Stabilisation of live mollicutes bacteria in liquid composition

Abstract

To provide stable liquid compositions containing live Mollicutes bacteria.SOLUTION: Provided is a liquid composition of live Mollicutes bacteria and a stabiliser, whereby the stabiliser is a natural deep-eutectic solvent (NADES). Preferably, the composition has a water activity of 0.85 or less, and the NADES comprises an organic salt and a polyhydric alcohol. In this liquid composition the live Mollicutes are stabilised without need for freezing or freeze-drying. This allows various advantageous uses in diagnostics and medicine, specifically as a liquid vaccine for use against infection or disease caused by Mollicutes bacteria, for human- or non-human animals.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present invention relates to the fields of bacteriology and vaccinology. In particular, the present invention relates to a live mollic Liquid composition of bacteria and stabilizer, preparation of said liquid composition, and use of stabilizer in said composition The present invention relates to the use of NADES in various medical applications of said compositions. [Background technology]

[0002] Viable bacteria are used in a wide variety of applications, some of the more well-known being: These include traditional and modern biotechnological processes, diagnostics and vaccines. Of importance in the field are the ability to stabilize such bacteria during storage and use, The common technique is to use glycerol or other glycerol-based stabilization agents to maintain the viability of the cells. They rely on deep-frozen storage using cryoprotectants such as nutrient media; or The viable bacteria can be lyophilized and maintained in lyophilized form under refrigerated or frozen conditions. However, all these stabilization methods have a number of drawbacks. This introduces ingredients of undefined and variable quality, such as hydrolysates and extracts. Such undefined components may have unpredictable effects on stability. The products require a laborious cold chain for storage and distribution. Even with these processes, the freezing step itself induces a significant decrease in viable cell numbers. Therefore, complete survival of viable bacterial cells is not achieved. Also, freeze-drying is very expensive and time-consuming. This process effectively stabilizes the viable cell count of bacteria during storage and use. Improved methods for doing so would be highly desirable.

[0003] A group of agriculturally and medically important bacteria is the class Mollicutes. They are classified into the taxonomic categories It is classified in the phylum Nericutes (Ludwig et al., 2009, Bergey's Man ual of Systematic Bacteriology,2nd edn,v ol.3.Springer:New York,USA,pp1-13). , which are derived from Gram-positive bacteria but have lost their cell wall. They are typically They are parasites or commensals of eukaryotic hosts.

[0004] Infection of animals with Mycoplasma bacteria as a primary or secondary pathogen can result in respiratory disease. often causing nausea and / or a variety of other symptoms, which together can range from mild to severe. It can cause severe illness and cause a lot of discomfort and economic loss to affected hosts. A reference on the impact of mycoplasma infections in agriculture is “The Mechanism of Mycoplasma Infections in Agriculture.” rck veterinary manual″(11th ed.,2016,ISB N-10:9780911910612).

[0005] Mycoplasma infections can be curatively treated with certain types of antibiotics, but are generally not treated with vaccines. Such vaccines consist of live attenuated strains or inactivated strains. It can be based on M. bovis (killed bacteria). A bacterin vaccine against B. s is available under the trade name MpB Guard (Agrilabs The most widely used commercial mycoplasma vaccines are for poultry. For example, A live attenuated vaccine for M. gallisepticum: Vaxsa fe® MG (Bioproperties) (frozen), and Nobilis MG 6 / 85 (MSD Animal Health) (lyophilized, cooled); M. galli Bacterin vaccine for M. gallisepticum: MG-Bac (Zoetis) and Nobilis (Methylparaben) MG inac (MSD Animal Health), both of which are emulsions containing oil adjuvants. A live attenuated vaccine for M. synoviae is available under the name Vaxsafe (registered trademark). MS (Bioproperties) (frozen) and Nobilis® MS Live (MSD Animal Health) (freeze-dried, cooled). Several live attenuated mycoplasma vaccines are known and are used on a more limited scale. For example, Contavax®, a ruminant antimicrobial agent for M. mycoides (Mm ycoides) is used in some African countries. In addition, attenuated M. hyopneumoniae (M. hyopneumoniae) has been used in China. e) live vaccines are used in pigs. For a general overview of Mycoplasma vaccination, see As follows: Whithear (1996, Rev. Sci. Tech. Off. nt.Epiz.,vol.15,p.1527-1553);Kleven(2008 ,Av.Dis.vol.52,p.367-374); and Perez-Casal et al. , (2017, Vaccine, vol. 35, p. 2902-2907).

[0006] Due to their association with the cell membrane and thus their invasion into host cells, the Mollicutes are a diverse group of organisms that infect a variety of eukaryotic cells. are known contaminants of in vitro cell cultures of live mollicutes. A few species are used for diagnostic and quality control purposes. These are listed in the European Pharmacopoeia and the US 9-CF It is also a formal requirement in biological production under government regulations such as R. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] WO2009 / 120839 [Patent Document 2] US8,247,198 [Patent Document 3] WO2011 / 155829 [Patent Document 4] WO2014 / 131906 [Patent Document 5] WO2016 / 108083 [Patent Document 6] WO2016 / 162703 [Patent Document 7] WO2017 / 222377 [Patent Document 8] WO2015 / 121463 [Patent Document 9] WO2014 / 029702 [Patent Document 10] WO2014 / 140239 [Non-patent literature]

[0008] [Non-Patent Document 1] Ludwig et al., 2009, Bergey's Manual of Systematic Bacteriology, 2nd edn, vol. 3. Springer: New York, USA, pp1-13. [Non-Patent Document 2] ″The Merck veterinary manual″, 11th ed., 2016, ISBN-10:9780911910612. [Non-Patent Document 3] Whithear,1996,Rev.Sci.Tech.Off.Int.Epiz.,vol.15,p.1527-1553. [Non-Patent Document 4] Kleven,2008,Av.Dis.vol.52,p.367-374. [Non-Patent Document 5] Perez-Casal et al., 2017, Vaccine, vol. 35, p. 2902-2907. Summary of the Invention [Problem to be solved by the invention]

[0009] Mollicutes are the smallest known self-replicating organisms. Bacteria from some species are directly They are only 0.2 micrometers in diameter, and some species have genomes of just 580 kbp. With so few genes, mollicutes are highly dependent on resources from the environment. Therefore, when culturing in vitro, a very rich medium is typically required. In addition, the absence of a cell wall exposes the cell membrane to the effects of the physical and chemical environment and can cause and reduced stability during use.

[0010] Thus, a key issue in the vaccine industry is the risk of infection during the shelf life of the final product and e.g. Such live Mollicutes bacteria may be used as bacterial stocks for use in diagnostics or vaccinations. One solution is to develop a non-live bacterin type vaccine. However, such vaccines are relatively highly available. They are relatively expensive due to the large amounts, repeated administrations, and the need for adjuvant use.

[0011] Vaccines based on live Mollicutes bacteria are deep-frozen or lyophilized and cultured in rich stabilised medium. As noted above, such media is less than desirable for a number of reasons. Additionally, the freezing process itself also causes a significant reduction in viable cell numbers, which can lead to cell death over several days. Within a maximum of 10 3 This results in a loss of colony forming units (CFU). Methods and materials for improved bacterial stabilization are highly desirable.

[0012] Deep eutectic solvents (DES) are known liquid compositions that combine many remarkable properties. Generally, these are mixtures of compounds that are liquid at ambient temperature but have a low water content. Also, the small amount of water present is tightly bound and may be involved in chemical or biological processes. This is reflected by the very low water activity values. S is, for example, as described in Abbott et al., 2003, Chem. Commun., vol. 1, p. 70-71, and WO2009 / 120839.

[0013] DES is highly ionic but at the same time has a very low water activity, making it an ideal solvent for polar hydrophobic compounds. It is very effective as a solvent. Examples include the extraction of gas, minerals or industrial bulk products. DES is used for the extraction and stabilization of biomolecules such as enzymes and RNA. These are also used in the synthesis of glycerol, as disclosed in US8,247,198 and WO2011 / 15582, respectively. See reference 9.

[0014] Since around 2011, special forms of DES made from natural substances have been under investigation. The so-called "natural DES" or NADES of Choi et al. are described, for example, in Choi et al., 2011, P. lant Physiol., vol. 156, p. 1701-1705; and Dai et al. , 2013, Anal. Chim. Acta, vol. 766, p. 61-68 NADES are currently being considered as "green" solvents for the extraction of natural products. They are of great interest as solvents for use in natural pesticides, for example.

[0015] WO2011 / 155829 describes the extraction of biological materials using NADES. One of the options listed is the use of protein materials such as subunit vaccines. It is an extraction.

[0016] Similarly, WO2014 / 131906 describes, inter alia, the detection of viral RNA for clinical diagnosis. Fixatives for viruses in blood or tissues with particular focus on extraction and stabilization are described. The fixing agent is a DES, and WO2014 / 131906 describes a number of possible DES combinations. The compounds actually used are urea or trimethylsilyl as proton acceptors. Only compositions containing fluoroacetamide, choline or betaine as a proton donor In WO2014 / 131906, the most preferred DES is choline chloride. It consists of trifluoroacetamide, so it is not a NADES, and Fluoroacetamide is not an acceptable pharmaceutical excipient.

[0017] WO2016 / 108083 describes the use of NADES for their bactericidal activity Several NADES have been reported to be effective against gram-positive and gram-negative bacteria, as well as Candida. It has been shown that the antibacterial properties of β-lactams such as β-lactams, ...

[0018] WO2016 / 162703 describes a method for extracting biomolecules, in particular from plant material, e.g. The use of NADES, for example consisting of betaine and organic acids or polyhydric alcohols, is described.

[0019] WO2017 / 222377 describes a method for producing a water source for animal feed that does not dry out under heat. The use of NADES gel in admixture with The path is for newly hatched chicks.

[0020] WO2015 / 121463 describes a method for producing a glycerol-based glycerol derivative comprising 5 to 40% sugar alcohol and 0.15 to 0.7% In a composition of 5M of an amino acid selected from arginine, glutamic acid, or glycine Specifically, liquid stable poultry vaccines for poultry viruses have been described. Also importantly, WO2015 / 121463 and WO2014 / 029702 and WO2 Similar publications such as 014 / 140239 do not disclose NADES and at least 6 Contains 0% (weight / volume) water and has a water activity (a w Regarding the aqueous solution containing Comparative data is provided in the Examples section.

[0021] Gutierrez et al. (2010, Angew. Chem. Int. Ed., vol. 49, p. 2158-2162) states that the solute is 20% (weight / volume) Gram using NADES diluent consisting of choline chloride and glycerol in a 1:2 molar ratio The cryopreservation of NA-negative bacteria Escherichia coli is described. To form the DES, a mixture of bacteria and a dilution of the NADES is freeze-dried.

