JPH0344054B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a highly active and highly safe pertussis vaccine and a method for producing an active ingredient of a pertussis vaccine. Since ancient times, pertussis infection caused by Bordetella pertussis in children has often caused serious effects, so vaccination in early childhood has been compulsory in Japan. Conventionally, vaccines prepared from killed B. pertussis bodies have been used to prevent pertussis infection, but there have been side effects thought to be caused by various components of B. pertussis (e.g., endotoxins, exotoxins, etc.). Because pertussis occurs with considerable frequency, improvements in pertussis vaccines are eagerly awaited. In recent years, pertussis HA vaccine has been developed with the aim of reducing the side effects of pertussis vaccine (Japanese Patent Application Laid-open No. 1983
-Refer to No. 141416). Pertussis HA vaccine is made by toxoidizing a protein with a molecular weight of approximately 110,000 to 130,000 with hemagglutinating activity among the many proteins produced by Bacillus pertussis with formalin. As a result of intensive research to develop a pertussis vaccine that is even more improved than the pertussis HA vaccine, the present inventors discovered that a protein substance produced by a microorganism belonging to the genus Bordetella that has insulin secretion-enhancing activity has been found to be effective against B. pertussis infection. The present invention was developed based on the discovery that it has activity. The vaccine of the present invention comprises a basic protein (hereinafter abbreviated as B-oligomer), which is one of the constituents of insulin-enhancing active factor (hereinafter abbreviated as IAP or N-IAP) produced by a microorganism belonging to the genus Bordetella. ) as the active ingredient. B-oligomer, which is the active ingredient of the pertussis vaccine of the present invention, is the same substance as the proteinaceous element CP-A, which is a basic protein, disclosed in JP-A-57-67591. The physical properties of B-oligomer are shown below. B-Oligomer physical properties: Molecular weight: SDS-gel electrophoresis 75000±5000 Basic protein amino acid analysis: Asp.6.4±0.7, Thr.7.3±0.8, Ser.8.2±0.9,
Glu.9.3±1.0, Pro.6.3±0.7, Gly.11.1±1.0,
Ala.9.0±0.9, Cys./21.2±0.3, Val.6.2±0.6,
Met.2.7±0.4, Ile.3.4±0.4, Leu.8.4±0.9,
Tyr.5.6±0.6, Phe.3.6±0.5, Lye.4.7±0.5,
His.1.1±0.3, Arg.4.9±0.5 Composition: Over 98% protein by Lowry method. The pertussis vaccine of the present invention comprises the purified B-
It is prepared by diluting the oligomer with a common diluent used in vaccine production, such as buffered saline. The vaccine of the present invention can confer immunity to children by administering 0.1 μg to 100 μg of B-oligomer once or several times. Vaccine administration methods include subcutaneous, intravenous, oral, intramuscular,
Although intradermal or rectal administration is possible, subcutaneous administration is most preferred. Examples of vaccine preparations for subcutaneous administration include the following formulations. Prescription: Mix the following ingredients with sterile distilled water to make a total volume of 1 ml. B-oligomer 20μg KH ₂ PO ₄ 12.2mg NaHPO ₄ 40.6mg NaCl 36.0mg Aluminum hydroxide 0.2mg Immunity can be conferred by subcutaneously administering 0.005ml to 0.5ml of the above-prescribed vaccine to children once or multiple times. obtain. The concentration of B-oligomer in the vaccine ranges from 1 to
200 μg/ml, preferably 10-50 μg/ml. In addition, the concentration of aluminum hydroxide or aluminum phosphate added as adjuvant is 0.01mg/
ml~10mg/ml, preferably 0.1mg/ml~1mg/ml
It is. Incidentally, according to measurements using mice, the LP ₅₀ of subcutaneous administration of B-oligomer is 100
mg/Kg or more, and the LD ₅₀ for intravenous injection is 50 mg/Kg.
