Culture media and methods for cultivating bordetella bacteria

Abstract

The present invention relates to culture media and methods for cultivating Bordetella bacteria, particularly for producing vaccines. In particular, the inventors investigated the effects of temperature and alkaline earth metals and transition metals compositions on the growth and virulence factor production of B. pertussis and B. bronchiseptica. They found that both bacteria grew better and expressed more antigens when cultured in MILNEZ medium at 35°C, which mimics the nasal environment of healthy individuals. They also showed that B. pertussis genes were differentially regulated by these conditions. Therefore, the present invention provides a new culture medium that mimics growth conditions in the nose. The new medium includes a source of carbon, nitrogen, salts, alkaline earth metals and specific transitions metals at concentrations close to those in mammalian nasal fluid cavity. The invention aims to improve the growth of Bordetella bacteria for vaccine production.

Description

[0001] CULTURE MEDIA AND METHODS FOR CULTIVATING BORDETELLA BACTERIA

[0002] FIELD OF THE INVENTION:

[0003] The present invention is in the field of medicine, in particular microbiology.

[0004] BACKGROUND OF THE INVENTION:

[0005] Bordetellae are Gram-negative, aerobic, rod-shaped bacteria belonging to the Alcaligenaceae family. They are primarily responsible for respiratory infections in humans and animals, such as whooping cough, bronchitis, and kennel cough. Bordetellae can inhabit the mucosal surfaces of the airways (upper and lower respiratory tract), including the nasal cavity, where they attach to epithelial cells and produce various toxins and virulence factors that damage host tissues and weaken the immune system. Infection of the airway epithelium by BordetellaQ can lead to inflammation, nasal discharge, and sneezing. In particular, Bordetella pertussis is the bacterial agent of whooping cough in humans. This bacterium is highly infectious and can cause severe respiratory symptoms, such as spasmodic cough, inspiratory whoop, and post-tussive vomiting. Bordetella bronchiseptica is the bacterial agent of kennel cough or infectious tracheobronchitis in dogs and other animals. This bacterium can also infect humans, especially those with weakened immunity or chronic lung diseases. Bordetella bronchiseptica can cause respiratory signs, such as coughing, sneezing, nasal discharge, and fever. One of the methods for preventing and controlling Bordetellae infections is vaccination. Two forms of vaccine are in use: acellular vaccine and whole-cell vaccines. Whole-cell vaccines were developed first as suspensions of the entire Bordetella organism that has been inactivated. These pertussis vaccines induce highly effective protection against the disease but the side effects associated with their administration have encouraged the development of a second generation that are less reactogenic, the acellular vaccines which do not contain live bacteria but only selected antigenic components to provide protection against the disease. The acellular pertussis vaccines are composed of proteins purified directly from Bordetella pertussis bacteria. Three to five bacterial proteins composed the acellular pertussis vaccines, including the pertussis toxin (PT), the filamentous hemagglutinin (FHA), the pertactin (PRN), and the fimbriae (FIM). Another vaccine type is composed of live attenuated vaccines made from weakened or modified strains of the bacterium that can still replicate in the host but do not cause disease. These vaccines can mimic natural infection and elicit a robust and long-lasting immune response, both humoral and cellular. Live attenuated vaccines can be administered nasally, which is more convenient and less invasive than the injection route. Moreover, live attenuated vaccines can potentially prevent colonization and transmission of the bacterium by inducing mucosal immunity in the respiratory tract. To grow Bordetella bacteria for producing the vaccines, appropriate culture media are needed to provide the necessary nutrients and environmental conditions for the bacterium. Some of the commonly used culture media include THUS culture medium, a synthetic liquid medium developed by (Thalen, Marcel, et al. "Rational medium design for Bordetella pertussis: basic metabolism." Journal of biotechnology 75.2-3 (1999): 147-159) for cultivating Bordetella bacteria, particularly for producing live attenuated vaccines. THUS culture medium has several advantages over other liquid media for Bordetella bacteria, such as Stainer-Scholte medium or Bordet-Gengou medium. For example, THUS culture medium does not require the addition of blood, serum, or charcoal, which can interfere with the quality and stability of vaccine strains. Moreover, THUS culture medium supports the growth of various species and strains of Bordetella, including Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica, and Bordetella avium. Furthermore, THUS culture medium allows for the production of high yields of live attenuated vaccine strains with reduced toxicity and enhanced immunogenicity. However, there is potential for improvement in the culture media used to grow Bordetella bacteria, particularly in identifying a culture medium that could mimic the growth conditions in the nose.

[0006] SUMMARY OF THE INVENTION:

[0007] The present invention is defined by the claims. In particular, the present invention relates to culture media and methods for cultivating Bordetella bacteria, especially for the production of vaccines.

[0008] DETAILED DESCRIPTION OF THE INVENTION:

[0009] The first object of the present invention relates to a culture medium that is suitable for cultivating Bordetella bacteria comprising a source of carbon, a source of nitrogen, a source of salts and a source of alkaline earth metals and characterized in that it further comprises a source of transition metals.

[0010] As used herein, the term “culture medium” is a liquid or solid substance that contains nutrients and other components that support the growth and maintenance of microorganisms, such as bacteria. Culture media can vary in their composition, pH, oxygen level, and sterility, depending on the type and purpose of the culture.

[0011] As used herein, the term “carbon source” refers to a substance that provides carbon atoms for the synthesis of organic molecules by bacteria. Carbon sources can be organic or inorganic, depending on whether they contain carbon-carbon bonds or not. Examples of organic carbon sources are sugars, such as glucose and fructose, alcohols, such as glycerol and ethanol, and amino acids, such as glutamate and aspartate.

[0012] In some embodiments, the culture medium of the present invention comprises an amount of lactate as a source of carbon. In some embodiments, the culture medium of the present invention comprises an amount of glutamate as a source of carbon. In some embodiments, the culture medium of the present invention comprises an amount of lactate and an amount of glutamate as a source of carbon.

[0013] In some embodiments, the culture medium of the present invention comprises one or more additional sources of carbon. In some embodiments, the culture medium of the present invention comprises an amount of Heptakis(2,6-O-dimethyl)beta-cyclodextrin. In some embodiments, the culture medium of the present invention comprises an amount of cysteine.

[0014] As used herein, the term "nitrogen source" refers to a substance that provides nitrogen atoms for the synthesis of amino acids, nucleotides, and other biomolecules by bacteria. Nitrogen sources can be organic or inorganic, depending on whether they contain carbon-nitrogen bonds or not. Examples of inorganic nitrogen sources are ammonium salts, such as ammonium chloride and ammonium sulfate, nitrate salts, such as sodium nitrate and potassium nitrate, and nitrogen gas, which can be used by some diazotrophic bacteria and archaea that can fix nitrogen through various metabolic pathways. In the culture medium of the present invention, the nitrogen source is typically an ammonium salt, such as ammonium chloride (NH4CI), which can be assimilated by most bacteria and does not interfere with the pH of the culture medium.

