JP5207380B2 - Whole cell bacterial vaccine with characteristics that do not return to toxicity even after long-term storage and its use - Google Patents

Description

  The present invention relates to a whole cell bacterial vaccine having characteristics that do not return to toxicity even in 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 mixed bacterial vaccine and others It is related with the combination vaccine with the vaccine. Moreover, it is related with those manufacturing methods and uses.

Pertussis is an infection that develops due to Bordetella pertussis infection and, when affected, has a characteristic cough that lasts for a long time. Severe in early childhood, may be accompanied by pneumonia and 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 give immunity by vaccine from the early stage 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 about whole cell pertussis vaccine and cell-free pertussis vaccine (KM Edwards, MD Decker, EA Mortimer Jr. Vaccine 3rd ed, SA Plotkin and WA Orenstein Ed, WB Saunders co 1999 pp.293-344).

  The whole cell pertussis vaccine has been used clinically since the 1930s, and since the 1950s, it has been used worldwide as a DTP triple vaccine. 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 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, undesirable side reactions such as fever after vaccination and hardening of the inoculated site are likely to occur, and safety issues have been pointed out.

  On the other hand, a cell-free pertussis vaccine was developed in Japan and used from the 1980s to today. The main purpose of developing the cell-free pertussis vaccine was to improve the safety-related drawbacks of whole cell pertussis vaccine. Numerous references have documented the effectiveness of cell-free pertussis vaccines. 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 the cell-free pertussis vaccine is manufactured by purifying and detoxifying the active ingredient as described above, toxic substances such as endotoxin derived from the cells contained in the vaccine are greatly increased compared to the whole cell pertussis vaccine. Has been 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.

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 production cost, were impaired. Against this background, the development of a whole cell pertussis vaccine that is safe and comparable to a cell-free pertussis vaccine while maintaining the effectiveness and low-cost manufacturing costs of a whole cell pertussis vaccine. 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 standard of the cell-free pertussis vaccine are different in each country, and no global standard is probable.
In addition, as with the pertussis vaccine, the whole bacterial bacterial vaccine derived from other bacteria is a whole bacterial bacterial vaccine that has safety comparable to that of a cell-free bacterial vaccine while maintaining low manufacturing costs. Development is currently being sought worldwide.

Prior art document information related to the invention of the present application is shown below.
JP-A-4-230328 JP 5-503628 KM Edwards, MD Decker, et al: "Vaccines", third ed., SA Plotkin and WA Orenstein (ed.), Pp.293-344.WB Saunders Co., 1999. KM Edwards, BD Meade, et al. Pediatrics 96: 548-557. 1995 M Kimura, Dev. Bio. Stand. 73: 5-9. 1991

  The present invention has been made in view of such circumstances, and an object of the present invention is to provide a whole cell bacterial vaccine having a feature that does not return to toxicity even during long-term storage and has improved safety. is there. Another object of the present invention is to provide a mixed bacterial vaccine obtained by mixing the above-mentioned vaccine with a bacterial component vaccine derived from the same bacteria, and to provide a safe and effective mixed vaccine comprising these mixed bacterial vaccine and other vaccine antigens. It is another object of the present invention to provide a production method and a usage method thereof.

In order to solve the above-mentioned problems, the present inventors have investigated the detoxification treatment method for whole cell pertussis vaccine, and examined the safety of a vaccine produced by mixing a plurality of vaccines.
First, the method for detoxifying the whole cell pertussis vaccine was improved. As a result, by performing detoxification treatment in the presence of amino acids or by carrying out lysis prevention treatment and simple purification treatment on pertussis cells, toxicity recovery is difficult and other safety is also improved. We found that we could produce a whole cell pertussis vaccine. Next, the whole cell pertussis vaccine and the cell-free pertussis vaccine with improved safety obtained by the above method were each mixed at a concentration below the effective amount, and the safety of the mixed vaccine was evaluated. . As a result, it has been found that a mixed pertussis vaccine having safety comparable to a cell-free pertussis vaccine can be produced.
In addition, in the case of other bacterial strains, it was found that when the vaccine was produced by the same production method as that of the pertussis vaccine, a whole cell bacterial vaccine having good storage stability was produced.

