WO2007111326A1

WO2007111326A1 - Whole cell vaccine suffering from no toxicity return even in prolonged storage and use thereof

Info

Publication number: WO2007111326A1
Application number: PCT/JP2007/056314
Authority: WO
Inventors: Takaaki Sato; Mitsuo Oishi; Katsuyo Abe; Chiharu Yoshino; Masaaki Nagai
Original assignee: The Kitasato Institute
Priority date: 2006-03-27
Filing date: 2007-03-27
Publication date: 2007-10-04
Also published as: JP5207380B2; JPWO2007111326A1

Abstract

Studies have been made on a method of improving the detoxification treatment of whole cell pertussis vaccine and the safety of a vaccine produced by mixing a plural number of vaccines. As a result, a pertussis vaccine having a higher safety than the conventional bacterial vaccines is successfully produced by a convenient procedure at a low cost. Also, it is found out that a vaccine having a favorable storage stability can be produced from a strain originating in another bacterium by the same production method.

Description

Specification

Whole cell bacterial vaccine with characteristics that do not return to toxicity even after long-term storage and its use

Technical field

[0001] The present invention relates to a whole cell bacterial vaccine having a characteristic that the toxicity does not return even after long-term storage, a mixed bacterial vaccine in which a bacterial component vaccine derived from the same bacteria as the whole cell bacterial vaccine is mixed, and the The present invention relates to a mixed vaccine of a mixed bacterial vaccine and another vaccine. Moreover, it is related with those manufacturing methods and uses.

Background art

[0002] Pertussis is an infectious disease that develops due to infection with Bordetella pertussis and has a characteristic cough that lasts for a long time. If you are affected in early childhood, it may become severe and may accompany pneumonia or encephalopathy. Pertussis can hardly be affected by maternal and child immunity, so it can be affected early in life. In order to prevent this, it is necessary to provide vaccine immunity from the early stages of infants.

There are a whole cell pertussis vaccine and a cell-free pertussis vaccine as vaccines used for prevention of pertussis. The following is a brief explanation of conventional theories of whole cell pertussis vaccine and cell-free pertussis vaccine (KM Edwards, MD Decker, EA Mortimer Jr. Vac cine 3rd ed, SA Plotkin and WA Orenstein Ed, (WB Saunders co, 1999 pp.293-344)

[0003] Whole cell pertussis vaccine has been used clinically since the 1930s, and the power of the 1950s has been used worldwide as a DTP triple vaccine worldwide. The effectiveness of the whole cell pertussis vaccine is described in many literatures (M Kimura, Dev. Bio. Stand. 73: 5-9, 1991). The whole cell pertussis vaccine is a vaccine produced by culturing pertussis bacteria and detoxifying the whole cell. Therefore, since the production method is simple and the production cost is low, it is still used all over the world. However, since the whole cell pertussis vaccine is produced by detoxifying the whole cell, a small amount of toxic substances such as endotoxin derived from the cell are mixed in the vaccine. And even if it was detoxified, there was a defect that toxicity return was likely to occur during long-term storage. For this reason, desirable side effects such as fever after vaccination and hardening of the inoculated site have been pointed out and problems related to safety have been pointed out.

[0004] On the other hand, a cell-free pertussis vaccine has been developed in Japan, and the power of the 1980s has been used to date. The main objective of developing a cell-free pertussis vaccine was to improve the safety-related drawbacks of whole cell pertussis cutin. The effectiveness of the cell-free pertussis vaccine has been described in many documents. A cell-free pertussis vaccine is a vaccine produced by culturing pertussis bacteria, collecting and purifying active ingredients from the culture, and detoxifying them. Therefore, the manufacturing method is complicated, it takes a long time to manufacture, and the manufacturing cost is expensive. However, since a cell-free pertussis vaccine is produced by purifying and detoxifying active ingredients as described above, it is more toxic, such as endotoxin derived from the cells contained in the vaccine, than the whole cell pertussis vaccine. Substances are greatly reduced. Therefore, it is a highly safe vaccine, and there are few side reactions such as fever after vaccination and hardening of the inoculated site. In recent years, demand has increased worldwide, and it has begun to be used globally, both in developed and developing countries, outside of Japan.

[0005] As described above, the safety that was a drawback of the whole cell pertussis vaccine was improved in the cell-free pertussis vaccine. However, the advantages of the whole cell pertussis vaccine, such as the simplicity of the production method and the low cost of production, were forgotten. Against this background, the effectiveness of the whole cell pertussis vaccine and the low cost of production are maintained, while the safety of the whole cell pertussis vaccine is as high as that of a non-cell pertussis vaccine. Development is required.

The World Health Organization (WHO) has created a quality standard for whole cell pertussis vaccines and is seeking to comply with governments. However, the manufacturing method and quality standards of the cell-free pertussis vaccine differ from country to country, and a global standard is probable!

In addition, as with the pertussis vaccine, the whole cell bacterial vaccine derived from other bacteria is a whole cell bacterial vaccine that has safety comparable to that of a cell-free bacterial vaccine while maintaining low production costs. Development is currently being sought worldwide.

[0006] Information on prior art documents related to the invention of the present application is shown below.

Patent Document 1: JP-A-4-230328 Patent Document 2: JP-A-5-503628

Non-Patent Document 1: KM Edwards, MD Decker, et al: "Vaccines", third ed., SA Plotkin an d WA Orenstein (ed.), Pp.293—344. WB Saunders Co., 1999.

Non-Patent Document 2: KM Edwards, BD Meade, et al. Pediatrics 96: 548-557. 1995 Non-Patent Document 3: M Kimura, Dev. Bio. Stand. 73: 5-9. 1991

Disclosure of the invention

Problems to be solved by the invention

[0007] The present invention has been made in view of such a situation, and an object of the present invention is to provide a whole microbial cell that has characteristics and improved safety even after long-term storage. To provide a bacterial vaccine. It is also an object to provide a mixed bacterial vaccine in which the above bacterial vaccine is mixed with a bacterial component vaccine derived from the same bacteria, and to provide a safe and effective mixed vaccine comprising these mixed bacterial vaccines and other vaccine antigens. To do. It is also an object to provide a method for producing and using them.

Means for solving the problem

[0008] In order to solve the above-mentioned problems, the present inventors have improved the detoxification treatment method for whole cell pertussis vaccine and studied the safety of a vaccine produced by mixing multiple vaccines. The

First, the method for detoxifying the whole cell pertussis vaccine was improved. As a result, it is difficult to recover from toxicity by carrying out detoxification treatment in the presence of amino acid, etc., or by performing lysis prevention treatment and simple purification treatment on pertussis cells. We have also found that an improved whole cell pertussis vaccine can be produced. Next, the whole cell pertussis vaccine and the cell-free pertussis vaccine with improved safety obtained by the above method are mixed at a concentration below the effective amount, respectively, to evaluate the safety of the combined vaccine. went. As a result, it was found that a mixed pertussis vaccine having safety comparable to a cell-free pertussis vaccine can be produced.

In addition, in other bacterial strains, it was found that vaccines were produced by the same production method as that of pertussis vaccine, and that whole cell bacterial vaccines with good storage stability were produced. That is, the present inventors have succeeded in producing a vaccine that is safer than conventional bacterial vaccines in a simple and inexpensive manner, thereby completing the present invention.

More specifically, the present invention provides the following [1] to [23].

[1] A whole cell bacterial vaccine obtained by the treatment described in (a) and (b) below, which does not cause a toxic return even in long-term storage.

