JP5121230B2 - Q fever vaccine preparation and method for producing the vaccine preparation - Google Patents

Description

  The present invention relates to a Q fever vaccine preparation containing as an active ingredient an antigen obtained by serum-free culture of Coccoliella and a method for producing the Q fever vaccine preparation.

  Q fever (Cokesiosis) is a zoonotic disease caused by infection with the bacterium Koksiella Bernetti. In humans, it is called Q fever, and it is suspected to be associated with chronic fatigue syndrome of unknown cause. In animals (including birds) it is called coccidiosis. Most of the animals are inapparent and are considered to be less lethal.

  The main causative agent of Q fever is a kind of bacteria, which is classified as Legionella sp. Like other Legionella bacteria, Cocciella is obligate intracellular parasites that only divide and proliferate in animal cells. This bacterium is Gram-negative, small gonococcus and large and small polymorphisms, and shows a phase mutation of phase I / II, similar to S / R (smooth / rough) mutation of E. coli. Phase I bacteria are found in freshly isolated bacteria and are highly toxic. The cell surface is covered with long-chain lipopolysaccharide (LPS). Phase II bacteria arise from subculture of phase I bacteria, are attenuated, and the cell surface is covered with short-chain LPS.

  The clinical picture of Q fever is diverse and can be divided into acute and chronic types. The acute form has many influenza virus-like infections characterized by fever, headache, muscle pain, joint pain, coughing, and the like. There is also a tendency to develop pneumonia and hepatitis. The prognosis is generally good, and fever and recovery usually takes about 2 weeks. On the other hand, the chronic symptom is often endocarditis, particularly with chronic hepatitis and myocarditis.

Antibiotics (tetracyclines) can be used to treat Q fever. However, in the current situation where there is no useful and simple diagnostic agent, it takes time until the diagnosis of Q fever is made, and then antibiotics are administered gradually, so the effect of antibiotics spreads the infection It is not enough to prevent this.
In addition, there are few effective and safe vaccines aimed at preventing and treating Q fever. As a Q fever vaccine, a vaccine (Patent Document 1) using an inactivated bacterial body of K. sierra is known.

  As described above, K. sierra is an obligate intracellular parasitic bacterium and divides and proliferates only in animal cells. Therefore, in the cell culture medium for K. sierra growth, the essential nutrient factor is the same as the medium used for virus propagation. Serum has been added and used so far. However, in the case of virus culture, there are cases where unknown factors mixed in serum act as virulence factors. For example, AIDS, hepatitis, mad cow disease causative factors should not be mixed. Urgent measures have been taken for the purpose.

  In addition, a serum-free medium has been developed for cell culture for virus strain growth and can be used for virus vaccine production. However, no studies have been conducted on serum-free media suitable for cell culture for the growth of Coccoliella. No Q-fever vaccine has been developed so far, in which K. sierra is grown in a serum-free medium and produced using this cell or an antigen obtained from each cell.

Prior art document information related to the invention of the present application is shown below.
WO97 / 33614 Kazar J. et al. Acta Virol. 31. 158-167. 1987 Schneer N. et al. Zentblat. Bakteriol. Microbiol. Hyg. 267.79-88. 1987

  The present invention has been made in view of such a situation, and the object of the present invention is to provide a Q-heat vaccine preparation containing an antigen obtained by serum-free culture of Coccoliella as an active ingredient, and the Q-heat vaccine preparation. It is an object of the present invention to provide a production method, a serum-free culturing method for Cocciella, and a utilization method thereof.

  In order to solve the above problems, the present inventors have developed an epidemiology of Q fever and a vaccine preparation. Since K. sierra is a kind of obligate intracellular parasite, normally, K. sierra is cultured using animal cell culture technology. Since serum is usually added to the animal cell culture medium, the serum-added culture method has also been used in the culture of Coccoliella. When the thus obtained culture was used for a vaccine, there was a risk of contamination with unknown pathogenic factors derived from serum.