That is, the present inventors have succeeded in producing a vaccine that is safer than conventional bacterial vaccines easily and inexpensively, thereby completing the present invention.
More specifically, the present invention provides the following [1] to [23].
[1] A whole bacterial cell vaccine obtained by the treatment described in the following (a) and (b), which has a characteristic that the toxicity does not return even in long-term storage.
(A) Cell detoxification treatment in the presence of amino acids, amines, and / or amides (b) Cell lysis prevention treatment [2] Long-term storage period is 1 year or more, preferably 3 years or more, More preferably, the whole bacterial cell vaccine according to [1], which is 5 years or longer and less than 6 years.
[3] The amino acid described in (a) is at least one selected from aspartic acid, γ-aminobutyric acid, alanine, β-alanine, arginine, glycine, glutamic acid, isoleucine, leucine, lysine, serine, threonine, and valine. The whole bacterial cell vaccine according to [1] or [2], which is 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 β- The whole bacterial cell vaccine according to any one of [1] to [3], which is at least one amide selected from alanylamide.
[5] The lysis prevention treatment according to (b) is at least one treatment method selected from acetone treatment, heating, γ-ray irradiation, electron beam irradiation, and laser light irradiation, [1] to [4] The whole bacterial cell 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 gene recombinant strain.
[7] A mixed bacterial vaccine comprising the whole bacterial cell vaccine according to any one of [1] to [6] and a bacterial component vaccine derived from the same bacteria.
[8] The mixed bacterial vaccine of [7], wherein the whole cell vaccine and / or the bacterial component vaccine alone is an effective amount or less.
[9] Bacteria are Bordetella pertussis, Bordetella parapertussis, Salmonella typhi, Escherichia coli, Vibrio cholera, Staphylococcus aureus The bacterial vaccine according to any one of [1] to [8], which is any one of (Staphylococcus aureus) or Streptococcus pneumoniae.
[10] A mixed vaccine comprising at least one bacterial vaccine according to [9] and at least one vaccine antigen derived from other bacteria and / or viruses.
[11] The vaccine antigen is at least one antigen selected from diphtheria toxoid, tetanus toxoid, H. influenzae vaccine, meningococcal vaccine, oral polio vaccine, inactivated polio vaccine, hepatitis vaccine, and Japanese encephalitis vaccine. [10] The combination 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 toxicity recovery does not occur even during long-term storage.
(A) Detoxification treatment of bacterial cells in the presence of amino acids, amines and / or amides (b) Bacterial lysis prevention treatment [13] The amino acid described in (a) is an aspartic acid, γ-amino acid Production of whole bacterial cell vaccine according to [12], which is at least one amino acid selected from butyric acid, alanine, β-alanine, arginine, glycine, glutamic acid, isoleucine, leucine, lysine, serine, threonine, and valine Method.
[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 β- The method for producing a whole cell bacterial vaccine according to [12] or [13], which is at least one amide selected from alanylamide.
[15] The lysis prevention treatment according to (b) is at least one treatment method selected from acetone treatment, heating, γ-ray irradiation, electron beam irradiation, and laser light irradiation, [12] to [14] A method for producing a whole bacterial cell vaccine according to any one of the above.
[16] The method for producing a whole cell bacterial vaccine according to any one of [12] to [15], wherein the bacterium is a clinical isolate, an artificial mutant thereof, or a gene recombinant 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] The method for producing a mixed bacterial vaccine of [17], wherein the whole cell vaccine and / or the cell-free vaccine alone is an effective amount or less.
[19] Bacteria are Bordetella pertussis, Bordetella parapertussis, Salmonella typhi, Escherichia coli, Vibrio cholera, Staphylococcus aureus The method for producing a mixed bacterial vaccine according to any one of [12] to [18], which is any one of (Staphylococcus aureus) or Streptococcus pneumoniae.
[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 / or viruses.
[21] The vaccine antigen is at least one antigen selected from diphtheria toxoid, tetanus toxoid, influenza virus vaccine, meningococcal vaccine, oral polio vaccine, inactivated polio vaccine, hepatitis vaccine, and Japanese encephalitis vaccine, [20] The method for producing a combination vaccine according to [20].
[22] The vaccine according to [1] to [11], which is used for diseases caused by bacteria and / or viruses.
[23] Bacteria are Bordetella pertussis, Bordetella parapertussis, Salmonella typhi, Escherichia coli, Vibrio cholera, Staphylococcus aureus The vaccine according to [22], which is either (Staphylococcus aureus) or Streptococcus pneumoniae.

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, “return 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 been known to frequently revert to toxicity during the storage process, and the absence of reversal of toxicity for a long period of time is necessary to maintain the safety of the vaccine. It is very important. In the present invention, the long-term storage period is preferably 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 parapertussis, Bordetella parapertussis, and Typhi. Mention may be made of fungi (Salmonella typhi), toxin-producing Escherichia coli, Vibrio cholera, Staphylococcus aureus, or Streptococcus pneumoniae. Any of clinical isolates, wild strains, artificial mutants and genetically modified strains can be used as long as the strain used for the production of these whole bacterial vaccines is antigenic.

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, pertussis bacteria (phase I) are inoculated into an appropriate medium (eg, Cohen Wheeler modified liquid medium), and cultured in a tank with aeration and agitation at around 35 ° C. for 1 day. Centrifuge the culture to collect the cells, suspend the cell fraction in an appropriate buffer to adjust the cell concentration, and then leave it in the presence of formalin around 0.5% (v / v) for several weeks. Detoxify by Thereafter, the bacterial cells were washed by centrifugation to remove formalin, and the bacterial cell concentration was adjusted to (10-20) × 10sup9 / mL to obtain a whole cell pertussis vaccine.