That's all. The vaccine of the present invention was found to be superior to the pertussis HA vaccine in tests using mice. That is, in a comparison of side effects, the current pertussis HA vaccine clearly exhibits leukocyte-proliferating effect and histamine-sensitizing effect at high doses, whereas the vaccine of the present invention does not substantially exhibit these side effects. In addition, regarding body weight changes after vaccination, the current pertussis HA vaccine causes weight loss more than the control, suggesting the presence of endotoxin in the vaccine, whereas the present vaccine contains endotoxin. Virtually no toxin was detected. Furthermore, in infection prevention tests, the vaccine of the present invention was shown to be effective against pertussis at low doses.
It showed stronger protection against infection than the HA vaccine. Next, an efficient method for producing B-oligomer will be described in detail. In the production method of the present invention, a microorganism belonging to the genus Bordetella, preferably Bordetella pertussis phase or Bordetella, is cultured, and the culture supernatant after culturing is treated with hydroxyapatite to increase insulin secretion enhancing activity. A crude fraction containing the factor IAP or N-IAP was obtained, and urea was added to the obtained crude fraction so that the final urea concentration was 3 to 6 M.
The B-oligomer is purified and isolated by incubating for ~48 hours and treating the post-incubation solution with puffed globin sepharose. In addition, as a similar manufacturing method, IAP or N-IAP
Desalt the salts in the crude fraction containing it by ultrafiltration method,
Add urea to the solution after desalination to make the final urea concentration in the solution 4-6M, adjust the pH of the solution to 7.5-10.0, incubate for 1-4 days, and then DEAE
There is a method of purification and isolation through a Sepharose column and a haptoglobin Sepharose column. Note that the final urea concentration in the present invention is the concentration of urea relative to the total liquid volume including the urea liquid volume. B-oligomers are also prepared as disclosed in JP-A-57-67591. In other words, IAP or N-IAP is produced by using the culture supernatant of a microorganism belonging to the genus Bordetella as a starting material through numerous purification steps.
and purified IAP or N-IAP from 4 to
24 in 5M urea-containing phosphate buffer (PH5.8-6.5)
After incubation for ~48 hours, the solution after incubation was purified using a CM-Sepharose column chromatogram equilibrated with the above-mentioned phosphate buffer, and then gel-filtered to obtain the B-oligomer as a single substance by disk electrophoresis. is obtained. As mentioned above, preparing purified IAP or N-IAP requires a purification process consisting of several steps, and further manufacturing operations such as gel filtration via CM-Sepharose chromatogram to obtain B-oligomer. It is complicated and the yield of B-oligomer is poor. For example, 114 mg of IAP (or N-IAP) was obtained from a hydroxyapatite eluate (crude fraction of IAP) containing 400 mg of protein, which was processed to yield 46 mg of B-IAP.
- Oligomers are obtained. The B-oligomer thus obtained can also be used as an active ingredient having a protective activity against B. pertussis infection in the highly active and highly safe pertussis vaccine of the present invention. On the other hand, in the production method of the present invention, the solution after incubation is passed through a haptoglobin cepharose affinity column in which haptoglobin is immobilized on cephalose, or through a DEDE cephalose column and then transferred to a haptoglobin cephalose affinity column. It is very easy to operate as it only needs to be passed through. Furthermore, the production method of the present invention is also excellent in terms of yield. For example, as much as about 150 mg of B-oligomer can be obtained from a hydroxyapatite eluate (crude fraction of IAP) containing 40 mg of protein.
That is, the production method of the present invention can significantly improve the yield of B-oligomer compared to conventional methods. Example 1 Bordetella per-tussis strain Higashihama
Bordet-
After culturing on Gengou plate medium at 37℃ for 2 days, Cohen-
Dispense 200ml of Wheeler's modified medium (CW medium).
One platinum loop was inoculated into a 500ml shaken Kolben, and the mixture was incubated at 37°C.
Shaking culture was performed for 20-22 hours. The amount of bacteria in this culture solution is
Measured with a spectrophotometer (wavelength 650 nm), so that the final concentration of bacteria when added was approximately 0.1 x ¹⁰⁹ cells/ml.