  In order to solve the above problems, the inventors of the present invention have studied the culture conditions of bacterial cells. As a result, the inventors found for the first time that C. sierra can be cultured by a serum-free culture method. By using this bacterial cell, It was clarified that vaccine production by a serum-free culture method is possible.

  That is, the present inventors succeeded in producing a Q-heat vaccine preparation containing an antigen obtained by serum-free culture of K. sierra bacteria as an active ingredient, thereby completing the present invention.

More specifically, the present invention provides the following (1) to (11).
(1) A Q fever vaccine preparation containing, as an active ingredient, an antigen obtained by serum-free culture of K. sierra bacteria.
(2) The Q fever vaccine preparation according to (1), wherein the antigen is at least one antigen selected from the following (a) and (b).
(a) Inactivated whole body cocciella obtained by inactivation treatment after serum-free culture of cocciella
(b) An inactivated antigen protein (3) antigen obtained by inactivating one or a plurality of antigen proteins derived from serum-free K. sierra is an antigen obtained by the following steps (a) and (b): The Q fever vaccine preparation according to (1).
(a) A step of culturing Kojiella bacteria serum-free to produce Kokusiella culture preparations
(b) a step of inactivating the whole cell body cocciella by adding an inactivating agent to the kokusiella culture preparation (4) the antigen is an antigen obtained by the following steps (a) and (b) The Q fever vaccine preparation according to (1).
(a) Step of serum-free culture of K. sierra
(b) A step of inactivating the antigen protein by adding an inactivating agent to one or a plurality of antigen proteins derived from serum-free K. sierra bacteria (5) K. sierra is a wild strain, a clinical isolate, or a source thereof The Q fever vaccine preparation according to any one of (1) to (4), which is an artificial mutant strain or a whole bacterial strain of a recombinant strain.
(6) The Q fever vaccine preparation according to any one of (1) to (5), wherein the K. sierra fungus is K. sierra burnetti.
(7) A method for producing a Q fever vaccine preparation according to any one of (1) to (6).
(8) The method for producing a Q fever vaccine preparation according to (7), comprising the following steps (a) to (c):
(a) A step of culturing Kojiella bacteria serum-free to produce Kokusiella culture preparations
(b) A step of inactivating all coccoliella bacteria by adding an inactivation agent to the coccierella culture preparation
(c) A step of mixing inactivated whole body coccyella and a pharmaceutically acceptable additive (9) comprising the following steps (a) to (c): a Q-heat vaccine preparation according to (7) Manufacturing method.
(a) Step of serum-free culture of K. sierra
(b) A step of inactivating the antigenic protein by adding an inactivating agent to one or a plurality of antigenic proteins derived from K. sierra bacteria cultured in serum-free culture
(c) A method for culturing K. sierra, comprising a step of mixing an inactivated antigen protein and a pharmaceutically acceptable additive (10) a step of culturing in a serum-free medium.
(11) A method for producing a kokusiella culture preparation, comprising a step of culturing kokusiella bacteria in a serum-free medium.

  The present invention provides a Q-heat vaccine preparation containing as an active ingredient an antigen obtained by serum-free culture of K. sierra. The present invention also relates to a method for producing the above-mentioned Q-heat vaccine preparation with improved safety.

  The Q-fever vaccine preparation of the present invention is produced by inactivating an antigen according to a general method for producing a bacterial vaccine after culturing and collecting a bacterial antigen.

  In the present invention, whole cells, cell components, proteins, DNA fragments and the like are used as antigens. Natural-type antigens or mutant antigens can also be used, and when antigenic proteins are produced in the culture solution, these can also be collected and used.

