AU2005253864B2 - Q fever vaccine preparation and method of producing the vaccine - Google Patents

Description

I DESCRIPTION Q FEVER VACCINE PREPARATION AND METHOD OF PRODUCING THE VACCINE 5 Technical Field The present invention relates to Q fever vaccine preparations comprising as an active ingredient an antigen obtained by growing a Coxiella strain in serum-free medium, and methods for producing the Q fever vaccine preparations. 0 Background Art Q fever (coxiellosis) is a zoonotic disease caused by infection with Coxiella burnetii bacteria. It is called Q fever in humans, and is suspected to be associated with chronic fatigue syndrome of unknown cause. In animals (including birds), it is called coxiellosis. It is mostly latent in animals and its lethality is considered to be low. 5 The main pathogen of Q fever is a bacterium, and is classified in the order Legionellales; family Coxiellaceae; genus Coxiella; species burnetii. Similar to other bacteria in the Legionelales family, the Coxiella strain is an obligate intracellular parasitic bacterium that divide and grow only inside animal cells. The bacteria of the present invention are Gram-negative small rod-shaped, and show size polymorphism and a Phase I/Phase II transition !0 similar to the S/R (smooth/rough) phase transition in E. coli. Phase I bacteria are observed among fresh isolates; they are highly virulent and their cell surface layer is covered with long-chain lipopolysaccharides (LPS). Phase II bacteria are produced in subcultures of Phase I bacteria; they are avirulent and their cell surface layer is covered with short-chain LPS. Various clinical manifestations of Q fever are reported, and they can be divided largely !5 into acute and chronic types. Most of the acute types show influenza virus-like infections characterized by fever, headache, muscular pain, joint pain, coughing, and such. Tendencies to develop pneumonia or hepatitis are also observed. The prognosis is generally favorable; fever is usually alleviated and the patient recovers in two weeks or so. On the other hand, symptoms of the chronic type are often endocarditis, and in particular, those accompanying chronic 0 hepatitis and myocarditis. Antibiotics (tetracyclines) can be used as a method for treating Q fever. However, under the current situation where useful and convenient diagnostic agents do not exist, it takes a rather long time before Q fever is diagnosed, followed by administration of antibiotics; therefore, the effect of antibiotics is insufficient for preventing the infection from spreading. 5 In addition, few vaccines are effective and safe for preventing and treating Q fever. Examples of known Q fever vaccines include a vaccine that uses inactivated Coxiella whole cells 2 (Patent Document 1). As mentioned above, the Coxiella strain is obligate intracellular parasitic bacterium and divide and grow only inside animal cells; therefore, serum has been added as an essential nutritional factor in media used for growing the Coxiella strain, as in the case of cell culture 5 media for growing viruses. However, in some virus cultures, unknown factors contained in the serum were found to act as pathogens; therefore, emergency measures have been taken to avoid contamination with factors causing, for example, AIDS, hepatitis, mad cow disease. Serum-free cell culture media were developed for growing virus strains, and they could also be used for producing viral vaccines. However, no research has been carried out on 0 serum-free cell culture media suitable for growing the Coxiella strain. Q fever vaccines produced by using the Coxiella strain grown in a serum-free medium or antigens obtained from such a medium have not been developed yet. Prior art documents related to the present invention of this application are shown below. [Patent Document 1] W097/33614 5 [Non-Patent Document 1] Kazar J. et al. Acta Virol. 31. 158-167. 1987 [Non-Patent Document 2] Schneer N. et al. Zentblat. Bakteriol. Microbiol. Hyg. 267. 79-88. 1987 Disclosure of the Invention 0 Problems to be Solved by the Invention The present invention was achieved in view of the above circumstances. An objective of the present invention is to provide Q fever vaccine preparations which comprise as an active ingredient an antigen obtained by growing a Coxiella strain in serum-free medium, and uses thereof, and methods for producing the Q fever vaccine preparations and for growing the 5 Coxiella strain in a serum-free medium. Means to Solve the Problems To solve the above-mentioned problems, the present inventors studied Q fever epidemiology, and developed vaccine preparations. Since Coxiella is a group of obligate intracellular parasitic bacteria, animal cell culture technology is usually used to culture Coxiella 0 bacteria. As media for animal cell culture are ordinarily supplemented with serum, serum-supplemented culture have also been used for culturing Coxiella strain. However, when cultures obtained by such methods are used as vaccines, risks that these cultures might be contaminated with unknown pathogenic factors derived from serum have to be considered. To solve the above-mentioned problems, the present inventors studied culture methods 5 for culturing bacteria and discovered for the first time that a Coxiella strain can be cultured in serum-free culture medium, and revealed that by using such cells, vaccines can be produced by 3 serum-free culture methods. Namely, the present inventors succeeded in producing Q fever vaccines comprising as an active ingredient an antigen obtained by propagating a Coxiella strain in a serum-free medium, thereby accomplishing the present invention. 