KR0180991B1 - Pseudomonas aeruginosa vaccine containing composite peptide and therapeutics made from it - Google Patents

Abstract

The present invention relates to vaccines containing synthetic peptides and therapeutic agents produced therefrom for preventing and treating infections of Pseudomonas aeruginosa. The vaccines of the present invention contain specific peptides as both main immune-epitopes and as neutralizing epitopes. The vaccine according to the present invention exhibits non-toxic and safe antibody production and also cross-reactivity against various Pseudomonas aeruginosa.

Description

Pseudomonas aeruginosa vaccine containing synthetic peptide and therapeutic agent produced therefrom

1 shows the synthesized overlapping peptides corresponding to amino acid residues of the C-terminal region of Pseudomonas aeruginosa OprF.

FIG. 2 is a graph showing the results of solid-state enzyme immunoassay (ELISA) of humans recovered after Pseudomonas aeruginosa infection using synthetic peptides as immunosorbent antigens. FIG.

3 shows titer profile of antigen-antibody by solid phase ELISA assay.

4 is a comparison of the amino acid sequence corresponding to the epitope of the present invention with the corresponding amino acid sequence in OprF derived from other Pseudomonas species.

The present invention relates to a synthetic peptide containing vaccine for preventing the infection of Pseudomonas aeruginosa and the therapeutic agent produced therefrom. More specifically, the present invention relates to synthetic peptides having an immunodominant epitope region that can be used as an antigen in the preparation of Pseudomonas aeruginosa vaccines and therapeutic agents, and vaccines and therapeutic agents made from the peptides.

Many attempts have been made to develop vaccines using common antigens for the prevention or treatment of P. aeruginosa pneumoniae. As an example, a method of isolating a specific part having an immunologically protective effect among the components constituting Pseudomonas aeruginosa and using it as an antigen. Common antigens of interest include toxin A toxoids, high molecular weight polysaccharides, detoxified LPS-protein conjugates, polysaccharide-toxin A conjugates, flagella, pili and outer membrane proteins. (Infect. Immun. 52, pp 161-165, 1986; J. Clin. Invest. 69, pp 303-308, 1982; and J. Biol. Chem. 256, pp 7305-7310, 1981).

Among the extracellular protein components, protein F, a kind of porin, is reported to be the most preferable substance. (J. Biol. Chem. 266, pp 770-779, 1991). Porin protein F (hereinafter referred to as OprF) is generally present in all strains of Pseudomonas aeruginosa and has immunologically cross-reactivity (J. Clin. Microbiol. 26, pp 1161-1165. 1988 And J. Imfect. Dis. 146, pp. 770-779, 1982). In this regard, OprF is a likely candidate for the development of vaccines and therapeutics against Pseudomonas aeruginosa infections.

However, one major drawback of using OprF is that it is a major common antigen in the prevention of Pseudomonas aeruginosa infection, but when used alone it does not effectively elicit a cross-immune response against all Pseudomonas aeruginosa with different serotypes. Therefore, the protective efficacy is relatively low compared to the case of using the whole outer membrane protein.

In other words, whether OprF is quantitatively insufficient to generate a sufficient immune response to prevent Pseudomonas aeruginosa infection, or if the neutralizing epitope is not exposed for survival of the bacterium itself, or if it requires some additional ingredients. There may be a cause (Infect. Immun. 33, 527-532, 1981).

Duchene et al. Cloned the gene for OprF and identified the nucleotide and amino acid sequences of the gene (J. Bacteriol. 170, pp. 155-162, 1988). Recently, studies have been conducted to find antigenic peptides in OprF that react with antisera obtained by immunizing OprF protein to animals based on the amino acid sequence of OprF, which Duken revealed.

De Mot et al. Performed the sequencing of OprF from P. fluorescens to present a structural model for OprF (Microbiology 140, pp 1377-1387, 1994).

According to the model, it was assumed that the estimated epitope would be present at positions 188 to 270 from the N-terminus, and most of the N-terminal region (1-187) was buried in the outer membrane. In addition, their report is consistent with the fact that the C-terminal region of Pseudomonas aeruginosa OprF was exposed to the membrane surface (Infect. Immun. 60, 3497-3503, 1992). However, Dimot et al. Do not present antigenic regions in both Pseudomonas aeruginosa and P. fluorescens.

Hughes et al. Identified the antigenic regions predicted by performing computer analysis using the amino acid sequence of Pseudomonas aeruginosa outer membrane protein F (OprF) published by Duken. That is, they analyzed 10 amino acid sequences of OprF and selected 10 peptides having the best antigenic index, surface exposure probability, and hydrophilicity (Infect. Immun. 60, pp 3497-3503, 1992). Mouse antibodies that specifically reacted to these peptides were obtained and reacted with whole cells of all immunotype strains of Pseudomonas aeruginosa. As a result, three synthetic peptides were reported to be able to produce antibodies capable of binding to whole cells of immunogenic strains of all Pseudomonas aeruginosa. These three regions are located at positions 219-232 (7th peptide), 261-274 (9th peptide) and 305-318 (10th peptide) from the N-terminus of 326 amino acid sequences identified by Duken. It is shown that this site is exposed outside the cell. The peptides presented by them are as follows.