The ion exchange resin-added CW medium 1 was added to the shaken Kolben 2 and cultured with shaking at 37° C. for 48 hours (shaking frequency: 97 times/min). The mycological properties of the above bacterial strain were consistent with the description in the following literature regarding pertussis bacteria.・ Bergy's Manual of Determinative
Becteriology 8th edition 1974Baltimore: The Willi-ams
& Willkns CO., ・ J.Exp.Med.129:523-550 (1969), ・ Bacteriology practice summary: 3rd edition, pages 6 onwards, 1967
(published by Maruzen Co., Ltd.) Table 1 Cohen-Wheeler's modified medium composition Casamino acids 10g Yeast extract 1g Potassium dihydrogen phosphate 0.5 Soluble starch 2g 0.5% copper sulfate solution 1ml 1% calcium chloride solution 1ml 4% magnesium chloride Solution 1 ml Polypeptone 5 g 1% cystine solution 2.5 ml 0.5% iron sulfate solution 1 ml Sodium chloride 2.5 g (Add distilled water to make a total volume of 1000 ml, 20%
After adjusting the pH to 7.2 with NaOH aqueous solution, anion exchange resin (Diaion SA-20AP; Mitsubishi Kasei Corporation)
After adding 3 g of the product, the mixture was sterilized with high-pressure steam at 121°C for 15 minutes and used. ) The obtained 48-hour shaking culture solution was heated at 56°C for 30 minutes, and then centrifuged at 4°C (15,000 rpm) to separate the culture supernatant and bacterial cells. The obtained culture supernatant was used as a starting material for the B-oligomer according to the present invention. After adjusting the culture supernatant of 200 to pH6.0 with 1N hydrochloric acid,
As the first purification step, the mixture was passed through a hydroxyapatite column (2.5 x 4 cm) at a flow rate of 200 ml/hour. Most of the proteins were not adsorbed and passed through the column as is. Next, for the adsorbed substances, first wash the column with 0.01M phosphate buffer (PH6.0), then increase the molar concentration of the phosphate buffer to 0.1 and PH to 7.0, respectively, to remove the adsorbed proteins. It eluted. However, N-IAP was still not eluted under these conditions, and was further eluted under the same conditions in a phosphate buffer containing 0.5 M NaCl. N-IAP could be efficiently recovered in accordance with the protein eluted under these conditions. The obtained N-IAP crude fraction 1 (400 mg protein,
300 mg of urea was added to the mixture (containing 14 g of phosphate and 30 mg of NaCl) and left in an atmosphere at a temperature of 4° C. for 5 hours. After standing, the solution was washed with Irons & mac Lennan (BBA1978,
Haptoglobin-Sepharose 4B column (10 x 25 cm) prepared according to the method of 580, 175-185)
added to. After washing the column with 300 ml of 0.1M phosphate buffer (PH7.0) containing 2M urea, the B-oligomer was eluted with 0.1M Tris-HCl buffer (PH10.) containing 3MKSCN. .
The B-oligomer fraction was converted to KSCN by ultrafiltration.
150 mg of B-oligomer was obtained by equilibration with 0.1M phosphate buffer (PH7.0) containing 2M urea. (Yield from protein contained in IAP crude fraction: 37.5%) The physical properties of the obtained B-oligomer were disclosed in JP-A-57
The physical properties were consistent with the physical properties of CP-A described in No.-142325. Example 2 15 mg of urea was added to 40 ml of N-IAP purified fraction (containing 200 mg of N-IAP, 0.56 g of phosphate, and 1.2 g of NaCl) and left in an atmosphere at a temperature of 4° C. for 5 hours. After standing, the solution was transferred to a haptoglobin sepharose column (4
×20cm). After the addition, it was washed with 160 ml of 0.1 M phosphate buffer (PH7.0) containing 2 M urea. After the protein content of the effluent washing solution became 50 μg/ml or less, the flow rate was 30 ml/hr with 100 ml of Tris-HCl buffer (PH 10.0) containing 3 MKSCN and 0.5 M NaCl.
