JPS61106518A - Cholera vaccine - Google Patents

Abstract

PURPOSE:A cholera vaccine composed of a composite material obtained by bonding a cholera toxin and a carboxylated lipopolysaccharide prepared from cholera bacillus. CONSTITUTION:The powder of lipopolysaccharide (LPSV) existing at the surface of cholera bacillus is suspended in a proper organic solvent (e.g. anhydrous pyridine), heated, and cooled. An aliphatic dicarboxylic acid or its reactive derivative is dissolved in the organic solvent same as above, and the solution is added to the above suspension. The mixed solution is reacted by heating, dialyzed with water and freeze-dried to obtain a modified LPSV. Separately, cholera toxin (CT) is dissolved in physiological saline water, and the above freeze-dried LPSV and a condensation agent for acid amide bond-formation are added to the solution to effect the reaction of CT with the modified LPSV. The reaction liquid is subjected to the gel-filtration to obtain a CT-LPSV complex. It is used as a cholera vaccine for oral administration with or without mixing with a proper carrier.

Description

[Detailed Description of the Invention] The present invention provides a cholera vaccine useful for the prevention of cholera disease, and more specifically, a cholera toxin and a carboxylated modified ribopolysaccharide obtained from cholera cells. Cholera vaccine consists of a complex that binds to the body and is particularly effective by oral administration.
It is a contagious diarrheal disease caused by cholera toxin (cholera toxin), which is a protein toxin produced by Vibrio cholerae.
It is said to be induced by CT (hereinafter abbreviated as CT). There is a lot of knowledge about its pathogenesis, and generally speaking, CT acts on the intracellular cyclic AMP system, increases the concentration of cyclic AMP, and thereby induces water leakage from the intestinal tract. It is thought that this results in specific diarrheal symptoms.

As a preventive method for such cholera disease, a killed bacterium vaccine in which cholera cells are inactivated with formalin or the like is currently used parenterally. However, since this type of vaccine has the drawbacks of insufficient efficacy and poor durability, there is a desire for the development of an even better vaccine.

In addition, considering that cholera disease is caused by CT produced by Vibrio cholerae, we thought that antitoxin immunization, which had been successful in treating tetanus and Nottilia, might also be effective against cholera. Parenteral immunization has also been attempted, but neither has achieved the desired preventive effect.

The reason why sufficient preventive effects cannot be obtained with the above-mentioned killed vaccines and antitoxin immunization is that, because they are administered parenterally, the immunization effect in the intestinal tract, which is the site of onset of cholerasis, is low.

Therefore, in order to overcome these drawbacks, it is necessary to develop a cholera vaccine that is effective by oral administration, and various studies are currently being carried out. Live bacterial vaccines have been developed as one of these, but they are difficult to use because they have problems with efficacy and side effects such as diarrhea (Myl.
on M, Levine et il, + In
infection and immunity 4
3+ 5 15-522. (1984)).

In addition, in an attempt to orally administer CT, N
For the purpose of suppressing diarrhea caused by
Mutsui et al. used Escherichia coli ribopolysaccharide (hereafter L
A complex with CT-LPSE (abbreviated as PSE) was prepared by the periodic acid cross-linking method, and such a CT-LPSE complex was detoxified to the extent that the toxicity of CT could be classified to a sufficient level and it could be administered orally. reported that anti-CT antibodies were produced after oral administration (Tsu Usui, Japan-US Medical Joint Conference, Cholera Subcommittee 19).
November 30, 1982, Kurashiki).

In addition to antibodies against CT, the serum of cholera patients usually contains ribopolysaccharide, which is present on the surface of Vibrio cholerae.
An increase in antibodies against LPSV (hereinafter abbreviated as LPSV) has been observed, and an increase in antibodies against (Anis S.

Majuldar et al. + Infect
ion and ranunity32.1 (19
(81)), it is thought that the production of antibodies against both of these is necessary for prevention against cholera infection.

On the other hand, it is said that patients who have recovered from cholera are almost never reinfected. These things suggest that a cholera vaccine needs to contain both CT and LPSV as immunizing antigens.

1 town” season
).

,Yo. Hey there, T. □1 Yu, □6,1.
In order to obtain a cholera vaccine suitable for oral administration that contains both 1T and LPSV, we prepared a vaccine containing CT and LPSV obtained from cholera cells, but found that it was difficult to use due to toxicity etc. When an attempt was made to prepare a composite by crosslinking CT and LPSV by a periodic acid crosslinking method according to the method of I et al., it was found that the desired crosslinking was not achieved. As a result of further research, we found that by modifying this LPSV by carboxylating it with an aliphatic dicarboxylic acid,
We have found that a T-LPS complex can be obtained, and that the toxicity of such a complex is well classified against CT, and that when administered orally, not only antibodies against CT but also antibodies against LPSV are produced. The present invention was completed based on this knowledge.

That is, the present invention provides a cholera vaccine suitable for oral administration, consisting of a complex of CT and LPSV carboxylated with an aliphatic dicarboxylic acid.