  In the present invention, as a strain used for culture, it can be used after being purified from field strains or clinical isolates of Cocciella, or artificial mutants of these strains can also be used. . As the bacterial species, Kokusiella Bernetti is preferable, and among these bacterial species, the Nine Mile strain is more preferable. The Coxiella Bernetti Nine Mile strain can be obtained as the VR615 strain from ATCC, a strain preservation organization. In the case of using a phase II bacterium of the Cokesiella Bernetti Nine Mile strain, it can be used by purifying and purifying the clinically isolated phase II bacterium. The present inventors used a strain distributed as a yolk sac emulsion by Dr. Kazar, J. (Slovakia) and then stored at the Kitasato Institute.

  In the method for producing a vaccine preparation of the present invention, a process for producing a culture product of coccoliella by serum-free culture of coccella is described.

  In the present invention, when cultivating K. sierra fungus as a bacterial species, animal cells are used as a substrate, the cells are grown, and K. sierra fungus is grown therein. Examples of animal cells to be used include BGM (buffalo green monkey) cells, Vero cells, L-929 cells, and CHO cells in the case of recombinants.

  The animal cell culture medium used for producing the vaccine of the present invention is a serum-free medium. The serum-free medium can be used in any composition that is suitable for the growth of the cells used. The medium can contain a carbon source, nitrogen source, phosphate source, trace metals, etc., but serum is not used. In addition, a commercially available product can be used as the serum-free medium. For example, VP-SFM, Opti-Pro SFM, CD-CHO, and the like are more preferable as examples of commercially available serum-free media that can be used for culturing K. sierra bacteria, but are not limited thereto.

  In the culture of bacterial cells in the present invention, a glass culture vessel, a stainless steel vessel, a plastic vessel, a commercially available fermenter, or the like can be used as a cell culture vessel.

  In the cell culture of the present invention, the culture temperature is usually 35 to 37 ° C., but the temperature is not particularly limited as long as it is a temperature at which growth is possible.

  In the cell culture of the present invention, the pH range is usually from 6.8 to 7.6, but is not particularly limited to this pH, and the culture can be performed under acidic or alkaline conditions where the cells do not die.

  As the culture method of the present invention, any known method such as stationary culture, roller bottle culture, microcarrier suspension culture, microchip culture, and floating cell culture can be used. The optimal culture period and culture conditions can be selected depending on the cells used.

When culturing using a serum-free medium, the number of coccyella is measured as necessary to confirm the progress of the culture. In this measurement, the inclusions are also measured and totaled. The number of bacteria can be measured after staining for a short period of time, after staining and measuring with a microscope, or with a turbidity of 650 nm. Recently, it can also be measured as a DNA copy number using quantitative PCR. That is, as a method for measuring the number (titer) of the body sierra germ, the following method is specifically used according to a known method (Schneider, W. Zentralbl. Bakteriol. 271: 77-84, 1989). I can do it. First, BGM cells are cultured in 24-well plates with coverslips. After inoculating a single layer sheet, inoculate 0.5 ml of each live suspension of C. sierra Bernetti, approximately 100 times, adsorb for 30 minutes, and then centrifuge at 1,500 rpm. After centrifugation, the supernatant is removed, 1 ml of MEM is added to each well, and cultured at 37 ° C. (CO ₂ 5%). On day 4 of culture, cover slips are collected and fixed with cold acetone. The number of inclusion bodies can be measured by the fluorescent antibody method (FA) or the enzyme antibody method. In the fluorescent antibody method, the serum of a mouse infected with C. sierra Bernetti is diluted with the primary antibody using phosphate buffered saline (PBS) and reacted at 37 ° C. for 60 minutes in a wet box. After washing 3 times with PBS for 5 minutes, FITC-labeled anti-mouse IgG sheep serum (manufactured by CAPPEL) is adjusted with PBS and reacted in the same manner. After washing, the reaction propidium iodide for 5 minutes. After washing, the cells are sealed with Slow Fade antifade Kits (Moleculer Probes), and the number of sealed pairs showing strong color development under a fluorescence microscope (BX-50-FLA, OLYMPUS) is measured.