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

The whole cell pertussis vaccine of the present invention solves this major problem. Of particular note is that the toxicity reversal phenomenon during long-term storage is significantly reduced. 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.
A first improvement in the detoxification treatment method of the present invention is the presence of amino acids and the like during the detoxification treatment. In the present invention, at the time of detoxification treatment of pertussis cells, an amino acid, an amine or an amide is present in a buffer together with a detoxifying agent (for example, formalin). This makes it possible to produce a whole cell pertussis vaccine that is unlikely to return to toxicity due to long-term storage. A method for controlling the degree of inactivation by the presence of amino acids, ammonia, amines, etc. during inactivation of protein toxins is known (Formaldehyde inactivation of foot-and-mouth disease virus. Conditions for the preparation of safe vaccine). SJ Barteling, R. Woortmeyer. Arch Virol. 1984; 80 (2-3): 103-17.).

Amino acids such as lysine react with formalin to capture excess formalin, neutralize the action of formalin, control excessive inactivation, or effectively stop inactivation Has been widely accepted. It has also been demonstrated that a Schiff salt is formed by the reaction of formalin and an amino group.
In producing a whole cell pertussis vaccine, it is desirable that the detoxification process proceeds as completely as possible, that is, the toxin is completely inactivated in order to remove toxicity and prevent reversion of toxicity. Therefore, amino acid addition, which has been considered to have an effect of preventing inactivation of toxins, has not been carried out in the past in the detoxification treatment of whole cell pertussis with formalin.

The present inventors examined the correlation with the degree of detoxification by changing the formalin concentration and the treatment time in the production of 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 toxicity recovery did not occur. Succeeded in developing a pertussis vaccine.
The addition effect of amino acids, amines and amides upon detoxification of whole bacterial vaccine is a new and useful finding found by the present inventors.

  The amino acid used in the detoxification treatment of the present invention may be any available amino acid, and among them, protein-constituting amino acids are preferred. Moreover, a basic amino acid is more preferable. Often, water-soluble amino acids are preferred, but water-insoluble amino acids such as valine 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, arginine, glycine, glutamic acid, isoleucine, leucine, lysine, serine, threonine, valine, peptides containing these, amino acid oligomers, and the like. However, it is not limited to these. Suitable amines and amides include alkylamines such as ethylamine, ethanolamine and propanolamine, amides such as urea, glycinamide and β-alanylamide, ammonia and inorganic salts of the above substances. However, it is not limited to these.

The concentration of amino acids or amines and amides used in the present invention is practically usable in the range of 0.005 M-0.5 M, and preferably in the range of 0.02 M-0.2 M. However, the optimum concentration varies depending on the concentration of whole cell pertussis, the pH of the buffer solution, the temperature of the detoxification reaction solution, etc., but methods for determining the optimum concentration are known to vaccine workers.
The method for adding amino acids, amines, or amides in this treatment may be added at once from the beginning, or may be added at intervals of about 1-2 times per 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 (reversal toxicity after detoxification) of the pertussis vaccine produced by the present invention is determined according to the biologics standards (mouse weight loss test, mouse leukocyte increase test, histamine sensitization test, and abnormal toxicity) It can be examined by negative tests). Among them, the detection sensitivity of the histamine sensitization test method is relatively high, and this is known to those who handle vaccines.

  In this treatment method, the mechanism of the inhibitory effect on the reversion of toxicity of amino acids, amines, and amides is considered as follows, using lysine as an example. A large polysaccharide structure such as endotoxin and other high molecular weight acidic molecules are present together with protein toxins on the surface of pertussis cells, so it is considered that a part of the toxin is masked. In addition, since formalin molecules also have an affinity for polysaccharides, they are trapped here and hardly reach a part of the free amino group of the toxin. Therefore, in the absence of amino acids, in the conventional method of detoxification, formalin inactivates the part of the toxin exposed on the cell surface, but cannot inactivate the hidden part of the toxin. It is thought that it remains hidden while holding. This is a semi-detoxified product that is prone to reversion of toxicity. Compared with the case of inactivating free protein toxins, when whole cells are treated, there is a high possibility that some toxins remain active. 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 also has affinity for some polysaccharides. Can bind to endotoxins and other large structures. At this time, it is easily imagined that a part of the formalin and the basic amino acid that have been bound so far are replaced. 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, exposing the toxin that was previously hidden. 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 view of this, 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 an amino acid or the like.

As a second improvement point in the detoxification treatment method of the present invention, a lysis preventing treatment is given to 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 will be described later, the specific method of the lysis preventing treatment may be substantially indistinguishable from the detoxification treatment. Therefore, it can be called a second detoxification process. However, since the present inventors performed the above-described treatment for the purpose of preventing lysis separately from the detoxification treatment and confirmed the lysis prevention effect, they will be referred to as lysis prevention treatment.