[0015] Typically, the ratio between the nitrogen source and the carbon source (N:C ratio) is 1 : 10.

[0016] In some embodiments, the concentration of glutamate is in the range of about 10 mM to about

[0017] 15 mM. In some embodiments, the concentration of glutamate is about 12.6 mM. In some embodiments, the concentration of lactate is in the range of about 14 mM to about 19 mM. In some embodiments, the concentration of lactate is about 16.6 mM.

[0018] In some embodiments, the concentration of ammonium chloride (NH4CI) is in the range of about 1.5 mM to about 2.5 mM. In some embodiments, the concentration of ammonium chloride (NH4CI) is about 2mM.

[0019] As used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value.

[0020] In some embodiments, the culture medium of the present invention comprises a phosphorus source. Phosphorus is an essential element for the growth and development of microorganisms, as it is involved in the formation and function of various biomolecules, such as nucleic acids, phospholipids, and ATP. The culture medium of the present invention can provide phosphorus to the microorganisms by including a phosphorus source, such as monopotassium phosphate (KH2PO4). Monopotassium phosphate is a salt that can dissolve in water and release potassium and phosphate ions. Potassium is another important nutrient for the microorganisms, as it can regulate the osmotic pressure, membrane potential, and enzyme activity of the cells. Phosphate is a form of phosphorus that can be readily assimilated by the microorganisms, and can be incorporated into various organic and inorganic compounds in the cells. By adding monopotassium phosphate or another phosphorus source to the culture medium of the present invention, the microorganisms can obtain sufficient amounts of phosphorus and potassium, and can grow more effectively.

[0021] In some embodiments, the culture medium of the present invention comprises a source of salts. The culture medium of the present invention can also contain potassium chloride and sodium chloride as the main salts. These salts can provide essential ions, such as potassium, sodium, and chloride, for the growth and metabolism of the microorganisms. Potassium is an important electrolyte that can regulate the osmotic balance, membrane potential, and enzyme activity of the cells. Sodium is another electrolyte that can maintain the fluid balance, pH, and nerve impulses of the cells. Chloride is an anion that can help to balance the charge of the cells, and also participate in the formation of hydrochloric acid in the stomach of some microorganisms. The concentration of potassium chloride and sodium chloride in the culture medium can vary depending on the type and strain of the microorganisms, the culture conditions, and the desired products. In some embodiments, the culture medium contains about 0.1% to about 5% (w / v) of potassium chloride and about 0.1% to about 5% (w / v) of sodium chloride.

[0022] In some embodiments, the culture medium of the present invention further comprises a source of nicotinate, also known as nicotinic acid or vitamin B3. Nicotinate is an organic compound that can act as a precursor for the synthesis of nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP), which are important cofactors for various enzymatic reactions in the bacteria. NAD and NADP are involved in the transfer of electrons in the processes of glycolysis, Krebs cycle, oxidative phosphorylation, and pentose phosp / hate pathway, which are essential for the production of energy and biomolecules in the cells. By adding nicotinate or another source of vitamin B3 to the culture medium of the present invention, the microorganisms can enhance their ability to cope with the environmental changes and challenges, and can improve their growth rate, survival, and productivity.

[0023] In some embodiments, the culture medium of the present invention further comprises a reducing agent. As used herein, the term “reducing agent” refers to a substance that can donate electrons to another substance, and thus reduce its oxidation state. This can be beneficial for the growth of microorganisms, as it can prevent or counteract the damage caused by oxygen and other oxidizing agents, such as reactive oxygen species (ROS). In some embodiments, the reducing agent is selected from the group consisting of sodium thioglycolate, L-cystein, and ascorbic acid. Sodium thioglycolate is a salt of thioglycolic acid, which is a thiol-containing compound that can react with ROS and form stable disulfide bonds. L-cystein is an amino acid that also contains a thiol group, and can act as a precursor for the synthesis of glutathione, another important reducing agent in the cells. Ascorbic acid, also known as vitamin C, is a water-soluble compound that can donate electrons to various oxidized molecules, such as iron, copper, and quinones, and regenerate them to their reduced forms. Ascorbic acid can also regenerate other antioxidants, such as vitamin E, glutathione, and uric acid, and thus enhance the overall antioxidant capacity of the cells. By adding one or more of these reducing agents to the culture medium of the present invention, the microorganisms can be more resilient to the oxidative stress induced by the presence of oxygen and other oxidizing agents in the culture environment. This can improve their growth rate, biomass yield, and metabolic efficiency, and thus enhance the production of the desired products. In some embodiments, the culture medium of the present invention further comprises an antioxidant, in addition to other components as above described. The antioxidant can be any compound that can prevent or reduce oxidative damage to the cells, such as by scavenging free radicals, chelating metal ions, or inhibiting lipid peroxidation. One example of an antioxidant that can be used in the culture medium of the present invention is glutathione, which is a tripeptide composed of glutamate, cysteine, and glycine. Glutathione can also act as a cofactor for various enzymes, such as glutathione peroxidase and glutathione reductase, that are involved in the protection of cells from oxidative stress. Glutathione can also participate in the synthesis and metabolism of various biomolecules, such as DNA, proteins, and lipids. Glutathione can also modulate the redox state of the cells, which can affect the expression and activity of various genes and proteins. By adding glutathione or another antioxidant to the culture medium of the present invention, the microorganisms can be more resistant to the harmful effects of oxygen and other oxidizing agents, and can grow faster and more efficiently.

[0024] In some embodiments, the culture medium of the present invention comprises a source of one or more alkaline earth metals.

[0025] As used herein, the term "alkaline earth metal" has its general meaning in the art and is used to describe a metal that belongs to the second group of the periodic table. These metals have two valence electrons in their outermost shell, and thus tend to form divalent cations (M2+) by losing these electrons. These metals are highly reactive and form ionic compounds with various anions. Some examples of alkaline earth metals are beryllium, magnesium, calcium, strontium, barium, and radium.

[0026] In some embodiments, the culture medium comprises a source of calcium ions (Ca2+), at a concentration close to that the one measured in the nasal fluid cavity of a mammal. The culture medium thus can contain a calcium salt, such as calcium sulfate, calcium chloride, or calcium acetate, to provide the source of calcium ions. The concentration of the calcium salt can vary depending on the type and purity of the salt, the pH and temperature of the culture medium, and the specific requirements of the cultivating the Bordetella bacteria. In general, the concentration of the calcium salt can range from about 0.5 pM to about 5 pM in the culture medium. In some embodiments, the calcium salt can range from about 1 pM to about 2pM in the culture medium. In some embodiments, the concentration is about 1.75 pM in the culture medium, which is close to the physiological concentration of calcium ions in the mammalian nasal fluid cavity.