5 More specifically, the present invention provides: 1. a Q fever vaccine preparation comprising as an active ingredient an antigen obtained from a Coxiella strain propagated inside an animal cell in a serum-free medium; 2. the Q fever vaccine preparation of 1, wherein the antigen is at least one of the antigens selected from: 10 (a) inactivated whole Coxiella cells obtained by inactivating a Coxiella strain propagated inside an animal cell in a serum-free medium; and (b) inactivated proteins obtained by inactivating one or more proteins derived from Coxiella cells propagated inside an animal cell in a serum-free medium; 3. the Q fever vaccine preparation of 1, wherein the antigen is obtained by the steps 15 of: (a) producing a Coxiella culture preparation by propagating a Coxiella strain inside an animal cell in a serum-free medium; (b) purifying the Coxiella culture preparation; and (c) inactivating whole Coxiella cells by adding an inactivating reagent to the 20 Coxiella culture preparation; 4. the Q fever vaccine preparation of 1, wherein the antigen is obtained by the steps of: (a) producing a Coxiella culture preparation by propagating a Coxiella strain inside an animal cell in a serum-free medium; 25 (b) purifying Coxiella cells from the culture preparation; and (c) adding an inactivating reagent to one or more proteins derived from the Coxiella cells to inactivate said antigen proteins; 5. the Q fever vaccine preparation of any one of 1 to 4, wherein the Coxiella strain is a wild-type strain, a clinical isolate, or an artificial mutant strain or a recombinant strain 30 derived from these strains; 6. the Q fever vaccine preparation of any one of 1 to 5, wherein the Coxiella strain is Coxiella burnetii; 7. a method for producing the Q fever vaccine preparation of any one of 1 to 6; 8. the method of 7, comprising the steps of: 35 (a) producing a Coxiella culture preparation by propagating a Coxiella strain inside an animal cell in a serum-free medium; 4 (b) purifying the Coxiella culture preparation; (c) inactivating whole Coxiella cells by adding an inactivating reagent to the Coxiella culture preparation; and (d) mixing the inactivated whole Coxiella cells with a pharmaceutically acceptable 5 additive; 9. the method of 7, comprising the steps of: (a) producing a Coxiella culture preparation by propagating a Coxiella strain inside an animal cell in a serum-free medium; (b) purifying Coxiella cells from the culture preparation; 10 (c) adding an inactivating reagent to one or more proteins derived from the Coxiella cells to inactivate said antigen proteins; and (c) mixing the inactivated antigen proteins with a pharmaceutically acceptable additive; 10. a method of culturing a Coxiella strain, which comprises propagating the Coxiella 15 strain inside an animal cell in a serum-free medium; 11. a method for producing a Coxiella culture preparation, which comprises propagating the Coxiella strain inside an animal cell in a serum-free medium. Best Mode for Carrying Out the Invention 20 The present invention provides Q fever vaccines, which comprise as an active ingredient an antigen obtained by growing a Coxiella strain in serum-free medium. Also, the present invention relates to methods for producing the above-mentioned Q fever vaccines with improved safety. The Q fever vaccine preparations of the present invention are produced by 25 culturing and collecting bacterial antigens and then inactivating the antigens, according to conventional methods of bacterial vaccine production. Whole bacteria cells, cell components, proteins, DNA fragments and such are used as antigens in the present invention. Natural-type antigens or mutant antigens can be used, and when antigen proteins are produced in culture, they can be collected and used. 30 In the present invention, bacterial strains of the wild type, clinical isolates or such of the Coxiella strains, or artificial mutants of such Coxiella strains may be used. The bacterial strain is preferably Coxiella burnetii, and of this bacterial strain, the Nine Mile strain is more preferable. The Coxiella burnetii Nine Mile strain can be obtained as VR615 strain from ATCC (an organization of culture collection). The Phase II Coxiella 35 burnetii Nine Mile strain can be used by refining and purifying the Phase II bacteria of a clinical isolate. The present inventors used a strain that was provided by Dr. Kazar, J.