(a) Detoxification treatment of bacterial cells in the presence of amino acids, amines, and Z or amides

(b) Bacterial lysis prevention treatment

[2] The whole bacterial cell vaccine according to [1], wherein the long-term storage period is 1 year or longer, preferably 3 years or longer, more preferably 5 years or longer and less than 6 years.

[3] The amino acid described in (a) is at least one in which aspartic acid, γ-aminobutyric acid, alanine, β-alanine, arginine, glycine, glutamic acid, isoleucine, leucine, lysine, serine, threonine, and balinka are also selected. The whole bacterial cell vaccine according to [1] or [2], which is one amino acid.

[4] The amines described in (a) are at least one amine whose ethylamine, ethanolamine, and propanolamine forces are also selected, or the amides described in (a) are urea, glycinamide, and The whole bacterial cell vaccine according to any one of [1] to [3], wherein j8-aralamidoca is also at least one amide selected.

[5] The lysis prevention treatment described in (b) is at least one treatment method in which acetone treatment, heating, γ-ray irradiation, electron beam irradiation, and laser beam irradiation power are also selected.] To [4] of

V Whole bacterial vaccine according to any one of the above.

[6] The whole bacterial cell vaccine according to any one of [1] to [5], wherein the bacterium is a clinical isolate, an artificial mutant thereof, or a genetically modified strain.

[7] A mixed bacterial vaccine comprising the whole bacterial vaccine according to any one of [1] to [6] and a bacterial component vaccine derived from the same bacteria.

[8] The mixed bacterial vaccine according to [7], wherein the whole cell vaccine and the sputum or bacterial component vaccine alone are less than the effective amount.

[9] Bacterial power Bordetella pertussis, Bordetella parapertu ssis, Salmonella typhi, Toxin producing Escherichia coli, Vibrio cholerae (Vibri o Bacterial vaccine according to any one of [1] to [8], which is any one of the following: [10] A mixed vaccine comprising at least one bacterial vaccine according to [9] and at least one vaccine antigen derived from other bacteria and Z or virus.

[11] Vaccine antigen potency Diphtheria toxoid, tetanus toxoid, Haemophilus influenzae vaccine, meningococcal vaccine, oral polio vaccine, inactive polio vaccine, hepatitis vaccine, and Japanese encephalitis vaccine The mixed vaccine according to [10].

[12] A method for producing a whole cell bacterial vaccine, which is obtained by the treatment described in (a) and (b) below and has a characteristic that toxic reversion does not occur even during long-term storage.

(b) Bacterial lysis prevention treatment

[13] The amino acid strength described in (a): aspartic acid, γ-aminobutyric acid, alanine, β-alanine, arginine, glycine, glutamic acid, isoleucine, leucine, lysine, serine, threonine, and balinca are also selected. The method for producing a whole bacterial vaccine according to [12], which is a single amino acid.

[14] The amine described in (a) is at least one amine selected from ethylamine, ethanolamine, and propanolamine, or the amide described in (a) is urea, glycinamide, and The method for producing a whole bacterial cell vaccine according to [12] or [13], which is at least one amide selected from j8-alanylamide.

[15] The lysis prevention treatment described in (b) is at least one treatment method selected from acetone treatment, heating, γ-ray irradiation, electron beam irradiation, and laser beam irradiation power. [14] A method for producing a whole bacterial cell vaccine according to any one of the above.

[16] The method for producing a whole bacterial cell vaccine according to any one of [12] to [15], wherein the bacterium is a clinical isolate, an artificial mutant thereof, or a genetically modified strain.

[17] A method for producing a mixed bacterial vaccine, comprising a step of mixing the whole bacterial cell vaccine according to any one of [1] to [6] and a cell-free bacterial vaccine derived from the same bacteria. [18] Whole cell vaccine and sputum or cell-free vaccine alone may not be effective [17] The method for producing a mixed bacterial vaccine according to [17].

[19] Bacteria are Bordetella pertussis, Bordetella parapert ussis, Salmonella typhi, Toxigenic Escherichia coli, Vibr io cholera ^ Color Butto ~~ ί | ^ ¾. | ¾ (Staphylococcus aureus) ^ 7 ί or any of Streptococcus cus pneumoniae according to any one of [12] to [18] A method for producing a mixed bacterial cutin.

[20] A method for producing a mixed vaccine comprising at least one bacterial vaccine according to [9] and at least one vaccine antigen derived from other bacteria and Z or virus.

[21] At least one vaccine antigen is selected from diphtheria toxoid, tetanus toxoid, Haemophilus influenzae vaccine, meningococcal vaccine, oral polio vaccine, inactive polio vaccine, hepatitis vaccine, and Japanese encephalitis vaccine The method for producing a mixed vaccine according to [20], which is an antigen.

[22] The vaccine according to any one of [1] to [11], which is used for diseases caused by bacteria and Z or viruses.

[23] Bacteria are Bordetella pertussis, Bordetella parapert ussis, Salmonella typhi, Escherichia coli toxin, Vibr io cholera ^ Colored pig ~~ ί | ^ ¾. | ¾ (Staphylococcus aureus) ^ The vaccine according to [22], which is one of Streptococcus cus pneumoniae.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention provides a whole cell bacterial vaccine having the characteristics that the toxicity does not return even after long-term storage and has improved safety. The present invention also relates to a method for producing the whole bacterial cell vaccine with improved safety.

In the present invention, “reversion to toxicity” is a phenomenon in which the original toxicity of pathogenic bacteria reappears in the preservation process after detoxification treatment. Even bacterial vaccines that have undergone detoxification of pathogenic bacteria have so far been known to frequently revert to toxicity during the storage process, and there is no long-term reversal of toxicity. It is very important to maintain. In the present invention, the long-term storage period is preferably Is a period of 1 year or more, more preferably a period of 3 years or more and less than 6 years. Examples of pathogenic bacteria from which the bacterial vaccine of the present invention is derived include pathogenic bacteria having an infectious ability, and preferably Bordetella pertussis, Bordetella parapertussis, Salmonella typhi ^ Toxin producing Escnerichia coli, Vibrio cholera ^ Colored butterfly Sk (Staphylococcus aureus) ^ 7 This is Streptococcus pneumoniae Can be mentioned. As long as the strain used for the production of these whole bacterial vaccines has antigenicity, any of clinical isolates, wild strains, artificial mutants and genetically modified strains can be used.

[0011] Here, the invention relating to the pertussis vaccine, which is one of the bacterial vaccines, will be described in detail.

The outline of the conventional whole cell pertussis vaccine production method is as follows. First, inoculate B. pertussis (phase I) into a suitable medium (eg, Cohenwihr's modified liquid medium) and incubate in a tank with aeration and agitation for 1 day before and after 35 ° C. The culture is centrifuged to suspend the cells, and the cell fraction is suspended in an appropriate buffer to adjust the cell concentration, and then several weeks in the presence of formalin around 0.5% (v / v). It is detoxified by leaving it alone. Thereafter, the cells were washed by centrifugation to remove formalin, and the cell concentration was adjusted to (10-20) x 10sup9 / mL to obtain a whole cell pertussis coutine.

[0012] The whole cell pertussis vaccine obtained by the conventional method as described above has an advantage that it is effective and suitable for mass production because the production method is simple. However, (1) toxic reversion is likely to occur during long-term storage! ヽ (2) lysis may occur during long-term storage, or quality is likely to deteriorate, (3) high endotoxin content is desirable, It has been pointed out that there are safety issues such as the likelihood of reactions.

