JPS6154769B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a protease toxoid produced from Pseudomonas aeruginosa and a method for producing the same. The present invention also relates to a method for preventing and treating protease-induced disorders of Pseudomonas aeruginosa by immunizing animals with the protease toxoid. Pseudomonas aeruginosa is a representative pathogen of opportunistic infections, which has received increasing attention due to the remarkable advances in medicine, and poses a variety of major problems in both the medical and veterinary fields. Infections with this fungus are likely to occur and are serious when the immune system is suppressed due to physiological immunodeficiency in newborns, cancerous leukemia, transplants, burn patients, or suppressed immune function due to medication such as steroids and immunotherapy. In the field of veterinary medicine, hemorrhagic pneumonia in mink caused by this fungus and mastitis in cattle caused by this fungus are major problems in the livestock economy. Although a few antibiotics against Pseudomonas aeruginosa have recently been developed, they are largely ineffective when an individual's immune system is dysfunctional or weakened. Furthermore, regarding the aforementioned bacterial infection in veterinary medicine, it is practically impossible to treat thousands to tens of thousands of mink with antibiotics, and it is also not viable from a cost perspective.
The situation is almost similar for bovine mastitis. Therefore, there is a strong desire for immunotherapy or a combination therapy of both in place of chemotherapy. As for immunotherapy, the common antigen OEP, which protects against infection by all Pseudomonas aeruginosa serotypes (currently over 13 types), has been isolated by Homma et al. and is already being applied [JYHomma et al.
Japan.J.Exp.Med., 45 , 355−360 (1975); J.
Y. Homma et al, Japan.J.Exp.Med, 42 , 23−
34 (1972); JY Homma, Microbial Drug
Resistance, Tokyo University Press,
Tokyo, (1975); JYHomma, The Fourth
International Congress of Animal, Plant and
Microbial Toxins, Plenum Publisher, London
(1975)]. On the other hand, Pseudomonas aeruginosa produces protease, elastase, and other substances outside the bacterial body, and it has been confirmed that these two enzymes act as pathogenic factors [JYHomma et al, Japan J.Exp.
Med., 45 , 79-88 (1975); JYHomma et
al, Japan.J.Exp.Med., 45 , 515 (1975); K.
Kawaharajo et al, Japan.J.Exp.Med., 44 , 435
−442 (1974); JYHomma et al, Japan.J.
Exp.Med., 45 , 89-100 (1975)]. It is fully estimated that there are high antibody titers for protease and elastase as well as OEP in the serum of chronic respiratory tract infections caused by this bacterium in humans and bovine mastitis [J.
Y. Homma, Japan.J.Exp.Med., 45 , 361−366
(1975)]. In fact, animal experiments have revealed that protease and elastase cause skin necrosis, corneal ulceration, and significant pathological changes in internal organs in small amounts [JYHomma et al, Japan.J.
Exp.Med., 45 , 79-88 (1975); JYHomma
et al, Japan.J.Exp.Med., 45 , 515 (1975);
K. Kawaharajo et al, Japan.J.Exp.Med., 44 ,
435−442 (1974); JYHomma et al, Japan.J.
Exp.Med., 45 89-100 (1975)]. Furthermore, when protease and elastase-producing bacteria and non-protease-producing bacteria are inoculated into the cornea of mice, ulcers are formed in the former, but
The difference in pathogenicity between the two was clarified by the fact that the latter did not form [JYHomma et al, Japan.