4a (Slovakia) as a yolk-sac emulsion and stored at Kitasato Institute. As for the methods for producing vaccines of the present invention, the step of producing Coxiella bacterial culture preparations obtained by growing a Coxiella strain in serum-free medium is described below. 5 When a Coxiella strain is cultured in the present invention, animal cells are used as substrates, and Coxiella cells are propagated inside the animal cells by growing the cells. Examples of animal cells that are used include buffalo green monkey (BGM) cells, Vero cells, and L-929 cells, and CHO cells for recombinants. Media for culturing animal cells to be used to produce vaccines of the present 10 invention 5 are serum-free media. Any composition may be used for the serum-free media, so long as the composition is suitable for growing the cells used. Carbon sources, nitrogen sources, phosphate sources, trace metals and such may be included in the media, but serum is not added. Commercially available serum-free media may also be used. Examples of commercially 5 available serum-free media that can be used to culture the Coxiella strain are preferably VP-SFM, Opti-Pro SFM, CD-CHO and such, but are not limited thereto. Glass container, stainless steel container, plastic container, commercially available fermentor or such can be used as a cell culture container for culturing the Coxiella strain in the present invention. 0 The incubation temperature for cell cultures of the present invention is usually 35-37*C, but it is not particularly limited to this temperature range so long as the cells can be grown. The pH range for cell cultures of the present invention is usually 6.8 to 7.6, but it is not particularly limited to this pH range, and cells can be cultured under more acidic or basic conditions provided that the cells are not killed. 5 Any one of the known culture methods, for example, static culture, roller bottle culture, microcarrier suspension culture, microchip culture, cell suspension culture, and such can be used for the culture of the present invention. The optimal period and conditions for culture are selected depending on the Coxiella strain used. When cells are cultured in a serum-free medium, the number of Coxiella bacterial cells 0 is counted as necessary to observe the progress of culture. In this measurement, inclusion bodies are also counted and summed. The number of bacterial cells may be measured on a microscope after a short incubation period and staining the cells, or it may also be determined by measuring the turbidity at 650 nm. Recently, it may also be determined from the number of DNA copies using quantitative PCR techniques, as follows. That is, the Coxiella cell count 5 (titer) can be measured according to a known method (Schneider, W. Zentralbl. Bakteriol. 271: 77-84, 1989), and specifically, the following procedures may be used. First, BGM cells are cultured on a 24-well plate with a cover slip. After formation of monolayer sheets, 0.5 mL of an approximately 100-fold diluted live Coxiella burnetii cell suspension is inoculated into each well, and after 30-minutes of adsorption, the cells are centrifuged at 1,500 rpm. After 0 centrifugation, the supernatant is removed, 1 mL of MEM is added to each well, and this is incubated at 37"C (5% CO 2 ). On the fourth day of incubation, the cover slips are collected, and the cells are fixed using cold acetone. The number of inclusion bodies can be measured by the fluorescent antibody (FA) method or enzyme antibody technique. In the fluorescent antibody method, the Coxiella burnetii-infected mouse serum, which is used as the primary antibody, is 5 prepared by dilution in phosphate buffered saline (PBS), and reaction is carried out in a humid box at 37*C for 60 minutes. After three 5-minute PBS washes, reaction is carried out with an 6 FITC-labeled anti-mouse IgG sheep serum (CAPPEL) prepared in PBS using a similar method. Washing is followed by 1200-fold dilution, and reaction with propidium iodide for five minutes. After washing, the cells are mounted using a Slow Fade Antifade Kit (Molecular Probes), and the number of strongly colored inclusion bodies is counted under a fluorescence microscope 5 (BX-50-FLA, OLYMPUS). Recombinant antigens of the present invention can be produced by recombinant organisms, and the following known method can be applied. Coxiella strains isolated from clinical specimens are used. Ordinarily, clinical isolates carry protein antigens derived from a Coxiella strain as well as protein antigens derived from heat-resistant Coxiella bacteria. First, 0 protein antigens derived from Coxiella bacteria are isolated. Genes encoding protein antigens derived from Coxiella bacteria (hereinafter referred to as "Coxiella antigen gene") (6.7 kbp) are excised from plasmids by restriction enzyme treatment and such, and are ligated into plasmids such as pBR322. Examples of Coxiella antigen genes that may be used include those disclosed in the following articles: Zhang,G. Q., et al. J. Clin. Microbiol. 