[0013] This major problem is solved in the whole cell pertussis vaccine of the present invention. Of particular note is the significant reduction in toxicity reversal during long-term storage. The method for producing an improved whole cell pertussis vaccine of the present invention is an improvement of the conventional detoxification treatment method. The improvement made in the present invention is to perform the following three treatments during detoxification.

The first improvement in the detoxification treatment method of the present invention is the presence of amino acids or the like during the detoxification treatment. In the present invention, the detoxification treatment of pertussis cells In this case, an amino acid, an amine or an amide is present in the buffer together with a detoxifying agent (for example, formalin). This makes it possible to produce a whole cell pertussis coughin that is difficult to recover from toxicity due to long-term storage. A method for controlling the degree of inactivation by the presence of amino acids, ammonia, amines, etc. during the inactivation of protein toxins is known (Form aldehyde inactivation of root-and-mouth disease virus. Onmtions for tne preparatio n of safe vaccine. SJ Barteling, R. Woortmeyer. Arch Virol. 1984; 80 (2— 3): 103— 17.

) o

[0014] Amino acids such as lysine react with formalin to capture excess formalin, neutralize the action of formalin, control the excessive progress of inactive 匕, or indicate inactive 匕It has been widely accepted that it has the effect of stopping. In addition, it has been proved that a Schiff salt is formed by the reaction between formalin and an amino group.

In producing a whole cell pertussis vaccine, in order to remove toxicity and prevent reversion, it is desirable that the detoxification process proceeds as completely as possible, that is, the toxin is completely inactivated. For this reason, it has been considered that no detoxification treatment of whole cell pertussis with formalin has been effective in preventing the inactivation of toxins, and no amino acid addition has been performed.

[0015] The present inventors examined the correlation with the degree of detoxification by changing the formalin concentration and the treatment time during the production of the whole cell pertussis vaccine. Among them, when amino acid was added as one method for controlling detoxification, it was found that toxin inactivation progressed as an unexpected new effect, and that detoxification was completely performed, and the recovery of toxicity was achieved. We succeeded in developing a whole cell pertussis vaccine that does not occur.

The present inventors have found that the addition effect of amino acids, amines and amides when detoxifying whole bacterial vaccines is a new and useful finding.

[0016] The amino acid used in the detoxification treatment of the present invention is preferably any amino acid as long as it is available. Also, basic amino acids are more preferred. Often, water-soluble amino acids are preferred, but water-insoluble amino acids such as Norin may be effective. Both natural type (L type) and non-natural type (D type) can be used. More preferred amino acids include aspartic acid, γ-aminobutyric acid, alanine, β-alanine, Luginine, glycine, glutamic acid, isoleucine, leucine, lysine, serine, thread ninin, norin, peptides containing these, amino acid oligomers, and the like are not limited to these. Suitable amines and amides include alkylamines such as ethynoleamine, ethanolamine and propanolamine, amides such as urea, glycinamide and β-aralamido, ammonia, inorganic salts of the above substances, However, it is not limited to these.

[0017] The concentration of amino acids or amines and amides used in the present invention can be practically used in the range of 0.005 Μ-0.5 、, and preferably in the range of 0.02 Μ-0.2 Μ. However, the optimal concentration differs depending on the concentration of the whole cell pertussis, the buffer pH, the temperature of the detoxification reaction solution, and the like. Methods for determining the optimal concentration are known to vaccine workers.

The method of adding amino acids, amines, or amides in this treatment can be added at once from the beginning, or divided addition can be performed once or twice a week.

The pertussis bacteria suitable for producing the pertussis vaccine of the present invention is not particularly limited. That is, any of clinical isolates (ie, wild strains), artificial mutants, and genetically modified strains can be used as long as they have the ability to exhibit antigenicity when used for vaccine production. The toxicity of the pertussis vaccine produced by the present invention (reversal toxicity after detoxification) is determined by the methods prescribed in the biopharmaceutical standards (mouse weight loss test, mouse leukocyte increase test, histamine sensitization test, and It can be examined by abnormal toxicity negative test). Among them, the relatively high detection sensitivity of the histamine sensitization test method is known to those who handle vaccines.

[0018] In the present treatment method, the mechanism of the effect of inhibiting the reversion of toxicity of amino acids, amines, and amides is considered as follows, using lysine as an example. On the surface of pertussis cells, protein toxins, large polysaccharide structures such as endotoxins and other high molecular weight acidic molecules are intricately present, so it is thought that some of the toxins are covered and hidden. In addition, since formalin molecules have affinity for polysaccharides, they are trapped here and are difficult to reach a part of the free amino group of the toxin. Therefore, in the absence of amino acids, formalin inactivates the toxins exposed on the cell surface, but cannot inactivate the hidden toxins when detoxified by the conventional method. Some toxins retain activity It is thought that it remains hidden. This is a semi-detoxified product that is prone to return to toxicity. Compared to inactivating free protein toxins, it is more likely that some toxins will remain active when whole cells are treated. Here, as in the present invention, when a basic amino acid such as lysine is added, lysine has a property of forming a salt with an acidic substance, and some polysaccharides have an affinity. Can bind to any forces of cell surface endotoxins and other large structures. At this time, it is easily imagined that V, a part of formalin, and a basic amino acid are interchanged until then. In fact, the present inventors observed that a part of formalin was released when formalin was added to a pertussis cell suspension and lysine was added after one week. When lysine is newly bound somewhere, a structural change occurs in the complex polysaccharide molecule on the surface of the cell, and so far, the part of the toxin is exposed. At this time, free formalin present in the vicinity binds to the newly exposed part. In other words, it is envisioned that formalin detoxification will become more complete. In this way, it can be explained that a whole cell pertussis vaccine that is difficult to recover from toxicity can be produced by treatment in the presence of amino acids.

[0019] As a second improvement in the detoxification treatment method of the present invention, a lysis preventing treatment is performed on the cells. That is, in the production of the whole cell pertussis vaccine of the present invention, lysis prevention treatment other than detoxification treatment with formalin or the like is performed.

The lysis prevention treatment may be a chemical treatment, a physicochemical treatment, or a combination thereof. As described later, the specific method of the lysis prevention treatment may be substantially indistinguishable from the detoxification treatment. Therefore, it can be called the second detoxification process. However, since the present inventors performed the above-mentioned treatment for the purpose of preventing lysis separately from the detoxification treatment and confirmed the lysis-preventing effect, it will be referred to as lysis-preventing treatment.

[0020] Conventionally, physicochemical treatment methods and chemical treatment methods have been used for the detoxification treatment of Bordetella pertussis. However, in chemical detoxification treatment with formalin or dartalaldehyde, or detoxification treatment with physicochemical means such as γ-ray treatment or ultraviolet light treatment, only one type of treatment is performed. No detoxification was carried out by combining these. Until now, in the case of detoxification treatment of whole cells, cell death and inactivation of toxins can be confirmed, and in the case of inactivation of free proteins, the disappearance of functional activity can be confirmed. It has been understood that inactivity will be completed if it comes. Therefore, it was considered useless to combine a plurality of detoxification treatments, and it was impossible to try it. The present inventors considered that lysis was one of the causes of the reversion of the toxicity of the whole cell pertussis vaccine produced by detoxification of chemical means, and examined the prevention method. As a result, it was found that in addition to the chemical detoxification treatment, additional lysis prevention treatment using physical and physical means is effective for quality stability of the kitchen.