[0022] Qiao et al. (2018, Appl. Microbiol. Biotech Nol., vol. 102, p. 5695-5705) uses a composition derived from NADES. We investigated the stabilization of lactic acid bacteria using lactic acid bacteria stabilization. These bacteria are gram-positive and contain cell walls. The survival rates of two species from the Lactobacillus genus were not stable or stable during cryopreservation. The use of S. thermophilus was also damaging. O. thermophilus has a 1:3 molar ratio of proline:glycerol. However, the NADES could be used to stabilize the nucleosomes in a frozen form. By applying a 1+1 volumetric mixture of hydrogen peroxide and bacterial culture, Qiao et al. The NADES was diluted until the bond network was intact. This was due to the melting of the ice crystals. and growth are evident from the differential scanning calorimetry (DSC) profile (Qiao et al., Figure 4). Although these are properties of aqueous compositions, intact NADES exhibit a glass transition temperature It is considered that.

[0023] Qiao et al. also showed that the presence of a cell wall reduces the susceptibility of bacteria to NADES toxicity. It has also been described that the cells become resistant to the effects of certain stresses, namely dehydration and damage to cell membranes.

[0024] The object of the present invention is to overcome the shortcomings of the prior art, to provide a method for preparing a freeze-dried liquid preparation that can be prepared without freeze-drying, It is not necessary to maintain the temperature, but sufficient stability is required when storing and handling at temperatures above 0°C. The present invention provides a method for the preparation of a composition comprising a live Mollicutes bacterium, The aim of this project is to meet the needs of the people who live in these regions. [Means for solving the problem]

[0025] Surprisingly, it is possible to stabilize live mollicutes without freezing or lyophilization. This object can be achieved by providing a liquid composition which can, as a result, It has now been discovered that one or more of the shortcomings of the prior art can be overcome by using NADE as a stabilizer. This is achieved by adopting S.

[0026] This discovery opens up many very promising fields of application. Bacteria can be stored without freezing or lyophilization, and such processes are labor-intensive. Furthermore, the liquid composition of the present invention requires the addition of water for stabilization, which is time-consuming and costly. Avoid using ingredients of uncertain or variable quality, such as decomposition products, and instead use ingredients of natural origin and chemically defined Furthermore, the liquid form makes it suitable for various applications such as diagnosis and quality control. This will not only facilitate use in the field, but also allow for use as a live attenuated vaccine. This is because NADES are acceptable pharmaceutical carriers. The chin can administer a liquid composition containing live mollicutes bacteria directly or after dilution. do.

[0027] It is unexpected that NADES can stabilize biomollicute bacteria: Strong ionic As solvents, NADES dissolve the delicate exposed bacterial cell membrane and release its cytoplasmic proteins. It was expected that the protein and nucleic acid would be extracted from the cells. They warn that wall-less bacteria are sensitive to the toxicity of NADES. 16 / 108083 (supra) employs NADES to actually kill bacteria.

[0028] Therefore, the present inventors have demonstrated that the NADES formulation can be produced in vivo without freezing or lyophilization. It was surprising to find that the method provided significant stabilization of viability of Lactobacillus bacteria. The efficacy of the antibacterial properties of M. synoviae was significantly superior to that observed in the aqueous liquid composition. (M. synoviae) can be cultured in a liquid composition according to the present invention at 4° C. for up to 24 weeks in 3 L. og 10 It can be maintained with less than 1 Log CFU loss, with some compositions losing less than 1 Log 10 CFU Even at room temperature, significant stabilization was observed for up to 8 weeks, with only 4Log 10 Although the culture in phosphate buffered saline showed a loss of CFU, Within a week it was completely gone.

[0029] How and why NADES are such effective stabilizers for biomollicute bacteria The inventors have proposed a theory that may explain these findings. Although we do not wish to be bound by any of these models, the amount of available water in NADES is small. They speculate that the lack of a cellular endothelium helps maintain the viability of the bacteria. Fungi appear to interact with NADES in a different way than do wall-bearing bacteria. There is no prior art on the stabilization of mollicutes in NADES, so this effect was not predictable.

[0030] Thus, in one embodiment, the present invention provides a method for preparing a stabilizer comprising the steps of: and bacterium and a stabilizer, [Brief description of the drawings]

[0031] [Figure 1]1 is a differential scanning calorimetry profile of a NADES formed with proline, sorbitol, and water in a molar ratio of 1:1:2.5. [Diagram 2] Stability results of Mycoplasma synoviae in liquid compositions containing different NADES. Panel A: incubated at room temperature; Panel B: incubated at 2-8 °C. [Diagram 3] Stability results of Mycoplasma gallisepticum in liquid compositions containing different NADES. Panel A: Incubated at room temperature; Panel B: Incubated at 2-8 °C. [Figure 4] 1 is a DSC profile of an aqueous stabilizer from the prior art, containing 30 wt. % sorbitol and 0.6 M arginine in 0.04 M PBS. [Diagram 5] Stability data for M. bovis in NADES at room temperature (A) and 2-8°C (B). [Figure 6] Long-term stability data for M. synoviae (A) and M. gallisepticum (B) in NADES at 2-8°C. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] The term "liquid" is used in its general sense and refers to a liquid that is A liquid refers to a composition that can flow. One method for determining liquid properties is to measure the composition at a particular temperature. The container containing the substance is tilted, and if a change in the morphology of the composition is detected after a certain time, the composition is identified. In the present invention, the composition is classified as a liquid by the tilt test. If it changes shape within 15 minutes of being tilted 90 degrees at room temperature, it is a liquid.

[0033] As used herein, the terms "comprising" (and "comprising") "se," "comprises," and "comprised" The term "variant" is used even if an element or combination is not explicitly recited. is covered or contained in the sentence, paragraph, claim, etc. in which the term is used. This refers to all elements and all possible combinations contemplated in the present invention. and does not exclude any such element or combination.

[0034] Accordingly, such statements, paragraphs, claims, etc., are "comprising" (or variations thereof) "consisting of" or "consist essentially of" The present invention relates to one or more embodiments in which the term "particularly of" is used instead of the term "particularly of" or the like. It is also possible.

[0035] The term "live" is used here in a general sense with respect to bacteria and refers to bacteria that, under appropriate conditions, This refers to the ability of bacteria to grow, move, take up and ferment nutrients, and / or replicate in the presence of Methods for determining whether a cell is alive or not are known in the art. The concentration of the β-amyloides can be determined by in vitro incubation, or more conveniently by culturing the β-amyloides in a suitable medium and measuring the concentration of the β-amyloides in the culture. The infection is determined by detecting an increase in optical density or by plating on suitable agar plates and counting the CFU. An alternative method is to use live / dead selective staining and counting.

[0036] The term "mollicutes" is based on taxonomic, morphological, genomic, and biochemical characteristics. Distinguishing features of the taxonomy, as well as biological features such as physiological, immunological or pathological behavior The same applies to the names of bacterial orders, families, genera, or individual bacterial species. The same applies to the reference.

[0037] The Mollicutes are divided into the following orders: Acholeplasmatida, Anaeloplasmatida, Entomoplasmatida, and The order Mycoplasmatales includes the order Acholeplasmatales, which includes Candidatus. s)) Known Mollicutes plant pests of the genus Phytoplasma. Entomoplasma The order Lepidoptera includes insect symbionts and plant pathogens of the genus Spiroplasma.

[0038] The best known are the Mycoplasmales, which are known members of the genus Mycoplasma. Human or animal pathogenic bacteria, e.g. (mainly) humans, ruminants, pigs, and M. pneumoniae and M. bovis infect birds and chickens (M. bovis), M. hyopneumoniae, M . meleagridis, and M. gallisepticum llisepticum).

[0039] As is known in the art, the classification of microorganisms within a particular taxonomic group The present invention is therefore based on a combination of such features. This includes species of Mollicute bacteria from the orders, families, genera, and species in which they are classified. It may be identified in any way, for example as a subspecies, strain, isolate, genotype, variant, subtype or subgroup. This refers to bacteria that are further classified into the following categories.

[0040] Furthermore, the particular order, family, genus or species of mollicute bacteria of the present invention is not currently a member of that group. Although species may be assigned to different taxonomic groups, new insights may be gained and new or different taxonomic groups may be assigned to different taxonomic groups. It is a classification that may change over time, possibly leading to reclassification. It will be apparent to those skilled in the art of the present invention that the present invention is a scientific classification. This does not change the bacterium itself or its antigenic repertoire, but rather its scientific name and classification. Since the only change is the structure of the reclassified bacteria, such reclassified bacteria remain within the scope of the present invention. For example, Gupta et al. (2018) Leeuwenhoek, vol. 111, p. 1583-1630) is the latest Based on this data, we propose a new framework for the Tenericutes phylum.

[0041] Samples of mollicute bacteria for use in the present invention can be obtained from a variety of sources. For example, they can be obtained from humans or from wild or farmed non-human animals, or from a variety of research as wild isolates from research centres, (repository) institutions or (veterinary) universities.

[0042] The term "stabilizer" in the present invention refers to a material that reduces the amount of oxidation that occurs due to handling or use and / or over a certain period of time. The present invention provides a method for reducing the loss of viable cell count of the mollicutes bacteria of the present invention due to storage at a specific temperature over a period of time. It is a composition that can reduce

[0043] "Stabilization" in the present invention refers to, for example, incubation at a specific temperature for a specific period of time. The effect of the composition containing live Mollicutes bacteria on the difference in CFU before and after a specific treatment was Thereby, the survival rate in different liquid compositions according to the present invention is expressed as a standard P BS buffer (10 mM Na2PO4, 1.8 mM KH2PO4, in water at pH 7.4, The survival rates were compared with those in the control group (2.7 mM KCl and 137 mM NaCl). The survival rate, which exceeds that observed in PBS under these storage conditions, is due to the liquid composition of the present invention. is considered to be the survival benefit provided by

[0044] "Deep eutectic solvents" (DES) are compounds that contain at least two compounds in a molar ratio that forms a eutectic mixture. and whereby the mixture The eutectic point of is significantly lower than the melting points of the individual compounds.

[0045] This decrease in the melting point of the mixture is caused by interactions between the compounds, one of which is hydrogen bonding. One acts as a hydrogen bond donor and the other acts as a hydrogen bond acceptor, which forms a stable hydrogen bond network. It provides a network of networks that incorporate small amounts of water that do not freeze. This allows the mixture to be heated to temperatures well above the melting points of the components. In general, "eutectic" means that it is easily soluble. Taste.