42:423-428 (1998); Nguyen, Sa 5 V., et al. FEMES Microbiol. Lett. 175:101-106 (1999); and Nguyen, Sa V., et al., Microbiol. Immunol. 43:743-749 (1999). Next, after amplifying the Coxiella antigen gene by methods such as PCR, the Coxiella antigen gene is ligated into a different expression vector. Next, site-directed mutagenesis is performed on this vector, and then the gene is amplified by PCR using selective primers to 0 produce a recombinant E. coli strain that produces protein antigens derived from a recombinant mutant of Coxiella bacteria. The recombinant E. coli were cultured according to standard methods to obtain Coxiella-derived protein antigens from culture medium. By performing site-directed mutagenesis in the above-mentioned genetic recombination procedure, recombinant E. coli that produce a Coxiella-derived mutant protein antigen can be 5 obtained. The E. coli can be cultured and subjected to purification procedures to obtain Coxiella-derived mutant protein antigen. For example, as described above, by applying site-directed mutagenesis or such, the amino acid residue at any site in the amino acid sequence of the Coxiella-derived protein antigen can be changed to another residue. Conversely, a particular amino acid residue at a particular site, for example a glutamic acid residue, can be 0 maintained unchanged. The same is essentially applied to sugar residues. Mutants having immunogenic activity are selected from the obtained mutants, and are used as Coxiella-derived protein antigen mutants of the present invention. The Coxiella bacterial antigenic activity of such mutants can be at any level. Several trial-and-error experiments will be necessary to obtain mutants, but the obtained recombinant mutant Coxiella antigens are generally 5 advantageous in that they are less likely to revert to natural form of Coxiella antigens. In the steps of producing Coxiella culture preparations, Coxiella cells are cultured in a 7 serum-free medium, and the whole bacterial antigens are washed, purified, and concentrated. The methods for washing, purification, and concentration are described below. After culturing in a serum-free medium, cells are collected and disrupted by freeze-thawing, sonication or such to collect Coxiella cells therefrom. Centrifugation, gel 5 filtration, salt precipitation and the like can be used in the present method. Ordinarily, bacterial cells are then washed and purified using phosphate buffered saline and such. Purification may be carried out using methods such as washing, centrifugation, stirring, concentration, and column methods, but not limited thereto. For example, purified bacteria can be obtained by the method of Example 1. Coxiella cells must be cultured, collected, washed and purified under 0 conditions of strict containment, conventionally at Biosafety Level 3 (BSL3). One or more antigen proteins derived from Coxiella bacteria cultured in a serum-free medium may also be collected in the present invention. One or more antigen proteins can be obtained by subjecting Coxiella cells to partial or complete disruption. For the production of subunit vaccines, effective antigen fractions may also be collected 5 by subjecting Coxiella cells to partial or complete disruption. Furthermore, when using antigen proteins produced and released into the culture, antigenic proteins may be concentrated and purified by combining effective methods such as centrifugation, concentration, and column methods. The timing of washing, purification, and concentration is preferably before inactivation, 0 but they may be carried out after inactivation. They may also be carried out both before and after inactivation. In the methods of the present invention for producing vaccines, the step of inactivating a Coxiella culture preparation or such by adding an inactivating regent to the Coxiella culture preparation or such will be described below. 5 Various methods can be used to inactivate antigens, but generally, the optimal inactivation method differs depending on whether the antigen is a whole bacterial cell or a protein. Inactivation can be carried out by various chemical treatments and physicochemical treatments. As an example, inactivation includes the following but is not limited thereto. Examples of chemical treatment substances that may be used to treat bacteria or 0 antigen-containing proteins for the purpose of inactivation include the following substances. Applicable concentrations are shown, but they are not limited thereto: formalin (0.1-10 v/v %), phenol (0.1-5 v/v %), chloroform (10-60 v/v %), acetone (10-80 v/v %), and SH reagent (1-100 mM); hydrogen peroxide (0.1-5%), peracetic acid (0.5-10 w/v %), and carbon dioxide (5-90 5 v/v %); and ozone (0.1-10 v/v %) and surfactant (0.01-5 w/v %).