J.Exp.Med., 45 , 515 (1975)]. Therefore, in order to ensure the completeness of immunotherapy, it is necessary to prevent the proliferation of OEP using the common infectious protective antigen OEP, such as that of Honma et al., and to neutralize the toxic effects of metabolites. For this purpose, protease and elastase must be toxoidized for immunization. The present inventors conducted research on the toxoidization of protease, and succeeded in obtaining a protease toxoid that retains antigenic activity but loses enzymatic activity, thereby completing the present invention. The Pseudomonas aeruginosa protease toxoid of the present invention is produced as follows. The method for producing the protease produced by Pseudomonas aeruginosa used in the present invention and its properties are
27315; K. Morihara et al, Biochim. Biophys.
Acta, 73 , 113-124, 125-131 (1963); K.
Morihara et al, Biochim.Biophys.Acta, 92 ,
351−360, 361−366 (1964); K. Morihara et
al, Arch.Biochem.Biophys., 114 , 158−165
(1966); K. Morihara et al, Biochim. Biophys.
Acta, 309 , 414−429 (1973); K. Morihara et
al, Agr. Biol. Chem., 38 (3), 621-626.
(1974). When protease is toxoidated, it may be toxoidated with formalin or oxymethane sulfinate in the presence of an amino acid (eg, lysine). The physicochemical properties of protease toxoid obtained by toxoidization with formalin in the presence of lysine are shown below. Molecular weight: 63,000 (gel filtration method) Ultraviolet absorption spectrum: maximum 280 mμ (E ²⁸⁰ ₁
%
= 9.27, 0.1M KCl) minimum 250mμ Isoelectric point: PH5.2 (focal electrophoresis) Amino acid composition: Amino acid residues (g/100g protein), aspartic acid (15.6), threonine (5.0), serine (7.6), Glutamic acid (9.5), Proline (2.1), Glycine (7.7), Alanine (8.5), Valine (5.0), Isoleucine (3.9), Leucine (8.7), Tyrosine (6.9), Phenylalanine (5.9), Lysine (4.1) ), Histidine (1.9), Arginine (2.3), Tryptophan (2.3), Ammonia (1.4) (Total 98.5g) Color of substance: Colorless powder Antigenic activity: Yes Enzyme activity: None Pseudomonas aeruginosa has the following physical and chemical properties Protease toxoids are new substances. Examples of the present invention are shown below, but the present invention is not limited to these Examples. Example 1 100 mg of Pseudomonas aeruginosa crystalline protease was dissolved in approximately 20 ml of 0.1 M Na ₂ HPO ₄ solution containing 0.2 M lysine.
Further formalin is added to give a final concentration of about 8%. After standing at room temperature for 3 days or more, it is dialyzed against water and then freeze-dried to obtain protease toxoid. Its yield is about 98-100%. The residual enzyme activity of protease after formalin treatment is shown in Tables 1, 2 and 3. Enzyme activity measurement method: 2% casein (PH7.4) 1ml
Mix with an appropriately diluted enzyme solution and react at 40℃ for 10 minutes. Immediately add 2 ml of reaction stop solution (a solution containing 0.1M trichloroacetic acid, 0.2M acetic acid and 0.2M sodium acetate), and incubate at that temperature for 20 minutes. After keeping the unreacted casein completely precipitated, filter it.
The amount of tyrosine in 1 ml of the filtrate was measured by the Foline method. That is, 5 ml of 0.4M Na ₂ CO ₃ to 1 ml of filtrate.
Phenol Reagent added and diluted to 1/5
Add 1 ml of Solution (acid concentration 1.8 N, phenol reagent, Hanui Chemical, Kyoto) and measure the absorbance at 670 mμ after 15 to 20 minutes. A sample treated in the same manner at 0 hours of reaction was used as a control, and the amount of free tyrosine due to the action of protease was calculated from the difference. Residual enzyme activity is expressed as relative activity (%). As is clear from Table 1, when protease is treated with formalin in the presence of lysine for 4 days, the enzyme activity is inactivated by about 90% or more, but even after 2 weeks of treatment, the enzyme activity is not completely inactivated. Protease is completely inactivated by treating it with formalin in the presence of lysine for about one day or more, dialyzing the resulting protease against water, lyophilizing it, and treating it again in the same manner.