Peptide 7: KFDFDKSKVKENSY (SEQ ID NO: 8)

Peptide 9: TDAYNQKLSERRAN (SEQ ID NO: 9)

Peptide 10: NATAEGRAINRRVE (SEQ ID NO: 10)

According to Hughes et al., The seventh synthetic peptide induced the Pseudomonas aeruginosa antibody at the lowest titer of the three peptides, and responded similarly to protein F-deficient KG 1077 cells, not a major epitope. Since the antigenic peptides of SEQ ID NO: 9 and 10 caused the Pseudomonas aeruginosa antibodies at the highest titers of all peptides tested, they were presented as antigens that are likely to be used as synthetic vaccines. They estimated that this high titer was due to the best exposure of the region of peptides to the cell. However, contrary to computer predictions, these peptides actually did not show a clear effect compared to the control in animal tests (Vaccine, 13, pp 1750-1753, 1995). In addition, when the inventors reacted these peptides with antibodies of humans recovered after infection with Pseudomonas aeruginosa, their reactivity with the antibodies was extremely low, and therefore, these sequences were not suitable for use as efficient immunogens.

It is an object of the present invention to provide immunologically significant specific antigenic peptides present in OprF, a common antigen of Pseudomonas aeruginosa.

A second object of the invention is to provide a vaccine prepared using the peptide according to the invention as an antigen.

It is a third object of the present invention to provide an antibody derived from a peptide according to the present invention and a therapeutic agent containing the same.

The above object of the present invention will become apparent from the following detailed description.

The above object of the present invention is achieved by providing a series of synthetic peptides essentially containing the amino acid sequence of LDVKFD (SEQ ID NO: 1).

As used herein, the term patient, or a patient infected with Pseudomonas aeruginosa, refers to a person who has recovered from infection with Pseudomonas aeruginosa and has an antibody against Pseudomonas aeruginosa. Throughout this specification, unless otherwise defined, these terms should be understood to mean those who have recovered after infection with Pseudomonas aeruginosa and have antibodies to Pseudomonas aeruginosa.

While identifying principal immune epitopes in OprF, the inventors found that peptides of a particular sequence, including new principal immune epitopes, which have not been known so far, react with specificity with a high enough titer with antibodies in the sera of patients infected with Pseudomonas aeruginosa. It became. Accordingly, the present invention has been completed by confirming that the peptide can be used as a novel main immune epitope for the production of excellent vaccines and therapeutic agents for preventing infection of Pseudomonas aeruginosa.

According to a first aspect of the present invention, there is provided a vaccine for the prevention of Pseudomonas aeruginosa infection, which comprises an antigenic peptide essentially containing a specific amino acid sequence as a main immune epitope.

According to a second aspect of the present invention, there is provided an antibody and a therapeutic composition for treating Pseudomonas aeruginosa infections, which are made of natural γ-globulin and obtained by using a peptide comprising a specific amino acid sequence as an antigen.

According to still another feature of the present invention, there is provided a use of a peptide having a specific amino acid sequence for the production of a Pseudomonas aeruginosa vaccine and therapeutic agent.

We isolated and purified OprF to select the C-terminal region of OprF as a target for peptide scanning in order to find the main immune epitope and neutralization reaction epitope against Pseudomonas aeruginosa.

The reason is that the N-terminal region is located in the outer membrane or in the periphery of the plasma, so the possibility of the presence of an antigenic determinant is very unlikely, whereas the C-terminal region is exposed to the outside from the outer membrane of Pseudomonas aeruginosa. This is because there is a high possibility that very effective epitopes exist.

Based on the amino acid sequence of OprF identified by Duken, we synthesized 15 peptides overlaid with 9 peptides on the C-terminus of OprF of Pseudomonas aeruginosa (see FIG. 1). Reactivity was assessed to identify major immune and neutralizing epitopes that could be candidates for prophylactic vaccines and therapeutics.

More specifically, using the reported 326 amino acid sequencing data for OprF of Pseudomonas aeruginosa, 15 overlapped peptides for the C-terminus as shown in the attached FIG. 1 were obtained by known methods [Sheppard, RC (1986). ) Synthesis using a commercially available synthesizer according to Science The LKB Instrument Journal 33, 9]. In the present invention, multisyntec (Germany, manufactured by Bochum) can be used.

Subsequently, in order to identify a major immune epitope from the synthetic peptide, the antisera and reactivity of the recovered human after infection with Pseudomonas aeruginosa are examined by ELISA. ELISA methods are well known in the art and are described in J. Clin. Microbiol. 15, pp 1054-1058, 1982. In the present invention it is investigated using a solid-phase ELISA method.

The absorbance at 490 nm was measured to determine the binding activity of each peptide and antibody by ELISA. 3 (SEQ ID NO: 2) and No. 4 (SEQ ID NO: 4) showed the highest absorbance, especially peptide No. The absorbance of 3 was higher.