[0027] In some embodiments, the culture medium comprises a source of magnesium ions (Mg2+), at a concentration close to that the one measured in the nasal fluid cavity of a mammal. The culture medium thus can contain a magnesium salt, such as magnesium sulfate, magnesium chloride, or magnesium acetate, to provide the source of magnesium ions. The concentration of the magnesium salt can vary depending on the type and purity of the salt, the pH and temperature of the culture medium, and the specific requirements of the cultivating the Bordetella bacteria. In general, the concentration of the magnesium salt can range from about 0.5 pM to about 5 pM in the culture medium. In some embodiments, the magnesium salt can range from about 1 pM to about 2pM in the culture medium. In some embodiments, the concentration is about 1.4 pM in the culture medium, which is close to the physiological concentration of magnesium ions in the mammalian nasal fluid cavity.

[0028] According to the present invention, the culture medium comprises a source of transition metals.

[0029] As used herein, the term "transition metal" has its general meaning in the art and refers to a metal that has incompletely filled d orbitals or that can give rise to cations with incompletely filled d orbitals. These transition metals have some common characteristics, such as having multiple oxidation states, forming complex coordination compounds, and acting as catalysts in various reactions. Some examples of transition metals are iron, copper, nickel, cobalt, zinc, manganese, chromium, silver, gold, platinum, and mercury.

[0030] According to the present invention, the culture medium of the present invention contains transition metals that are similar in composition and concentration to those found in the nasal cavity of healthy subjects. As used herein, the term “subject” is interchangeable with the term “individual”. In some embodiments, the subject is a mammal. Non-limiting examples of mammals include rodents (e.g., mice and rats), primates (e.g., lemurs, bushbabies, monkeys, apes, and humans), rabbits, dogs (e.g., companion dogs, service dogs, or work dogs such as police dogs, military dogs, race dogs, or show dogs), horses (such as race horses and work horses), cats (e.g., domesticated cats), livestock (such as pigs, bovines, donkeys, mules, bison, goats, camels, and sheep), and deer. In some embodiments, the mammal is a human. In some embodiments, the culture medium of the present invention comprises one or more transition metal ions selected from the group consisting of Co2+, Cu2+, Zn2+, Fe3+and Mn2+.

[0031] In some embodiments, the culture medium comprises a source of cobalt ions (Co2+), at a concentration close to that the one measured in the nasal fluid cavity of a mammal. The culture medium thus can contain a cobalt salt, such as cobalt sulfate, cobalt chloride, or cobalt acetate, to provide the source of cobalt ions. The concentration of the cobalt salt can vary depending on the type and purity of the salt, the pH and temperature of the culture medium, and the specific requirements of the cultivating the Bordetella bacteria. In general, the concentration of the cobalt salt can range from about 0.5 pM to about 5 pM in the culture medium. In some embodiments, the cobalt salt can range from about 0.5 pM to about 1 pM in the culture medium. In some embodiments, the concentration is about 0.68 pM in the culture medium, which is close to the physiological concentration of cobalt ions in the mammalian nasal fluid cavity.

[0032] In some embodiments, the culture medium comprises a source of copper ions (Cu2+), at a concentration close to that the one measured in the nasal fluid cavity of a mammal. The culture medium thus can contain a copper salt, such as copper sulfate, copper chloride, or copper acetate, to provide the source of copper ions. The concentration of the copper salt can vary depending on the type and purity of the salt, the pH and temperature of the culture medium, and the specific requirements of the cultivating the Bordetella bacteria. In general, the concentration of the copper salt can range from about 1 pM to about 10 pM in the culture medium. In some embodiments, the copper salt can range from about 5 pM to about 8 pM in the culture medium. In some embodiments, the concentration is about 6.5 pM in the culture medium, which is close to the physiological concentration of copper ions in the mammalian nasal fluid cavity.

[0033] In some embodiments, the culture medium comprises a source of zinc ions (Zn2+), at a concentration close to that the one measured in the nasal fluid cavity of a mammal. The culture medium thus can contain a zinc salt, such as zinc sulfate, zinc chloride, or zinc acetate, to provide the source of zinc ions. The concentration of the zinc salt can vary depending on the type and purity of the salt, the pH and temperature of the culture medium, and the specific requirements of the cultivating the Bordetella bacteria. In general, the concentration of the zinc salt can range from about 10 pM to about 15 pM in the culture medium. In some embodiments, the zinc salt can range from about 11 pM to about 13 pM in the culture medium. In some embodiments, the concentration is about 12 pM in the culture medium, which is close to the physiological concentration of zinc ions in the mammalian nasal fluid cavity.

[0034] In some embodiments, the culture medium comprises a source of iron ions (Fe3+), at a concentration close to that the one measured in the nasal fluid cavity of a mammal. The culture medium thus can contain an iron salt, such as iron sulfate, iron chloride, or iron acetate, to provide the source of iron ions. The concentration of the iron salt can vary depending on the type and purity of the salt, the pH and temperature of the culture medium, and the specific requirements of the cultivating the Bordetella bacteria. In general, the concentration of the iron salt can range from about 0.01 pM to about 0.03 pM in the culture medium. In some embodiments, the concentration is about 0.02 pM in the culture medium, which is close to the physiological concentration of iron ions in the mammalian nasal fluid cavity.

[0035] In some embodiments, the culture medium comprises a source of manganese ions (Mn2+), at a concentration close to that the one measured in the nasal fluid cavity of a mammal. The culture medium thus can contain a manganese salt, such as manganese sulfate, manganese chloride, or manganese acetate, to provide the source of manganese ions. The concentration of the manganese salt can vary depending on the type and purity of the salt, the pH and temperature of the culture medium, and the specific requirements of the cultivating the Bordetella bacteria. In general, the concentration of the manganese salt can range from about 0.5 pM to about 1 pM in the culture medium. In some embodiments, the concentration is about 0.7 pM in the culture medium, which is close to the physiological concentration of manganese ions in the mammalian nasal fluid cavity.