8 Physicochemical treatment method for inactivation can be carried out by taking the following measures. Examples of applicable conditions are shown below, but they are not limited thereto: Heating (temperature: 30-70*C; heating time: 10-120 minutes), y-ray irradiation 5 (radiation source: cobalt 60; 5-50 kGy (kiloGray)), laser light irradiation (light source: various laser irradiation equipment; wavelength: 500-700 nm; intensity of light: 0.01-1 J (Joule)/cm2 electron beam irradiation (microwave oven), ultrasound irradiation and the like. The treatment conditions are not fixed, and the amount of bacteria cells, temperature, buffer pH, treatment period, and such can be varied to set up suitable conditions. It is generally 0 carried out under sterile conditions. These inactivation methods can be used singly or in combination for whole bacteria cells or proteins derived from bacteria cells (including culture medium-derived antigenic proteins). Since the presence of amino acids or amines during inactivation may result in improvement and stabilization of the quality of inactivated preparations, these substances may be 5 supplemented as necessary. Also, in addition to inactivation by formalin, additional application of organic solvent treatment, heating, and y-ray irradiation treatment can yield inactivated whole bacteria that have stable quality because of less bacteriolysis activity. An example of an inactivation procedure is as follows. First, 1% (v/v) formalin is added to a live cell suspension of a Coxiella strain, which are left to stand for 5 days at room 0 temperature for inactivation. Thereafter, the suspension is centrifuged at 13,000 rpm for 15 minutes, the supernatant is removed, and the cells are suspended in an equal amount of phosphate buffered saline (0.1 M, pH6.8) to produce an inactivated Coxiella burnetii suspension. Completion of inactivation can be confirmed by withdrawing a portion of the inactivated sample and confirming that the bacterium do not grow when incubated. After inactivation treatment, 5 chemical reagents used for the inactivation are removed by washing with phosphate buffer or such. Then, the number of bacteria is adjusted so that the turbidity at 650 nm is about 1-10 (approximately 1-10 x 10 9 cells/mL). Alternatively, the antigen protein concentration is adjusted. A fixed amount (for example, 0.5 mL, 1.0 mL, or 10 mL) of the aforementioned 0 Coxiella suspension or antigen protein solution is poured into each vial or syringe to produce the vaccines. An example of a conventional Q fever vaccine composition is as follows, but is not limited to such composition. Antigen: inactivated whole Coxiella bacteria; protein concentration of 50-100 mcg/mL 5 Buffer: 0.01 M phosphate buffer supplemented with salt pH: 7.0 9 Osmotic pressure: 1 Volume: 0.6 mL Preservatives: 2% sorbitol and 5% lactose Pharmaceutically acceptable additives may be added to the vaccine preparations of the 5 present invention. Herein, "pharmaceutically acceptable additives" refer to pharmaceutically acceptable materials that are substances different from antigen (or immunogen) and that can be administered with an antigen during vaccination. Examples include vaccine additives such as adjuvants, preservatives, and stabilizers, but are not limited thereto. Without limitation, adjuvants such as aluminum phosphate, aluminum hydroxide, and MF59 may be used as 0 approved adjuvants for use in human vaccine. Without limitation, about 0.2% gelatin or dextran, 0.1-1.0% sodium glutamate, approximately 5% lactose, approximately 2% sorbitol, or such may be used as a stabilizer. Without limitation, about 0.01% thimerosal, about 0.1% beta-propionolactone, about 0.5% phenoxyethanol or such may be used as a stabilizer. 5 Injections are prepared by adding pH-control agents, buffers, stabilizers, preservatives or such if necessary and made into subcutaneous, intramuscular, and intravenous injections by common procedures. An injection can be prepared as a solid formulation before use by, for example, freeze-drying a solution stored in a container. A single dose can be filled in a container, or multiple doses may be filled in one container. 