These results are completely different from the results reported by Hughes et al. Hughes et al synthesized peptides presumed to be epitopes by computer analysis and immunized these synthetic peptides to mice to test whether they induce antibodies in all serotypes of Pseudomonas aeruginosa. As a result, it has been reported that the antigenic peptides of SEQ ID NOs. 9 and 10 caused the Pseudomonas aeruginosa antibodies with the highest titers among all peptides tested. However, when the inventors reacted the peptides reported by Hughes et al. With antibodies of humans recovered after being infected with Pseudomonas aeruginosa, their reactivity with the antibodies was extremely low. That is, these peptides 9 and 10 are peptide No. 1 of FIG. 9 and No. It was included in 14 and did not show immunoreactivity. These different results were estimated by epidemiology by computer analysis in Hughes et al. And tested for antibodies in mice, whereas in the present invention, each synthetic peptide was directly assayed against antibodies of humans recovered after Pseudomonas aeruginosa infection. It seems to come from. Moreover, Hughes et al. Only used peptide analysis of the present invention by computer analysis. It is very likely that you have overlooked the region containing the sequence of 3. Another reason is that in the case of other researchers, peptide no. According to the results of previous studies that did not yield significant values of antiserum assays obtained from animals for the region comprising the sequence of 3, or that the region containing peptide 3 was probably present in the periplasm region No. It seems to overlook 3.

Based on these results, peptide No. The 18 amino acids for 3 were analyzed using data from each data bank, showing the highest surface exposure potential and hydrophilicity of the sequence of LDVKFD (SEQ ID NO: 1).

Peptide No. 3 (SEQ ID NO: 2) and peptide no. A part of the sequence of SEQ ID NO: 4 (SEQ ID NO: 4) was substituted with another amino acid to examine the reactivity with serum. Although the sequence of KSKVKE (SEQ ID NO: 7) of 4 was most likely an epitope, peptide No. 4, 4-1 and 4-3 (described in Table 2 for these sequences) had low reactivity with serum.

These results indicate that peptide No. 3 (SEQ ID NO: 2) shows that it is useful as a principal immune epitope against Pseudomonas aeruginosa.

Peptides according to the invention useful as major immune epitopes contain essentially the amino acid sequence of LDVKFD (SEQ ID NO: 1).

These peptides have the following amino acid sequences.

GAPAVAEVVRVQLDVKFD --- (SEQ ID NO: 2)

GAPAAAAVVRVQLDVKFD --- (SEQ ID NO: 3)

RVQLDVKFDFDKSKVKEN --- (SEQ ID NO: 4)

VRVQLDVKFDFD --------- (SEQ ID NO: 5)

AVAEVVRVQLDVKFDFDKSKVKE --- (SEQ ID NO: 6)

Synthetic peptides according to the present invention can be used as is to prepare a vaccine for preventing Pseudomonas aeruginosa infection. Alternatively, the synthetic peptides of the present invention dissolve 125 mg per ml in a known KLH solution (keyhole limpet hemocyanine, available from Calbiotec, San Diego, California; USA; 50% glycerol solution to increase the antigenicity of the vaccine). ) May be coupled. Vaccines of the present invention are pharmaceutically acceptable excipients, for example aluminum hydroxide (Al (OH) ₃ ), aluminum phosphate (AlPO ₄ ), immunostimulating complex (ISCOM), Syntex Adjuvant Formulation-1 (SAF-1), or SAFm (Modified Syntex Adjuvant Formulation) and the like.

When used as a vaccine against Pseudomonas aeruginosa infection, the amount used depends on the age, weight, sex, and general state of health of the subject to which Pseudomonas aeruginosa infection is concerned. The route of administration is preferably intramuscular administration.

Immunoglobulins, which are used as therapeutic agents to treat Pseudomonas aeruginosa, are obtained by administering an antigen containing peptides to an experimental animal in an amount capable of inducing immune antibodies. The therapeutic agent uses a pure antibody obtained by purifying antisera obtained by collecting and separating blood from the heart. Alternatively, it may be used in the form of a composition containing a purified gamma globulin component and optional components such as physiological saline, aqueous glucose solution or phosphate buffer.

The immunoglobulins according to the present invention may be formulated in liquid or lyophilized form, if desired, and may optionally contain pharmaceutically acceptable carriers such as stabilizers, preservatives, isotonic agents and the like. Pharmaceutically acceptable carriers include, for example, mannitol, lactose, saccharose, and human albumin in the case of lyophilized preparations, and physiological saline, water for injection, phosphate buffer, aluminum hydroxide, and the like, for liquid preparations. have. However, it is not limited only to these.

The dosage of immunoglobulins will vary depending on the age, weight, sex, general state of health, extent of Pseudomonas aeruginosa infection, and the components of the immunoglobulin administered. In the case of intravenous administration, the immunoglobulin preparations of the present invention are generally administered at 0.1 to 1000 mg, preferably 1 to 100 mg per kg body weight per day for adults.

Hereinafter, the present invention will be described in more detail by way of example. However, the present invention is not limited to these examples.

Example 1

Preparation of Serum from Persons Recovered After Pseudomonas Aeruginosa Infection