[0036] In some embodiments, the culture medium of the present invention comprises an amount of Mg2+, Ca2+, Co2+, Cu2+, Zn2+, Fe3+and Mn2+according to the following concentrations:

[0037] In some embodiments, the culture medium of the present invention comprises an N:C ratio of 1 : 10 and an amount of Mg2+, Ca2+, Co2+, Cu2+, Zn2+, Fe3+and Mn2+according to the following concentrations: In some embodiments, the culture medium consists of the THUS culture medium in which the metallic elements already present in the culture medium are substituted by the metal present in the nasal fluid cavity as described in the EXAMPLE. In some embodiments, the culture medium consists of the THUS culture medium in which the metallic elements already present in the culture medium are substituted by an amount of one or more metal ions selected from the group consisting of Mg2+, Ca2+, Co2+, Cu2+, Zn2+, Fe3+and Mn2+. In some embodiments, the culture medium consists of the THUS culture medium in which the metallic elements already present in the culture medium are substituted by an amount of Mg2+, Ca2+, Co2+, Cu2+, Zn2+, Fe3+and Mn2+according to the following concentrations:

[0038] The optimal pH range for the culture medium is from 6.5 to 7.5, with 7.0 being the most preferred value. The pH affects the growth and viability of the cells in the culture.

[0039] The culture medium of the present invention is a liquid medium that can support the proliferation and differentiation of the cells. The culture medium is sterile to prevent any contamination or infection of the bacteria. The culture medium of the present invention can be prepared by any conventional method. A preferred method is to dissolve all the ingredients of the culture medium in de-ionised water and then filter-sterilise the solution to obtain the culture medium of the invention. The preparation of the culture medium is exemplified in the EXAMPLE.

[0040] The present invention also provides the use of the culture medium of the invention for cultivating Bordetella bacteria.

[0041] As used hereon, the term “cultivating” as used herein means the maintenance of, and preferably the growth of, bacteria. Bacterial growth is herein defined as an increase in bacterial biomass.

[0042] As used herein, the term "Bordetella bacteria" refers to a group of Gram-negative, aerobic, rod-shaped bacteria of the genus Bordetella, which are mainly found in the respiratory tract of mammals and birds. Bordetella bacteria are known to cause respiratory infections and diseases in humans and animals, such as pertussis, bronchitis, kennel cough, and atrophic rhinitis. Some examples of Bordetella bacteria are Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica, Bordetella avium, Bordetella holmesii and Bordetella hinzii.

[0043] In some embodiments, the culture medium of the present invention is particularly suitable for cultivating Bordetella pertussis.

[0044] As used herein, the term “Bordetella pertussis has its general meaning in the art and refers to a Gram-negative, aerobic, pathogenic, encapsulated coccobacillus of the genus Bordetella, and the causative agent of pertussis or whooping cough.

[0045] In some embodiments, the present invention relates to a culture medium that is capable of assisting the development of bacteria of the Bordetella genus, and in particular, assisting the simultaneous production of bacterial antigens that include but are not limited to Pertussis Toxin (PTX), Filamentous Hemagglutinin (FHA), Pertactin and the Fimbriae with a high yield.

[0046] Thus a further object of the present invention relates to a cultivating Bordetella bacteria, comprising inoculating the culture medium according to the present invention with a strain of Bordetella bacteria.

[0047] The culture, properly speaking, is carried out by seeding a fermenter with a bacteria concentration in the order of 108or 109bacteria / ml. The bacteria concentration is evaluated by reading the optical density by spectrophotometric analysis at 650 nm.

[0048] The culture is carried out at 35-°37° C., and preferably at 35°C.

[0049] In some embodiments, the culture is carried out with stirring and with aeration.

[0050] The evolution of the biomass is estimated by measuring during the course of time the optical density at 650 nm and by measuring the opacity using a standard OMS equivalent to 10 "Opacity units" per ml, a unit corresponding to a billion bacteria / ml.

[0051] A variety of methods can be used to measure the amount of antigens produced by the culture. For example, an ELISA test can be performed to estimate the levels of pertussis toxin antigens and FHA in the supernatant, using antibodies that recognize FHA and pertussis toxin. Alternatively, other assays known to those skilled in the art can be applied to quantify the antigens. The antigens produced by the culture can be used to compose acellular vaccines or live attenuated vaccines against Bordetella infections.

[0052] The duration of the culture operation depends on the microbial development and expression in the culture supernatant of bacterial antigens such as pertussis toxin and FHA. This period lasts between 30 and 72 hours, and more often between 40 and 45 hours.

[0053] One of the advantages of the cultivating method of the present invention is that it can be suitable for the production of different types of vaccines against Bordetella infections, such as acellular vaccines or whole-cell vaccines including live attenuated vaccines.

[0054] As used herein, the term "vaccine" refers a composition administered to humans or animals to elicit an immune response that may provide protection against a disease or pathogen. This immune response can result in a production of antibodies or simply in the activation of certain cells, in particular antigen-presenting cells, T lymphocytes and B lymphocytes. In some embodiments the vaccine is capable of producing an immune response that leads to the production of neutralizing antibodies in the patient with respect to the antigen provided in the vaccine. The vaccine can be a composition for prophylactic purposes or for therapeutic purposes, or both.

[0055] As used herein, the term “acellular vaccine” has its general meaning in the art and refers to a vaccine that contain purified antigens, that can be inactivated, from a pathogenic microorganism, rather than the whole microorganism.

[0056] As used herein, the term “whole-cell vaccine” refers to a vaccine that contains whole cells of a pathogenic microorganism, such as Bordetella, that have been killed or attenuated by chemical, physical, or genetic means. In particular, whole-cell vaccines include live attenuated vaccine.

[0057] The culture medium of the present invention can support the growth and expression of both the live bacteria and the antigens, and can be easily adapted to different scales and modes of production. Therefore, the cultivating method of the present invention can provide a versatile and efficient way to produce various kinds of vaccines against Bordetella infections. For instance, for preparing an acellular vaccine by the cultivating method of the present invention, the Bordetella bacteria are first grown in the culture medium as described above, under conditions that allow the expression at a high yield of the desired antigens. The antigens can be selected from the list of known virulence factors of Bordetella, such as pertussis toxin, filamentous hemagglutinin, pertactin, fimbriae, or any combination thereof. After a sufficient growth period, the bacteria are harvested and lysed to release the antigens. The antigens can then be purified by standard techniques, such as precipitation, chromatography, filtration, or electrophoresis. The purified antigens can be formulated with suitable carriers and adjuvants, as discussed below, to obtain the final acellular vaccine product. Alternatively, the antigens can be partially purified or used as crude extracts, depending on the desired potency and purity of the vaccine. The acellular vaccine can also comprise one or more heterologous antigens from other pathogens that cause common or co-infections with Bordetella, such as tetanus toxoid and diphtheria toxoid. These antigens can provide additional protection and convenience for the patients, as they can reduce the need for separate vaccinations against these diseases. The heterologous antigens can be formulated with the Bordetella antigens in the same or different vials, depending on the stability and compatibility of the components. The heterologous antigens can also be selected based on the epidemiology and prevalence of the corresponding infections in different regions and populations.