Elution treatment was carried out under conditions of 1 fraction/3 ml. The elution state is shown in Figure 1. Next, the eluted B-oligomer-containing fraction was treated in the same manner as in Example 1 to obtain 142 mg of B-oligomer. Example 3 IAP crude fraction 1 prepared in the same manner as Example 1
(contains 400mg of protein), add 400mg of urea,
After adjusting the pH to 9 using about 24 ml of 1N NaOH, it was left at room temperature for 1 day. After standing, the solution was applied to a DEAE Sepharose column (10 x 10 cm), and the fraction of unadsorbed protein that was not adsorbed to the column was collected. Collected fraction solution 1.6 to 0.1M
After adding 1 part of phosphate buffer (PH7.0) and stirring thoroughly, the mixture was left at room temperature for 1 day. After standing, the protein-containing solution was applied to a haptoglobin sepharose column (4
x 20cm) and washed with 0.1M phosphate buffer containing 0.5M NaCl, then 0.1M containing 3MKSCN.
The B-oligomer was eluted with Tris-HCl buffer (PH 10.0). Hereafter, B was treated in the same manner as in Example 1.
- 160 mg of oligomer was obtained. Comparative Example 1 Based on the examples and methods described in JP-A-57-67591, N-IAP crude fraction 1 (containing 400 mg of protein) was obtained from 200 mg of culture supernatant, and then isolated and
114 mg of purified N-IAP was obtained, and 114 mg of isolated and purified N-IAP was processed to obtain 46 B-oligomers.
I got mg. The yield was 11.5% based on the protein contained in the N-IAP crude fraction. Example 4 20 μg of B-oligomer produced in Example 1,
KH ₂ PO ₄ 12.2mg, NaHPO ₄ 40.6mg, NaCl 36.0mg
and 0.2mg of aluminum hydroxide in a container,
Further, sterile distilled water was added to bring the total volume to 1 ml, and the mixture was thoroughly mixed to produce a pertussis vaccine for subcutaneous injection. Example 5 A pertussis vaccine was produced in the same manner as in Example 4, except that 0.2 mg of aluminum phosphate was used instead of aluminum hydroxide. Comparative Example 2 A pertussis HA vaccine was prepared as follows based on the example described in JP-A-55-141416. Pertussis bacteria (Higashihama strain) was cultured for 5 days in a Cohen-Wheeler liquid medium, and ammonium sulfate was added to the culture centrifugation supernatant to 50% saturation to remove the resulting precipitate.
Collected by centrifugation at 10,000 rpm for 30 minutes, added 1M NaCl to 0.05M
Suspended and dissolved in phosphate buffer. This 1M NaCl solution was centrifuged at 3900 rpm for 20 hours on a 10-30% sucrose density gradient, and the HA active fraction was collected. This HA fraction
To inactivate LPF and HSF activities, formalin was added to a concentration of 0.1% and kept at room temperature (22℃).
After standing for 7 days, formalin was further added to a concentration of 0.2%, and the mixture was left standing at a temperature of 37°C for 2 days. Buffered saline (PH7.0) was added to the solution after it was allowed to stand, and the final protein concentrations were adjusted to 12.5, 25, 50, and 12.5, respectively.
100μg/ml, aluminum hydroxide 0.2
Four types of pertussis HA vaccines containing mg/ml were prepared. Test Example 1 Weight change: B-oligomer 12.5 μg, 25 μg in 1 ml of sterile physiological saline containing 0.2 mg/ml aluminum hydroxide
4 groups (10 mice per group) of ddY mice (male, 4 weeks old, average weight 19.8 g) were each intraperitoneally administered with 0.4 ml of the vaccine of the present invention containing 50 μg, 50 μg, and 100 μg, respectively. The presence or absence of endotoxin in the vaccine of the present invention was examined based on the degree of change in body weight 24 hours after administration. Additionally, 0.4 ml of the four types of pertussis HA vaccines prepared in Comparative Examples were administered to four other groups of mice in the same manner. The group to which 0.4 ml of sterile physiological saline was administered was used as a control group. The changes in body weight of the group administered with the vaccine of the present invention and the group administered with the pertussis HA vaccine are shown in Table 2 below. [Table] Since there was no decrease in body weight in the group administered with the vaccine of the present invention compared to the control group, it was found that the vaccine of the present invention was substantially free of endotoxin. On the other hand, the group receiving the conventional pertussis HA vaccine lost more weight than the control group.