About 20 different kinds of blood collected from patients confirmed to be infected with Pseudomonas aeruginosa were aseptically divided into about 1 to 5 ml each and left at room temperature for 1 hour, and then stored in a refrigerator at 4 ° C. overnight. Subsequently, the solids were removed by centrifugation at 1500 g at 4 ° C. for 10 minutes to obtain serum of the supernatant. Serum obtained above was added to a tryptic soy broth culture plate to which 1.0-1.5% agar was prepared in advance. 1.15 g of Na ₂ HPO ₄ per liter. 0.2 g of KCl. 0.2 g of KH ₂ PO ₄ After diluting each of them by diluting at a ratio of 1:10 using NaCl 8.766g), they were incubated for at least 12 hours in an incubator in aerobic conditions maintained at 37 ° C. The culture thus obtained was transferred to a new culture plate and microorganisms were isolated using known methods of pure microbial separation (Biology of Microorganisms, 5th Edition, Thomas D. Brock, Michael T. Madigan, pp 28-34, 1988). Isolated in pure form. The serological-immunological classification of isolated Pseudomonas aeruginosa is known (see Bergey´s Manual) and 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 650 nm was maintained at about 2.0 or more. 1 ml of this culture solution was aseptically aliquoted, 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 pancreas) were respectively added to the prepared 96-well microplates, and then 15 μl of the microbial suspension obtained above was added to each serum and mixed, and agglutination was performed between 10 and 30 minutes. It was observed whether this happened. At this time, the strain of the well in which the positive aggregation reaction occurs can be easily distinguished since it has a serotype consistent with the serum added thereto. Here, the patients recovering from Pseudomonas aeruginosa infection were selected from the patients with positive reactions, blood was collected, and the antisera was separated and stored at −20 ° C. until used for biochemical or immunological analysis according to the aforementioned method.

Example 2

Passed Synthesis and Bonding

Duchene et al, J. Bacteriol. 170, 155-162, 1988] 15 overlapping peptides were synthesized for the C-terminal region of Pseudomonas aeruginosa using the 326 amino acid sequencing data of OprF. (multisyntec, Bochum, Germany) (Fig. 1). In FIG. 1, cysteine (C) of mature OprF was replaced with alanine (A) in the synthetic peptide. The numbers in parentheses indicate the position of Pseudomonas aeruginosa in maturation OprF. In order to identify the main immune epitope, the reaction with the sera of the patients infected with Pseudomonas aeruginosa prepared in Example 1 using 15 overlapping peptides as antigen was confirmed by performing a solid phase ELISA. ELISA analysis method is as follows.

First, synthetic peptides formulated at a concentration of 20 μg / ml with coating buffer (0.05 M carbonate buffer, pH 9.6) were diluted and added to 100 μl in each well of a 96-microplate. Each plate was allowed to react for 2 to 12 hours at room temperature or in a humidifying chamber at 4 ° C. for 12 to 16 hours to attach the peptides to the plates, followed by removal of the aqueous solution and blocking 1% soot to block the unbound portions of each plate. Iii) After adding 200 μl of serum albumin (BSA), it was allowed to react for 2 hours to block the non-binding portion of the protein. After washing three times with PBS (PBST, pH 7.2) containing 0.05% Tween 20, 100 μl of serum diluted 1:50 with PBS was added to each well of each micropeptide coated with synthetic peptides. And reacted for 2 hours. At this time, the negative control group used serum of healthy humans with basal low titer in Pseudomonas aeruginosa. After completion of the reaction, the resultant was washed 4 to 5 times with the same phosphate buffer wash. 100 μl of goat-anti human IgG-peroxidase conjugate as a secondary antibody was added thereto and reacted at room temperature for 1 hour, followed by 5 or more times with the same phosphate buffer wash. Washed vigorously. To the citrate-phosphate buffer (0.1 M, pH 5.0), ortho-phenylenediamine dihydrochloride was added thereto at a concentration of 0.3 to 0.4 mg / ml, and 50 µl was added thereto, followed by reaction under light shielding for 20 minutes. 50 µl of 1N sulfuric acid was added to stop the reaction. Then, each reaction solution was determined by measuring the absorbance at 490 nm using a spectrophotometer for binding activity of the peptide and the antibody. The results are shown in FIG.

As shown in Figure 2, peptide No. 1 out of 15 synthetic peptides analyzed. 3 (SEQ ID NO: 2) and peptide no. Only two peptides of SEQ ID NO: 4 (SEQ ID NO: 4) showed a reaction with specificity. 4 is peptide No. It showed almost 50% reactivity compared to 3.

According to the same solid-phase ELISA method, known extracellular membrane proteins (OMP, CFC-101), peptide No. 3 and peptide no. 4 was used as an antigen to determine the binding capacity of the patients infected with Pseudomonas aeruginosa. At this time, the concentrations of the antigens used were all 20 µg / ml, and the human antibody was diluted 1:30. The results are shown in FIG. 3 and Table 1. In Figure 3, curve a is OMP and b is peptide no. 3, c is peptide No. 4 Each point in the curve represents the average of three statistical points. Table 1 below shows the immunological response of various antigens as immunosorbents and serum from Pseudomonas aeruginosa infection patients. In Table 1, a is ELISA unit (A each assay sample-A negative control) / (A positive control-A negative control) x 160 (where 160 is the inverse function of serum dilution and A is absorbance at 490 nm Positive control group is the most active serum of Pseudomonas aeruginosa infected patients (in peptide No. 3), and the negative control group is the serum of a normal blood donor with the lowest concentration of natural antibody against Pseudomonas aeruginosa. Both positive and negative controls were used in ELUSA. Values are expressed as mean ± standard deviation of the statistics for 10 patients. b is the p value and c is the Outer Membrane Protein (OPM).

As shown in FIG. 3 and Table 1, all of the antigens used exhibited strong reactivity, among which peptide No. The absorbance value of 3 was similar to that of mixed OMP, and the same high reactivity was observed for nine different human serums used in the experiment (Table 1). The results of this experiment indicate that, unlike previously known peptides that are major immune epitopes and may be good immunogens, the peptides may be peptide. It is contained in the sequence of 3 (GAPAVAEVVRVQLDVKFD: SEQ ID NO: 2).