[0046] NADES for use in the present invention are those that have intact hydrogen bond networks. This can be easily confirmed by thermal analysis of the phase transition profile using DSC. As a result, NADES for use in the present invention can be characterized by a DSC spectrometer that exhibits a glass transition temperature. Has a profile.

[0047] Determination of DSC profiles is known and can be performed using standard equipment. An example profile is shown in FIG.

[0048] A limited level of dilution is possible due to the hydrogen bond network. If the solution is diluted too much, the DES will turn into a normal aqueous solution. In that case, the DSC profile will be: As in Qiao et al., supra, Fig. 4, the enthalpy of fusion and the enthalpy of crystallization This displays the following:

[0049] The individual compounds (excluding water) used to form the NADES of the present invention are approximately 80 0 C and the resulting NADES has a melting point below about 40° C. For example, The melting points of betaine and sorbitol are 310°C and 95°C, respectively. The NADES formed with a molar ratio of 1:1:2.5 of perfluoroacetate and water had a glass transition temperature of - 50°C and exhibiting no ice crystallization and / or ice melting points in its DSC profile. There wasn't.

[0050] The term "natural" is used to define natural DES (NADES) in the present invention. The compound to be studied is derived from biological sources such as plants or animals in amounts well above trace amounts under normal conditions. These are useful for identifying organic compounds that can be found in these materials. Such natural compounds are derived from primary metabolic products present in particular materials of plant or animal origin. As will be appreciated by those of skill in the art, the term "naturally occurring" is used herein to refer to a was used solely to characterize the original origin of the compounds used in the NADES of the present invention. It does not characterize the actual source of the compounds used and to be used in the present invention. Thus, natural compounds may also be used in the present invention when derived by (semi)synthetic production. It is also possible.

[0051] Examples of naturally occurring compounds that can be used to form NADES in the present invention include Quaternary ammonium salts such as organic acids, amines, sugars, sugar alcohols, amino acids, and choline derivatives. It's salt.

[0052] Within the embodiments disclosed herein of the invention, other combinations of A combination of certain types of NADES and certain types of Mollicutes bacteria is more favorable than a combination of the two. It will be apparent to those skilled in the art of the present invention that there may be It will be understood that those skilled in the art can easily implement the present invention using only routine methods and materials with the information disclosed herein. The combinations and compositions according to the present invention can be easily selected, optimized and fine-tuned. There will be.

[0053] Further details of preferred embodiments and further aspects of the invention are set forth below.

[0054] The water present in the NADES used in the present invention is a hydrogen bond network structure of the NADES. are tightly bound to the mollicutes, and chemically linked to the mollicutes that may affect their stability. This characteristic strongly reduces the availability of water for environmental and biological processes. In practice, it is expressed by showing the total water volume and the amount of free water. The availability of that water (free water) is , symbol a w The water activity is expressed as the upper limit of pure water, 1 Water activity varies between 0.0 and a lower limit of 0. Water activity is generally calculated by comparing the vapor pressure of a test composition to the vapor pressure of pure water. Atmospheric pressure and water activity are measured by comparing with a known saturated salt solution (at the same temperature). This is seen, for example, in FAO Agricultural Service Bulletin No. 149 (Canovas et al., FA O, Rome, 2003, ISBN 92-5-104861-4) The various handbooks, catalogues and manuals on preservation are available at the "Fu ndamentals of water activity”, Decagon D evices Inc.,Washington,2015(http: / / pdf.d irectindustry.com / pdf / decagon-devices-in c / fundamentals-water-activity / 64142-6344 33.html).

[0055] Below a water activity of approximately 0.8, most bacterial growth ceases, below 0.7 At a water activity of less than 0.4, most yeast and mold growth stops, and most enzymes are lost. Activity is effectively stopped.

[0056] Instruments and procedures for measuring water activity are known, for example, headspace pressure analysis. It can be utilized by using.

[0057] Effective stabilization of Mollicutes bacteria in liquid compositions according to the present invention occurs when the composition is about 0.8 It has been found that this can be achieved with a water activity of less than 5.

[0058] Thus, in one embodiment of a liquid composition according to the present invention, the composition has a water activity of 0.85 or less. It has sexuality.

[0059] In the present case, the indicated water activity is the maximum water activity, e.g., as provided by commercial producers. It refers to the water activity of the liquid composition according to the invention in the form of the final product, in which form it has a long shelf life. This composition can be used to dilute or mix with other ingredients. , which may result in a change in water activity value.

[0060] In one embodiment of the liquid composition according to the invention, the water activity of the composition is less than about 0.8; More preferably, it is less than about 0.7, about 0.6, about 0.5, about 0.4, about 0.3, or even is less than about 0.2, with the order of preference increasing.

[0061] Preferably, the water activity of the liquid composition according to the present invention is 0.8 to 0.1. Or, in this order of priority, 0.7~0.1, 0.6~0.1, 0.5~0.1, 0.4~ 0.1, 0.3 to 0.1, or even 0.2 to 0.1, with the order of preference being higher. become.

[0062] For the present invention, the indication of a range is intended to include the stated end values.

[0063] In the present invention, "about" means that a numerical value may vary within ±10% around the stated value. Preferably, "about" means ±9% around the value, more preferably " "About" means ±8, 7, 6, 5, 4, 3, 2, 1, or even 0% around the value , in that order of preference.

[0064] The water used in the liquid compositions according to the invention is preferably (multiple) distilled water or reverse osmosis water. More preferably, the water is of pharmaceutical grade and is suitable for parenteral administration. The nature of such water is typically sterile and essentially pyrogen-free. For example, water for injection (WFI).

[0065] NADES for use in liquid compositions according to the present invention are formulated with limited amounts of water. By varying the water content of the liquid composition, its properties can be altered, for example, by changing the viscosity of the liquid composition. It can be adjusted and optimized for viscosity. The organic composition can be made with a total water content of up to 50% by weight (Dai et al., 2015, vol. (I.187, p.14-19). The relatively large amount of water is the hydrogen bond network of NADES. This could disrupt the network.

[0066] Thus, in one embodiment of the liquid composition according to the invention, the composition contains no more than 50% by weight water. Has quantity.

[0067] The water content of the liquid composition according to the present invention is expressed as a percentage of weight per weight (w / w). Expressed as cents, this is the percentage of the liquid composition according to the present invention by weight of the liquid composition. The water content is the weight of the total amount of water in the sample. The water content can be determined, for example, by the Karl Fisher method known in the art. It can be measured by gel titration using a variety of procedures.

[0068] With respect to water activity, a liquid composition in this context refers to the water content of the final product prior to any mixing or dilution. The composition is in the form of:

[0069] Preferably, the liquid composition according to the invention contains no more than 40% by weight, more preferably 30, 25 , 20, 15, 10, 8, 7, 6, or 5 weight percent or less of water content, in that order of preference. will become higher.

[0070] The lower limit of the water content in the liquid composition according to the present invention is, for example, How little water is enough to solubilize the material and also to provide an acceptable level of viscosity for the NADES. Practical factors such as the amount of water needed to reach and make it conveniently treatable or usable. is determined by.

[0071] Thus, in one embodiment of the liquid composition according to the present invention, the water content in the liquid composition is 5 0 to 0.5% by weight; 40 to 1% by weight; 30 to 1.5% by weight; 20 to 2% by weight; or even is 10 to 3% by weight, with the preference increasing in this order.

[0072] Furthermore, by mixing NADES with a sample of live mollicute bacteria, a certain amount of of water is introduced into the mixture to prepare a liquid composition according to the present invention. Liquid compositions should not be overly diluted.

[0073] Thus, in one embodiment of the liquid composition according to the invention, the microbial samples to be combined The ratio of the volume of the NADES to the volume is preferably at least 3:1. more preferably, at least a 5:1 volume ratio of NADES:bacteria sample. or even a NADES:bacteria sample volume ratio of at least 10:1, Note: A 10:1 ratio is 9 parts NADES and 1 part bacterial saponin. For example, proline, sorbitol, and sorbitol in a molar ratio of 1:1:2.5. The NADES of ethanol and water has a total water content of 13.1% by weight. The obtained Mycoplasma bacteria of the present invention were mixed with a sample of the Mycoplasma bacteria of the present invention in a ratio of 0.1 to 0.2. The total amount of water in the liquid composition is 21.8% by weight.

[0074] In order to limit excessive dilution of the NADES of the present invention into the aqueous composition, The bacterial sample may need to be concentrated.

[0075] In the liquid composition according to the present invention, NADE is effective as a stabilizer of live Mollicutes bacteria. S may be formed from various combinations and ratios of organic salts and / or polyhydric alcohols; It has been found to be formed from several polyhydric alcohols.

[0076] Thus, in one embodiment of the liquid composition according to the present invention, the NADES comprises an organic salt and a polyhydric alcohol. Contains alcohol.

[0077] In another embodiment of the liquid composition according to the present invention, the NADES are all polyhydric alcohols. It contains two or more compounds.

[0078] In some of these embodiments, the hydrogen bond network acts as a hydrogen bond donor. The ionic species is an organic salt, and the hydrogen bond acceptor is a polyhydric alcohol. Alternatively, if the NADES contains only polyhydric alcohols, the network will consist of these It is formed by hydrogen bonds between molecules.

[0079] In the present invention, a "polyhydric alcohol" is an organic compound that contains two or more hydroxyl groups. However, there are very large polyhydric alcohols, such as cellulose, that fall under the definition of polyhydric alcohols. mers are preferred because they are less effective at forming NADES as defined herein. As a result, the polyhydric alcohols used in the present invention Preferably, the molecular weight is less than about 10,000 grams / mole.

[0080] In a more preferred embodiment, the polyhydric alcohol used in the present invention has a molecular weight of 500 It may be less than 0, or even less than 1000 grams / mole.

[0081] Compounds for use in the NADES used in the present invention are available from a variety of commercial suppliers. The compounds are readily available in a variety of purity and quality. Preferably, the compounds are of pharmaceutical grade quality. Such excipients are used in accordance with government regulations, such as the European Pharmacopoeia and the US 9 CFR. and The Handbook of Pharmaceutical Excipie nts(R.Rowe et al., Pharmaceutical press 2012, IS BN 0857110276);Remington:the science and practice of pharmacy(2000,Lippincott,USA ,ISBN:683306472), and "Veterinary vaccinolo gy″(P. Pastoret et al., ed., 1997, Elsevier, Amster This information is included in handbooks such as the 'Dam' Handbook, ISBN 0444819681.

[0082] The preferred polyhydric alcohols for use in the present invention are sugars or sugar alcohols. is a versatile component that allows for the production of a variety of effective NADEEs for use in the present invention. Because it has been proven.