【table】

[Table] Measuring gender

[Table] Measurement of existing activity As is clear from Table 2, the higher the formalin concentration, the better. However, if the enzyme concentration is higher than necessary (>4 mg/ml), precipitation will occur during the reaction, which is unsuitable. A suitable reaction pH is 6 to 9, and oxymethane sulfinic acid can also be used instead of formalin (oxymethane sulfinic acid 0.5M, lysine 0.05M, activity decreases to 15% or less after 3 days at room temperature. Oxymethane sulfinic acid 2.5M, lysine 0.05M, 3
Completely inactivated by day treatment). As seen in Tables 1 and 3, the effect of lysine is significant and toxoidation is not possible without it. Other alternatives such as arginine, leucine, tyrosine, and glutamic acid were considered as substitutes, but none were expected to be as effective as lysine.
Thus, we succeeded in obtaining protease toxoid by formalin treatment in the presence of lysine. The following experimental examples demonstrate that the Pseudomonas aeruginosa protease toxoid of the present invention has antigenic activity. Experimental Example 1 Protease Toxoid Rabbit Immunology Test 1 Experimental Method: Three New Zealand White rabbits were given 1 mg of protease toxoid in Freund.
is injected subcutaneously with Freund's Incomplete Adjuvant and, two weeks later, 1 mg of protease toxoid is injected subcutaneously with Freund's Incomplete Adjuvant. Furthermore, 2 weeks-2
Inject 1 mg of protease toxoid intramuscularly at weekly - 3 week intervals. As shown in Table 4, blood is collected from the ear veins of rabbits over time. Separate the serum, inactivate it, and measure the HA titer. The method for measuring HA value is Japan.J.Exp.Med.Vol45,
No. 5, p. 361-365, 1975. That is, the method was a passive hemagglutination reaction using protease as an antigen. 2 Experimental results: The experimental results are shown in Table 4. H.A.
The value has increased 250 to 4000 times.

[Table] Experimental example 2 Protease toxoid mouse immunity test 1 Experimental method: DDY (female, 5 weeks old) mouse 3
Animals were injected subcutaneously with protease toxoid 10γ together with Freund's incomplete adjuvant, and then 2 weeks later with protease toxoid 10γ.
10γ is injected subcutaneously with Freund's Complete Adjuvant. Furthermore, protease toxoid 10γ is injected intraperitoneally at 2-week intervals. Blood is collected from the tail vein of the mouse over time. Separate the serum, inactivate it, and measure the HA titer. The method for measuring the HA value is described in Experimental Example 1. 2 Experimental results The HA titer in mice was 1024 ~ 2 weeks after the final immunization.
It has increased 2048 times. Experimental Example 3 Protease toxoid 1000gamma was subcutaneously injected together with an adjuvant (potash alum) to 13 mink (Saphia), and when 500gamma was reinjected about two weeks later, the protease-HA value increased 64 to 512 times. Protease in mink serum before injection
No HA value was observed. Experimental Example 4 Protease neutralizing activity of anti-protease toxoid rabbit serum (1) Protease neutralizing activity was determined by enzyme activity. (a) Test serum: Rabbit immune serum immunized with protease toxoid and having a protease-HA titer of 7860 was used. Elastase-HA, OEP-
The HA reaction was negative in both cases. Normal rabbit serum (all negative for protease-HA, elastase-HA, and OEP-HA) was used as a control. Furthermore, elastase toxoid antiserum was obtained by immunizing a rabbit, and the protease-HA titer and OEP-HA titer were all negative. (b) Measurement method: Serum was inactivated at 56°C for 30 minutes before use. Dissolve a certain amount of protease in 1/15M phosphate buffer (PH7.4), add 0.2 ml of test serum to 0.2 ml, add physiological saline, and make the entire volume.
The volume was 3.0ml. The remaining enzyme activity was measured after keeping at 37°C for 60 minutes. Enzyme activity was measured according to the method described in Example 1. (c) The results are shown in Table 5.