Example 3

Localization of Principal Immune-Epitope in the Extracellular Membrane of Pseudomonas aeruginosa Opr F by Amino Acid Sequencing

Analysis of amino acid sequences for OprF of Pseudomonas aeruginosa species was obtained from the new TBLASTX program, PDB / SwissProt / PIR / SPUpdate / GenPept / GPUpdate peptide sequence data libraries, via the NCBI BLAST e-mail service. Analysis of the selected peptides was performed on IBM computers with various programs included in the PC / GENE genetic analysis package (Intelligene-tics). Peptide No. 18 amino acid sequences corresponding to 3 were analyzed by the method of Hop and woods (P.N.A.S U.S.A. 781, 3824-3828, 1981) using PC / GENE software.

As a result of analysis, peptide No. The sequence of LDVKFD (SEQ ID NO: 1) of 3 showed the highest surface exposure possibility and hydrophilicity. This portion, predicted as a major immune epitope, is the peptide No. 3 and peptide No. Despite being common to 4, peptide No. 4 is peptide No. Compared to 3, the results showed relatively low reactivity with antibodies in the sera of patients infected with Pseudomonas aeruginosa, indicating that the region of LDVKFD (SEQ ID NO: 1), presumed to be a major immune epitope, was weakly exposed to the surface in intact cells or was easily exposed. It is assumed to have a native configuration that is inaccessible. It may also be assumed that lipo- or exo-polysaccharides in the extracellular membrane are due to the spatial hinderance of the binding of the antibody to the LDVKFD region in OprF. Therefore, the antibody in the serum of a patient infected with Pseudomonas aeruginosa was expressed by peptide No. In the tertiary structure of 3, the LDVKFD (SEQ ID NO: 1) region protrudes out, so that the epitope can be easily recognized. In the case of 4, this epitope will be present in the internal region of the tertiary structure, and therefore is not considered to be in easy contact with the antibody.

Example 4

Identification of main immune epitopes

In order to prove that the LDVKFD (SEQ ID NO: 1) is a major immune epitope, the modified 5 peptides for this portion were synthesized and reacted with antibodies isolated from the sera of Pseudomonas aeruginosa infected human patients, the method described in Example 2. Reactivity was investigated in the same manner as The sequence of the synthesized peptide is shown in Table 2.

First, when KFD presumed to be a major immune epitope is replaced with AAA (Nos. 3-1 and 4-1), no reactivity of the peptide with the antibody in human serum appears (No. 3-1), or It was found to decrease considerably (No. 4-1). However, peptide No. When the other part of No. 3 (No. 3-2: VAE → AAA; the second hydrophilic part of peptide No. 3) was substituted, or the length of the peptide was changed while LDVKFD was preserved (No. 4). -2, and 4-3) was not affected much. In addition, peptide No. 3-peptide No. As a result of analyzing the entire sequence corresponding to 4 by computer analysis, the most hydrophilic part was peptide no. 4 shows the sequence of KSKVKE (SEQ ID NO: 7), but the peptide No. Reactivity to human serum of 4, 4-1, 4-3 was determined by peptide No. It was lower than 3. Thus, peptide No. It can be seen that LDVKFD (SEQ ID NO: 1) of 3 is a more important principal immune epitope.

Example 5

Comparative Analysis of Amino Acid Sequences of Major Immune Epitopes

Peptide No. 3 and peptide No. The amino acid sequence corresponding to 4 was compared with the OprF sequences of various known Pseudomonas spp. Pseudomonas species and sequences to be compared are shown in FIG. In Figure 4, the number indicates the position of the mature OprF. Candidates of the neutralizing epitopes proposed by the inventors' experimental results are indicated by bold lines.

As can be seen from FIG. 4, the main immune epitope region (LDVKFD: SEQ ID NO: 1) in OprF in Pseudomonas aeruginosa was completely conserved in the corresponding portion in OprF of other Pseudomonas species. Synthetic peptide No. 3 and peptide no. The peptide sequence of OprF of Pseudomonas aeruginosa corresponding to 4 is underlined. In the case of Pseudomonas aeruginosa, the C-terminal region is exposed to the surface, and the exposed loop is found to be a very variable region. (See, J. Bacteriol. 174, 4977-4985, 1992). In particular, it has been reported that the cysteine domain is exposed to the bacterial surface (Mol. Microbiol. 10, 283-292, 1993). Thus, it is assumed that the LDVKFD region, the main immune epitope in OprF, is exposed out of the outer membrane.

Example 6

Preparation of the antigen

In order to increase the antigenicity of the synthetic peptide, a known method of peptide-KLH coupling was performed. The specific method is as follows. The synthetic peptide was dissolved in sterile distilled water and adjusted to a final concentration of 10 mg / ml. Use a magnetic bar with 80 μl KLH solution (dissolve 125 mg per ml in 50% glycerol solution), 520 μl distilled water, and 100 μl 1M sodium phosphate (pH 7.0) in 300 μl (3 mg) synthetic peptide. Mix slowly. 25% glutaraldehyde was properly diluted to prepare a final concentration of 21 mM, followed by mixing with 0.5 ml dropwise. This solution was reacted at room temperature for 14-16 hours to be used as a prophylactic vaccine or stored at −20 ° C. until used as an antigen for the production of antibodies.