[0058] The vaccines prepared by the method of the present invention can require the addition of one or more pharmaceutical acceptable carrier(s). As used herein, the term “pharmaceutically acceptable carrier” includes any and all solvents, diluents, or other liquid vehicle, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington's Pharmaceutical-Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Typically, the vaccine can comprise a pharmaceutically acceptable excipient, carrier, buffer, stabilizer, or other materials well known to those skilled in the art. Such materials should typically be nontoxic and should not typically interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material can depend on the route of administration, e.g., oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, or intraperitoneal routes.

[0059] The vaccines prepared by the method of the present invention, in particular the acellular vaccines, can require the addition of one or more adjuvant(s). As used herein, the term “adjuvant” refers to a compound that can induce and / or enhance the immune response against an antigen when administered to a patient or an animal. It is also intended to mean a substance that acts generally to accelerate, prolong, or enhance the quality of specific immune responses to a specific antigen. In the context of the present invention, the term "adjuvant" means a compound, which enhances both the innate immune response by affecting the transient reaction of the innate immune response and the more long-lived effects of the adaptive immune response by activation and maturation of the antigen-presenting cells (APCs) especially Dendritic cells (DCs). Examples of adjuvants that may be effective include but are not limited to: aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl- L-alanyl-D-isoglutamine, MTP-PE and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene / Tween 80 emulsion. Other examples of adjuvants include DDA (dimethyldioctadecylammonium bromide), Freund's complete and incomplete adjuvants and QuilA. In addition, immune modulating substances such as lymphokines (e.g., IFN-[gamma], IL-2 and IL-12) or synthetic IFN-[gamma] inducers such as poly EC or poly ICLC (Hiltonol) can be used in combination with adjuvants described herein.

[0060] The method of the present invention can be used to prepare vaccines that are suitable for different modes of administration. For example, the acellular vaccines can be formulated for intramuscular or subcutaneous injection, which are common routes of immunization. The vaccines can also be formulated for oral administration, which can induce both mucosal and systemic immunity. Oral formulations can include enteric coatings, microencapsulation, or other methods to protect the antigens from degradation in the gastrointestinal tract. The vaccines can also be formulated for transdermal or intradermal administration, which can exploit the presence of antigen-presenting cells in the skin. These formulations can involve patches, microneedles, or other devices that deliver the antigens across or into the skin. In some embodiments, the live attenuated vaccine according to the present invention is formulated as to be compatible for nasal administration. As used herein, the term “nasal administration” refers to any form of administration whereby an active ingredient is propelled or otherwise introduced into the nasal passages of a patient so that it contacts the respiratory epithelium of the nasal cavity, from which it is absorbed into the systemic circulation. Nasal administration can also involve contacting the olfactory epithelium, which is located at the top of the nasal cavity between the central nasal septum and the lateral wall of each main nasal passage. The region of the nasal cavity immediately surrounding the olfactory epithelium is free of airflow. Thus, specialized methods must typically be employed to achieve significant absorption across the olfactory epithelium. However, the choice of the mode of administration can depend on various factors, such as the availability, cost, safety, efficacy, and patient preference of the vaccine. The method of the present invention can provide flexibility and versatility in developing and delivering vaccines against Bordetella infections.

[0061] The vaccines prepared by the method of the present invention may be provided as a pharmaceutical composition comprising the vaccine of the present invention in admixture with a pharmaceutically acceptable carrier and optionally adjuvants as mentioned above.

[0062] The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

[0063] FIGURES:

[0064] Figure 1: Growth Monitoring of B. pertussis B1917 in various media and temperatures.

[0065] Bacterial growth kinetics in A) minimal medium THUS at 37 °C, 35 °C, and 32 °C, and B) in vivo mimicking medium MILNEZ at 37 °C, 35 °C, and 32 °C. T75 flasks containing overnight cultures of B. pertussis B1917 inoculated in media at an initial OD of 0.15 units / ml, were connected to the Elocheck device (an inline optical density measurement device designed for continuous analysis of cell suspensions) for precise measurement of the biomass production. The cultures were incubated at 37 °C, 35 °C, and 32 °C. Optical density was measured every minute for 72 hours. The absorbance units on the y-axis are specific to the Elocheck device. C) Comparison of growth curves of B. pertussis B1917 cultured in MILNEZ at 37 °C and 35 °C. Optical density (OD) measurements were taken using a spectrometer. The graph shows the average optical density (OD) at 600 nm from three independent cultures. The statistical analysis was performed using GraphPad Prism software. *** p-value < 0.001.

[0066] Figure 2: Growth Monitoring at 35°C of B. pertussis B1917 cultured in MILNEZ versus THUS.

[0067] A) Growth kinetics of B. pertussis cultured in MILNEZ at 35 °C and THUS 35 °C. The growth monitoring was performed in 96-well plates. Overnight cultures were diluted to an initial OD of 0.15 units / ml and 200 pl of suspension is loaded into the center 60 wells of a 96-well plate. The plate was placed in the Tecan instrument (Tecan, Mannedorf, Switzerland). OD readings were taken at 600 nm every 30 minutes for 72 hours. B) Area under growth curves of B. pertussis cultured in MILNEZ and THUS. Statistical analysis was performed using Unpaired t Test on GraphPad Prism software, *** (p < 0.001).

[0068] Figure 3 : Transcriptomic analysis of B. pertussis B1917 cultured in MILNEZ.

[0069] Bacterial cultures of B. pertussis B1917 in MILNEZ at 35 °C and THUS at 37 °C were harvested at mid-exponential phase (OD=2). Total RNA was extracted followed by library construction and Illumina sequencing. The RNAseq data of each sample were analyzed using Rockhopper v2.0.3 with the default parameters. A) Volcano plot representing the differential gene expression in MILNEZ compared to a standard THUS 37°C culture condition. B) Scatter plot representing an Over-Representation Analysis (ORA) of pathways in MILNEZ at 35°C compared to THUS at 37°C.

[0070] Figure 4: Relative Quantification of Two Major Vaccine Antigens Under Nasal- Mimicking Conditions.

[0071] Overnight cultures of B. pertussis B1917 were inoculated into T75 flasks containing MILNEZ and THUS media at an initial optical density (OD) of 0.15 units / mL. The cultures were incubated at 35°C until they reached an OD of 2, corresponding to the mid-exponential phase. Bacterial lysates were prepared from these aliquots. To visualize the antigens FHA and PTX, Western blot analysis were performed using antibodies specific for FHA (monoclonal antibody Fl; gift of H. Sato; Japan; Leinninger et al. Immunodominant domains present on the Bordetella pertussis vaccine component filamentous hemagglutinin. J. Infect. Dis. (1997), 175, 1423- 1431.) and for the SI subunit of PT (monoclonal antibody 1B7; gift of H. Sato; Japan ; Sato et al. Monoclonal antibody against pertussis toxin: effect on toxin activity and pertussis infections. Infect. Immun. (1984), 46, 422-428.), and protein bands were relatively quantified using Fiji software. A) Western blot corresponding to FHA (first line) and PTX (second line). B) Barplot corresponding to relative quantification of FHA and PTX bands using Fiji software.