[Table] As is clear from this result, the normal rabbit serum and solvent-only controls do not contain neutralizing antibodies.
*Elastase toxoid rabbit antiserum also does not contain neutralizing antibodies. In contrast, only protease toxoid-immunized rabbit antiserum neutralized protease activity. (2) Protease neutralization activity is determined by the presence or absence of corneal ulcer. K. Kawaharajo et al.
Japan.J.Exp.Med., 44 , 435-442 (1974).
Judgment is made based on the strength and presence of lesions such as ulcer formation. As a result, protease toxoid antiserum has a concentration of about 100γ to 1ml, although it varies depending on the HA titer.
200γ protease activity was neutralized. (3) Protease neutralizing activity was determined based on various lesions in the internal organs of mice and survival. When a reaction solution of protease and protease toxoid antiserum and its precipitate are injected into the peritoneal cavity of a mouse, various lesions in the internal organs that occur when no antiserum is added do not occur, and the lethal dose is neutralized and the mouse survives. . No neutralizing effect was observed even when normal rabbit serum was added instead of antiserum. Experimental Example 5 Serum of mink immunized with OEP single vaccine and triple vaccine containing OEP, protease toxoid, and elastase toxoid
HA titer and infection protective effect OEP vaccine is currently known as a vaccine for Pseudomonas aeruginosa infection. When a three-way combination vaccine was produced by mixing this OEP antigen with protease toxoid and the elastase toxoid of the present invention, it was revealed that this three-way combination vaccine was more effective in preventing and treating Pseudomonas aeruginosa infection than a single OEP vaccine. Summer. 1 Experimental method Animal: Mink (Saphia species), 5-6 month old male. Vaccine administration method: Vaccines were administered subcutaneously or intramuscularly. Challenge test: Pseudomonas aeruginosa No.5 strain was used. Infection with live bacteria was performed by injecting 0.5 ml of bacterial solution into the nasal cavity through a vinyl tube under ether anesthesia. Method of infection and immunization: Date of immunization, dose, date of infection, and date of autopsy are shown in Table 6. Production method of OEP vaccine and triple vaccine:

[Table] (1) Protease toxoid-potassium alum solution: 100mg of protease toxoid
24.8 ml phosphate buffered saline (M/15, PH
7.4) Dissolve in (PBS) and add 2.5ml to this solution.
10% potassium alum [K ₂ Al ₂ (SO ₄ ) ₄ .
24H ₂ O]. In addition, 20%
Add 2.5ml of Na ₂ HPO ₄ 12H ₂ O to adjust the pH to 6.5.
Complete precipitation. Finally 0.3ml 1
% Thimerosal as a preservative (1mg
Protease toxoid/0.3ml). (2) *Elastase toxoid-potassium alum solution: Produced by the same method as above except that elastase toxoid is used instead of protease toxoid. (3) OEP-potassium alum solution: 100mg of OEP
Dissolve in 5ml of 0.01N NaOH, add 28ml to this
Add PBS. Then add 3.3ml of this solution to 10
Add % potassium alum and add another 3.3 ml of 20
% _{Na2HPO4 was} added. Finally, 0.4 ml of 1% thimerosal was added (1 mg OEP/0.4 ml). (4) Immediately before use, mix the protease toxoid-potassium alum solution, elastase toxoid-potassium alum solution, and OEP-potassium alum solution. This mixture
500 gamma protease toxoid in 0.5 ml,
500γ elastase toxoid, 500γ
Including OEP. *Elastase toxoid: Elastase solution (10-15mg enzyme protein/ml, 5M salt,
(containing 10mM sodium acetate, 2mM calcium chloride, and 0.1mM zinc chloride) was diluted with borate buffer (PH9) to an enzyme concentration of 2-5mg/ml (final borate concentration approximately 0.1M).
Then add formalin to a final concentration of 1% and keep at room temperature. The inactivated elastase solution is dialyzed against water and then lyophilized to obtain elastase toxoid. 2. Experimental results Mink were infected with live bacteria (challenge test).
The immune effects of OEP and the three-way combination vaccine were investigated and the results are shown in Table 7. In the case of potash alum alone, LD ₅₀ is approximately 3.0×10 ⁶
It was hot. In the case of OEP alone, LD ₅₀ ≦3.6×10 ⁶ and no difference from potash alum was observed. However, the LD ₅₀ of the three-way mixed immunization group C was >1.9×10 ⁹ , which clearly showed a difference from the groups B and A. The LD ₅₀ was 3.4×10 ⁵ without treatment. Potash alum alone or OEP and potash alum immunity at most 10
Becomes able to withstand twice the amount of viable bacteria. However, when used as a three-way vaccine with protease toxoid and elastase toxoid added to it, about 10,000 times the dose can be tolerated.