[0083] In a preferred embodiment, the sugar is a non-reducing sugar.

[0084] In one embodiment of the non-reducing sugar used in the liquid composition according to the present invention, the non-reducing sugar is sucrose. The sugar is selected from among glycerol, glycerol, and trehalose.

[0085] Preferably, the non-reducing sugar is trehalose. Trehalose is preferably represented by the CAS number The number is 6138-23-4.

[0086] In the present invention, a "sugar alcohol" is a hydrogenated sugar containing three or more carbon atoms, and is a monosaccharide. The sugars may be based on disaccharides or polysaccharides.

[0087] In one embodiment of the sugar alcohol used in the liquid composition according to the present invention, the sugar alcohol is , erythritol, xylitol and sorbitol.

[0088] Erythritol is preferably CAS number 149-32-6. Xylitol is preferred. or CAS number 87-99-0. Sorbitol is preferably D-sorbitol. , CAS number 50-70-4.

[0089] In one embodiment of the sugar alcohol used in the liquid composition according to the present invention, the sugar alcohol is , xylitol and sorbitol.

[0090] In a more preferred embodiment, the sugar alcohol is sorbitol.

[0091] The polyhydric alcohols used in the liquid compositions according to the invention may be in the form of different salts, isomers, hydrates or The preferred glycerols for use in the present invention may be used in the form of anhydrous glycerol, etc. It is entirely possible to select and test various forms of polyhydric alcohols.

[0092] In the present invention, "organic salt" refers to the natural compounds as presented herein above. Within the scope of the definition and capable of forming deep eutectic solvents for the invention described herein , zwitterions, and the like. Those skilled in the art will appreciate that organic salts for the purposes of the present invention are of any suitable organic acid or base salt. It is entirely possible to select and apply it to form a NADES.

[0093] In one embodiment of the liquid composition according to the invention, the organic salt is betaine, proline, choline, One or more selected from salts of methionine and arginine.

[0094] In the present invention, "betaine" is preferably the compound N,N,N-trimethylglycine. betaine, CAS number 107-43-7, which is also known as glycine betaine Proline is preferably CAS number 609-36-9. Choline is preferably CAS Methionine is preferably L-methionine, CAS number 63- The score is 68-3.

[0095] In a preferred embodiment, the choline is choline chloride, CAS number 67-48-1.

[0096] As with the polyhydric alcohols mentioned above, the organic salts may be in the form of different isomeric forms, hydrates or anhydrous salts. Those skilled in the art will be able to easily determine the appropriate form of organic salt for use in the present invention. can be fully selected and tested.

[0097] In one embodiment of the liquid composition according to the present invention, when the organic salt is arginine, Arginine is L-arginine (CAS number 74-79-3) and L-arginine-HC l (CAS number 1119-34-2), thereby Nin-HCl constitutes the majority of the mixture.

[0098] Such a mixture of arginine compounds may be used, for example, by pre-concentrating the composition prior to use as a vaccine. The pH of the liquid composition according to the present invention when diluted or deposited for counting This helps bring the values ​​closer to physiological values.

[0099] Preferably, the molar ratio of L-arginine:L-arginine-HCl is 1:1 to 1:10. 0; more preferably, the molar ratio of L-arginine:L-arginine hydrochloride is 1:1 to 1:50, and more preferably, the molar ratio is about 1:19.

[0100] In one embodiment of the liquid composition according to the present invention, the NADES comprises three polyhydric alcohols. .

[0101] In a preferred embodiment, the three polyhydric alcohols are sorbitol, xylitol and and trehalose.

[0102] In one embodiment of the NADES used in the liquid composition according to the present invention, the organic salt is beta- the polyhydric alcohol is selected from the group consisting of salts of xylitol, proline, choline and arginine; The monomer is selected from among ethanol and sorbitol.

[0103] In the present invention, the organic salt and the polyhydric alcohol or the plurality of polyhydric alcohols are mixed in different They can be applied in molar ratios to provide NADES with different properties. The method of the present invention is described in detail below. It can be achieved.

[0104] Thus, in one embodiment of the liquid composition according to the present invention, a mixture of an organic salt and a polyhydric alcohol is All molar ratios, as defined herein, are between 1:5 and 5:1.

[0105] In a preferred embodiment of the liquid composition according to the present invention, a molar ratio between an organic salt and a polyhydric alcohol is The ratio of the amines may be from 1:4 to 4:1, from 1:3 to 3:1, or more preferably from 1:4 to 4:1, all as defined herein. The ratio is 1:2 to 2:1, with the preference increasing in that order.

[0106] In one embodiment of the liquid composition according to the present invention, three polyhydric alcohols, xylitol, The molar ratios between any of the above-mentioned glycerol, trehalose and sorbitol are all as defined herein. As a result, the ratio of sorbitol:xylitol:trehalose can be 1 to 5 times. The molar ratios of the bases were 1:1:1, 1:1:5, 1:5:1, 5:1:1, 1:5:5, 5 :1:5, 5:5:1 and 5:5:5.

[0107] One type of advantageous use of the liquid composition according to the invention is as a vaccine. In such applications, mollicutes are used as the active ingredient in live bacterial vaccines. , attenuated nature so as not to induce an unacceptable vaccination response in the target animal. It must be of the same quality as the original document.

[0108] Thus, in one embodiment of the liquid composition according to the invention, the mollicutes bacteria are It has been attenuated with respect to its pathogenicity.

[0109] The term "attenuated" in the present invention is used in its general sense and Infectivity in a host compared to unmodified, non-attenuated or "wild type" Mollicutes bacteria such as and / or reduce the rate of replication, thereby causing a lower level of pathogenic effect Such attenuated live bacteria are also called "modified live bacteria."

[0110] Attenuation of Mollicutes bacteria can be achieved in a variety of ways, including, for example, As is known, the method involves passaging wild-type bacteria in laboratory animals or cell cultures, using mutagens, This can be achieved by recombinant DNA techniques.

[0111] The liquid composition according to the present invention is effective in stabilizing the viability of various bacteria from the class Mollicutes. It is effective.

[0112] Thus, in one embodiment, the Mollicutes bacteria is from the bacterial orders: Acholeplasmales, Anaerobic One or more selected from the orders Plasmatales, Entomoplasmales and Mycoplasmales.

[0113] In one embodiment, the Mollicutes bacterium of the invention is selected from the group consisting of Acholeplasmataceae, Anaeroplasma and selected from the bacterial families Entomoplasmataceae, Spiroplasmataceae and Mycoplasmataceae. The value is 1 or greater.

[0114] In one embodiment, the mollicutes bacteria of the present invention are Acholeplasma, Phytoplasma, Anaeroplasma, Asterolplasma, Entomoplasma, Mesoplasma, Spiroplastes Plasma, Hepatoplasma, Marinoplasma, Moeniplasma, Mycoplasma and Urea The bacterial genera are selected from the group consisting of plasma.

[0115] Preferably, the Mollicutes bacteria of the present invention are Acholeplasma, Spiroplasma and and Mycoplasma.

[0116] More preferably, the Mollicutes bacterium of the present invention is derived from a bacterium of the genus Mycoplasma. do.

[0117] In one embodiment of the liquid composition according to the present invention, the mollicutes bacterium is M. pneumoniae. M. pneumoniae, M. bovis, M. hyopneumoniae M. hyopneumoniae, M. meleagridis gridis, M. gallisepticum, and M. schidigera. noviae (M. synoviae).

[0118] In a preferred embodiment, the Mollicutes bacterium is M. gallis epticum and / or M. synoviae.

[0119] In an embodiment of the liquid composition according to the present invention, the Mollicutes bacterium is M. galliseptica M. gallisepticum and M. synoviae be.

[0120] Experiments have shown that M. gallisepticum and M. synovi M. synoviae can be combined and stored in the liquid composition according to the present invention, When tested at ~8°C for up to 23 weeks, no significant effect of one on the stability of the other was detected. In such a mixture, the same mixture sample was divided into two different types of Agar plates (plates containing NAD (nicotinamide adenine dinucleotide) and NA D) and comparing the colony counts, two types of bacteria were identified. It was possible to detect and count. As described in Vian Dis., vol. 16, p. 387-396, M M. synoviae requires NAD for its growth, so NAD On plates without M. galli, all colonies observed were M. galli septicum).

[0121] In a preferred embodiment, the liquid composition according to the present invention comprises the stabilization of live Mollicutes bacteria. Thus, the number of Mollicutes CFUs increased after 12 weeks of incubation at 4°C. After incubation, 3 Log 10 Even more preferably, the The number of CFU of the class Cutelidae increased by 7 Log after 8 weeks of incubation at room temperature. 10 Exceeding The key point is that it does not decrease.

[0122] Room temperature is a temperature of 20±5°C, and 4°C is 2 to 8°C.

[0123] In an embodiment of the liquid composition according to the invention, one or more selected from the group consisting of: All terms and conditions apply.

[0124] The water activity of the liquid composition is less than or equal to 0.85.

[0125] The water activity of the liquid composition is less than about 0.8; more preferably less than about 0.7, more preferably less than about 0.6 , about 0.5, about 0.4, about 0.3, or even less than about 0.2, in that order of preference. will become higher.

[0126] The water activity of the liquid composition is preferably 0.8 to 0.1; more preferably 0.7 to 0.1, more preferably 0. 6 to 0.1, 0.5 to 0.1, 0.4 to 0.1, 0.3 to 0.1, or even 0.2 to 0.1, and the order of preference increases accordingly.

[0127] The water content of the liquid composition is less than or equal to 50% by weight.

[0128] The liquid composition is preferably at most 40% by weight, more preferably at most 30, 25, 20, 15, 10, It has a moisture content of no more than 8, 7, 6, or 5% by weight (in that order of increasing preference).

[0129] The water content in the liquid composition is 50 to 0.5% by weight; 40 to 1% by weight; 30 to 1.5 % by weight; 20 to 2% by weight; or even 10 to 3% by weight, in this order of preference. become.

[0130] The ratio of the volume of the NADES to the volume of the bacterial sample to be combined is preferably small. At least 3:1 (by volume) NADES:bacteria sample; more preferably, at least 5: 1 (by volume) NADES:bacteria sample; or at least 10:1 (by volume) NADES : Bacteria sample, in order of increasing preference.

[0131] -NADES includes organic salts and polyhydric alcohols.

[0132] -NADES includes two or more compounds that are all polyhydric alcohols.

[0133] The polyhydric alcohols used in the present invention have a molecular weight of less than 5000 g / mol, or even is less than 1000 g / mol.