0 Various known methods can be used for administering bacterial vaccines of the present invention. Vaccines are administered by preferably subcutaneous injection, intramuscular injection, intranasal administration, oral administration, percutaneous administration and such, and more preferably by intramuscular injection, but are not limited thereto. Examples of intranasal administration include intranasal sprays, powder sprays, drops, swabs and the like. 5 Among these methods of vaccination, oral inoculation and intranasal inoculation are more preferable, because the respiratory tract and digestive tract mucous membranes are important sites at early stages of infection. By inoculating a vaccine comprising appropriate adjuvants having strong immunity-enhancing activities, immune mechanism is induced at local mucous membrane and protective effects can be exhibited at the earliest possible stages of infection. 0 A suitable vaccination method is determined by considering the type of vaccine antigen, dosage form, timing of antibody expression, duration of antibody persistence, age of the subject receiving vaccine, and such. Besides humans, examples of the subject to be inoculated include pet animals, domestic animals, wild animals and birds, which are considered to be sources of infection. The method of vaccination is ultimately determined through clinical testing by 5 specialists, and these methods are well known to those skilled in the art. The product may be for single inoculation or for multiple inoculations. The dosage changes depending on the 10 weight and age of the patient, the method of administration and such, but those skilled in the art can appropriately select a suitable dose. Determination of vaccine efficacy is important in vaccine development. While these tests are well known to vaccine professionals, a conventional method is described below. 5 Antigenic materials which possibly comprise a protective antigen or an antigen that prevents disease onset are inactivated, and then laboratory animals are immunized. Next, the immunized animals are challenged with a virulent Coxiella strain, and the effectiveness of the test material is determined from the viability of the laboratory animals, decrease in the number of infectious Coxiella cells, and the like. Since Coxiella strains are zoonotic, they can infect many types of 0 laboratory animals. Methods that are more suitable for laboratory animals are determining their viability, or measuring the degree of Coxiella infection in the organs. All prior art references cited herein are incorporated by reference into this description. Examples 5 Hereinbelow, the present invention will be specifically described with reference to the Examples, but is not limited thereto. [Example 1] Serum-free culture of Q fever bacterium A type culture strain, Coxiella burnetii Nine Mile strain (ATCC VR 615), was used as 0 the bacterial strain. Vero cells (ATCC CCL-8 1) were used to culture Coxiella cells, and VP-SFM, a serum-free medium, was used as the cell growth medium. Coxiella bacteria were inoculated onto a monolayer sheet of Vero cells cultured in a large flask or into a microcarrier culture, and after incubation at 37*C for five to seven days, development of cytopathogenic effect (CPE) was confirmed, and then the culture was collected. 5 The culture was filtered using a filter with a pore size of 0.45 pm. All subsequent procedures were carried out in a facility environment that allows containment of microorganisms. The collected Coxiella culture was centrifuged at 7,500 rpm for 60 minutes at 4'C, and the sediment was suspended in PBS. The cell suspension was centrifuged at 1,500 rpm for ten 0 minutes at 4*C, and the supernatant was collected. The sediment was suspended in a suitable amount of PBS, and then centrifuged at 1,800 rpm for five minutes at 4*C, and the supernatant was collected. The above-described operation was repeated again and all of the collected supernatants