Example 7

Infection protection effect test by active immunity

In order to examine the infection protection effect on the purified synthetic peptides, experiments were conducted using healthy male ICR mice (20-25 g) at 5 weeks of age. The antigen prepared according to the method of Example 6 was administered intraperitoneally three times at two dose intervals of 20 animals per group at a dose of 150 μg / kg each time, and Pseudomonas aeruginosa GN11189 (purchased from the Japanese Episome Institute) 5 days after the final administration. Was intraperitoneally infected and the survival rate was observed for 7 days. Synthetic peptides used as negative control are peptide No. 10 (SEQ ID NO: 14), and 20 animals were used under the same conditions. After immunization with synthetic peptides, the results for infection protection effect in mice are shown in Table 3.

As can be seen in Table 3 above, peptide No. In the test group using the vaccine containing 3, the survival rate was 100%, indicating a good protective effect of infection, whereas the negative control group had a 0% survival rate. According to the experimental results, peptide No. Existing reports presumably lacking antigenic and protective effects when using 3 [Infect. Immun. 60, pp 3497-3503, 1992], which shows very different results.

Example 8

Cross-immune Defense Effects against Pseudomonas Aeruginosa of Different Serotypes

In order to confirm the cross-infection protection effect on the purified synthetic peptides, experiments were conducted using healthy male ICR mice (20-25 g) at 5 weeks of age. Antigens prepared according to the method of Example 6 were administered intraperitoneally three times, two days apart, 20 per group, at a dose of 150 μg / kg each time, and after 5 days of final administration, each of the seven different serotypes Corresponding Pseudomonas aeruginosa was infected intraperitoneally, followed by survival for 7 days. Synthetic peptide used as negative control was peptide No. 10 (SEQ ID NO: 14), and experiments were conducted using 20 animals under the same conditions. Synthetic peptide No. After immunization using the three antigens, the results of the prophylactic efficacy test of mice infected with the various immunotype strains are shown in Table 4. The Pseudomonas aeruginosa strains F1 to F7 used in Table 4 are according to the Fisher-Devlin immune type (J. Bacteriol. 1969, 98, 835-837).

As can be seen in Table 4 above, peptide No. The survival rate of the test group with the vaccine containing 3 was 100% for the F1, F2, F3, and F6 types, showing a good protection against infection, whereas 90% for the F4, F5, and F7 types. It showed the above protective effect. However, for the negative control (peptide No. 10, SEQ ID NO: 14) showed a low survival of less than 15% in all groups. Therefore, according to this experimental result, peptide No. It can be seen that the antigen including 3 is a vaccine that can effectively protect against infection of Pseudomonas aeruginosa of various serotypes.

Example 9

Toxicity Testing of Peptide-Containing Vaccines

In order to use the vaccine containing the synthetic peptide prepared in Example 6 as a prophylactic against Pseudomonas aeruginosa infection, the safety of the vaccine product as well as the cross-immune defense effect against Pseudomonas aeruginosa of other serotypes must be ensured.

The vaccine of the present invention was tested for toxicity after intramuscular administration using ICR male and Sprague-Dawley (SD) mice weighing 20-25 g as experimental animals. The dose of the vaccine was set at 25 mg / kg for the highest dose group and acute phase (12.5 mg / kg) with the azeotropy of 0.5, taking into account drug solubility and expected clinical application. 10 ml / kg was observed and observed for a week. The results are shown in Table 5 below.

As can be seen from Table 5, when the vaccine was administered to mice and mice by muscle administration, mortality and specific symptoms were not observed in all administration groups (maximum dose: 25 mg / kg) and the control group. In addition, there was no significant difference in weight change between the control group and the vaccine-administered group.

Therefore, LD was 25 mg / kg or more under all conditions of this test, confirming that it is a very safe substance, about 1500 times the average dose expected for clinical use.

Example 10

Mouse Antibody Preparation Against Synthetic Peptides

Healthy synthetic ICR mice (20-25 g) of 5 weeks old were immunized to obtain antiserum with antigens KLH coupled to each synthetic peptide prepared in Example 6. Appropriately diluted with 10 mM complete phosphate solution (pH 7.2), 1 ml (0.5 mg / kg) per mouse was administered intraperitoneally twice a week, and again 1 ml (0.25 mg / kg) 1 week before blood collection. Intravenous injection. After administration three times at 7-day intervals, blood was collected one week later to determine whether sufficient antibodies were produced by ELISA, and then the antisera obtained from the blood was isolated from the heart, and then -20 until used for biochemical or immunological analysis. Store at ° C.

Example 11

Preparation of Rabbit Antibodies Against Synthetic Peptides

Rabbits were immunized to obtain antiserum with antigens with KLH coupled to each synthetic peptide prepared in Example 6. The rabbits used a 3-month-old male Japanese white rabbit weighing 2.5 kg. Appropriately diluted with 10 mM phosphate buffer solution (pH 7.2), subcutaneously administered three times per week at 1 ml (100 μg / kg) per rabbit and after 4 weeks, one week before blood collection to obtain a large amount of antibody Again 1 ml (50 μg / kg) was injected intravenously. After final administration, blood samples were collected to determine whether enough antibodies were produced by ELISA, and then antiserum obtained from blood samples was isolated from the heart and stored at −20 ° C. until used for biochemical or immunological analysis.