  The recombinant antigen of the present invention can also be produced by a recombinant cell organism, and the following known methods can be applied mutatis mutandis. As the microbial cells, Kokusiera bacteria isolated from patients is used. Usually, clinical isolates have both a protein antigen derived from Coccoliella and a protein antigen derived from heat-resistant Cocciella. First, a protein antigen derived from K. sierra is isolated. A gene encoding a protein antigen derived from K. sierra fungus (hereinafter referred to as “koku sierra antigen gene”) (6.7 kbp) is excised from the plasmid by restriction enzyme treatment or the like and ligated to a plasmid such as pBR322. For example, the following paper (Zhang, GQ, et al. J. Clin. Microbiol. 42: 423-428 (1998), Nguyen, Sa V., et al. FEMES Microbiol. Lett. 175: 101-106 (1999), Nguyen, Sa V., et al, Microbiol. Immunol. 43: 743-749 (1999)) can be used.

  Next, after the K. sierra antigen gene is amplified by PCR or the like, the K. sierra antigen gene is ligated to another expression vector. Next, position-directed mutagenesis is performed on this vector, and then amplified by PCR using selective primers to produce a recombinant Escherichia coli strain that produces a protein antigen derived from the recombinant mutant Koksiella. . This recombinant Escherichia coli can be cultured according to a conventional method, and a protein antigen derived from Coccoliella can be obtained from the culture solution.

  Recombination Escherichia coli producing a protein antigen mutant derived from Coccoliella can be obtained by performing position-directed mutagenesis during the above gene recombination operation. If this is cultured and purified, a protein antigen mutant derived from Coccella can be obtained. For example, as described above, by applying a position-specific mutagenesis method, it is possible to change an amino acid residue at any site to another residue in the amino acid sequence of a protein antigen derived from Bacillus subtilis. . Conversely, it is possible to retain a specific amino acid residue at a specific site, for example, a glutamic acid residue without change. The same applies to sugar residues. Among the obtained mutants, those having immunogenic activity are selected and used as protein antigen mutants derived from the body sierra fungus of the present invention. It should be noted that the mutant sicillus antigen activity of this mutant can be at an arbitrary level. In order to obtain a mutant, some trial and error experiments are required. However, the obtained recombinant mutant cocceria antigen generally has an advantage that it is difficult to return to a natural coccella antigen.

  In the process of producing the Kokusiella culture preparation, the whole cell antigen is washed, purified and concentrated after being cultured and produced in a serum-free medium. The cleaning, purification, and concentration methods will be described below.

  After completion of the culture in the serum-free medium, the cells are collected, disrupted by freezing and thawing, sonication, etc., and then Cokesiella is collected from them. Centrifugation, gel filtration, salting out, etc. can be used for this method. Usually, after that, the cells are washed and purified with a phosphate buffer solution added with salt. A purification method, a centrifugal method, a stirring method, a concentration method, a column method, and the like can be used for purification, but the purification method is not limited to these. For example, purified bacteria can be obtained by the method described in Example 1. Cultivation, collection, washing, and purification of Coccella must be performed in an environment that allows for strict containment. Usually, a Biosafety Regulation Level 3 (BSL3) environment is required.

  In the present invention, one or a plurality of antigenic proteins derived from Coccellaella cultured in serum-free culture may be collected. One or a plurality of antigen proteins can be collected by performing a treatment for partially destroying or completely destroying the body of the body sierra germ.

  In the case of producing a subunit vaccine, an effective antigen fraction may be collected by performing a treatment for partially destroying or destroying the body of the body sierra germ. Furthermore, when using the antigen protein produced in the culture solution, the antigen protein may be concentrated and purified by a combination of an effective centrifugation method, concentration method, column method and the like.

  The period of washing, purification, and concentration is preferably before inactivation, but can be performed after inactivation. You may implement both before and after inactivation.

  In the vaccine production method of the present invention, the step of adding an inactivating agent to the coccoliella culture preparation or the like to inactivate the coccierella culture preparation or the like will be described below.