[0134] The polyhydric alcohol is a sugar or a sugar alcohol.

[0135] The sugar is a non-reducing sugar.

[0136] - the non-reducing sugar is selected from sucrose and trehalose; preferably, the non-reducing sugar is trehalose trehalose is preferably CAS number 6138-23-4.

[0137] The sugar alcohol is selected from erythritol, xylitol and sorbitol. That's all.

[0138] - erythritol preferably has the CAS number 149-32-6; xylitol sorbitol is preferably D-sorbitol; Tol, CAS number 50-70-4.

[0139] The sugar alcohol is at least one selected from the group consisting of xylitol and sorbitol. Yes; in a more preferred embodiment, the sugar alcohol is sorbitol.

[0140] the organic salts are selected from the group consisting of betaine, proline, choline, methionine and arginine salts; The number is 1 or more.

[0141] Betaine is preferably the compound N,N,N-trimethylglycine, CAS number 10 7-43-7, also known as glycine betaine. Proline is the preferred or CAS No. 609-36-9; choline is preferably CAS No. 62-49 -7; methionine is preferably L-methionine, CAS number 63-68-3. Arginine is preferably L-arginine, CAS number 74-79-3.

[0142] -Choline is choline chloride, CAS number 67-48-1.

[0143] - If the organic salt is arginine, the arginine is L-arginine (CAS number 74-79 -3) and L-arginine hydrochloride (CAS number 1119-34-2). can be.

[0144] The molar ratio of L-arginine to L-arginine-HCl is preferably 1:1 to 1:100; Preferably, the molar ratio of L-arginine to L-arginine hydrochloride is 1:1 to 1:50. More preferably, the molar ratio is about 1:19.

[0145] The organic salts are selected from the salts of betaine, proline, choline and arginine; The cholesteryl is selected from xylitol and sorbitol.

[0146] The molar ratio of the organic salt to the polyhydric alcohol is 1:5 to 5:1.

[0147] The molar ratio of the organic salt to the polyhydric alcohol is 1:4 to 4:1, 1:3 to 3:1, or even 1: :2 to 2:1, with preference increasing in that order.

[0148] -NADES comprises three polyhydric alcohols; in a preferred embodiment, The cholesteryl compounds are sorbitol, xylitol and trehalose.

[0149] -Sorbitol:xylitol:trehalose molar ratios of 1:1:1 and 1:1:5; 1:5:1, 5:1:1, 1:5:5, 5:1:5, 5:5:1, and 5:5:5 It is Reka.

[0150] - Mollicutes bacteria are attenuated with respect to their pathogenicity.

[0151] - Mollicutes bacteria are classified as Acholeplasmales, Anaeroplasmales, and Entomoplasmales. and one or more selected from the bacterial orders Mycoplasmales.

[0152] - Mollicutes bacteria include Acholeplasmataceae, Anaeroplasmataceae, and Entomoplasmataceae , Spiroplasmataceae and Mycoplasmataceae.

[0153] - Mollicutes bacteria include Acholeplasma, Phytoplasma, Anaeroplasma, and Aspergillus. Terolplasma, Entomoplasma, Mesoplasma, Spiroplasma, Hepatoplasma Selected from the bacterial genera: The number of bits selected is 1 or more.

[0154] - Mollicutes bacteria are the bacterial genera Acholeplasma, Spiroplasma, and Mycoplasma. One or more of the following may be selected:

[0155] - Mollicutes are bacteria of the genus Mycoplasma.

[0156] - Mollicutes bacteria include M. pneumoniae, M. bovis M. bovis, M. hyopneumoniae , M. meleagridis, M. gallisepticum (Mg allisepticum and M. synoviae The value is 1 or greater.

[0157] - Mollicutes bacteria include M. gallisepticum and / or M. synoviae.

[0158] - Mollicutes bacteria include M. gallisepticum and The species is M. synoviae.

[0159] In one embodiment of the liquid composition according to the invention, the liquid composition has a water activity of 0.85 or less, and has a water content of 50% by weight or less; NADES comprises an organic salt and a polyhydric alcohol; Here, the polyhydric alcohol is xylitol or sorbitol, and the organic salt is betaine. , proline, choline and arginine; Mollicutes bacteria have The mollicutes bacteria are M. pneumoniae, iae), M. bovis, M. hyopneumoniae eumoniae), M. meleagridis, M. galli septicum (M. gallisepticum), and M. synoviae (M. synoviae) iae).

[0160] In a preferred embodiment, the Mollicutes bacterium is M. gallis epticum and / or M. synoviae, and NADE S includes betaine and sorbitol.

[0161] In one embodiment of the liquid composition according to the invention, the liquid composition has a water activity of 0.85 or less, and has a water content of 50% by weight or less; NADES are sorbitol, xylitol and thiamine. It contains three polyhydric alcohols of lehalose; Mollicutes bacteria are weakly The mollicutes bacteria are M. pneumoniae ), M. bovis, M. hyopneumoniae oniae, M. meleagridis, M. gallisepti M. gallisepticum and M. synoviae ) is one or more selected from the following.

[0162] As explained, the liquid compositions according to the present invention can be prepared using conventional techniques and materials. Such a method, use or the like for preparing a liquid composition according to the present invention may be Process details and examples are described herein and such procedures may be performed using routine materials and methods. can be readily utilized by one skilled in the art using the techniques described herein.

[0163] For example, the NADES of the present invention can be produced on an industrial scale and then used in raw materials. The liquid composition according to the invention is prepared by mixing with a preparation of Lactobacillus subtilis. The mixture is then filled into containers of suitable size. The various steps in the manufacturing process are carried out The quality and quantity of bacteria can be monitored by appropriate tests, e.g. biochemical or immunological tests. This can be done.

[0164] Thus, in a further aspect, the present invention provides a method for the preparation of a liquid composition according to the present invention. hand, a. Preparing a NADES; and b. mixing the NADES with live Mollicutes bacteria The present invention provides a method comprising:

[0165] Conveniently, the two steps of the method according to the invention are separated in time and / or space. This can be done in a controlled manner and can involve storage, transportation, or even further steps. This provides operational flexibility in planning and logistics.

[0166] The manufacture of the NADES used in the method according to the invention uses routine tools and methods. One convenient method is to mix the combined compounds in an autoclave, for example. The process involves heating the mixture to a temperature at which it can be mixed and solubilized, as in the case of the bromination process. However, some compounds or compositions of the NADES used in the present invention may be may not be able to tolerate high temperatures of up to 100°C; those skilled in the art will be able to optimize and fine-tune such processes. The heating can be performed in combination with, for example, stirring or ultrasonic treatment. can.

[0167] In addition, one of the components that form NADES is first dissolved in water in a somewhat concentrated form. (a) can be mixed with additional ingredients; and (b) can be mixed with additional ingredients. The NADES structure is formed by removing water. Preferably, such removal is achieved by freeze-drying. Another method is, for example, drying in a water bath at 70°C under reduced pressure for several hours. The method is to subject the mixture to rotary evaporation.

[0168] The composition comprising live Mollicutes bacteria for use in the method according to the invention may be cultured in vivo. From culture, but preferably from in vitro cultivation of bacteria in a suitable medium, various methods can be used Such bacterial cultures can be induced to grow using automated control and monitoring techniques. Large industrial size fermenters for the production of fermented food products of any desired size or volume up to several thousand liters. It can be done.

[0169] The bacteria are then isolated from the culture as a supernatant, cell pellet, or culture medium depending on the characteristics of the bacteria. The whole culture is harvested. The bacteria are then harvested either directly or by first dissolving the bacteria in a liquid, e.g., a buffer. Or, the NADES are mixed indirectly by mixing with the medium.

[0170] The bacterial compositions used in the methods of the invention may be further concentrated, for example by passage through an ultrafiltration membrane. The bacterial composition can then be prepared using process steps comprising: All this is known to those skilled in the art.

[0171] The mixing of the NADES and the bacterial composition in the method according to the invention is preferably carried out using aseptic techniques. This is done using.

[0172] The order of mixing may vary in that the NADES may be added to the bacterial composition or in other forms. The method and method are not critical. Preferably, a suitable method is used to prevent unnecessary shear forces on the bacteria. Mechanical mixing is low speed and low impact. Additionally, mixing and / or filling is performed in a manner that reduces oxygen exposure. To achieve this, the reaction can be carried out in a protective atmosphere of an inert gas (eg, nitrogen).

[0173] A simple device for mixing and filling relatively viscous liquids is available for various supplies of process equipment. Available from the supplier.

[0174] In a further aspect, the present invention relates to a stabilizer in a liquid composition comprising live Mollicutes bacteria. The present invention provides uses of the NADES described herein.

[0175] "Stabilizer", "liquid composition" and "live mollicutes bacteria" are all used herein with respect to the present invention. As defined in any one or more of the embodiments described herein.

[0176] As explained, the advantageous use provided by the present invention is the use of the present invention as a bacterial vaccine. The medical use of the liquid composition according to the invention.

[0177] Thus, in a further aspect, the present invention relates to a method for the treatment of malaria infection caused by Mollicutes bacteria. A method according to the invention or produced according to the invention for use as a vaccine against an infection or disease. A liquid composition obtainable by the method is provided.

[0178] Similarly, in a further aspect, the present invention relates to a method for treating a bacterial infection caused by a Mollicutes bacterium. A liquid vaccine for use against an infectious disease or disease is provided, the vaccine comprising a The present invention also includes liquid compositions which are obtainable by the method according to the present invention.

[0179] And in a further aspect, the present invention relates to a pharma- ceutical acceptable carrier in a liquid vaccine according to the present invention. The present invention provides the use of the NADES described in the present invention as an acceptable carrier.

[0180] A "vaccine" is known to be a composition having a medical effect. The term "immunologically active ingredient" includes an immunologically active ingredient and a pharma- ceutically acceptable carrier. The "vital component" is represented by live Mollicutes bacteria. Vaccines are produced by the immune system of the target animal. The virus is recognized by the target's innate immune system and / or immune system, which then triggers a protective immune response. They may originate from the adaptive immune system and may be of the cellular and / or humoral type.

[0181] Vaccines generally serve to, for example, reduce the number of pathogens present or to prevent their development in a host animal. These drugs are effective in reducing the severity of infection by shortening the replication period of the virus.

[0182] Additionally, or perhaps as a consequence thereof, vaccines generally prevent such infection or replication. Diseases that may be caused by or in response to infection or replication of a target It is effective in reducing or ameliorating the (clinical) symptoms of.

[0183] The "pharmaceutically acceptable carrier" in the present invention is a liquid of high purity, preferably sterile. In this case, the support is a NADES as defined herein. The support may also include stabilizers, oxidizers, Further additives such as inhibitors or preservatives may be included.

