Iii) separating and purifying extracellular membrane proteins from E. coli or P. aeruginosa ; (Iii) mixing the lipopolysaccharide obtained in (ii) and the extracellular membrane protein obtained in (iii): Method for producing a vaccine according to claim 1 comprising a. E. coli CFCEC 97415 strain (Accession No. KFCC-11276), which is attenuated and produces a modified lipopolysaccharide lacking O-lipopolysaccharide.