  Various methods can be used to inactivate the antigen. In general, the optimum inactivation method varies depending on whether the antigen is a whole cell or a protein. Inactivation can be carried out by various chemical treatment and physicochemical treatment methods. Examples thereof include, but are not limited to, the following.

The following substances can be mentioned as chemical treatment substances used for treating bacterial cells or antigen-containing proteins for the purpose of inactivation. Although the concentration which can be used is shown together, it is not limited to these.
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 for inactivation. Although an example of the conditions which can be used is shown below, it is not limited to these.

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, and the like.
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.

  These inactivation methods may be used alone or in combination of a plurality of methods, regardless of whether they are whole cells or cells derived from cells (including antigenic proteins derived from culture broth). Also good. In the case of inactivation, coexistence of amino acids and amines may improve the stability of quality after inactivation, and these substances can be added as necessary. Moreover, in addition to formalin inactivation, by carrying out organic solvent treatment, heating treatment, and γ-ray irradiation treatment, it is possible to obtain stable inactivated whole cells with a quality that hardly causes lysis.

An example of inactivation is as follows. First, 1% (v / v) formalin is added to a live suspension of K. sierra fungus and left at room temperature for 5 days to inactivate. Thereafter, the mixture is centrifuged at 13,000 rpm for 15 minutes. After removing the supernatant, the suspension is suspended in an equal amount of physiological saline-added phosphate buffer (0.1 M, pH 6.8) to obtain an inactivated Coxiella burnetti suspension. Confirmation of inactivation can be confirmed by the fact that after inactivation treatment, a part of the sample is taken and cultured, the bacteria do not grow. After the inactivation treatment, the chemicals used for inactivation are removed by washing with a phosphate buffer or the like. Thereafter, the number of bacteria is adjusted so that the turbidity at 650 nm is about 1-10 (about 1-10 × 10 ⁹ cells / mL). Alternatively, the concentration of the antigen protein is adjusted.

A vaccine is manufactured by dispensing a predetermined amount (for example, 0.5 mL, 1.0 mL, 10 mL) of the aforementioned K. sierra fungus suspension or antigen protein solution into vials or syringes.
The general composition of the Q fever vaccine is exemplified below, but the vaccine preparation composition is not limited to the following.

Antigen: Inactivated whole cell body Coccoliella protein concentration 50-100 mcg / mL
Buffer: 0.01M phosphate buffer with saline
pH: 7.0
Osmotic pressure: 1
Capacity: 0.6 mL
Preservative: 2% sorbitol, 5% lactose

  A pharmaceutically acceptable additive may be added to the vaccine preparation of the present invention. Here, the pharmaceutically acceptable additive means a pharmaceutically acceptable material that is a substance different from the antigen (or immunogenicity) itself and can be administered together with the antigen in vaccine administration. For example, vaccine additives such as adjuvants, preservatives, and stabilizers can be exemplified, but are not limited thereto. Examples of adjuvants that can be used for human vaccines include, but are not limited to, aluminum phosphate, aluminum hydroxide, and MF59.

  As a stabilizer, about 0.2% gelatin or dextran, 0.1-1.0% sodium glutamate, or about 5% lactose or about 2% sorbitol can be used, but it is not limited thereto. . As a preservative, about 0.01% thimerosal, about 0.1% betapropionolactone, about 0.5% phenoxyethanol, and the like can be used, but are not limited thereto.

  When preparing injections, add pH adjusters, buffers, stabilizers, preservatives, etc., if necessary, and make subcutaneous, intramuscular, and intravenous injections by conventional methods. The injection may be a solid preparation or a preparation prepared at the time of use by lyophilization after the solution is stored in a container. One dose may be stored in a container, and the dose may be stored in the same container.