  Conventionally, physicochemical treatment methods and chemical treatment methods have been used in the detoxification treatment of Bordetella pertussis. However, in chemical detoxification treatment with formalin and glutaraldehyde, or detoxification treatment by physicochemical means such as γ-ray treatment and ultraviolet treatment, only one type of treatment is performed and detoxification is performed by combining multiple methods. Was never done. Until now, it has been understood that inactivation is completed if cell death and toxin inactivation can be confirmed in the case of detoxification treatment of whole cells, and if loss of functional activity can be confirmed in inactivation of free protein. It was. Therefore, it is considered useless to carry out a combination of a plurality of detoxification processes, and no attempt has been made. The present inventors considered that the whole cell pertussis vaccine produced by detoxification of chemical means is one of the causes of reversion of virulence, and examined the prevention method. As a result, it was found that in addition to the chemical detoxification treatment, additional lysis prevention treatment by physicochemical means is effective in stabilizing the quality of the vaccine.

The lysis prevention treatment can be carried out by various chemical treatments and physicochemical treatment methods. Examples of chemical treatment substances used for treating bacterial cells for the purpose of preventing lysis and the following chemicals and usable concentration examples 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%), chloroform
(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%),

Moreover, the following means can be used for the physicochemical processing method. An example of conditions that can be used is shown together, 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), gamma irradiation (radiation source: cobalt 60; 5-50 kGy (kilo gray)), laser light irradiation (light source: various laser irradiation devices) ; Wavelength: 500-700 mn; Light quantity: 0.01-1 J (joule) / cm ² ), electron beam irradiation (microwave oven), ultrasonic irradiation In addition, apparently prevent lysis by microencapsulating whole cells It is also possible. One or more of these methods can be used in combination. The same method may be repeated several times.

The treatment conditions are not fixed, and suitable conditions can be set by changing the amount of cells, temperature, pH of the buffer solution, treatment time, and the like. It is usually operated under aseptic conditions.
Depending on the lysis prevention treatment conditions, the antigen protein may be excessively denatured and lose antigenicity as a result of the treatment. Therefore, the immunogenicity and other properties are examined after the treatment, and the optimum means and the optimum treatment conditions are comprehensively selected.
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.

The lysis-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.

The mechanism of the effect of the lysis preventing treatment of the present invention is considered as follows. In the chemical detoxification treatment of the whole cell body, the functional protein in the cell surface layer is inactivated and the cell dies, but the autolysis activity in the vicinity of the cell membrane May be lower than untreated cells, but may remain partly active. The remaining autolytic activity does not develop sufficiently under low temperature conditions where the vaccine is stored, but acts gradually during long-term storage over several months to several years, and finally kills dead cells. On the other hand, when additional treatment is performed by a physicochemical method after the chemical detoxification treatment, the residual autolysis activity is further reduced, and as a result, lysis is not caused for a long period of time. As a fact that supports this estimation, heating 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 enzyme proteins.

  An improvement in the third detoxification treatment method of the present invention is that the cells are subjected to purification and purification 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.

The present invention provides a mixed bacterial vaccine in which a bacterial component vaccine derived from the same bacteria as the above 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 bacterial vaccines, will be described in detail.
In the following description, ellipsis is often used, and its meaning is as follows. The whole cell pertussis vaccine is written as “wP”. The acellular pertussis vaccine is referred to as “aP”. Also, 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) of an effective amount or less and a cell-free pertussis vaccine (aP) of an effective amount or less. As described in Examples 4 and 5, awP has the same efficacy as whole cell pertussis vaccine and safety comparable to cell-free pertussis vaccine.

As the wP used in the production of the awP of the present invention, an improved whole cell pertussis vaccine obtained by improving the detoxification method described above is suitable. However, whole cell pertussis vaccine produced by a conventional method can also be used.
The method described in the well-known literature can be utilized for the manufacturing method of aP used for manufacture of awP of this invention. An outline of the method employed by the present inventors as an example of a conventional production method is shown below.
Inoculate Bordetella pertussis (Phase I) into a suitable medium (eg Cohen Wheeler modified liquid medium) and incubate statically at around 35 ° C. for 4-5 days. Centrifuge the culture and add a salting-out agent to the supernatant. In other words, ammonium sulfate is added so that it is about 50% saturated, or alcohol is added in an amount that is about 60% (v / v), and the resulting precipitate is collected by centrifugation at a speed of about 10,000 rpm for about 30 minutes, This precipitate is extracted with a sodium chloride addition buffer, and the extracted fraction is subjected to sucrose density gradient centrifugation to recover a 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.

  The amount of wP and aP used in the production of the awP of the present invention will be described. Approved 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 comprises (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, the effectiveness is reduced as it is, but the toxicity due to endotoxin associated with the whole cell vaccine can be reduced. In particular, when the improved whole cell pertussis vaccine of the present invention is used, the improvement effect is remarkable.

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

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 safety comparable to aP. Moreover, since wP is included, the manufacturing cost is low. Considering the manufacturing cost, it is cheaper when the aP content is low.
In the present invention, the method for mixing wP and aP is not particularly limited. Usually, each bulk solution is manufactured and then physically mixed to the desired ratio. After that, dispensing and stoppering complete the vaccine. The vaccine may be in the form of a solution or, if necessary, powdered by freeze drying. The quality of the new pertussis vaccine awP must meet the quality standards approved by the supervisory authority.