[0072] Figure 5 : Growth Monitoring of Bordetella bronchiseptica RB50 in in vivo mimicking medium compared to a standard medium.

[0073] A) Growth curves of B. bronchiseptica cultured in MILNEZ and THUS 35 °C. T75 flasks containing overnight cultures of B. bronchiseptica RB50 inoculated in MILNEZ media, at an initial OD of 0.15 units / ml, were connected to the Elocheck measurement cells. The cultures were incubated at 35 °C. Optical density (OD) measurements were taken using a spectrometer. The graph shows the average optical density (OD) at 600 nm from three independent cultures.

[0074] B) Area under growth curves of B. bronchiseptica VJoOcultured in MILNEZ and THUS. Statistical analysis was performed using Unpaired t Test on GraphPad Prism software, ** (p < 0.01).

[0075] Figure 6 : Murine nasal and lung colonization by B. pertussis after nasal administration of bacteria cultured in nasal mimicking conditions compared to standard media.

[0076] C57BL / 6 mice were intranasally infected with 105CFU of B. pertussis B1917 strain grown either on BG solid medium (•), in THUS liquid medium at 37 °C (■), or in MILNEZ liquid medium at 35 °C (▼). Bacteria were harvested during mid-logarithmic phase and maintained at their respective culture temperatures prior to infection. At 4, 24, 48, and 72 hours postadministration, mice (n = 5 per group per time point) were sacrificed and CFUs were enumerated in homogenized tissues. A) Bacterial load (logw CFU) in nose. B) Bacterial load (logw CFU) in lungs. Each bar represents the mean bacterial count, and each point represents an individual mouse. Data were analyzed using a non-parametric permutation-based ANOVA followed by Conover’s post-hoc test to assess statistical differences between groups. Groups that do not share a common letter are significantly different from each other (p < 0.05).

[0077] EXAMPLE:

[0078] Material & Methods

[0079] Preparation of nasal Mimicking Medium “MILNEZ”

[0080] Composition of in vivo mimicking medium (IVM) for culture of Streptococcus pneumoniae was generously provided by M. de Jonge (Radboud Centre for Infectious Diseases, Nijmegen, Netherlands). This medium is a minimum growth medium for S. pneumoniae containing alkaline earth metals and transition metals concentrations adjusted to those found in the human nasal cavity (van Beek LF, et al. Exploring metal availability in the natural niche of Streptococcus pneumoniae to discover potential vaccine antigens. Virulence. 2020; 11: 1310- 1328). We have adapted this medium to meet the growth requirements of B. pertussis. The Thalen-IJssel (THUS) medium is a standard minimum medium used for the growth of B. pertussis strain (Thalen M., van den IJssel J., Jiskoot W., Zomer B., Roholl P., de Gooijer C., et al. ( 1999). Rational medium design for Bordetella pertussis: basic metabolism. J. BiotechnoL 75 147-159). The MILNEZ medium is based on THUS in which the metallic elements already present in the THUS media were substituted by metal element present in the nasal fluid cavity (Table 1). Briefly, from standardized metal ion solutions of Mg2+, Ca2+, Co2+, Cu2+, Zn2+, Fe3+ and Mn2+ (Titrisol®, Merck, Amsterdam, The Netherlands), stock solutions of alkaline earth metals and transition metals adjusted to the concentrations found in the human nasopharynx fluid were prepared. The pH of the metal mixture was adjusted to pH 1.9 with 5 M NaOH. The metal mixture was then added to the standard THUS medium. The pH of the MILNEZ medium was adjusted to 7.2 with 5 M NaOH, then filtered through 0.22 pm filters (Millipore Express®, Merck, Darmstadt, Germany) and stored at 4 °C.

[0081] Table 1:

[0082] Alkaline earth metals and Transition metals solutions lOOx (Vf = 25 mL)

[0083] Standard copper solution Titrisol® (Merck) : 1000 mg Cu in water

[0084] Standard cobalt solution Titrisol® (Merck) : 1000 mg Co in water

[0085] Standard zinc solution Titrisol® (Merck): 1000 mg Zn in 0.06% hydrochloric acid

[0086] Standard magnesium solution Titrisol® (Merck): 1000 mg Mg in 6% hydrochloric acid

[0087] Standard calcium solution Titrisol® (Merck): 1000 mg Ca in 6.5% hydrochloric acid

[0088] Standard iron solution Titrisol® (Merck): 1000 mg Fe in 15% hydrochloric acid All standards are diluted in 50 mL of the indicated buffer.

[0089] Top up to 20 mL with Milli-Q water (6.68 mL)

[0090] Adjust to 1.9 with 5M NaOH

[0091] Top up to 25 mL with Milli-Q water

[0092] Manganese solution lOOOx (Vf=10 mL)

[0093] Standard manganese solution Titrisol® (Merck): 1,000 mg Mn diluted in 50 mL of water

[0094] Top up to 10 mL with Milli-Q water

[0095] Strain and culture conditions

[0096] Bordet Gengou blood-agar medium (Difco, Detroit, USA), supplemented with 1% glycerol, 10% fresh defibrinated blood and with the appropriate antibiotics was used to culture the Bordetella bronchiseptica and B. pertussis strains. Bacteria were then harvested and inoculated into either THUS or MILNEZ medium. These liquid precultures were incubated for 5 hours at 37°C, 35°C, and 32°C, followed by optical density (OD) measurements. A volume of each culture was taken to inoculate new liquid cultures in THUS or MILNEZ media, with an initial OD of 0.15 units per ml.

[0097] Bordetella cultures monitoring

[0098] Growth monitoring was performed using either an Elocheck measurement cell (Biotronix, Hennigsdorf, Germany), an automated photometer for continuous optical density measurements, or a Tecan Microplate Reader Spark® (Tecan Trading AG, Mannedorf, Switzerland). For the growth monitoring using the Elocheck, Overnight cultures were used to inoculated T75 flasks containing THUS or MILNEZ media inoculated at an initial OD of 0.15 units / ml. The flasks were then connected to the Elocheck. The cultures were incubated at 37 °C, 35 °C, and 32 °C. The optical density at 600 nm was continuously measured for 72 hours. The OD measurement data were collected in Excel datasets and modeled into growth kinetic curves. For Tecan Microplate Reader, the growth monitoring was performed in 96-well plates. Overnight cultures were diluted to an initial OD of 0.15 units / ml and 200 pl of suspension is loaded into the center 60 wells of a 96-well plate. The plate was placed in the Tecan instrument (Tecan, Mannedorf, Switzerland). OD readings were taken at 600 nm every 30 minutes for 72 hours. The turbidities measured using the Elocheck and TECAN devices, which use different wavelengths and pathlengths, cannot be directly compared.