  Various known methods can be used as the method for inoculating the bacterial vaccine of the present invention. As the inoculation method, subcutaneous injection, intramuscular injection, nasal inoculation, oral inoculation, transdermal inoculation and the like are preferable, and intramuscular injection is more preferable, but it is not limited thereto. Nasal inoculation includes intranasal spray, powder spray, instillation, application and the like. Of these inoculation methods, oral inoculation and nasal inoculation are more preferred because the respiratory tract and gastrointestinal mucosa are important sites in the early stages of infection. Inoculation with a vaccine containing an appropriate adjuvant with strong immunopotentiating activity can induce an immune mechanism in the local mucosa and exert a protective effect as early as possible in the infection.

  Which inoculation method is appropriate is determined in consideration of the type of vaccine antigen, dosage form, antibody expression time, antibody duration, age of the inoculation subject, and the like. In addition to humans, examples of inoculations include pets, domestic animals, outdoor animals, and birds that are considered as sources of infection. Inoculation methods are finalized through expert clinical trials and these methods are well known to those skilled in the art. A single inoculation product or multiple inoculation products may be used. The dose varies depending on the weight and age of the patient, the administration method, etc., but those skilled in the art can appropriately select an appropriate dose.

It is important to determine vaccine effectiveness for vaccine development. These tests are well known to vaccine workers, but general methods are described below. After inactivating antigenic material that may contain protective or prevention antigens, the experimental animal is immunized. Next, the immunized animal is attacked with highly virulent cocciella, and the effectiveness of the test material is determined by the life and death of the experimental animal or the decrease in the number of infected coccella. Since Kokusiera is a zoonotic infection, many laboratory animals can be infected. In experimental animals, it is more appropriate to determine whether the animal is alive or dead, or to measure the degree of infection with Cocciella in an organ.
In addition, all prior art documents cited in the present specification are incorporated herein by reference.

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.
[Example 1] Serum-free culture of Q thermophile As a strain, the standard strain Kokusiella Nine Mile strain (ATCC VR 615) was used. Vero cells (ATCC CCL-81) were used for cell culture of Coccoliella, and VP-SFM, which is a serum-free medium, was used for the cell growth medium.

  A large flask with Vero cells in serum-free culture in a single layer, or Vero cells cultured with microcarriers was inoculated with K. sierra fungus and cytopathic effect (CPE) appeared after culturing at 37 ° C for 5-7 days After confirming this, the culture solution was recovered. The culture solution was filtered using a filter having a pore size of 0.45 μm.

All subsequent operations were performed in a facility environment where microbial containment was possible.
The collected K. sierra fungus culture solution was centrifuged at 4500C for 7,500 rpm for 60 minutes, and the precipitate was suspended in PBS. The suspension was centrifuged at 1,500 rpm for 10 minutes at 4 ° C., and the supernatant was collected. The sedimentation was suspended in an appropriate amount of PBS, centrifuged at 1,800 rpm for 5 minutes at 4 ° C., and the supernatant was collected. The above operation was repeated again, and all the collected supernatants were centrifuged at 7,500 rpm for 60 minutes at 4 ° C. The obtained precipitate was suspended in a small amount of PBS, layered on PBS containing 7.6% urografin (Nihon Schering) containing 30% sucrose, and centrifuged at 4 ° C. and 10,000 rpm for 60 minutes using an RPD40T rotor (Hitachi). After removing the supernatant, the cloudy layer formed at the bottom of the tube was collected. The collected cloudy layer was layered on PBS, and centrifuged at 14,000 rpm for 60 minutes at 4 ° C. using the same rotor. After removing the supernatant, the precipitate was suspended in PBS to obtain a crudely purified bacterium.