  The amount of 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 mcg PN / mL for aP. Nevertheless, the awP of the present invention exhibits the same degree of immunogenicity as conventional concentrations of vaccine. The reason is explained as follows. Since this vaccine contains pertussis cells and pertussis toxin, it is considered that a state closer to natural infection is produced for the recipient. It can also be explained from the relationship between toxin protein concentration and toxin activity. Toxin protein dose response curves are often not linearly related. 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 contained in wP decreases geometrically with decreasing dose. The dose-response curve of wP also changes geometrically. If aP is increased by a small amount, there is a range where the antigenic action increases exponentially. 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. However, the addition of aP restores immunogenicity while maintaining the reduced toxicity. The awP vaccine of the present invention has an effect equivalent to that of wP, and that the toxicity associated with wP is reduced as explained above.

It explains from the nature of the toxin protein contained. Pertussis toxin contained in aP used in the present invention is a protein having an action of activating adenylate cyclase activity in 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 be applied to bacterial vaccines derived from other bacteria.
The present invention provides a safe and effective combination vaccine comprising at least one type of mixed bacterial vaccine described above and at least one other type of vaccine antigen. Moreover, this invention relates to the manufacturing method of said mixed vaccine.

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. Examples of the fourth vaccine antigen include, but are not limited to, influenza virus vaccine, meningococcal vaccine, oral polio vaccine, inactivated polio vaccine, hepatitis vaccine, and Japanese encephalitis vaccine.

  It is known to add effective adjuvants to vaccines to enhance the effectiveness of the vaccines. Therefore, an appropriate adjuvant can be added to the vaccine of the present invention (including a combination vaccine). Examples of the adjuvant used in the vaccine of the present invention include aluminum hydroxide and its inorganic salts, hydrocarbons such as squalene and oil, bacterial toxins such as cholera toxin, and saponins, but are not limited thereto. is not. In general, there is no regularity in the type of adjuvant effective for enhancing 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.

Cell-free pertussis vaccine (aP) is usually bound to aluminum gel. The aP used for the vaccine awP of the present invention may be combined with an aluminum adjuvant. In the awP of the present invention, the aluminum gel is present in the awP, but the amount of aluminum is also reduced in proportion to the amount of aP reduced from the antigen amount of the approved vaccine. Moreover, aP used for the vaccine awP of the present invention may be free. When free aP is used, aluminum is not present in awP. A vaccine with reduced aluminum content is expected to have the effect of reducing undesirable side reactions that are believed to be based on aluminum. For example, there are few lumps at the site of inoculation, and there is a low possibility of neurotrophic action that is sometimes said to be due to aluminum.
As a customary vaccine, the vaccine product of the present invention may contain a stabilizer (gelatin), a preservative (thimerosal, phenoxyethanol), a colorant (phenol red), etc. in addition to the antigen.

Various known methods can be used as the method for inoculating the bacterial vaccine of the present invention. 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 also be inoculated by subcutaneous injection or intramuscular injection. Furthermore, nasal inoculation, oral inoculation, and percutaneous inoculation are possible if an appropriate dosage form is selected.
It should be noted that all prior art documents cited in the present specification are incorporated herein by reference.

[Example 1] Production of whole cell pertussis vaccine detoxified in the presence of amino acids and the like 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, and is toxic during this period. We examined whether a return occurred. The test implementation method is as follows.
Strain Bordetella pertussis (Phase I)
Medium: Cohen Wheeler Modified liquid medium Culture: 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 sodium chloride-added phosphate buffer (PBS) (0.01 M, pH 7) so that the turbidity at 650 nm was 20. This 100 mL was transferred to a 500 mL glass container. (The height of the liquid level is about 2 cm). One kind of each of various amino acids or other test substances having a concentration of 0.05 M was added thereto. Then, it was detoxified by leaving it to stand in 0.5% (v / v) formalin for 5 weeks according to the method for producing 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. Thereafter, thimerosal (0.01 w / v%) was added, and 1.3 mL each was dispensed into vials to prepare a whole cell pertussis vaccine.
Each vial was stored in a cold place at 2-10 ° C. After 6 months and 2 years, the vials were opened, and 5 sample vials prepared under the same conditions were combined to make one sample. Using 5 specimens, quality control tests were conducted in accordance with Japanese biopharmaceutical standards. Table 1 shows the results of histamine sensitization tests (measured values after heating at 37 ° C.) that can detect reversion of toxicity.

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 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 that was detoxified with no amino acid added (control). 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 was only a slight reversal of toxicity. . However, toxic reversion was significant in untreated subjects.