[0184] For details regarding "infections or diseases caused by Mollicutes bacteria," please refer to the technical paper The methods are well known in the art and are described, for example, in the Merck Veterinary Manual (supra), and in Diseases o f Poultry,12th edition,2008,Wiley-Blackw This information is contained in handbooks such as ISBN:0813807182.

[0185] The liquid vaccine according to the present invention comprises an immunologically effective amount of a live Mollicute bacterium.

[0186] Those skilled in the art of the present invention will recognize that, for example, by monitoring immune responses after vaccination, Thus, the live Mollicutes bacteria in the liquid vaccine according to the invention can be used to induce such immunologically effective It is also possible to determine and optimize the amount of the antibody in question, in the case of non-human animal targets. The study involves infecting animals with 100,000 gram-negative serovar IgE and then monitoring the disease symptoms, clinical scores, and spread and prevalence of the pathogen in the target population. The results were compared with those of the vaccination challenge seen in mock-vaccinated animals. The response can be compared to the

[0187] Methods for determining a vaccine dose or the amount of live bacteria in an animal sample are known in the art. This method typically involves the use of bacterial culture and counting techniques. The amount of such bacteria can be expressed as a number, for example, in CFU.

[0188] The target of the liquid vaccine according to the present invention is the specific live Mollicutes bacteria contained in the vaccine. Humans requiring vaccination against infections or diseases caused by pathogenic forms The vaccine may be administered to humans or non-human animals. The age, weight, sex, immunological status and and other parameters are less important, but vaccinating healthy, uninfected targets Therefore, it is preferable to vaccinate as early as possible to prevent field infection.

[0189] The liquid vaccine according to the invention may further comprise an additional antigen or microorganism, a cytokine, or a non-methylated It may further include other compounds such as immunostimulatory nucleic acids including CpG, adjuvants, and the like. Alternatively, a liquid vaccine according to the present invention may itself be added to the vaccine.

[0190] The liquid vaccine according to the invention can advantageously contain one or more further antigens or, for example, It may be combined with a microorganism derived from a microorganism pathogenic to a human or non-human animal target. Such additional antigens may themselves be live, attenuated, or inactivated. The infectious microorganism may be a purified infectious microorganism, or may be a subunit, extract, fraction, or the like. The antigen may be a biological molecule such as a protein, carbohydrate, lipopolysaccharide, lipid or nucleic acid molecule. Or it can consist of synthetic molecules.

[0191] The liquid vaccine of the present invention contains thimerosal, merthiolate, phenolic compounds, and and / or gentamicin. Preferably, no preservatives are used.

[0192] The liquid vaccine according to the invention may contain a stabilizer, but preferably such a stabilizer is , are not variable or undefined compounds as described herein.

[0193] The liquid vaccine according to the invention may contain ascorbic acid, citrate, methionine or tocopherol. The composition may contain an antioxidant such as rol.

[0194] The liquid vaccine according to the invention can be used as a prophylactic or therapeutic treatment, or both. It is possible.

[0195] The liquid vaccine according to the present invention functions as an effective priming vaccination. This can be followed by one or more booster vaccinations using the same or different vaccines. Vaccination can be performed.

[0196] The liquid vaccine according to the present invention can be prepared by a known method incorporating the method for producing the liquid composition according to the present invention. It can be prepared using techniques.

[0197] Therefore, in a further aspect, the present invention provides a method for the preparation of a liquid vaccine according to the invention. The present invention provides the use of NADES as described above.

[0198] Such production includes microbiological testing for sterility and the absence of adventitious substances; and finally The vaccine incorporates vaccination trials to confirm efficacy and safety. Quality, quantity, and sterility After safety and efficacy testing is completed, the vaccine product can be released.

[0199] All of these are known to those skilled in the art and are suitable for vaccine production under known standards of pharmaceutical manufacturing. General techniques and considerations that apply include, for example, government directives and regulations (Pharmacopeia, 9 CFR ) and in known handbooks (Pastoret, Lippincott, both cited above). It is written.

[0200] The liquid vaccine according to the invention can be used in human or non-human animals, for example in the form supplied by a pharmaceutical company. However, in certain embodiments of the liquid vaccine according to the invention, Depending on the particular composition of the embodiment, it may be preferable to perform dilution of the liquid vaccine. This may facilitate the actual administration to the target, for example by reducing the viscosity, or may reduce local reactions upon vaccination of the target.

[0201] The diluent for such dilution is typically a liquid, and the liquid vaccine according to the invention Preferably, the diluent is provided separately from the composition, e.g., as a kit of parts. It will be provided together with the liquid vaccine by Ming.

[0202] Thus, in a further aspect, the present invention comprises at least two containers (containing the liquid vaccine according to the invention). and one container containing a liquid diluent. .

[0203] For diluting a liquid vaccine according to the invention, e.g. for inclusion in a kit of parts according to the invention The "liquid diluent" is a liquid suitable for diluting the liquid vaccine according to the present invention. Thus, the liquid diluent can be water or an aqueous solution such as a buffer. Due to the low water content of liquid vaccines according to the invention, the diluent may also be oil-based or oil-based. The emulsion may be an oil-in-water emulsion, a water-in-oil emulsion, or a double emulsion.

[0204] In one embodiment, the liquid diluent is itself a liquid vaccine, e.g., a reconstituted lyophilized live vaccine. It may be a cutin or emulsion vaccine.

[0205] For example, a liquid vaccine according to the invention can be diluted for inclusion in a kit of parts according to the invention. The choice of liquid diluent for the vaccine depends on the intended use of the diluted vaccine; The pathogenicity of bacteria depends on several factors, such as the intended human or non-human animal target of vaccination. Those skilled in the art are fully capable of selecting, preparing and applying such diluents. be.

[0206] In one embodiment of the kit of parts according to the invention, the kit comprises the following components: In a preferred embodiment, the instructions include instructions for use, e.g., for the manufacture of the kit. provided by the supplier or distributor on or together with one or more of the components of the kit; or in electronic form, such as information available for viewing or downloading from an Internet website. Reference is made to the instructions provided.

[0207] In one embodiment, the kit of parts includes at least two containers, on or with a box: or instructions for use provided on an information carrier (e.g., card or booklet) contained within the box It is a box containing a book.

[0208] In one embodiment of the kit of parts, the kit also comprises a vaccine for use in the vaccination method according to the invention. components (related to commercial sales) on internet websites for It could also be a donation of goods.

[0209] A liquid vaccine according to the invention, or which can be produced by the method or use according to the invention. or can be prepared using a kit of parts according to the invention. Liquid vaccines in diluted form are suitable for use in humans or non-human animal targets requiring such vaccination. Vaccination is intended to be administered to a subject in need thereof using non-attenuated, live Mollicutes bacteria of the invention. To reduce infection and / or disease symptoms that may be caused by infection with the counterpart. This then induces an increased immune response in the target human or non-human animal, which in turn reduces the number, severity, and / or severity of lesions. or resulting in a reduction in the severity and clinical signs of disease caused by bacterial infection. Such vaccines are colloquially called vaccines "against" specific mollicutes bacteria. or "mollicutes vaccine."

[0210] Thus, in a further aspect, the present invention provides a live morivirus of a pathogenic type contained in a liquid vaccine. Human or non-human animal targets for infection and / or disease caused by C. cerevisiae bacteria The present invention also provides a liquid vaccine according to the present invention for use in a method for protecting a target.

[0211] Such a "pathogenic live Mollicutes bacterium contained in the liquid vaccine" is, for example, The non-attenuated parent mollicutes from which the attenuated forms of the mollicutes bacteria of the present invention were generated. Alternatively, such pathogenic types of bacteria may be derived from various sources, as described herein. Those skilled in the art of the present invention can easily derive the above-mentioned compounds from the above-mentioned sources, for example, from the above-mentioned sources. -Comparative tests in vitro or in vivo will determine whether the Mollicutes are pathogenic or not. can be determined completely.

[0212] The "method of protection" includes administering a liquid vaccine according to the present invention to said human or non-human animal target. The vaccine "captures" (i.e., protects) its favorable immunological effect in the target. This includes establishing infection and demonstrating it.

[0213] In a further aspect, the present invention relates to an infection or disease caused by a Mollicutes bacterium. provides a method for vaccinating a human or non-human animal target against a disease, the method comprising the steps of: or by using a kit of parts according to the invention. nothing.

[0214] In the method of vaccination according to the invention, the liquid vaccine according to the invention is administered to a human or non-human The liquid vaccine according to the present invention may be used for direct administration to human animal targets. The vaccination method according to the present invention can be administered to a human or non-human animal target in the form of a vaccine. This latter embodiment of the invention has a reduced viscosity, making it easier to handle and administer by injection. Alternatively or additionally, administration in a diluted form may be used to improve, for example, the composition at the site of inoculation. The present invention may reduce local reactions in human or non-human animal targets due to tissue irritation.

[0215] Vaccination according to the method of the invention can be carried out by the parenteral route, i.e. through the skin, e.g. For example, the administration can be intramuscular, intraperitoneal, intradermal, submucosal or subcutaneous. The preferred route of parenteral administration of the cutin composition is by intramuscular or subcutaneous injection.

[0216] Furthermore, the liquid vaccine according to the invention can be administered by enteral or mucosal route, i.e. eye drops, nasal drops. It can be administered orally, enterally, by oral nasal drops, spray or aerosol.

[0217] Alternatively, the liquid vaccine according to the present invention can be administered via drinking water, coarse spray, atomization, feeding, etc. A further alternative is to administer the compound to the target, such as via a mass administration method. It is administered to fetal birds via the oral route.

[0218] Needless to say, the optimal route of administration of the liquid vaccine according to the invention depends on the vaccine used. The details of the administration route and the specific characteristics of the target will determine whether such a route is appropriate. The method can be fully selected and optimized.

[0219] The liquid vaccine according to the present invention can be administered parenterally in a volume acceptable to the target human or non-human animal. It can be administered by bronchoscopy, for example in an amount of about 0.01 mL to about 10 mL. Preferably, one dose has a volume of about 0.05 mL to about 5 mL, more preferably One dose is 0.1 mL to 3 mL.

[0220] When administered intramuscularly, the volume of one dose is preferably about 0.5 mL to about 3 mL, more preferably about 0.5 mL to about 3 mL. The amount is preferably about 1 mL to about 2 mL.

[0221] When administered intradermally, the volume of one dose is preferably about 0.1 mL to about 0.5 mL. More preferably, it is about 0.2 mL.