Next, density gradient centrifugation of the roughly purified bacteria was performed. 76% urografin was diluted to 25, 30, 35 and 40% with PBS and layered gently. After standing for 2 hours, the crudely purified bacteria were overlaid, and density gradient centrifugation was carried out at 24,000 rpm for 60 minutes at 4 ° C. using the same rotor. After centrifugation, the layers formed between 25-30%, 30-35% and 35-40% were recovered. The collected bacteria were layered on PBS and centrifuged at 14,000 rpm for 60 minutes at 4 ° C. using the same rotator. After removing the supernatant, the precipitate was suspended in a small amount of PBS to obtain purified bacteria.
The concentration of the purified cells was adjusted to 1 mg / mL (protein concentration).
When the microbial cells in the culture supernatant and the purified microbial cells were compared with those cultured in the presence of serum, no substantial difference was observed when examined by electrophoresis.

[Example 2] Production, inoculation, and antibody confirmation of Q-fever vaccine Formalin 0.5% was added to the viable cell suspension of Example 1 (100 mL in PBS) and left at 4 ° C for 2 weeks for inactivation. Thereafter, dialysis was performed with PBS to remove formalin.
Thereafter, the protein amount was diluted to 60 to 100 μg / ml, and a stabilizer was added to make a vaccine stock solution.
As the stabilizer, those used in general biopharmaceuticals such as lactose and sorbitol were used as they were.

  The vaccine was injected into 5 Balb / C mice (5 weeks old) and immunized again after 4 weeks. Blood was collected from the mice 2 weeks after the second vaccination. When the anti-Coxiella antibody was measured in the serum, it was found to be antibody positive (IFA antibody titer 64 times or more). From this result, it was shown that this vaccine retains sufficient immunogenicity.

  Until now, there was a vaccine preparation manufactured by serum-added culture as a Q fever vaccine, but it is possible that an unknown pathogenic factor contained in the serum was mixed, and that it is highly safe I could not say. The Q fever vaccine preparation containing as an active ingredient an antigen obtained by serum-free culture of the coccyellae of the present invention is a mixture of unknown pathogenic factors contained in the serum by growing the cocciellae in a serum-free medium. It is safer than conventional vaccines and is very useful in the medical and pharmaceutical fields.

  These highly safe Q fever vaccine preparations can be applied not only to human Q fever prevention but also to the prevention and treatment of pets, domestic animals, outdoor animals and birds, which are considered to be the source of human infection. It is also useful in the field of livestock industry.

Claims (3)

A Q fever vaccine preparation containing, as an active ingredient, an antigen obtained by serum-free culture of the Coxsiella burnetti Nine Mile strain , wherein the antigen is the following antigen (a) :
(a) Inactivated Kokusiera obtained by inactivating Vero cells as cultured cells and VP-SFM medium as serum-free medium, inoculating Kokusiella Bernetti Nine Mile strain with serum-free culture and formalin treatment ・ Balletti Nine Mile strain A Q fever vaccine preparation containing, as an active ingredient, an antigen obtained by serum-free culture of Coxiella burnetti nineine mile strain , wherein the antigen is an antigen obtained by the following steps (a) to (c): A Q fever vaccine formulation.
(a) A step of producing a culture preparation of Coxiella burnetti nine mile strain by serum-free culture of Coxiella burnetti nine mile strain using Vero cells as cultured cells and VP-SFM medium as serum free medium.
(b) A process for purifying the cells of the Koxiella Bernetti Nine Mile strain from the culture preparation
(c) The process of inactivating all cells of the Koxiella barnetti nine mile strain by adding formalin to the Kokusiella bacteria culture preparation A method for producing a Q fever vaccine preparation comprising the following steps (a) to (d):
(a) A step of producing a culture preparation of Coxiella burnetti nine mile strain by serum-free culture of Coxiella burnetti nine mile strain using Vero cells as cultured cells and VP-SFM medium as serum free medium.
(b) A process for purifying the cells of the Koxiella Bernetti Nine Mile strain from the culture preparation
(c) The process of inactivating all cells of the Koxiella barnetti nine mile strain by adding formalin to the Kokusiella bacteria culture preparation
(d) mixing the inactivated whole body of Cokesiella Bernetti Nine Mile strain with pharmaceutically acceptable additives