[Example 2] Bacteriolytic treatment of pertussis Bacterial lysis during the long-term storage of a product that had been subjected to detoxification treatment and lysis prevention treatment for B. pertussis was examined. 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 the whole cell pertussis vaccine production, and suspended in 0.01M PBS (pH 7) to a turbidity of 20. did. This 100 mL was transferred to a 500 mL glass container. (The height of the liquid level is about 2 cm). In Table 2, treatment method 1, the chemicals described were added to the final concentrations as described. In the treatment method 2, it was left in a carbon dioxide incubator. In the processing method 3-6, it was processed 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 suspended again in the same buffer solution. Thereafter, it was detoxified with formalin in the presence of 0.05 M lysine as described in the table. The bacterial suspension of each treatment was dispensed in 1.3 mL vials according to the vaccine production method after removing formalin. Thus, a whole cell pertussis vaccine to which the treatment of 1-7 was added was prepared.

  Each vial was stored in a cold place at 2-10 ° C. Two years later, the vial was opened, and the samples for 5 vials prepared under the same processing conditions were combined into one sample. After appropriately diluting three samples of seven kinds 1-7, turbidity was measured at a wavelength of 650 nm. The value was compared with the turbidity of Control 2 immediately after production. The results are shown in Table 2.

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,
-: Colorless and transparent.
From the above results, it has been clarified that the decrease in turbidity after 2 years is reduced when acetone treatment, heating treatment, or the like is performed. 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 performed.

[Example 3] Manufacture of cholera vaccine Whole cell cholera vaccine was manufactured and the quality was examined.
Test strains: Vibrio cholera Ogawa strain (type S), Inaba strain (type S)
Culture medium: Ordinary agar medium Culture: Culture at 37 ° C for 20 hours Wash the resulting cells as in the case of whole cell cholera vaccine production and suspend in 0.015M PBS (pH 7) to a turbidity of 10 did. This 100 mL was transferred to a 500 mL glass container. (The height of the liquid level is about 2 cm). It processed by the processing method 1-11 as it was. In either case, the glass container being treated was appropriately lightly stirred. After completion of the treatment, the cells were washed by centrifugation and resuspended in PBS. 0.5% (v / v) formalin was added and detoxified according to the method for producing whole cell cholera vaccine. Three weeks later, formalin was removed by centrifugation. According to the cholera vaccine manufacturing method, phenol was added to the bacterial suspension of each treatment, and 10 mL was then dispensed into vials to prepare 1-11 treated whole cell cholera vaccine.
Each vial 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 a 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.

PCMB description: Abbreviation for p-choloro-mercuribenzoate. It is a kind of SH reagent (that is, a reagent that reacts strongly with the SH group of a protein).
The symbols in the turbidity column have the following meanings. Turbidity at a wavelength of 650 nm is +++: 0.8-1.2; ++: 0.5-0.8; +: 0.2-0.5,
±: 0.2 or less; —: Colorless and transparent.
From the above results, it was clarified that the decrease in turbidity after 2 years was less when untreated (control 1) when acetone treatment, warming treatment, etc. were performed (Table 3). In the case of no treatment (control 1), lysis was remarkable.

  Next, a vaccine was prepared by treatment method 4 in Table 3 and administered to a human. The results of comparing the two are summarized in Table 4.

  From the above results, it was revealed that the safety of the cholera vaccine of the present invention was improved (Table 4).

[Example 4] Production of new pertussis vaccine awP Whole cell pertussis vaccine wP and cell-free pertussis vaccine aP were mixed to produce novel pertussis vaccine awP of the present invention. The result of investigating safety is shown.
As the whole cell pertussis vaccine wP, an improved whole cell pertussis vaccine produced by formalin detoxification treatment in the presence of lysine prepared in Example 2 (Table 2, Test No. 3) was used. 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 examine the effectiveness and safety of the vaccine, a quality control test was conducted by the method stipulated in the 2004 Japanese Biopharmaceutical Standards. The results are shown in Table 5. In the table, “#” and “*” indicate whether each measurement value is in conformity (#) or non-conformity (*), compared to the Japanese biopharmaceutical standard for some cell-free pertussis vaccine. .

ワクチン A1-A3： 無細胞百日せきワクチン、使用量は蛋白チッソ量（mcg PN/mL） で示す。
ワクチンW1-W2： 全菌体百日せきワクチン、使用量は細胞数(単位はビリオン(billion), 10sup9)で示す。
ワクチン AW1-AW2:：無細胞百日せきワクチンと全菌体百日せきワクチンを混合した百日せきワクチンawPを示す。
ＤＴｗＰ：ジフテリアー破傷風―全菌体百日せき 三種混合ワクチン（市販品）。

Vaccine A1-A3: Cell-free pertussis vaccine, the amount used is expressed in protein nitrogen (mcg 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).