were combined and centrifuged at 7,500 rpm for 60 minutes at 4*C. The obtained sediment was suspended in a small amount of PBS, overlaid on 7.6% Urografin (Japan 5 Schering)-containing PBS comprising 30% sucrose and centrifuged at 10,000 rpm, for 60 minutes at 4*C using a RPD40T rotor (Hitachi). After removing the supernatant, the cloudy 11 layer formed at the bottom of the tube was collected. The collected cloudy layer was overlaid on PBS, and was centrifuged at 14,000 rpm, 60 minutes, 4*C using the same rotor. After removing the supernatant, the sediment was suspended in PBS to produce partially purified bacterial cells. 5 Next, density gradient centrifugation was carried out on the partially purified bacterial cells. Seventy-six percent of Urografin was diluted to 25, 30, 35, and 40% using PBS, and were gently layered. After the Urografin was left to stand for 2 hours, the partially purified bacterial cells were overlaid, and this was subjected to density gradient centrifugation at 24,000 rpm, 60 minutes, 4*C using the same rotor. After centrifugation, each of the layers that formed 0 between 25-30%, 30-35%, and 35-40% was collected individually. The collected bacterial cells were overlaid on PBS and then centrifuged at 14,000 rpm, 60 minutes, 4*C using the same rotor. After removing the supernatant, the sediments were suspended in a small amount of PBS to produce purified bacterial cells. The concentration of the purified bacterial cells was adjusted to 1 mg/mL (protein 5 concentration). When the cells in the culture supernatant and purified bacterial cells were compared electrophoretically with the bacterial cells cultured in the presence of serum, no substantial differences were found. 0 [Example 2] Production and inoculation of the Q fever vaccine and detection of antibody formation To the live bacterial cell suspension (100 mL in PBS) obtained as in Example 1, 0.5% formalin was added, and this was left to stand for two weeks at 4"C for inactivation. Then, formalin was removed by dialysis against PBS. 5 This was followed by dilution to a protein concentration of 60-100 jig/mL and addition of a stabilizer to produce a vaccine stock solution. Lactose, sorbitol, and such substances used in conventional biological preparations were used as stabilizers without further modification. The vaccine was injected into five Balb/C mice (five-weeks old), and a booster 0 inoculation was given four weeks later. Blood was drawn from the mice two weeks after the second vaccination. When the anti-Coxiella antibody in the sera was measured, the sera were found to be antibody positive (IFA antibody titer of 64 folds or greater). The results showed that the present vaccine possessed sufficient immunogenicity. 5 Industrial Applications Q fever vaccine preparations produced by serum-containing cultures were available, but 12 they were not considered safe because there is a possibility that unknown pathogens might be contaminated in the serum. In the Q fever vaccine preparations of the present invention, which comprise as an active ingredient an antigen obtained by growing a Coxiella strain in serum-free culture, contamination by unknown pathogenic factors mixed in the serum was 5 prevented by growing the Coxiella strain in a serum-free medium; therefore, the products are very safe compared to conventional vaccines and are very useful in the fields of medicine and pharmacy. Such high-safety Q fever vaccine preparations can be applied not only to prevention of Q fever in humans, but also to prevention and treatment of the disease in pet 10 animals, domestic animals, wild animals, and birds which are considered to be sources of infection for humans, and are useful in the fields of livestock industry and such. Comprises/comprising and grammatical variations thereof when used in this specification are to be taken to specify the presence of stated features, integers, steps or components or groups thereof, but do not preclude the presence or addition of one or more 15 other features, integers, steps, components or groups thereof.