[0021] The lysis prevention treatment can be carried out by various chemical treatments and physicochemical treatment methods. The following chemicals and chemicals that can be used as chemical treatment substances used to treat bacterial cells for the purpose of lysis prevention treatment are shown below, but are not limited thereto. Chemically treated substances and their concentrations>

Formalin (0.1-10 v / v%), phenol (0.1-5 v / v%), black mouth form

(10-60 v / v%), acetone (10-80 v / v%), SH reagent (1-100 mM),

Hydrogen peroxide (0.1-5%), peracetic acid (0.5-10 w / v%), carbon dioxide (5-90 v / v%), ozone (0.1-10 v / v%), surfactant (0.01 -5 w / v%),

[0022] In addition, the following means can be used for the physicochemical treatment. An example of conditions that can be used is also shown, but the present invention is not limited to these.

Physicochemical treatment method and conditions>

Heating treatment (temperature: 30-70 ° C; heating time: 10-120 minutes), γ-ray irradiation (source: cobalt 60; 5-50 kGy (kilo gray)), laser light irradiation (light source: various laser irradiations) Equipment; Wavelength 500-700 mn; Light intensity: 0.01-1 J (joule) / cm ² ), electron beam irradiation (microwave oven), ultrasonic irradiation

It is also possible to apparently prevent lysis by microcapsulating the whole cell. One or more of these methods can be used in combination. You may repeat the same method multiple times.

[0023] The treatment conditions can be set appropriately by changing the amount of bacterial cells, temperature, pH of the buffer solution, treatment time, etc. that are not fixed. It is usually operated under aseptic conditions.

Depending on the lysis prevention treatment conditions, the antigenic protein may be excessively denatured and lose its antigenicity as a result of the treatment. Therefore, the immunogenicity and other properties should be examined after treatment, and the optimum means and optimum treatment conditions should be considered comprehensively. In the vaccine production process of the present invention, the lysis treatment time is preferably before detoxification. However, it can be carried out even after detoxification.

[0024] The bacteriolysis-preventing treatment of the present invention performs at least one of a chemical detoxification treatment and a physicochemical detoxification treatment. When detoxification is performed by combining both treatments, physicochemical detoxification treatment may be performed after chemical detoxification treatment, or chemical detoxification treatment is performed after physicochemical detoxification treatment. May be.

Whether or not lysis occurs during storage of the whole cell pertussis vaccine product produced according to the present invention can be determined by measuring the turbidity of the product. This method is well known to specialists dealing with microorganisms.

[0025] The mechanism of the lysis preventing treatment effect of the present invention is considered as follows.

In the process of chemical detoxification of all cells, the cell surface functional protein is inactivated and the cells die, but the autolysis activity in the vicinity of the cell membrane is lower than that of untreated cells but remains partly active. There is a case. The remaining autolytic activity does not fully develop under the low temperature conditions in which the vaccine is stored, but it acts little by little during long-term storage over several months to several years, eventually lysing dead cells. On the other hand, when chemical detoxification treatment is followed by additional treatment using a physicochemical method, the remaining autolytic activity is further reduced, and as a result, no lysis is expected to occur over a long period of time. The facts that support this presumption are that heat treatment, γ-ray treatment, and PCMB treatment, which have been confirmed to have a treatment effect, are common in that they have the effect of causing irreversible changes in the enzyme protein.

[0026] An improvement in the third detoxification treatment method of the present invention is that the cells are subjected to a purified pure treatment. That is, in the production of the whole cell pertussis vaccine of the present invention, purification and purification treatment is performed before or after detoxification.

[0027] The present invention provides a mixed bacterial vaccine in which a bacterial component vaccine derived from the same bacteria as the above-described improved whole cell vaccine is mixed. The present invention also relates to a method for producing the above mixed bacterial vaccine.

Here, the invention relating to the pertussis vaccine, which is one of the above bacterial vaccines, will be described in detail.

The power to use ellipsis often in the following descriptions: The The whole cell pertussis vaccine is written as “wP”. The cell-free pertussis vaccine is referred to as “aP”. In addition, the mixed pertussis vaccine of the whole cell pertussis vaccine and the cell-free pertussis vaccine of the present invention is referred to as “awP”.

The mixed pertussis vaccine (awP) of the present invention will be described. The awP of the present invention is produced by mixing a whole cell pertussis vaccine (wP) below the effective amount and a cell-free pertussis vaccine (aP) below the effective amount. As described in Examples 4 and 5, awP has the same efficacy as the whole cell pertussis vaccine and the safety comparable to the cell-free pertussis vaccine.

[0028] The improved whole cell pertussis vaccine obtained by improving the detoxification method described above is suitable as the wP used in the production of awP of the present invention. However, whole cell pertussis vaccine produced by the conventional method can also be used.

The method for producing aP used for the production of awP of the present invention is described in publicly known literature and can be used. An outline of the method adopted by the present inventors as an example of the conventional production method is shown below.

Inoculate Bordetella pertussis (Phase I) into a suitable medium (eg, Koenwihr modified liquid medium) and incubate at 35 ° C for 4-5 days. Centrifuge the culture and add salting-out agent to the supernatant. In other words, ammonium sulfate is added so that it becomes about 50% saturated, or alcohol is added in an amount that becomes about 60% (v / v), and the resulting precipitate is added at a speed of about 10,000 rpm. In this case, the precipitate is collected with a buffer solution containing sodium chloride and sodium chloride, and the extracted fraction is subjected to sucrose density gradient centrifugation to recover the protein fraction containing pertussis toxin and the like. Next, the collected protein fraction is detoxified with formalin to obtain a cell-free pertussis vaccine. If necessary, it can be adsorbed on aluminum gel.

[0029] The amount of wP and aP used in the production of awP of the present invention will be described. Authorized !, wP usually contains approximately 20 X 10 sup 9 cells / mL. According to WHO standards, wP should contain no more than 20 X 10 sup 9 cells per mL, and the titer should be at least 8 international units / mL. In contrast, the vaccine of the present invention contains (1-20) × 10 sup 9 cells / mL bacterial cells. That is, it may be less than the number of cells in the approved vaccine. As a result, it is possible to reduce the effectiveness of the product as it is. Can reduce the toxicity. In particular, when the improved whole cell pertussis vaccine of the present invention is used, the improvement effect is remarkable.

[0030] On the other hand, the approved aP usually contains about 15 mcgPN / mL of protein containing toxin (as protein nitrogen content). According to Japanese biopharmaceutical standards, aP should contain no more than 20 mcg / mL as protein nitrogen (referred to as PN), and the titer must be at least 8 international units / mL. On the other hand, the vaccine (awP) of the present invention is mixed so that ap is contained in 0.1-20 mcgPN / mL. In other words, the amount contained in a vaccine approved in accordance with the Japanese Biopharmaceutical Standard can be used. This ensures safety while maintaining the effectiveness of aP.

[0031] The mixing ratio of wP and aP can be freely changed as long as the effectiveness as a vaccine is maintained. The awP of the present invention thus produced has the same effectiveness as wP and the safety equivalent to aP. Also, since wP is included, the manufacturing cost is low. When manufacturing costs are taken into consideration, a P content is low, and in some cases it is inexpensive.

In the present invention, the method for mixing wP and aP is not particularly limited. Usually, each Balta solution is produced and then physically mixed to achieve the desired ratio. Afterwards, dispensing and stoppering will complete the vaccine. The vaccine may be in solution or, if necessary, powdered by lyophilization. The quality of the new pertussis vaccine awP must meet the quality standards approved by the supervisory authority.

[0032] wP and aP used in the vaccine of the present invention alone may be an effective amount or less.