[0099] RNA extraction, libraries construction and illumina RNA sequencing

[0100] Bacteria cultivated in MILNEZ and THUS at different temperatures were harvested at midexponential phase (OD=2) by adding 2 ml of a 5:95 phenol / ethanol (v / v) mix to 8 ml of bacterial suspension. Samples were centrifuged for 15 minutes at 4000 rpm at 4°C. The supernatant was removed, and total RNA was extracted from each bacterial pellet using TRI-Reagent (Invitrogen, California, USA) according to the manufacturer's instructions. Briefly, cell lysis was performed using a solution of 1 ml of TRI-Reagent per 107cells (a monophasic solution of phenol and guanidine thiocyanate) and 10 mg / ml lysozyme diluted in lx TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.00). The cell homogenates were incubated for 5 minutes at room temperature to ensure dissociation of nucleoprotein complexes. The supernatant recovered after centrifugation was separated into two phases using centrifugation with a chloroform solution. Nucleic acids partitioned into the aqueous phase, while proteins remained in the organic phase. Nucleic acids were precipitated by adding an equal volume of isopropanol to the aqueous phase (v / v), followed by centrifugation at 12,0007g at 4°C. The pellet was then washed with 75% ethanol and dried. The extracted nucleic acids were resuspended in RNase-free water and treated with DNase I (Sigma-Aldrich), followed by purification using the Ampure XP kit (Beckman Coulter) to remove genomic DNA and contaminating solvents. The concentration and purity of the total RNA were assessed using a NanoDrop® UV-Vis spectrophotometer (Thermo Fisher Scientific, USA), and the integrity of the extracted RNA was evaluated using an Agilent 2100 Bioanalyzer with the RNA 6000 Nano LabChip® kit (Agilent Technologies, Palo Alto, California, USA). Only samples with an RNA integrity number (RIN) > 7 were selected for further experiments. Ribo-Zero rRNA Removal Kit (Illumina, San Diego, USA) was used to deplete ribosomal RNA. The rRNA-depleted RNA was then used to build the Illumina library using the TruSeq RNA Library Preparation Kit, followed by sequencing on an Illumina NextSeq 500 benchtop sequencer on SRI 50 high output run mode.

[0101] Relative quantification of vaccinal antigens

[0102] Bordetella pertussis B1917 was cultured overnight and then diluted to an initial OD600 of 0.15 OD units / ml in MILNEZ and THUS media. Cultures were incubated at 37°C with continuous agitation until they reached an OD600 of 2. Aliquots were collected, centrifuged, and pellets were resuspended in 10 mM Tris buffer pH=8. Bacterial cells were lysed using a Ribolyser instrument at speed 6 for 40 secondes. Lysates were clarified by centrifugation at 10,000 x g for 15 minutes at 4°C. Protein concentrations were determined using the BCA assay. Equal amounts of protein were separated by SDS-PAGE, transferred to nitrocellulose membranes, blocked, and probed with primary antibodies against FHA and PTX. HRP-conjugated secondary antibodies and ECL detection were used to visualize bands, which were quantified using Fiji software to compare protein levels between MILNEZ and THUS conditions.

[0103] Mice infection with B. pertussis B1917

[0104] Female C57BL / 6 mice (7 weeks old, Janvier Labs, France) were used for intranasal infection. Mice were anesthetized and intranasally inoculated with 20 pL of bacterial suspension containing 105CFU. For each condition and time point, five mice were used. Mice were euthanized at 4, 24, 48, and 72 hours post-infection. All animal experiments were performed at the animal facility of the Institut Pasteur de Lille (number A59-35-064, Lille, France) according to the rules of the European Community Council guidelines (86 / 609ZEEC) for laboratory animal experimentation. The animal protocol was approved by the local institutional review board (Comite d'Ethique en Experimentation Animale Nord-Pas-De-Calais, CEEA75 10 / 2024) and authorized by the French ministry of Higher Education and Research under APAFIS #51236.

[0105] The Bordetella pertussis B1917 GRstrain was used in this study [1], Bacteria were initially cultured at 37 °C on Bordet-Gengou (BG) agar (Difco Bordet-Gengou Agar Base), supplemented with 1% glycerol and 10% defibrinated sheep blood, and 10 pg / ml gentamycin. For the BG solid medium condition, the bacteria were harvested after 40 h of growth at 37°C, by scraping the plates and resuspended in sterile PBS to a final concentration of 5 * 10.6 CFU / mL. For the liquid medium conditions, 40-hours BG-grown bacteria were harvested and used to inoculate into Stainer-Scholte (SS) medium. After incubation for 17 hours at either 37 °C or 35 °C, the optical density (OD) of the cultures was measured to inoculate fresh liquid cultures at an initial OD600nm of 0,15 units / ml in : THUS medium at 37 °C or MILNEZ medium at 35 °C. After incubation for 17 hours, bacterial cultures were harvested during mid- logarithmic growth phase, and the bacterial suspensions were adjusted to a final concentration of 5 x 10.6 CFU / mL in pre-warmed THUS or MILNEZ medium. To maintain physiological conditions, bacterial suspensions were kept at their respective growth temperatures in a heating block until administration.

[0106] At each time point, noses and lungs were aseptically collected as described in [2], Organs were homogenized in 1 mL of sterile IX PBS using an Ultra-Turrax tissue homogenizer. Serial dilutions of the homogenates were plated on BG agar plates supplemented with gentamicin (10 pg / mL) to select for the resistant strain and prevent contamination. Plates were incubated at 37 °C for 3 to 5 days. Colony-forming units (CFUs) were then enumerated to determine bacterial loads in each organ. Bacterial load data were represented as bar plots and statistical comparisons between groups were assessed using a non-parametric permutation-based ANOVA followed by Conover’s post-hoc test to determine significant differences between conditions.