The meanings of the abbreviations indicating the test results are as follows.
Toxicity index 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.
Effectiveness index Ab (antibody titer) self-standard P (percentage of pertussis vaccine by mouse brain attack method) Japanese biologics

  The novel pertussis vaccine AW1 of the present invention is half the maximum bacterial quantity of 20 x 10 sup 9 / mL allowed by the WHO biopharmaceutical standard for wP, and 1 / of the amount of toxin protein nitrogen in the vaccine approved for aP. A vaccine containing 5 doses. As is clear from Table 5, AW1 is a Japanese biological product of a cell-free pertussis vaccine in LP (mouse leukocyte increased toxicity), HS (mouse histamine-sensitive toxicity), and Tox (guinea pig abnormal toxicity negative test), which are toxicity indicators. The measured values meeting the standard were shown. It can also be seen that the titer (mouse brain attack method) is also suitable. However, BWD (mouse weight loss toxicity), which is supposed to reflect toxicity due to endotoxin and the like, was not compatible. It can be seen that the commercially available DTwP used as a control does not meet some Japanese biopharmaceutical standards (2004 edition) regarding the quality of the cell-free pertussis vaccine.

[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 method.
The spray infection method was adopted to examine the effectiveness of this awP. The intracerebral attack method used to examine the titer in Example 4 was adopted as the standard method for measuring the titer of whole cell bodies and acellular therapy pertussis vaccine in Japanese biopharmaceutical standards. Not used in other countries. There is no standard titration method in Western countries or WHO biopharmaceutical standards, but the spray infection method is one of the standard assay methods. Therefore, this method was adopted.

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, Iyama S, Sato T, Nagai M. Efficacy of pertussis components in an acellular vaccine, as containing in a murine model of respiratory infection and a murine model of intracerebral infection. Vaccine 2002; 20: 1429-1434.
(2) Watanabe M, Nagai M. Reciprocal protective immunity against Bordetella pertussis and Bordetella parapertussis in a murine model of respiratory infection. Infect Immun 2001; 69: 6981-6986.)

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 placed in a safety cabinet. On the other hand, Bordetella pertussis (18-232 strain) cultured at 37 ° C. for 30 hours was suspended in ice-cooled PBS to prepare 10 × 10 sup 9 cells / mL. The prepared bacterial suspension was placed in a nebulizer and connected to a spray infection 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 fungus aerosol. After spraying, filtered sterilized air was introduced for 30 minutes to remove the aerosol in the spray infection box, and the mouse was removed. After aseptically raising the mice for 2 weeks, the lungs were aseptically removed from the cervical dislocation mice and homogenized in 10 mL of ice-cold PBS. This solution was used as a 10sup (-1) dilution, and 10-fold serial dilution was performed. The diluted solution was smeared on a Bordeaux-Jung medium, cultured at 37 ° C. for 4 days, and the number of viable bacteria in the lung was calculated from the number of colonies produced. The number of living bacteria in the lung was expressed in colony-forming unit (CFU).
The results are shown in Table 6.

Test number 3 (W20) contains the same amount as the approved whole cell pertussis vaccine.
Test number 8 (A15) contains an equivalent amount of the approved cell-free pertussis vaccine. When these two were inoculated, the number of viable bacteria decreased by about 2 logs from the non-inoculated control (test number 9). When this is an effective level, it can be determined that test number 5 (W10 + A3) is effective.

[Example 6] DTwP triple-mixed vaccine According to the production method of DTaP manufactured by Kitasato Institute (Japan), wP produced by the method of Example 2 (Table 2, treatment method 3) was used instead of aP. A ternary 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.

  The results all met Japanese biopharmaceutical standards in the potency tests tested.

[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. The Japanese encephalitis vaccine (ccJE) used a prototype manufactured by Kitasato Institute (Japan). The titer of each antigen component was measured by the method of Japanese biopharmaceutical standards. The results are shown in Table 8.

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

Example 9 Production of Whole Bacterial Bacterial Vaccine Test Bacteria: Pertussis and Vibrio cholerae used vaccine production strains. For other strains, laboratory stocks were used.
Culturing method: Pertussis and Parapertussis were obtained by culturing as in Example 1. Vibrio cholerae was cultured as in Example 3. The other bacteria were smeared on a normal nutrient agar medium and cultured at 33 ° C. for 16-24 hours. Each bacterial cell obtained was 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). It was suspended in. This 100 mL 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: The heating treatment was carried out 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. In 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 was removed by washing and centrifugation, and the treated cells were suspended again at 650 nm in sodium chloride-added phosphate buffer (PBS) (0.001M, pH 7). . 1.3 mL was dispensed into each vial and stored in a cold place.

Stability measurement method: Each vial was stored in a cold place at 2-10 ° C. One year later, the vial was opened, and a sample of five vials prepared under the same processing conditions was combined into one specimen. 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 cells subjected to various treatments (immediately after the start of storage) was intraperitoneally administered to mice and inoculated three times at intervals of 3 weeks. One month after the last inoculation, blood was collected from the mice to obtain antiserum.
Antibody detection method: Antiserum was taken, 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 sedimentation reaction was observed, and the antibody titer was calculated from the dilution ratio of the antiserum. Antibody titers are shown in Table 9.