The cholera vaccine of the present invention is composed of a complex of CT and LPSV that has been carboxylated with an aliphatic dicarboxylic acid, and the cholera vaccine is made of a complex of CT and LPSV that has been carboxylated with an aliphatic dicarboxylic acid or a reactive derivative thereof. and the obtained modified LPSV
can be obtained by reacting with CT.

The CT used in the present invention is isolated by a conventional method from a culture solution obtained by culturing Vibrio cholerae by a conventional method. It is obtained by gel filtration and is usually stored in the form of a lyophilized product.

LPSV can also be obtained by culturing Vibrio cholerae in a conventional manner, collecting the cells, and extracting and isolating them with an appropriate solvent such as water or phenol (C, Ga1ano
s, O, Li1doritz and O.

WesLphal+ Eur, J., Bioehe
m, + 1.245 (1969)), LP
Examples of aliphatic dicarboxylic acids or reactive derivatives thereof used for modification of SV include aliphatic nocarboxylic acids or reactive derivatives thereof, such as malonic acid, succinic acid, geltaric acid, and adipic acid, particularly acid anhydrides, For example, succinic anhydride, adipic anhydride, etc. are preferred.

If these aliphatic dicarboxylic acids or their reactive derivatives are used in excess, the immunogenicity of the resulting complex against Vibrio cholerae tends to decrease;
．． It is preferable to use about 1 to 2.5 parts by weight.

The CT-LPSV complex of the present invention can be prepared as follows.

The LPSV powder obtained by the method described in the above-mentioned Wes Lphal et al. paper is suspended in a suitable organic solvent (eg, anhydrous pyrinone), heated, and then cooled.

To this, a solution of an aliphatic dicarboxylic acid or its reactive derivative (e.g. succinic anhydride) dissolved in the same organic solvent (e.g. pyridine anhydride) was added, and the mixture was heated and reacted. 1. The reaction solution was dialyzed with water. + (Later, lyophilized to obtain a lyophilized product of modified LPSV. Separately, a lyophilized product of CT was dissolved in physiological saline, and the above modified LPSV was added to this solution.)
A lyophilized SV product and a condensing agent (for example, a carbodiimide reagent) commonly used for acid amide bonding are added, and CT and modified LPSV are reacted. The desired CT-LPSV complex is obtained by subjecting this reaction solution to a deluer filtration using a conventional method.

In the above method, CT and modified LPSV are usually 1:
The reaction is carried out at a ratio of 0.75 to 1:1.5 (weight ratio).

According to the results of a blood sampling test after administering the CT-LPSV complex of the present invention intraperitoneally to mice, the anti-CT antibody titer in the blood was superior to that of formalin-inactivated CT, and furthermore, it was not observed in formalin-inactivated CT. It was also observed that some anti-cholerae immunity was expressed.

Furthermore, when the CT-LPSV complex was administered intragastrically to mice, production of anti-CT antibodies and anti-Vibrio cholerae antibodies, which were not observed in formalin-inactivated CT, was observed in the blood, indicating that the CT-LPSV complex was superior. It was confirmed that the drug had immunogenicity. This is also clear from the experimental results described later. That is, according to the in-gel precipitation reaction between the CT-LPSV complex and anti-CT serum (see Figure 2), anti-CT
A sedimentation line was observed between the serum, CT, and CT-LPSV complex, indicating that the CT-LPSV complex clearly retains the antigenicity of CT.

Furthermore, according to the results of in-gel precipitation reaction between the CT-LPSV complex and anti-cholerae serum (see Figure 3), a single sedimentation line was observed between each, and the CT-LPSV complex was It is found that it has antigenicity of LPSV.

In addition, as a toxicity test for the created CT-LPSV complex, a skin capillary permeability test was conducted using rabbits, and the results showed that the toxicity of the CT-LPSV complex was about 1/100th that of CT. confirmed. That is, it can be seen that the inactivation of CT is satisfied by conjugating CT with CT-LPSV.

As mentioned above, the cholera vaccine of the present invention consists of CT and modified L.
It consists of a complex bound to PSV, and since it is obtained from LPSV used for binding or from cholera cells, the antigen contains CT and components of cholera cells,
It has been found that antibodies generated by this antigen are extremely effective in protecting against Vibrio cholerae infection. More LPs
SV became able to bind to CT for the first time through modification, and binding of modified LPSV to CT also helped inactivate CT, and modified LPSV
It was found that SV shows the 7-dijuvant effect of a CT-based vaccine. Thus, binding the modified LPSVLCT exhibits a fully expected effect as a cholera vaccine.

The cholera vaccine thus obtained can be administered orally, which is an extremely easy vaccine administration method. In the case of cholera, oral administration is a more desirable immunization method than parenteral administration because it can be expected to have a direct immune effect, considering its pathogenic mechanism.

In addition, in the case of oral administration, it is very convenient to administer as it can be administered in the form of tablets or enteric-coated capsules, and it has the advantage of producing IgA in the mucus of the intestinal tract, which has an extremely large immune effect. has.