[0222] When administered intraembryo, the volume of one dose is preferably about 0.01 mL to about 0.5 mL. and more preferably about 0.05 mL.

[0223] The method, timing and volume of administration of the liquid vaccine according to the present invention are preferably In order to reduce the stress on the target human or non-human animal and to reduce labor costs, These other vaccines will be integrated into the existing vaccination schedule. The drugs may be administered simultaneously, simultaneously, or sequentially in a manner compatible with their registered uses. It is possible.

[0224] Further optimization of the liquid vaccine according to the present invention is within the skill of the art. In general, this involves fine-tuning the efficacy of the vaccine to further improve the immune protection it provides. This includes, for example, adjusting the dose, volume, or antigen content of the vaccine. or by application via different routes, methods or administration methods. All of these are within the scope of the present invention.

[0225] The invention will now be further illustrated by the following non-limiting examples. EXAMPLES

[0226] Example 1: Methods and Materials 1.1: Preparation of NADES formulations NADES allows for the mixing of the desired ingredients, which can be accelerated by heating and / or the addition of small amounts of water. The NAD can be prepared by the following methods. ES samples were prepared by dissolving relevant compounds in water via sonication in a water bath. The required amount of compound was added to the flask. The flask was briefly shaken or vortexed to The powders were mixed. The required amount of water was then added. This mixture was stirred until the NADES were formed. The mixture was then placed in a sonication-water bath set at 45° C. and sonicated for up to several hours. During sonication temperatures up to 80°C could be reached.

[0227] 1.2: Measurement of water activity sample For biocontainment purposes, samples of NADES preparations thus free of viable bacteria However, to form a liquid vaccine composition according to the present invention, the water activity of If the amount of aqueous bacterial composition added to the NADES is known, the relationship between the NADES and the bacterial composition can be determined. The resulting water activity of the combination was calculated by Dai et al. (2015, Food Chem., vol. .187, p.14-19).

[0228] Sample preparation Transfer a volume of NADES sample into a 10 mL glass vial and fill approximately 1 / 4 of the vial. For each sample, coded vials were filled in duplicate. Close the vial using an aluminum crimp cap with a PTFE stopper. Equilibrium between the sample and the headspace was allowed to reach by standing at room temperature for 12 hours.

[0229] Device The water vapor pressure in the headspace of the sample was measured using the Lighthouse FMS- The measurements were performed using a 1400 moisture / pressure analyzer. The sample was placed directly in the sample chamber. Before the sample measurement, the sample was inserted into the w Calibrated headspace pressure standards in the range The system was calibrated using saturated salt standards. w Nonlinear range of was calibrated and recalculated.

[0230] calibration a w To calibrate the machine over the full range of 0 to 1 and create a baseline for water activity, several Several standard samples were measured: pure water, followed by five samples of specific saturated salt solutions, six Each of the four standard duplicate samples was measured in triplicate. The pure water reading was 1.000. By setting and a of 0.1131 w Other standard values ​​were determined using the standard. A series of certified vapor pressure standards were also measured.

[0231] a w The results of the measurements are included in Table 1.

[0232] 1.3: Examination of DSC profile of NADES Thermal Analysis software was used. Differential scanning calorimetry (DSC) analysis was performed on a Q2000 DSC instrument from For the SC profile, samples were first equilibrated at 40 °C and then cooled to -90 °C at 5 °C / min. After cooling and subsequent equilibration at -90°C for 5 min, the samples were again incubated at 5°C / min to 40°C. This cooling and heating cycle was repeated a second time on the same sample to measure the phase hysteresis. The lysis was investigated by software from the heating curve by the midpoint of the stepwise shift in the baseline. Sample T by software g This is due to the increase in thermal conductivity caused by the increase in mobility during the glass transition. It shows a change.

[0233] For NADES formed with a molar ratio of proline, sorbitol, and water of 1:1:2.5, A typical DSC profile of the NADES of the present invention is shown in FIG.

[0234] Figure 1 shows a sudden shift in the baseline, here from -41 to -45°C, which is This corresponds to the characteristics of the effective NADES of the present invention. The profile was reported by Qiao et al. (supra) in their Figure 4 as The solution was diluted to an aqueous composition and showed an essentially flat baseline, indicating no evidence of water-ice crystal formation and and melting: the rise and fall of heat flow during so-called "cold-crystallization" It is essentially different from what has been

[0235] 1.4: Mixing of NADES and bacterial compositions The NADES preparation was mixed with the bacterial composition in a 9+1 (volume ratio) in a laminar flow cabinet. Mix the samples gently using a roller bank; vortex; or reduce the size of the sample. Depending on the size of the samples, they were briefly stirred using a sterile loop. Fill 1 mL of the pull into a 3 mL glass vial and cap with a rubber stopper and metal cap.

[0236] 1.5: Stability assay For each test sample, a unique vial was prepared and stored under refrigerated (2-8°C), frozen (-2 After a certain period, the vials were opened and diluted with 10% PBS. 10x dilution -1 ~10 -10 Dilutions were prepared in at least three dilutions. In duplicate, plate 50 μL samples onto agar plates that support the growth of specific bacteria. Plates were incubated at 37 °C and 5% CO until colonies were large enough to count. The samples were incubated at 25°C for 5–8 days in 024°F (100°C). From this, the titer of Mollicutes bacteria was calculated and 30 to 300 colonies were counted. The titer was calculated as the number of colonies per mL of sample. The data were expressed as colony forming units (CFU / mL). From these data points, graphs of the stability data were generated, as shown in Figures 2 and 3.

[0237] Example 2: Composition of NADES used in the present invention A number of different compounds, different molar ratios and different water contents have been prepared for use in the present invention. Different NADES compositions were prepared.

[0238] Table 1 below lists many of the most effective NADES formulations; these are used at room temperature (approximately The compositions and water contents of the fluids were calculated using the formula: Details are provided. The water activity of some of the prepared samples was determined and Includes results.

[0239] Table 1: Compositions and properties of a typical number of NADES of the invention [Table 1]

[0240] Example 3: Bacterial stability assay results M. synoviae and M. Stability assays were performed on M. gallisepticum bacteria. The experiments were carried out over an extended period at temperatures between 2 and 8°C and at room temperature (approximately 20°C).

[0241] Incubation at room temperature for 8 weeks is more effective than incubation at 4°C for 1-2 years. This can therefore serve as a very important indicator of bacterial viability. This was a significant attack and not all samples were expected to survive these conditions. It was.

[0242] Conclusions from stability results M. synoviae and M. gallis epticum, the inherent low stability during storage in both standard phosphate buffered saline (PBS) and This was demonstrated by a control, a 1+9 (by volume) mixture in 1000 mL of ... For details, see Figures 2 and 3. This control liquid composition was tested in the first time. At this point, bacterial colonies were no longer detectable in any of the dilutions examined. This applied to both storage conditions of 2-8°C and room temperature.

[0243] From these control samples, M. synoviae in PBS or There was no detectable survival of any M. gallisepticum. It was concluded that

[0244] Surprisingly, and in stark contrast to the PBS control, M. synoviae ( Both M. oviae and M. gallisepticum were It showed remarkable survival when combined with NADES in a liquid composition according to the invention. 1+9 When mixed with various NADES (volume ratio), it can be measured after storing at 20℃ for up to 8 weeks. Good titers were obtained.

[0245] In the case of M. synoviae, the stabilizing effect at 20°C was S betaine:sorbitol:WFI molar ratio 1:1:2.5, sorbitol:xylitol The strongest reaction was observed at a molar ratio of 2:2:1:10 for thorose:trehalose:WFI. Storage for up to 6 months showed significant viability, with some NADES showing 3% viability. Log 10 There was a loss of less than CFU / mL.

[0246] In the case of M. gallisepticum, the stabilization effect at 20°C was The results showed that the molar ratio of arginine:sorbitol:WFI in NADES was 1:2:6, and that the molar ratio of proline was 1:2:6. The strongest reaction was observed at a molar ratio of 1:1:2.5 for sorbitol and WFI. After months of storage, significant viability titers were observed, up to 3 Log for some NADES. 10 There was a loss of less than CFU / mL.

[0247] Example 4: Administration of NADES to experimental animals Evaluate the ease of administration and safety of the NADES used in the present invention in target animals To do so, examples of NADES were administered to laboratory animals and post-mortem material.

[0248] To this end, two variants of proline:sorbitol-based NADES were Two types of NADES 3A-based high viscosity liquids were prepared and tested: one with high viscosity (NADES 3A) and one with low viscosity. These are the two extremes of the viscosity spectrum that are the focus of this invention. This was useful for comparing the administration characteristics of NADES in the current study. The characteristics of NADES 3A are shown in Table 1. Furthermore, their viscosities were measured at 30°C at 60 rpm in a standard measuring cup of approximately 80 mL. 2 seconds, using spindle type No. 62, Brookfield (trade name) DV-I +Measured using a viscometer using standard conditions at approximately 20°C.

[0249] handling High viscosity NADES 3A is injected into a hypodermic needle of size 21G or less, i.e., an outer diameter of 0.8 mm or more. I was able to push it into the needle.

[0250] Table 3: NADES formulations tested by administration to animals [Table 2]

[0251] In vivo subcutaneous administration NADES formulations 3B and 3C were administered by subcutaneous injection in a safety study in dogs. Particular attention should be paid to any side effects that may occur following local or systemic administration of sedative preparations. I did.

[0252] As previously described, proline, sorbitol, and water for injection were heated and sonicated in a sealed vial. Both formulations were prepared by microwave processing to give a clear, homogenous liquid. They were then filter sterilized and their bioburden and endotoxin levels were analyzed. Both formulations were below the minimum dilution standard of 1 unit / mL, which is within the general acceptable range for sterile formulations. The NADES 3C formulation was administered in a volumetric dose of 1 part NADE Proline:sorbitol:WFI was prepared by diluting S3A with 9 parts by volume of WFI. The formulation contained NAD in a ratio of 1:1:5 and had a viscosity of 289 mPa s. The viscosity of ES 3B is 33.4 mPa·s.

[0253] Six healthy beagle dogs aged 7 months were divided into two groups of three dogs each. S formulation, subcutaneously using a 1 mL syringe and a 21G 5 / 8 needle for 2 weeks. Dogs in Group 1 received two doses of 1 mL of the assigned formulation, spaced at intervals. Dogs in group 1 were administered high viscosity NADES 3B, and dogs in group 2 were administered high viscosity NADES 3C. All dogs were vaccinated. Patients were clinically monitored daily throughout the study, including for local reactions at the site.