[0107] Results:

[0108] The temperature combined with the composition of alkaline earth metals and transition metals, both of which mimicking the nasal environment, improve the growth rate of B. pertussis

[0109] In contrast to the body temperature which is at 37°C, the temperature in the nasal cavity is between 32°C and 35°C. B. pertussis B1917 was cultured in THUS (Figure 1A) or in MILNEZ (Figure IB) at 32°C or 35°C and the bacterial growth rate was compared with that measured at 37°C. The optimal growth temperature of B. pertussis was 35 °C in both media. The MILNEZ medium contains alkaline earth metals and transition metals which are similar in composition and concentration to those found in the nasal cavity of healthy individuals (van Beek et al. 2020). The influence of the metal composition on bacterial growth rate was analysed at 35°C. A significant increase in biomass, visualized by an increased OD at 600nm, was observed when B. pertussis was cultured in MILNEZ at 35°C compared to THUS medium 24h after the start of the bacterial culture (Figure 1C). The increase in bacterial biomass in MILNEZ compared with THUS medium was confirmed by another method and proved to be statistically significant when measuring the area under the growth curve (Figures 2 A and 2B).

[0110] Genes expression in B. pertussis is regulated by temperature and alkaline earth metals / transition metals present in MILNEZ

[0111] Transcriptomic analysis of B. pertussis B1917 grown in MILNEZ at 35°C showed that 270 genes were up-regulated and 146 genes were down-regulated compared to bacteria cultured in THUS medium at 37°C (Figure 3A). Particularly, genes involved in type III secretion system (T3SS) and genes encoding other virulence factors were up-regulated in nasal mimicking conditions, MILNEZ at 35°C (Figure 3B).

[0112] Increased production of the filamentous hemagglutinin (FHA), and the pertussis toxin, (PT) by B. pertussis cultured in MILNEZ at 35°C

[0113] Among the virulence factors, FHA is the major adhesin produced by B. pertussis and was shown to play a key role during nasal infection (Holubova, Jana, et al. "The Fim and FhaB adhesins play a crucial role in nasal cavity infection and Bordetella pertussis transmission in a novel mouse catarrhal infection model. " PLoS Pathogens 18.4 (2022): e 1 10402). A 3-fold increase in the production of FHA was measured when B. pertussis was cultured at 35°C in MILNEZ compared to THUS (Figures 4A and 4B). Similarly, the production of the SI subunit of the PT, the major toxin produced by B. pertussis, was increased when bacteria were grown in MILNEZ.

[0114] The growth rate of Bordetella bronchiseptica is increased when cultured at 35°C in MILNEZ compared to THUS medium

[0115] B. bronchiseptica is a zoonotic pathogen closely related to B. pertussis. Increased biomass production was measured at 24h and 48h after the start of the culture when B. bronchiseptica was grown in MILNEZ compared to THUS medium at 35°C (Figure 5A). This increase in biomass is statistically significant as shown by the calculation of the area under the growth curve (Figure 5B).

[0116] Pre-culturing B. pertussis in MILNEZ at 35°C increases subsequent nasal and pulmonary colonization

[0117] C57BL / 6 mice were infected intranasally with 105CFU of the B. pertussis B1917 strain, which was cultured on either a BG solid medium, in THUS liquid medium at 37 °C, or in MILNEZ liquid medium at 35 °C. The kinetics of nasal and lung colonization were monitored for 72 hours (Figures 6A and 6B). Significantly higher bacterial loads were measured at all time points, in the noses and in the lungs of mice infected with B. pertussis bacteria cultured in nasalmimicking conditions (MILNEZ at 35°C) compared to bacteria grown in standard media prior to their nasal administration.

[0118] REFERENCES:

[0119] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

Claims

CLAIMS:

1. A culture medium for cultivating Bordetella bacteria, comprising a source of carbon, a source of nitrogen, a source of salts, a source of alkaline earth metals and a source of transition metals, wherein the transition metals are present at concentrations close to those in mammalian nasal fluid cavity.

2. The culture medium according to claim 1 wherein the ratio between the nitrogen source and the carbon source (N:C ratio) is 1 : 10.

3. The culture medium according to claim 1 or 2 that comprises glutamate as a source of carbon wherein the concentration of glutamate is in the range of about 10 mM to about 15 m, preferably about 12.6 mM.

4. The culture medium according to any one of claims 1 to 3 that comprises lactate as source of carbon wherein the concentration of lactate is in the range of about 14 mM to about 19 mM, and about 16.6 mM.

5. The culture medium according to any one of claims 1 to 4 that comprises ammonium chloride (NH4C1) as a source of nitrogen wherein the concentration of ammonium chloride (NH4C1) is in the range of about 1.5 mM to about 2.5 mM, and about 2mM.

6. The culture according to any one of claims 1 to 5 that comprises one or more metal ions selected from the group consisting of Mg2+, Ca2+, Co2+, Cu2+, Zn2+, Fe3+and Mn2+.

7. The culture medium according to any one of claims 1 to 6 wherein the concentration of the magnesium salt ranges from about 0.5 pM to about 5 pM, preferably from about 1 pM to about 2pM, more preferably about 1.4 pM.

8. The culture medium according to any one of claims 1 to 7 wherein the concentration of the calcium salt ranges from about 0.5 pM to about 5 pM, preferably from about 1 pM to about 2pM, more preferably about 1.75 pM.

9. The culture medium according to any one of claims 1 to 8 wherein the concentration of iron salt can range from about 5 pM to about 10 pM, preferably from about 6 pM to about 8 pM, more preferably about 7 pM in the culture medium.

10. The culture medium according to any one of claims 1 to 9 wherein the concentration of the cobalt salt ranges from about 0.5 pM to about 5 pM, preferably from about 0.5 pM to about 1 pM, more preferably about 0.68 pM.

11. The culture medium according to any one of claims 1 to 10 wherein the concentration of the copper salt ranges from about 1 pMto about 10 pM, preferably ranges from about 5 pM to about 8 pM, and more preferably about 6.5 pM.

12. The culture medium according to any one of claims 1 to 11 wherein the concentration of the zinc salt ranges from about 10 pM to about 15 pM, preferably from about 11 pM to about 13 pM, and more about 12 pM.

13. The culture medium according to any one of claims 1 to 12 wherein the concentration of the manganese salt can range from about 0.5 pM to about 1 pM in the culture medium, preferably about 0.7 pM.

14. The culture medium according to any one of claims 1 to 13 that comprises an amount of Mg2+, Ca2+, Co2+, Cu2+, Zn2+, Fe3+and Mn2+according to the following concentrations:

15. A method for cultivating Bordetella bacteria, comprising inoculating the culture medium according to any one of claims 1 to 14 with a strain of Bordetella bacteria and incubating the inoculated culture medium at a temperature of 35°C.

16. A method for producing an acellular vaccine against Bordetella infection comprising cultivating Bordetella bacteria by the method of claim 15, and harvesting and lysing the bacteria and finally purifying the antigens as the vaccine.

17. A method for producing a whole-cell vaccine against Bordetella infection, comprising cultivating Bordetella bacteria by the method of claim 15 and harvesting the bacteria as the vaccine.

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