Meaning of treatment symbol: H Warming treatment; A. Acetone treatment; F Formalin inactivation Lysis mitochondrome: Turbidity at wavelength 650 nm is 0.8-1.2, and control turbidity is 1.0 It shows that.
From the results in Table 9, it was revealed that a whole cell bacterial vaccine with good storage stability was produced in any of the tested strains.

Even bacterial vaccines manufactured by detoxifying pathogenic bacteria have been shown to frequently revert to toxicity during the storage process, and the fact that reversion to toxicity does not occur during long-term storage is It was very important in maintaining sex. 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 with these pathogens is there. 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 efficacy as the whole bacterial cell vaccine produced conventionally, can be produced 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 (19)

The following (a) and (b) obtained by the process according to long-term even in storage having the features toxicity return does not occur whole cell Pertussis Vaccine:
(A) amino, amine, and / or detoxification treatment of pertussis bacteria in the presence of amides (b) one hundred days lysis prevention processing cough bacteria. The amino acid described in (a) is at least one amino acid selected from aspartic acid, γ-aminobutyric acid, alanine, β-alanine, arginine, glycine, glutamic acid, isoleucine, leucine, lysine, serine, threonine, and valine. there, whole cell pertussis vaccine according to claim 1. The amine described in (a) is at least one amine selected from ethylamine, ethanolamine, and propanolamine, or the amide described in (a) is selected from urea, glycinamide, and β-alanylamide at least, one of amides, whole cell pertussis vaccine according to claim 1 or 2 which is. lysis preventing process according to (b) is acetone treatment, heating, gamma irradiation, electron beam irradiation, and at least one of the processing method is selected from the laser beam irradiation, according to any one of claims 1 to 3 whole-cell pertussis vaccine of. Pertussis bacteria clinical isolates, the artificial variant or whole cell pertussis vaccine according to any one of claims 1 to 4 is a genetically modified strain. Whole cell pertussis vaccine according to any one of claims 1 to 5, and whooping mixed bacterial vaccine containing bacterial components derived vaccines cough bacteria. Whole-cell pertussis vaccine and / or one hundred days mixed bacterial vaccine according to claim 6, wherein the bacterial component vaccines from cough bacteria alone are less effective amounts. A mixed vaccine comprising at least one bacterial vaccine according to any one of claims 1 to 7 , and other vaccine antigens derived from bacteria and / or viruses. Vaccine antigen is at least one antigen is selected from Diphtheria toxoid, tetanus toxoid, Haemophilus influenzae vaccine, meningococcal vaccine, oral poliomyelitis vaccine, inactivated poliomyelitis vaccine, hepatitis vaccine, and Japanese encephalitis vaccine according to claim 8 Combination vaccine described in 1. The following (a) and (b) obtained by the process described in, for whole-cell pertussis vaccines also have a characteristic toxicity return does not occur during long-term storage manufacturing method:
(A) amino, amine, and / or detoxification treatment of pertussis bacteria in the presence of amides (b) one hundred days lysis prevention processing cough bacteria. The amino acid described in (a) is at least one amino acid selected from aspartic acid, γ-aminobutyric acid, alanine, β-alanine, arginine, glycine, glutamic acid, isoleucine, leucine, lysine, serine, threonine, and valine. there, whole cell whooping method for producing cough vaccine according to claim 10. The amine described in (a) is at least one amine selected from ethylamine, ethanolamine, and propanolamine, or the amide described in (a) is selected from urea, glycinamide, and β-alanylamide at least, one of amides, whole cell whooping method for producing cough vaccine according to claim 10 or 11 which is. lysis preventing process according to (b) is acetone treatment, heating, gamma irradiation, electron beam irradiation, and at least one of the processing method is selected from the laser beam irradiation, according to any of claims 10 to 12 whole cell hundred days method of manufacturing a cough vaccine of. Pertussis bacteria clinical isolates, whole cell whooping method for producing cough vaccine according to the any artificial mutants or claim 10 which is a genetically modified strain, 13. Whole cell pertussis vaccine according to any one of claims 1 to 5, and a hundred days mixing the acellular bacterial vaccine from cough bacteria, the production method of the mixed bacterial vaccine. Method for producing a mixed bacterial vaccine of claim 15 which whole cell pertussis vaccine and / or one hundred days acellular vaccine derived from cough bacteria characterized in that alone is less effective amounts. A method for producing a mixed vaccine comprising the step of mixing at least one bacterial vaccine according to any one of claims 1 to 7 and other bacterial and / or virus-derived vaccine antigens. Vaccine antigen is at least one antigen is selected from Diphtheria toxoid, tetanus toxoid, Haemophilus influenzae vaccine, meningococcal vaccine, oral poliomyelitis vaccine, inactivated poliomyelitis vaccine, hepatitis vaccine, and Japanese encephalitis vaccine according to claim 17 The manufacturing method of the mixed vaccine as described in 1 .. The vaccine according to any one of claims 1 to 9, which is used for diseases caused by bacteria and / or viruses.