Example 12

Infection Protection Effect Test by Passive Immunity

Experiments were performed using 5-week old healthy male ICR mice (weight: 20-25 g) to determine the effect as a therapeutic agent by passive immunization with antiserum of mice and rabbits on the synthetic peptides obtained in Examples 10 and 11 It was. Antiserum for each synthetic peptide antigen was administered intraperitoneally at a dose of 0.3 ml of 20 mice per group, and after 2 hours, the invasive bacterium Pseudomonas aeruginosa GN11189 was infected intraperitoneally and observed for 7 days of survival. Synthetic peptide used as negative control was No. Antisera for 10 was used and 20 animals were tested under the same conditions. Results for the therapeutic efficacy by passive immunization of anti-rabbit and anti-mouse serum against synthetic peptide vaccines are shown in Table 6.

As can be seen in Table 6 above, peptide No. In the test group using the antiserum against 3, the survival rate was excellent as 90% (mouse antibody) and 80% (rabbit antibody), while the control group peptide No. In case of 10, all survival rates were lower than 10%. According to the experimental results, peptide No. In the case of using 3, the treatment effect against Pseudomonas aeruginosa was shown to have a very good survival rate through animal experiments. Thus, the peptide-containing sequence indicates that it can be used as an antigen for producing an effective antibody with high therapeutic rate.

Example 13

Formulation of Immunoglobulins

The Pseudomonas aeruginosa immunoglobulin prepared in Examples 10 and 11 was formulated using a suitable pharmaceutically acceptable carrier described below to administer 1-100 mg / kg body weight. Formulated immunoglobulins were administered to mice with Pseudomonas aeruginosa infection-induced mice, and immunoglobulins according to the types of carriers were administered to mice with Pseudomonas aeruginosa infection-induced differences in the efficacy of immunoglobulins according to the types of carriers. As a result, immunoglobulin preparations using physiological saline or phosphate buffer solution as carriers were found to be effective in the case of liquid preparations, and mannitol, saccharose and lactose were used as carriers in the case of lyophilized preparations. Indicated. On the other hand, in the case of lyophilized formulations, the effectiveness was rather low when human albumin was used in combination, and the effect was inferior when aluminum hydroxide was used as a liquid formulation. The efficacy of Pseudomonas aeruginosa immunoglobulins according to pharmaceutical carriers is shown in Table 7 below.

Example 14

Efficacy test of formulated immunoglobulin

The phosphate buffer formulation confirmed to be high in Example 13 was used to confirm the effectiveness of the formulated immunoglobulin as a prophylactic and therapeutic agent for secondary skin infections in laceration, burns, trauma and the like. Known burn test methods [J. Infect. Dis. 131, 688-691, 1975], and then sprayed into the burn site of the mice of the test group by formulating a spray with a liquid formulation containing 0.5 to 1 mg of Pseudomonas aeruginosa immunoglobulin per ml of phosphate buffer. The effect was determined by comparing with the state in the untreated group. The results of the image test are shown in Table 8 below.

As can be seen from Table 8, the mouse population sprayed with the immunoglobulin-phosphate buffer spray showed superior survival compared to the non-immunoglobulin treated group. Therefore, it can be seen that the Pseudomonas aeruginosa immunoglobulin-containing preparation according to the present invention exhibits an excellent prophylactic and therapeutic effect against infection of Pseudomonas aeruginosa.

Sequence table

(1) General Information:

(i) Applicant: CheilJedang Co., Ltd.

(ii) Address: 500 Namdaemun-ro 5-ga, Jung-gu, Seoul

(iii) Name of the Invention: Pseudomonas aeruginosa vaccine containing synthetic peptide and therapeutic agent produced therefrom

(iv) SEQ ID NO: 14

(v) Computer readable:

(A) Medium form: floppy disk

(B) Computer: IBM PC Compatibility

(C) operating system: PC-DOS / MS-DOS

(D) Software: PatentIn Release # 1.0, Version # 1.25

(2) Information about SEQ ID NO: 1:

(i) sequence properties:

(A) Length: 6 amino acids

(B) Form: Amino Acid

(D) topology: straight chain

(ii) molecular form: peptide

(xi) Sequence description: SEQ ID NO: 1

Leu Asp Val Lys Phe Asp

1 5

(2) Information about SEQ ID NO: 2:

(i) sequence properties:

(A) Length: 18 amino acid

(B) Form: Amino Acid

(D) topology: straight chain

(ii) molecular form: peptide

(xi) Sequence description: SEQ ID NO: 2

Gly Ala Pro Ala Val Ala Glu Val Val Arg Val Gln Leu Asp Val Lys

1 5 10 15

Phe asp

(2) Information about SEQ ID NO:

(i) sequence properties:

(A) Length: 18 amino acid

(B) Form: Amino Acid

(D) topology: straight chain

(ii) molecular form: peptide

(xi) sequence description: SEQ ID NO: 3

Gly Ala Pro Ala Ala Ala Ala Val Val Arg Val Gln Leu Asp Val Lys

1 5 10 15

Phe asp

(2) Information about SEQ ID NO: 4:

(i) sequence properties:

(A) Length: 18 amino acid

(B) Form: Amino Acid

(D) topology: straight chain

(ii) molecular form: peptide

(xi) sequence description: SEQ ID NO: 4

Arg Val Gln Leu Asp Val Lys Phe Asp Phe Asp Lys Ser Lys Val Lys

1 5 10 15

Glu asn

(2) Information about SEQ ID NO:

(i) sequence properties:

(A) Length: 12 amino acids

(B) Form: Amino Acid

(D) topology: straight chain

(ii) molecular form: peptide

(xi) sequence description: SEQ ID NO: 5

Val Arg Val Gln Leu Asp Val Lys Phe Asp Phe Asp

1 5 10

(2) Information about SEQ ID NO: 6:

(i) sequence properties:

(A) Length: 23 amino acids

(B) Form: Amino Acid

(D) topology: straight chain

(ii) molecular form: peptide

(xi) Sequence description: SEQ ID NO: 6

Ala Val Ala Glu Val Val Arg Val Gln Leu Asp Val Lys Phe Asp Phe

1 5 10 15

Asp Lys Ser Lys Val Lys Glu

20

(2) Information about SEQ ID NO: 7:

(i) sequence properties:

(A) Length: 6 amino acids

(B) Form: Amino Acid

(D) topology: straight chain

(ii) molecular form: peptide

(xi) sequence description: SEQ ID NO: 7

lys Ser Lys Val Lys Glu

1 5

(2) Information about SEQ ID NO: 8:

(i) sequence properties:

(A) Length: 14 amino acids

(B) Form: Amino Acid

(D) topology: straight chain

(ii) molecular form: peptide

(xi) sequence description: SEQ ID NO: 8

Lys Phe Asp Phe Asp Lys Ser Lys Val Lys Glu Asn Ser Tyr

1 5 10

(2) Information about SEQ ID NO: 9:

(i) sequence properties:

(A) Length: 14 amino acids

(B) Form: Amino Acid

(D) topology: straight chain

(ii) molecular form: peptide

(xi) sequence description: SEQ ID NO: 9

Thr Asp Ala Tyr Asn Gln Lys Leu Ser Glu Arg Arg Ala Asn

1 5 10

(2) Information about SEQ ID NO: 10:

(i) sequence properties:

(A) Length: 14 amino acids

(B) Form: Amino Acid

(D) topology: straight chain

(ii) Molecular Form: Teptide

(xi) sequence description: SEQ ID NO: 10

Asn Ala Thr Ala Glu Gly Arg Ala Ile Asn Arg Arg Val Glu

1 5 10

(2) Information about SEQ ID NO 11:

(i) sequence properties:

(A) Length: 27 amino acids

(B) Form: Amino Acid

(D) topology: straight chain

(ii) molecular form: peptide

(xi) sequence description: SEQ ID NO: 11

Gly Ala Pro Ala Val Ala Glu Val Val Arg Val Gln Leu Asp Val Lys

1 5 10 15

Phe Asp Phe Asp Lys Ser Lys Val Lys Glu Asn

20 25

(2) Information about SEQ ID NO: 12:

(i) sequence properties:

(A) Length: 18 amino acid

(B) Form: Amino Acid

(D) topology: straight chain

(ii) molecular form: peptide

(xi) sequence description: SEQ ID NO: 12

Gly Ala Pro Ala Val Ala Glu Val Val Arg Val Gln Leu Asp Val Ala

1 5 10 15

Ala Ala

(2) Information about SEQ ID NO: 13:

(i) sequence properties:

(A) Length: 18 amino acid

(B) Form: Amino Acid

(D) topology: straight chain

(ii) molecular form: peptide

(xi) sequence description: SEQ ID NO: 13

Arg Val Gln Leu Asp Val Ala Ala Ala Phe Asp Lys Ser Lys Val Lys

1 5 10 15

Glu asn

(2) Information about SEQ ID NO: 14:

(i) sequence properties:

(A) Length: 18 amino acid

(B) Form: Amino Acid

(D) topology: straight chain

(ii) molecular form: peptide

(xi) sequence description: SEQ ID NO: 14

Lys Leu Ser Glu Arg Arg Ala Asn Ala Val Arg Asp Val Leu Val Asn

1 5 10 15

Glu tyr

Claims (5)

A vaccine for the prevention of Pseudomonas aeruginosa infection comprising a peptide essentially containing the amino acid sequence of SEQ ID NO: 1 as a main immune epitope. LDVKFD --- (SEQ ID NO: 1) The vaccine of claim 1, wherein the peptide essentially containing the amino acid sequence of SEQ ID NO: 1 is selected from peptides having the amino acid sequence of SEQ ID NOs: 2 to 6 below. GAPAVAEVVRVQLDVKFD --- (SEQ ID NO: 2) GAPAAAAVVRVQLDVKFD --- (SEQ ID NO: 3) RVQLDVKFDFDKSKVKEN --- (SEQ ID NO: 4) VRVQLDVKFDFD --- (SEQ ID NO: 5) AVAEVVRVQLDVKFDFDKSKVKE --- (SEQ ID NO: 6) An antibody obtained by using a peptide comprising a natural gamma globulin and having the amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 6 according to claim 1 or 2 as a fragrance. A therapeutic agent for Pseudomonas aeruginosa infections containing the antibody of claim 3. Peptide for use in the production of a vaccine for preventing Pseudomonas aeruginosa infection and the antibody for treatment comprising the amino acid sequence of any one of SEQ ID NOs 1 to 6 of claim 1.