In the present invention, the effective amount indicates a range of 1-20 × 10 sup 9 cells / mL for wP and 0.1-20 meg PN / mL for aP. Nevertheless, the awP of the present invention exhibits the same degree of immunogenicity as conventional concentrated vaccines. The reason is explained as follows. Since this vaccine contains pertussis cells and pertussis toxin, it is considered that the vaccine will be closer to natural infection. It can also be explained from the relationship between toxin protein concentration and toxin activity. Toxin protein dose response curves are often not linear. In the case of pertussis toxin, the change is sigmoidal. To a certain extent, antigenicity is substantially maintained despite dose reduction, with further reduction in geometrical series. The dose-response curve of endotoxin in wP decreases geometrically with decreasing dose To do. The dose-response curve of wP also changes geometrically. Increase aP by a small amount! In other words, there is a range where the antigenic action increases geometrically. Now, when two vaccines of wP and aP are mixed in small amounts, within a certain appropriate concentration range, the decrease in wP will reduce immunogenicity and toxicity. Addition of aP restores immunogenicity while maintaining reduced toxicity. The awP vaccine of the present invention has an effect equivalent to wP, and the decrease in toxicity associated with wP is explained by the above consideration.

[0033] The nature of the toxin protein contained will be described. Pertussis toxin contained in aP used in the present invention is a protein having an action of activating adenylate cyclase activity on target cells, and has a strong adjuvant action. Considering this point, it is explained that the effectiveness is remarkably recovered by adding a small amount.

The above method for producing a mixed bacterial vaccine can also be applied to bacterial vaccines derived from other bacteria.

The present invention provides a safe and effective mixed vaccine comprising at least one mixed bacterial vaccine described above and at least one other vaccine antigen. The present invention also relates to a method for producing the above combination vaccine.

[0034] The improved whole cell bacterial vaccine of the present invention and the mixed bacterial vaccine of the present invention can be mixed with another vaccine antigen and used as a mixed vaccine.

For example, conventional pertussis vaccine may be used as a DTP triple vaccine mixed with tetanus toxoid and diphtheria toxoid. Similarly, wP and awP of the present invention can be used as a DTawP vaccine after being mixed with tetanus toxoid and diphtheria toxoid. In addition, the DTP vaccine can be used in combination with a fourth antigen. The fourth vaccine antigen includes, but is not limited to, influenza virus vaccine, meningococcal vaccine, oral polio vaccine, inactive polio vaccine, hepatitis vaccine, and Japanese encephalitis vaccine.

[0035] It is known to add an effective adjuvant to a vaccine to enhance the effect of the vaccine. Therefore, an appropriate adjuvant can be added to the vaccine of the present invention (including a combination vaccine). Adjuvants used in the vaccine of the present invention include hydroxyaluminum and its inorganic salts, hydrocarbons such as squalene and oil, bacterial toxins such as cholera toxin, sabo- Examples include, but are not limited to. In general, there is no regularity in the type of adjuvant effective to enhance the immunity of a vaccine antigen. The type of effective adjuvant varies depending on the administration method. However, the type and concentration of vaccine effective for the vaccine can be determined by trial and error methods known to those skilled in the art.

[0036] The cell-free pertussis vaccine (aP) is usually bound to aluminum gel. The aP used for the vaccine awP of the present invention may be bound to an aluminum adjuvant. The awP of the present invention has a migel present in the awP, but contains aP reduced in amount from the antigen amount of the approved vaccine, so the amount of aluminum is reduced proportionally. Moreover, aP used for the vaccine awP of the present invention may be a free form. When free aP is used, there is no aluminum in awP. A vaccine with reduced aluminum content is expected to have the effect of reducing undesirable side reactions that may be based on aluminum. For example, there are few lumps at the site of inoculation, and there is little possibility of neurotrophic effects that are sometimes said to be due to aluminum.

As a customary vaccine, the vaccine product of the present invention can contain a stabilizer (gelatin), a preservative (thimerosal, phenoxyethanol), a colorant (phenol red) and the like in addition to the antigen.

[0037] As a method for inoculating the bacterial vaccine of the present invention, various known methods can be used. That is, since the whole bacterial cell vaccine and the cell-free bacterial vaccine are inoculated by subcutaneous injection or intramuscular injection, the vaccine of the present invention can be inoculated by subcutaneous injection or intramuscular injection. Furthermore, nasal inoculation, oral inoculation and transdermal inoculation are possible if an appropriate dosage form is selected.

All prior art documents cited in this specification are incorporated herein by reference.

Example

[0038] [Example 1] Production of whole cell pertussis vaccine detoxified in the presence of amino acids and the like An improved whole cell pertussis vaccine obtained by detoxification in the presence of amino acids and the like is stored for a long period of time. During this time, it was examined whether or not toxic reversion occurred. The test implementation method is as follows.

Strain Bordetella pertussis (Phase I)

Medium: Koenweiler Modified liquid medium Culturing: The test strain was inoculated into the medium and cultured in a tank with aeration and agitation at around 35 ° C for 1 day.

The obtained cells were washed by centrifugation, and suspended in a phosphate buffer solution containing sodium chloride and sodium chloride (PBS 0.01M, pH 7) so that the turbidity at 650 nm was 20. This lOOmL was transferred to a 500 mL glass container. (The height of the liquid surface is about 2cm). One amino acid or other test substance at a concentration of 0.05 M was added thereto. After that, it was detoxified by leaving it in 0.5% (v / v) formalin for 5 weeks as per the manufacturing method of whole cell pertussis vaccine. Next, after removing formalin and added substances by centrifugation, the number of bacteria was adjusted to 20 × 10sup9 / mL in PBS. After that, thimerosal (0.01 w / v%) was added, and 1.3 mL was dispensed into vials to prepare a whole cell culture vaccine.

Each knob was stored in a cold place at 2-10 ° C. The seals were opened after 6 months and 2 years, and a total of 5 vials prepared under the same conditions were combined into one sample. Using 5 specimens, quality control tests were conducted in accordance with Japanese biopharmaceutical standards. Table 1 shows the results of the histamine sensitization test that can detect reversion of toxicity (measured values after heating at 37 ° C).

[0039] [Table 1]

[0040] The numbers displayed in the histamine sensitization test results indicate the following ranges, and the unit is HSU / mL. 0: 0.1 or less

1: 0.1-0.2

2: 0.2-0.4

3: 0.4 or more

From the above results, it was revealed that the preparations detoxified with formalin in the presence of amino acids such as glycine, lysine or threonine are stable during storage (Table 1: after 2 years). Toxicity recovery was observed in the product (control) that was detoxified without the addition of amino acids. Even when detoxified in the presence of amines, amides, or other amino acids (alanine, γ-aminobutyric acid, arginine, glutamic acid, leucine, isoleucine, or serine), there is only a slight reversal of toxicity. It was. However, reversion to toxicity was prominent in untreated subjects.

[0041] [Example 2] Bacteriolytic treatment of Bordetella pertussis

We examined the lysis during long-term storage of products that had been subjected to detoxification treatment and lysis prevention treatment for B. pertussis. The test method is as follows.

The cells obtained by culturing in the same manner as in Example 1 were washed in the same manner as in the case of whole cell pertussis vaccine production, and suspended in 0.01M PBS (pH 7) to a turbidity of 20. did. This lOOmL was transferred to a 500 mL glass container. (The height of the liquid surface is about 2cm). In Table 2, treatment method 1, the chemicals listed were added to the final concentrations as described. In treatment method 2, it was left in a diacid-carbon incubator. In treatment method 3-6, the treatment was performed as it was. In either case, light agitation was appropriately performed during the treatment. After the treatment, the treated cells were washed twice with a buffer solution and resuspended in the same buffer solution. Thereafter, as indicated in the table, it was detoxified with formalin in the presence of 0.05 M lysine. The bacterial suspension of each treatment was dispensed in 1.3 mL vials after removing formalin according to the vaccine production method. In this way, a whole cell pertussis vaccine with the treatment of 1-7 was prepared.