[Table] The toxicity of the protease toxoid of the present invention is as follows. No acute toxicity was observed even when 1 mg/mouse was administered intraperitoneally (minimum lethal dose of protease).
0.2 mg/mouse). As is clear from the above experimental examples, in animals inoculated with the protease toxoid of the present invention, HA
An increase in the HA titer and neutralizing antibody titer was observed, but no HA titer or neutralizing antibody titer was observed in uninoculated animals. Therefore, it is clear that the protease toxoid of the present invention is indispensable for the production of antibodies (antisera) for preventive vaccines and therapeutics against Pseudomonas aeruginosa infections in animals and humans. Specific application examples of the protease toxoid of the present invention are shown below. Inoculating animals and humans with protease toxoid to produce serum or antibodies containing protease toxoid antibodies, and using the antiserum or antibodies to protect and treat protease-induced disorders (such as corneal ulcers) of Pseudomonas aeruginosa. (serum therapy). In addition, protease toxoids or protease toxoids containing adjuvants are administered as vaccines to animals and humans (subcutaneously, intradermally,
(intramuscular injection, etc.) can be used to prevent and treat protease-induced disorders of Pseudomonas aeruginosa. A vaccine using protease toxoid as an antigen can be produced according to the method for producing ordinary vaccines for animals and humans. That is, a solvent may be added to the protease toxoid to form a solution, and if necessary, an adjuvant and/or preservative may be added. Solvents include, for example, distilled water, saline or phosphate buffered saline. Adjuvants include, for example, aluminum hydroxide, aluminum phosphate, calcium phosphate, alum or Freund's complete adjuvant. Preservatives include thimerosal, phenol, carbolic acid, or formalin. Furthermore, the protease toxoid of the present invention is a very effective substance for producing a highly active and effective Pseudomonas aeruginosa vaccine.

Claims (1)

[Claims] 1 Obtained by toxoidizing protease produced from Pseudomonas aeruginosa; Molecular weight: 63,000 (gel filtration method) Ultraviolet absorption spectrum: maximum 280 mμ (E ²⁸⁰ ₁
%
= 9.27, 0.1M KCl) minimum 250mμ Isoelectric point: PH5.2 (focal electrophoresis) Amino acid composition: Amino acid residues (g/100g protein), aspartic acid (15.6), threonine (5.0), serine (7.6), glutamic acid (9.5), Proline (2.1), Glycine (7.7), Alanine (8.5), Valine (5.0), Isoleucine (3.9), Leucine (8.7), Tyrosine (6.9), Phenylalanine (5.9), Lysine (4.1) , histidine (1.9), arginine (2.3), tryptophan (2.3), ammonia 1.4 (total 98.5g) Substance color: Colorless powder Antigenic activity: Yes Enzyme activity: A Pseudomonas aeruginosa protease toxoid with physicochemical properties of no. 2. A method for producing a toxoid of Pseudomonas aeruginosa protease, which comprises toxoidizing a protease produced from Pseudomonas aeruginosa with formalin or oxymethanesulfinic acid in the presence of lysine. 3. A method for preventing and treating protease-induced disorders of Pseudomonas aeruginosa, which comprises inoculating an animal with a sufficient amount of protease toxoid to form antibodies.