The lyophilized CT-LPSV complex of the present invention can be used as a cholera vaccine for oral administration, either as it is, or by mixing it with a suitable pharmaceutical carrier used in conventional oral preparations and formulating it by a conventional method.

In addition to solid preparations such as tablets, capsules, granules, fine granules, and powders, the preparations can also be used as liquid preparations such as emulsions and suspensions. It is preferable to formulate the CT-LPSV complex so that each dosage unit contains several grams to several tens of mg.

EXAMPLES Next, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited thereto.

Example (1) Preparation of modified LPSV 30+sg of LPSV powder extracted from 01 type Fr.
Gin 2s, el: Suspend and heat at 100°C for 1 minute.

After cooling to room temperature, dry succinic anhydride with viridi 22
Add the dissolved solution to the ring and heat at 11'C for 3
Heat for 0 seconds. This reaction solution is dialyzed against water and then freeze-dried to obtain modified LPSV. The results shown in Table 1 were obtained by varying the dose of succinic anhydride.

Table 1 (2) Preparation of CT-LPSV complex 20 g of lyophilized CT was dissolved in 6-ml physiological saline.
Dialyze 5 times against approximately 50 times the volume of physiological saline. The above modified LP'5V15@g and 100 mg of water-soluble carbodiimide are added to this solution, and the mixture is reacted at 20° C. for 5 hours.

Add this reaction solution to physiological saline! .

0. IM) Lis-H (4 buffer (DH8,0,0,
Dialyze against 1M NaCl (containing 0.01% NaN3). A column (3.2 cm x 100
cm). Using the above Tris-HC (buffer) as the eluent, elute for 39-7 hours and fractionate into 3 ml each. Use a differential refractometer as a detector. The CT-LPSV complex is obtained from the first eluted fraction.The results are shown in Table 2.

Table 2 Gel filtration chromatography for removing unreacted CT from the CT-LPSV complex is shown in FIG.

The following experiment was conducted on the CT-LPSV complex obtained above.

Experiment (1) Properties of CT-LPSV complex Protein (CT) and modified LPS of each complex obtained in the above examples
Table 3 shows the content of V.

(2) Precipitation reaction of CT-LPSV complex and anti-CT serum in tonogel.
This was done using the CHTERLONY method.

The results are shown in Figure 2.

In Figure 2, if anti-CT serum (i-c”r) and CT (
71,:CT-LPSV complex r, II, III,
Since a sedimentation line between IV is observed, it can be seen that the CT-LPSV complex clearly retains the antigenicity of CT.

(3) In-gel precipitation reaction between CT-LPSV complex and anti-cholerae serum A precipitation reaction between the CT-LPSV complex and anti-cholerae serum (i-VC) was performed. The results are shown in FIG. Third
As shown in the figure, a sedimentation line was observed between the knee VC and each CT-LPSV complex, and the CT-LPSV complex was
It can be seen that it has SV antigenicity.

, 1'((")"'°"'"10w' The CT-LPSV complex was intraperitoneally administered to mice to examine its sensitization.
1 og (as protein amount) dissolved in physiological saline 0.5
-ρ to mice (weight 20 g, 10 mice per group) on the first day.
A second intraperitoneal administration was performed on the 21st day, and one blood sample was taken immediately before and on the 28th day, and the anti-CT antibody titer was measured by CT-attached latex agglutination reaction. In addition, Vibrio cholerae immunity was measured by the Vibriocyglu test. The results are shown in Table 4.

Table 4 1) Maximum dilution factor log of CT-attached latex agglutination reaction
Displayed in 2.

2) Unable to detect at 1:8 dilution rate.

As shown in the above results, in terms of anti-CT antibody titers, complexes I to 1v showed almost the same high values, but in terms of anti-cholera antibody titers, complexes I and II had extremely low values1, while complexes Iv showed the highest value.

(4) Toxicity test In order to investigate the toxicity of the CT-LPSV complex, a skin capillary permeability test was conducted using a tsugi (Cra
ig+ J, P,, Nature 20yu, 614
-616 (1965)).

As a result, the CT-LPSV complex was 1/1 compared to CT.
It was confirmed that the toxicity had decreased to about 0.00.

[Brief explanation of the drawing]

@1 Figure 1 is a diagram showing the relative refractive index intensity and 7-raction distribution of the tcT-LPSV composite IV. Figure 2 is CT
- A reproduction of an in-gel sedimentation reaction photograph showing the reactivity between the LPSV complex and anti-CT serum. Figure 3 is a reproduction of an in-gel sedimentation reaction photograph showing the reactivity between the CT-LPSV complex and anti-cholerae serum. It is a diagram. Figure 1 Figure 2 T 743 Figure U≧TEV

Claims (3)

[Claims] (1) A cholera vaccine comprising a complex of cholera toxin and a modified carboxylated ribopolysaccharide with aliphatic dicarboxylic acid obtained from cholera cells. (2) The vaccine according to item (1) above, wherein the aliphatic dicarboxylic acid is an aliphatic dicarboxylic anhydride. (3) The vaccine according to item (2) above, wherein the aliphatic dicarboxylic anhydride is selected from succinic anhydride and adipic anhydride.