[0042] Each nozzle was stored in a cold place at 2-10 ° C. Two years later, the vial was opened, and five vials prepared under the same processing conditions were combined into one sample. After seven samples of seven kinds of 17 were appropriately diluted, turbidity was measured at a wavelength of 650 nm. This value was compared with the turbidity of Control 2 immediately after production. The results are shown in Table 2. [0043] [Table 2]

[0044] Turbidity at a wavelength of 650 nm is as follows:

+ + +: 0. 8- 1. 0

+ +: 0. 5-0. 8,

+: 0. 2-0. 5,

±: 0.2 or less,

1: Shows colorless and transparent.

From the above results, it has been clarified that the decrease in turbidity after 2 years is reduced when acetone treatment or heating treatment is applied. In the case of no treatment (control 1), lysis was remarkable. Comparison of treatment methods 3 and 4 and treatment methods 5 and 6 revealed that lysis was reduced when both lysis prevention treatment and detoxification treatment were applied.

[Example 3] Production of cholera vaccine

Whole cell cholera vaccine was manufactured and the quality was examined.

Test strain: Vibrio cholera Ogawa strain (type S), Inaba strain (type S) Medium: Ordinary agar medium

Cultivation: 37 ° C for 20 hours

The obtained microbial cells were washed in the same manner as in the case of whole microbial cholera vaccine production, and suspended in 0.015M PBS (pH 7) to a turbidity of 10. This lOOmL was transferred to a 500 mL glass container. (The height of the liquid surface is about 2cm). It processed with the processing method 1-11 as it was. In either case, the glass container being treated was gently stirred as appropriate. After treatment, the cells are washed by centrifugation and resuspended in PBS. It was. According to the method for producing whole cell cholera vaccine, 0.5% (v / v) formalin was added to make it nontoxic. Three weeks later, formalin was removed by centrifugation. The bacterial suspension of each treatment was added with phenol according to the cholera vaccine production method, then 10 mL was dispensed into vials to prepare 1-11 treated whole cell cholera vaccine.

Each knob was stored in a cool place around 5 ° C. Two years later, the vial was opened, and a sample prepared under the same conditions was used as the specimen. Two specimens were used for each treatment method, diluted as appropriate, and turbidity was measured at a wavelength of 650 nm. Table 3 shows the result compared with the turbidity of the control immediately after production.

[0046] [Table 3]

[0047] PCMB description: Abbreviation for p-choloro-mercuribenzoate. It is a type of SH reagent (ie, a reagent that reacts strongly with the SH group of proteins).

The symbols in the turbidity column have the following meanings. Turbidity at 650 nm is + + +: 0. 8- 1. 2; + +: 0. 5-0. 8; +: 0. 2-0. 5,

±: 0.2 or less; 1: Colorless and transparent.

Based on the above results, no treatment (control 1) was observed when acetone treatment or heating treatment was applied. In comparison, the decrease in turbidity after 2 years was reduced (Table 3). No treatment (Control 1

In the case of), the lysis was remarkable.

[0048] Next, a vaccine was prepared by the method of treatment method 4 in Table 3 and administered to humans. Table 4 summarizes the results of comparing the two.

[0049] [Table 4]

[0050] From the above results, it became clear that the safety of the cholera vaccine of the present invention has been improved (Table 4).

[Example 4] Production of new pertussis vaccine awP

A whole cell pertussis vaccine wP and a cell-free pertussis vaccine aP were mixed to produce the novel pertussis vaccine awP of the present invention, and the results of examining its effectiveness and safety are shown.

Whole cell pertussis vaccine wP used the improved whole cell pertussis vaccine prepared in Example 2 (Table 2, Test No. 3) and prepared by a formalin detoxification treatment in the presence of lysine. The cell-free pertussis vaccine aP used an aluminum adsorption product manufactured by Kitasato Institute (Japan).

A new pertussis vaccine awP was produced by mixing wP and aP in various amounts. In order to investigate the effectiveness and safety of the vaccine, a quality control test was conducted using the method specified in the 2004 Japanese Biologics Standards. The results are shown in Table 5. In the table, “#” and “*” indicate whether each measurement value conforms (#) or does not conform (*) compared to the Japanese biologics standard for acellular pertussis vaccine (partly self-standard). .

[0052] [Table 5] Fuku wP aP BWD IP HS Tox Tox Ab Ab P Chin 10sup9 meg / mL / mL / mL Dl D7 PT FHA IU

cells PN / mL ML

/ mL

A1 3 6 # 0.05 # 0.06 # 5 35 8 * 15 * 7 *

A2 7.5 8 # 0.08 # 0.09 # -7 31 11 * 19 * 31 #

A3 15.0 6 # 0.09 # 0.15 # -8 30 12 # 30 # 39 #

W1 10 137 * 0.11 # 0.12 # -7 34 13 # 13 * 11.6 #

W2 20 350 * 0.14 # 0.18 # -15 17 10 * 17 * 13.6 #

AW1 10 3 245 * 0.15 # 0.15 # -23 31 15 # 26 # 24 #

AW2 20 3 209 * 0.12 # 0.14 # -29 22 12 # 23 # 28 #

DTwP 1083 * 5.1 * 4.5 * -35 -6 2 * 321 # 24.9 # Reference value 20 15 10 0.5 0.4 -8 20 12 20 8

Below Below Below Below Above Above Above Above Above Vaccine A1-A3: Cell-free pertussis vaccine, the amount used is expressed in protein nitrogen (meg PN / mL).

Vaccine W1-W2: Whole cell pertussis vaccine, the amount used is indicated by the number of cells (unit: billion, 10sup9).

Vaccine AW1-AW2 :: A pertussis vaccine awP in which a cell-free pertussis vaccine and a whole cell pertussis vaccine are mixed.

DTwP: diphtheria, tetanus, whole cell pertussis, triple vaccine (commercially available).

[0053] The meanings of the abbreviations indicating the test results are as follows.

Toxicity indicators

BWD (mouse weight loss toxicity) Japanese biologics

LP (Mouse leukocyte increase toxicity) Japanese biologics

HS (mouse histamine sensitive toxicity) Japanese biologics

Tox (Negative toxicity test in guinea pigs) Japanese biologics, body weight values are shown.

Effectiveness metrics

Ab (antibody titer) self-standard

P (Title of pertussis vaccine by mouse brain attack method) Japanese biologics

[0054] The novel pertussis vaccine AW1 of the present invention is acceptable according to WHO biopharmaceutical standards for wP This vaccine contains half the maximum bacterial quantity of 20 x 10 sup 9 / mL and 1/5 of the amount of toxic protein nitrogen in vaccines approved for aP. As is clear from Table 5, AW1 is a toxic indicator of LP (mouse leukocyte increased toxicity), HS (mouse histamine sensitive toxicity), and Tox (guinea pig abnormal toxicity negative test). The measured values that met the formulation criteria were shown. Also, it can be seen that the titer (mouse brain attack method) is also suitable. However, BWD (mouse weight loss toxicity), which is considered to reflect toxicity due to endotoxin, etc., was not suitable. It can be seen that the commercial DTwP used as a control does not meet some Japanese biopharmaceutical standards (2004 edition) regarding the quality of the cell-free pertussis vaccine!