[0254] Vaccinations were administered at the nape of the neck: the first vaccination was administered on the left side, the second vaccination on the right side. Vaccination was performed on the right side of the neck. Before each dose, the vaccination site was shaved and the injection site was marked with a marker pen. No discomfort was observed during any of the injections. Four hours after injection, the injection site was palpable. There was little or no palpable material at the injection site, and no swelling or redness was observed. Ta.

[0255] -3, -2, -1 days before each vaccination; on the day of vaccination: immediately before and after vaccination Rectal temperatures were measured 4 hours after vaccination; and then daily for 7 days after vaccination. .

[0256] No significant increases in body temperature were observed in any of the dogs throughout the study, and no clinical side effects were observed. I couldn't.

[0257] Conclusion: Inoculation of dogs with NADES formulations 3C or 3B via the subcutaneous route resulted in both local and systemic toxicity. In general, it did not induce any adverse inoculation reactions.

[0258] Intranasal The spread pattern of the drug on the nasal mucosal surface after intranasal administration was investigated using two NADES. Formulations: 3A and 3B were tested. Both NADES formulations were tested neat and without adding water. As a result, the viscosity of NADES 3A was 5197 mPa s. The viscosity of S3B is 33.4 mPa·s. The tip of the spatula is colored "patent blue." The agent was added to 30 mL of each NADES formulation to obtain a deep blue color. I used S.

[0259] Calf heads were obtained postmortem from calves approximately 2 to 4 weeks of age. To simulate this, dosing was performed while the head was held in an upright position with the nasal passages tilted slightly upwards.

[0260] The liquid was then administered according to standard procedures for intranasal vaccination, thereby A 2.5 mL syringe without a needle containing 0.5 mL is placed in the nostril and the liquid is then squirted in one steady stream. The head was then divided into two halves, the septum removed, and the intranasal passages examined for the presence of blue color. .

[0261] In all samples, the blue liquid was observed to move easily through the nasal passages. The low viscosity of the BS sample allowed for easy injection, but all liquid passed through quickly. A blue color was observed throughout the nasal cavity, including the beginning of the trachea.

[0262] The injection of NADES 3A, which has a very high viscosity, requires a little force to eject from the syringe. Also, as expected, it took a relatively long time for the blue liquid to appear under his head. After the head was incised, this viscous liquid also spread evenly throughout the nasal passages, forming a thick layer. The vaccine appeared to be delivered to the patient via the nasal cavity, and more material was observed to remain in the nasal cavity. When administered to live calves at the time of vaccination, retention is likely to be improved and there is less upper respiratory tract penetration. This shows that there is something.

[0263] The effect of administration of the low-viscosity NADES 3B formulation was compared with that of a PBS sample in which the liquid was passed through the nasal cavity. The effects were very similar to those of

[0264] Conclusion: Monitoring the distribution of colored formulations of different viscosities after intranasal injection into the head of a calf The distribution of liquid, as determined by the blue color, was comparable between the two NADES preparations and PBS. However, there are some differences in ease of administration and amount of residue. The agent was slightly more difficult to expel, but resulted in a thicker layer of residue in the nasal passages. This may result in sustained release of antigen from such viscous NADES formulations. This suggests that...

[0265] Example 5: Comparative measurements of aqueous stabilizers WO with 30% sorbitol + 0.6M arginine in 0.04M PBS An aqueous stabilizer composition was prepared as described in US Pat. No. 6,2015 / 121463. The properties such as water activity and DSC profile are examined to determine whether the composition meets the above definition. It was revealed that the NADES does not have the characteristic properties that would make it a NADES as defined and outlined above.

[0266] water activity a w The measurement was performed using a head-scratch CT scanner according to the method described in Example 1 of WO2020 / 122329. Water activity was measured by vapor pressure measurement. The water activity results were measured in duplicate and averaged. w is 0.8 9. Clearly, this high level of available water indicates that this composition is not a NADES. This indicates that

[0267] DSC Profile Measurement of the DSC profile of a prior art aqueous stabilizer is described herein in Example 1.3. This was done according to the method described.

[0268] The results are shown in Figure 4. According to this, the upper horizontal part of the graph (as in Figure 1 here) shows the cooling period (from right to left) from 40 °C to -90 °C at 5 °C / min, with The horizontal section shows the heating phase (from left to right) from -90°C to 40°C at 5°C / min.

[0269] During cooling, at a temperature of about -27°C, extensive and sudden crystallization of free water into ice can be observed. Then, when it is reheated, the typical ice crystal melting profile peaks at -5.5°C. and was observed.

[0270] Furthermore, in Figure 4, during the heating period, the horizontal baseline after ice melting is at the same level as before it melted. This means that the heat capacity of the sample has not changed. This is clearly different from the gradual baseline shift typically observed in the case of a glass transition. .

[0271] Also, the ice melting temperature in Figure 4 is much higher than the glass transition temperature (-45°C) in Figure 1. -5℃), which is also important because the samples examined in Figure 4 are deep eutectic mixtures. This shows that it was not an object.

[0272] In FIG. 4, both peaks, the high melting temperature, and the baseline all returned to their previous levels. This indicates the formation and melting of ice crystals and therefore the presence of a large amount of free water in the compositions examined. This indicates that it was not NADES.

[0273] However, in contrast, in the sample of FIG. 1 (the NADES according to the present invention), no ice crystals were formed. No melting occurred and a clear baseline glass transition shift was observed.

[0274] Example 6: Additional Stability Results: M. bovis Further species of Mollicutes: Mycoplasma bovis ovis) was tested for stabilization of the liquid composition according to the invention. Specifically, M. synoviae and M. galli septicum) according to the methods described in Examples 1 to 3.

[0275] Different compositions according to the invention and the results of stability assays at either room temperature or 2-8° C. The results are shown in Figure 5.

[0276] Panel A: Room temperature, up to 10 weeks; and Panel B: Incubation at 2–8°C for up to 26 weeks.

[0277] The initial titer was 9.9 Log 10 CFU / mL. PBS (negative control) was used at room temperature and in cold storage. Some survival was observed after two weeks of incubation in both cultures. However, in samples incubated for longer periods in PBS, viable cells no longer migrated at all. I couldn't collect it.

[0278] At room temperature, several NADES compositions show good initial stability (2–4 weeks of incubation). However, the arginine:sorbitol:water (2:1:6) Best results are obtained using 100% ethanol, which stabilizes the bacteria at room temperature for at least 10 weeks. I was able to make it happen.

[0279] Similar images were observed with refrigerated incubation, with some of the compositions examined Good initial stabilization was observed for 6-13 weeks in The incubation mixture was arginine:sorbitol:water (2:1:6), but The differences observed between the different compositions were not as great as with incubation at room temperature.

[0280] In general, M. bovis is freeze-dried in the liquid composition according to the invention. The glycerol could be effectively stabilized at various storage temperatures above freezing without the need for freezing.

[0281] Example 7: Results of an expanded stability study M. synoviae and M. gallice in the liquid composition according to the present invention Stability test results for M. gallisepticum showed that it can be stored at 2-8℃ for up to one year. These data were obtained from continuous incubation of The results were obtained from the same set of samples as those described above and are shown in Figures 2B and 3B herein. This is an extension of the above.

[0282] The enlarged results are shown in FIG.

[0283] Panel A: M. synoviae, 2–8°C, up to 54 weeks; Beauty Panel B: M. gallisepticum, 2–8°C, maximum Long 52 week incubation.

[0284] In the case of M. synoviae, liquid compositions tested according to the invention Three of the products stabilize the viability of these bacteria at 2-8°C for at least one year. This allows the synthesis of proline:sorbitol:water (1:1:2.5) and betaine. :sorbitol:water (1:1:2.5) gave the same end result.

[0285] In the case of M. gallisepticum, the live bacteria is betaine: Sorbitol:water (1:1:2.5) at 2-8°C for up to 52 weeks of incubation It just wasn't retained.

Claims

1. A liquid composition comprising a live Mollicutes bacterium and a stabilizer, the stabilizer being a natural deep eutectic. A liquid composition that is a solvent (NADES).

2. 10. The liquid composition of claim 1, wherein the composition has a water activity of 0.85 or less.

3. 3. The liquid composition of claim 1, wherein the composition has a water content of 50% by weight or less.

4. The method according to any one of claims 1 to 3, wherein the NADES comprises an organic salt and a polyhydric alcohol. The liquid composition described above.

5. 5. The method according to claim 1, wherein the NADES comprises two or more compounds, each of which is a polyhydric alcohol.

13. The liquid composition according to claim 12 .

6. The liquid composition according to claim 4 or 5, wherein the polyhydric alcohol is a sugar or a sugar alcohol. 。

7. The sugar is selected from sucrose and trehalose, or the sugar alcohol is erythritol.

7. The method according to claim 4, wherein the glycerol is one or more selected from the group consisting of tetritol, xylitol and sorbitol.

2. The liquid composition according to claim 1 .

8. The organic salt is selected from the group consisting of salts of betaine, proline, choline, methionine and arginine. The liquid composition according to claim 4 , wherein the liquid composition is one or more of the following:

9. 9. The method according to claim 1, wherein the mollicutes bacteria are attenuated with respect to their pathogenicity. The liquid composition according to any one of claims 1 to 5.

10. A method for producing the liquid composition according to any one of claims 1 to 9, comprising the steps of: - preparing said NADES, and - mixing said NADES with live Mollicutes bacteria The method includes:

11. A method according to any one of claims 1 to 9 as a stabilizer in a liquid composition comprising live Mollicutes bacteria. Use of NADES as described in paragraph 1.

12. Used as a vaccine against infections or diseases caused by Mollicutes bacteria. The method according to any one of claims 1 to 9 or according to claim 10, Possible, liquid composition.

13. Liquid vaccines used against infections or diseases caused by Mollicutes bacteria The method according to any one of claims 1 to 9 or according to claim 10.

5. A vaccine comprising a liquid composition capable of being administered to a patient.

14. 14. The compound according to claim 1 as a pharma- ceutically acceptable carrier in the liquid vaccine according to claim 13. Use of a NADES according to any one of claims 1 to 9.

15. A method for producing a liquid vaccine according to claim 13, comprising the steps of: Use of NADES.

16. At least two containers, namely one containing a liquid vaccine according to claim 13, and and a container containing a liquid diluent.

17. Infection caused by the pathogenic type of live Mollicutes bacteria contained in said liquid vaccine and / or for use in a method for protecting a human or non-human animal target against disease.

3. A liquid vaccine according to claim 3.

18. Human or non-human animal targets for infection or disease caused by Mollicutes bacteria A method for vaccination of a target comprising administering to said target a liquid vaccine according to claim 13. or the use of a kit of parts according to claim 16.

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