Claims (14)

1. A Q fever vaccine preparation comprising as an active ingredient an antigen obtained from a Coxiella strain propagated inside an animal cell in a serum-free medium. 5

2. The Q fever vaccine preparation of claim 1, wherein the antigen is at least one of the antigens selected from: (a) inactivated whole Coxiella cells obtained by inactivating a Coxiella strain propagated inside an animal cell in a serum-free medium; and 10 (b) inactivated proteins obtained by inactivating one or more proteins derived from Coxiella cells propagated inside an animal cell in a serum-free medium.

3. The Q fever vaccine preparation of claim 1, wherein the antigen is obtained by the steps of: 15 (a) producing a Coxiella culture preparation by propagating a Coxiella strain inside an animal cell in a serum-free medium; (b) purifying the Coxiella culture preparation; and (c) inactivating whole Coxiella cells by adding an inactivating reagent to the Coxiella culture preparation. 20

4. The Q fever vaccine preparation of claim 1, wherein the antigen is obtained by the steps of: (a) producing a Coxiella culture preparation by propagating a Coxiella strain inside an animal cell in a serum-free medium; 25 (b) purifying Coxiella cells from the culture preparation; and (c) adding an inactivating reagent to one or more proteins derived from the Coxiella cells to inactivate said antigen proteins.

5. The Q fever vaccine preparation of any one of claims 1 to 4, wherein the Coxiella strain 30 is a wild-type strain, a clinical isolate, or an artificial mutant strain or a recombinant strain derived from these strains.

6. The Q fever vaccine preparation of any one of claims 1 to 5, wherein the Coxiella strain is Coxiella burnetii. 35

7. A method for producing the Q fever vaccine preparation of any one of claims 1 to 6. 14

8. The method of claim 7, comprising the steps of: (a) producing a Coxiella culture preparation by propagating a Coxiella strain inside an animal cell in a serum-free medium; (b) purifying the Coxiella culture preparation; 5 (c) inactivating whole Coxiella cells by adding an inactivating reagent to the Coxiella culture preparation; and (d) mixing the inactivated whole Coxiella cells with a pharmaceutically acceptable additive. 10

9. The method of claim 7, comprising the steps of: (a) producing a Coxiella culture preparation by propagating a Coxiella strain inside an animal cell in a serum-free medium; (b) purifying Coxiella cells from the culture preparation; (c) adding an inactivating reagent to one or more proteins derived from the 15 Coxiella cells to inactivate said antigen proteins; and (c) mixing the inactivated antigen proteins with a pharmaceutically acceptable additive.

10. A method of culturing a Coxiella strain, which comprises propagating the Coxiella 20 strain inside an animal cell in a serum-free medium.

11. A method for producing a Coxiella culture preparation, which comprises propagating the Coxiella strain inside an animal cell in a serum-free medium. 25

12. A method of medical treatment or prophylaxis of a subject suffering from Q fever wherein the subject is administered an effective amount of the preparation according to any one of claims 1 to 9.

13. A Q fever vaccine preparation substantially as hereinbefore described with reference 30 to any one of the examples of the invention in the description.

14. A method for producing a Q fever vaccine preparation substantially as hereinbefore described with reference to any one of the examples of the invention in the description. 35