[0055] [Example 5] Measurement of effectiveness of awP by spray infection method

AwP was prepared in the same manner as in Example 4. The effectiveness of awP was examined by spray infection. The spray infection method was adopted to examine the effectiveness of this awP. The intracerebral aggression method used to examine the titer in Example 4 was adopted as the standard method for measuring the titer of whole cell bodies and cell-free pertussis vaccine in the Japanese Biologics Standards. Not used in other countries. There is no standard titration method in Western countries or WHO biopharmaceutical standards! However, the spray infection method is one of the standard assay methods. Therefore, this method was adopted.

[0056] The outline of the titer measurement method by spray infection is as follows. Details of the method are described in the literature (1) Watanabe M, Komatsu E, Abe K, lyama S, Sato T, Nagai M. Efficacy of pertussis components in an acellular vaccine, as containing in a murine model of re spiratory infection and a murine model of intracerebral infection. Vaccine 2002; 20: 1429-1434.

(2) Watanabe M, Nagai M. Reciprocal protective immunity against Bordetella pertu ssis and Bordetella parapertussis in a murine model of respiratory infection. Infect I mmun 2001; 69: 6981-6986.)

[0057] Mice (3.5 weeks old) were inoculated intraperitoneally with 0.5 mL of test vaccine diluted in phosphate buffered saline (PBS) per mouse. Three weeks later, spray infection was carried out by the following method. The mouse was placed in a net cage and suspended in an acrylic spray box installed in a safety cabinet. On the other hand, Bordetella pertussis (18-232 strain) cultured at 37 ° C for 30 hours is suspended in ice-cold PBS. Prepared to 0 X 10 sup 9 cells / mL. The prepared bacterial suspension was placed in a nebulizer and connected to a spray-contamination box, and then aerosol was generated through filtered sterilized air (3.5 to 4.0 L / min). This aerosol was introduced into a spray infection box and inhaled by mice. The cage was rotated 90 degrees once every 5 minutes so that the mouse evenly inhaled the aerosol of the fungus solution. After spraying, filtered sterilized air was introduced for 30 minutes to remove the aerosol in the spray infection box, and the mouse was removed. Mice were bred aseptically for 2 weeks, and then the cervical dislocation mouse force was removed aseptically and homogenized in 10 mL of ice-cold PBS. This solution was used as a lOsup (-l) diluted solution, and 10-fold serial dilution was performed. The diluted solution was smeared on Borde's DiYoung medium and cultured at 37 ° C for 4 days. The number of viable bacteria in the lung is 7 colonies forming unit (CFU).

The results are shown in Table 6.

[0058] [Table 6]

[0059] Test No. 3 (W20) is approved and contains an equivalent amount of the whole cell pertussis vaccine.

Test number 8 (A15) is approved and contains the same amount as a cell-free pertussis vaccine. When these two were inoculated, the number of viable bacteria decreased by about 21 og compared to the non-inoculated control (Test No. 9). If this is the effective level, it can be determined that test number 5 (W10 + A3) is effective.

[Example 6] DTwP triple vaccine In accordance with the DTaP manufacturing method manufactured by Kitasato Institute (Japan), wP manufactured by the method of Example 2 (Table 2, processing method 3) was used instead of aP. A triple mixed vaccine DTwP containing the pertussis vaccine wP of the present invention, diphtheria toxoid (D), and tetanus toxoid (T) was prepared. The titer of each component antigen was measured by the method of Japanese biopharmaceutical standards. The results are shown in Table 7.

[0061] [Table 7]

[0062] The results were based on the tested titer tests and all met Japanese biopharmaceutical standards.

[0063] [Example 7] Combination vaccine of DTawP and Japanese encephalitis vaccine (ccJE)

AwP was produced by the method of Example 4 (Table 5, test symbol AW1). According to the DTwP production method, the above-mentioned awP was used in place of wP, and a triple vaccine DTawP containing awP, diphtheria toxoid (D), and tetanus toxoid (T) was prepared. Furthermore, a mixed vaccine (DtawP-ccJE) was prepared by mixing this with the Japanese encephalitis vaccine (ccJE) produced by the cell culture method. For the Japanese encephalitis vaccine, a prototype manufactured by Kitasato Institute (Japan) was used. The titer of each antigen component was measured by the method of Japanese biopharmaceutical standards. The results are shown in Table 8.

[0064] [Table 8] Test item Japanese biopharmaceutical standard Measured value Judgment Toxicity test Abnormal toxicity denial test No abnormality No abnormality Applicable mouse histamine 0.4 unit or less 0.08 unit ® „Sensitization test

Diphtheria No abnormalities No abnormalities

? S

Tetanus toxin No abnormalities No abnormalities Conformity Detoxification test

Titer test Pertussis vaccine 8 units or more 33 units Applicable diphtheria toxoid 47 units or more 71 units Applicable tetanus toxoid 27 units or more 32 units Applicable

JE titer Standard product or higher Standard product (Beijing Co.) Fit (after mixing) or higher

JE titer More than standard product Standard product (Beijing Co.)

(Before mixing)

[0065] From the above results, it was clarified that all the titer tests tested conformed to the Japanese biopharmaceutical standards of DTaP. The titer of the Japanese encephalitis vaccine (ccJE) after mixing was substantially unchanged or somewhat higher than before mixing.

[Example 9] Production of whole bacterial cell vaccine

Test bacteria: Pertussis and Vibrio cholerae used vaccine production strains. For other strains, laboratory-stored strains were used.

Culture method: Bordetella pertussis and parapertussis were obtained by culturing as in Example 1. Vibrio cholerae was cultured as in Example 3. Other bacteria were smeared on a normal nutrient agar medium and cultured at 33 ° C for 16-24 hours. The bacterial cells obtained were washed by centrifugation, and the turbidity at 650 nm was 10 (approximately 1x10 sup 9 cells / mL) in phosphate buffered saline (PBS 0.001M, pH 7). Suspended. This lOOmL was transferred to a 500 mL glass container. This was treated by the following treatment method to obtain a whole bacterial cell vaccine solution.

Treatment method: Heating was performed at 55 ° C for 30 minutes. Acetone treatment was performed by adding an equal volume of acetone and treating with stirring at room temperature for 12 hours, and this was repeated twice. Formalin treatment, formalin was added to a final concentration of 0.5% (v / v) and left at room temperature for 3 weeks. In either case, after treatment, the treatment agent is removed by washing and centrifugation, and the treated cells are washed with sodium chloride-added phosphate buffer (PBS) ( Resuspended at 0.001M, pH 7) at 650 nm. Each vial was dispensed 1.3 mL and stored in a cool place.

[0067] Stability measurement method: Each vial was stored in a cold place at 2-10 ° C. One year later, the vial was opened, and five samples of samples prepared under the same processing conditions were combined to make one sample. After each specimen sample was diluted, turbidity was measured at a wavelength of 650 nm. The value was compared with the turbidity of the control immediately after production. The results are shown in Table 9.

Immunization of mice: 1 mL of a suspension of bacterial cells that had been subjected to various treatments (immediately after the start of storage) was intraperitoneally administered to mice and inoculated three times at 3-week intervals. The final inoculation force and the mouse force were collected 1 month later to obtain antiserum.

Antibody detection method: Antiserum was removed, a dilution series was prepared with PBS, and 0.05 mL was dispensed into a 96-well microplate. To this, 0.05 mL of detoxified bacteria (number of cells: about 10 sup 8 / mL) used as an antigen was added, the precipitation reaction was observed, and the antibody titer was calculated from the antiserum dilution rate. Antibody titers are shown in Table 9.