It has been discovered that endotoxins are present in vaccines. Test Example 2 Leukocytosis effect: Same as Test Example 1, 0.4 ml of 4 types of vaccines of the present invention with different contents of B-oligomer or 4 types with different contents of toxoidized HA fraction protein as a comparative example. Pertussis HA vaccine was intraperitoneally administered to mice in each group, and the increase and decrease in peripheral leukocytes was examined 3 days after administration. The results are shown in FIG. As is clear from the graph in Figure 2, the amount of leukocytes in the vaccine of the present invention increases only slightly even when the dose of B-oligomer increases, whereas the comparative pertussis HA vaccine contains only a toxoidized HA fraction. As the protein dose increased, the white blood cell count increased significantly. Test Example 3 Histamine sensitization effect: In the same manner as Test Example 1, 0.4 ml of four types of vaccines of the present invention with different contents of B-oligomer or four types with different contents of toxoidized HA fraction protein as a comparative example were prepared. Pertussis HA vaccine was administered intraperitoneally to mice in each group, and 3 days after administration, 0.2 ml of physiological saline containing 1 mg of histamine was intraperitoneally administered to examine the survival rate 2 hours later. The results are shown in Figure 3. As is clear from the graph in Figure 3, the vaccine of the present invention survived 100% even when the dose of B-oligomer was increased, whereas the vaccine for pertussis in comparison
The survival rate of the HA vaccine decreased significantly as the dose of toxoidated HA fraction protein increased. Test Example 4 Infection prevention test: 0.25 μg of B-oligomer in 1 ml of sterile physiological saline containing 0.2 mg/ml of aluminum hydroxide.
0.4 ml of the vaccine of the present invention containing 2.5 μg and 25 μg, respectively, was intraperitoneally administered to three groups (16 mice per group) of ddY mice (male, 4 weeks old), and 2 weeks after administration, one mouse Bacillus pertussis per animal 18~
4 x 10 ⁴ live bacteria of strain 323 (competitive fungus) were inoculated into the brains of mice, and the survival rate was determined by observing whether they were alive or dead for two weeks after the inoculation. The results are shown in Table 3. On the other hand, toxoidized HA was added to 1 ml of sterile physiological saline containing 0.2 mg/ml of aluminum hydroxide.
A pertussis HA vaccine prepared by adding and mixing 0.25 μg, 2.5 μg, and 25 μg of fractionated proteins, respectively, was prepared in the same manner as in Comparative Example 2, and three types of pertussis were prepared.
0.4 ml of HA vaccine was administered intraperitoneally to each of the three groups of mice, and live Bordetella pertussis was inoculated into the brain in the same manner as in the group administered with the vaccine of the present invention, and the survival and death of the mice was observed for 2 weeks after inoculation. , the survival rate was determined. The results are shown in Table 3. The control group consisted of mice treated in the same manner as the vaccine administration group, except that only 0.4 ml of sterile physiological saline was administered intraperitoneally. [Table] [Table] As is clear from Table 3, the vaccine of the present invention exhibited superior infection-preventing action than the pertussis HA vaccine in the infection-prevention test.

[Brief explanation of drawings]

In the accompanying drawings, FIG. 1 shows an elution graph for the haptoglobin Sepharose column in Example 2, FIG. It is a figure which shows the graph of the survival rate regarding a sensitization effect.

Claims (1)

[Claims] 1. Molecular weight by SDS-gel electrophoresis method is 7500±
5000; Protein by Lowry method is 98% by weight or more, and the amino acid composition and composition ratio (μM/100μM) of the protein component are Asp.6.4±0.7, Thr.7.3±0.8,
Ser.8.2±0.9, Glu.9.3±1.0, Pro.6.3±0.7,
Gly.11.1±1.0, Ala.9.0±0.9, Cys./21.2±0.3,
Val.6.2±0.6, Met.2.7±0.4, Ile.3.4±0.4,
Leu.8.4±0.9, Tyr.5.6±0.6, Phe.3.6±0.5,
Lys.4.7±0.5, His.1.1±0.3, and Arg.4.9±0.5; and a pertussis vaccine containing B-oligomer, which is a basic protein, as an active ingredient. 2. The pertussis vaccine according to claim 1, which contains aluminum phosphate or aluminum hydroxide as an adjuvant.