[0068] [Table 9]

Strain Treatment Stability After 1 year in mice Antibody titer Pertussis H F Lysate> 1 2 8

Bordetella pertussis

Bordetella, a parapertussis of paralysis> 3 2 parapertussis

Salmonella typhimunum> 1 6 Salmonella typhimunum

Escherichia coli THEC A / F lysis mitomimes> 3 2 Escherichia coli

Vibrio cholera> 3 2 Vibrio cholera

Yellow bud staphylococcus H / F lysate> 6 4 Staphylococcus aureus

A / F lysis mitomime> 6 4

Bacillus brevis

Streptococcus pneumoniae H / F lysis mitome> 3 2

Streptococcus pneumoniae [0069] Meaning of treatment symbols: H warming treatment; A. Acetone treatment; F formalin inactivation Bacterial mitochondrome: Turbidity at wavelength 650 nm is 0.8-1.2 and turbidity of control is 1. Indicates 0.

From the results in Table 9, it became clear that a whole cell vaccine with good storage stability was produced in any of the tested strains.

Industrial applicability

[0070] Even bacterial vaccines manufactured by detoxifying pathogenic bacteria have often been used to restore toxicity in the course of storage, and have returned to toxicity in long-term storage. The absence was very important in maintaining vaccine safety. The whole bacterial cell vaccine of the present invention has a property that safety is easily ensured even when stored for a long period of time, compared to conventionally produced vaccines, and is very useful in the medical and pharmaceutical fields. Since it can be stored for a long time, the vaccine of the present invention can be used all over the world. In the case of pathogens that are violent for a long time, the pathogens may mutate over time, and it is necessary to store various types of vaccines for a long time in order to cope with the prevention of infection by these pathogens. There is. The vaccine production method of the present invention can be said to be very effective when there is a need for such long-term storage.

In addition, the mixed bacterial vaccine of the present invention has the same or higher effectiveness as the whole bacterial vaccine produced in the past, can be manufactured at low cost, is suitable for mass production, and is comparable to the cell-free bacterial vaccine. It is expected to be a safe vaccine.

Claims

The scope of the claims

[1] A whole cell bacterial vaccine obtained by the treatment described in (a) and (b) below, which does not return to toxicity even after long-term storage!

(b) Bacterial lysis prevention treatment

[2] The whole bacterial cell vaccine according to claim 1, wherein the long-term storage period is 1 year or more, preferably 3 years or more, more preferably 5 years or more, and less than 6 years.

[3] Amino acid strengths described in (a): aspartic acid, γ-aminobutyric acid, alanine, β-alanine, arginine, glycine, glutamic acid, isoleucine, leucine, lysine, serine, threninin, and balinka are also selected The whole bacterial cell vaccine according to claim 1 or 2, which is at least one amino acid.

[4] The amine described in (a) is at least one amine selected from ethylamine, ethanolamine, and propanolamine, or the amide described in (a) is urea, glycinamide, and The whole bacterial cell vaccine according to any one of claims 1 to 3, which is at least one amide selected from j8-alanylamide.

[5] The lysis prevention treatment according to (b) is at least one treatment method in which acetone treatment, heating, γ-ray irradiation, electron beam irradiation, and laser light irradiation power are also selected. 4. The whole bacterial cell vaccine according to any one of 4 above.

[6] The whole bacterial cell vaccine according to any one of claims 1 to 5, wherein the bacterium is a clinical isolate, an artificial mutant thereof, or a genetically modified strain.

[7] A mixed bacterial vaccine comprising the whole bacterial vaccine according to any one of claims 1 to 6 and a bacterial component vaccine derived from the same bacteria.

[8] The mixed bacterial vaccine according to claim 7, wherein the whole bacterial vaccine and the sputum or bacterial component vaccine alone are less than the effective amount.

[9] Bordetella pertussis, Bordetella parapertussis, Salmonella typhi, Escherichia coli, Vibrio cholerae (Vibrio c holera) The bacterial vaccine according to any one of claims 1 to 8, which is any one of ~ Staphylococcus aureus ^ or 7 (Streptococcus pneumoniae).

[10] A mixed vaccine comprising at least one bacterial vaccine according to claim 9, and at least one vaccine antigen derived from other bacteria and Z or viruses.

[11] Vaccine antigen power Diphtheria toxoid, tetanus toxoid, H. influenzae vaccine, meningococcal vaccine, oral polio vaccine, inactive polio vaccine, hepatitis vaccine, and Japanese encephalitis vaccine are also at least one antigen selected The combination vaccine according to claim 10.

[12] A method for producing a whole cell bacterial vaccine having the characteristics that is obtained by the treatment described in the following (a) and (b), and does not return to toxicity even in long-term storage!

(b) Bacterial lysis prevention treatment

[13] Amino acid strengths described in (a): aspartic acid, γ-aminobutyric acid, alanine, β-alanine, arginine, glycine, glutamic acid, isoleucine, leucine, lysine, serine, threninin, and balinka are also selected 13. The method for producing a whole bacterial vaccine according to claim 12, wherein the whole bacterial vaccine is at least one amino acid.

[14] The amine described in (a) is at least one amine selected from ethylamine, ethanolamine, and propanolamine, or the amide described in (a) is urea, glycinamide, and 14. The method for producing a whole cell bacterial vaccine according to claim 12 or 13, which is at least one amide selected from j8-alanylamide.

[15] The lysis preventing treatment according to (b) is at least one treatment method in which acetone treatment, heating, γ-ray irradiation, electron beam irradiation, and laser light irradiation power are also selected. 14's

V, A method for producing a whole bacterial vaccine according to any one of the above.

[16] The bacterium is a clinical isolate, an artificial mutant thereof, or a genetically modified strain.

6. A method for producing a whole bacterial cell vaccine according to any one of 5 above.

[17] A method for producing a mixed bacterial vaccine, comprising the step of mixing the whole bacterial cell vaccine according to any one of claims 1 to 6 and a cell-free bacterial vaccine derived from the same bacteria as those.

[18] The method for producing a mixed bacterial vaccine according to [17], wherein the whole cell vaccine and the sputum or cell-free vaccine alone are less than the effective amount.

[19] Bacterial power S. Bordetella pertussis, Bordetella parapertussis ), Salmonella typhi, Escherichia coli, Vibrio c holera (Staphylococcus aureus) ^ 7 This is the moon of the moon 巿! Streptococcus pneumoniae 19. The method for producing a mixed bacterial actin according to any one of claims 12 to 18, which is any of the above.

[20] A method for producing a mixed vaccine comprising at least one bacterial vaccine according to claim 9, and at least one vaccine antigen derived from other bacteria and Z or viruses.

[21] Vaccine antigen power Diphtheria toxoid, tetanus toxoid, H. influenzae vaccine, meningococcal vaccine, oral polio vaccine, inactive polio vaccine, hepatitis vaccine, and Japanese encephalitis vaccine are also at least one antigen selected The method for producing a mixed vaccine according to claim 20.

[22] The vaccine according to any one of claims 1 to 11, which is used for diseases caused by bacteria and Z or viruses.

[23] Bacterial power Bordetella pertussis, Bordetella parapertussis, Salmonella typhi, Escherichia coli, Vibrio cholerae (Vibrio c holera) 23. The vaccine according to claim 22, wherein the vaccine is Staphylococcus aureus ^ 7 is a moon frog flame ¾ (Streptococcus pneumoniae).