Stabilization of live Mollicutes bacteria in liquid compositions

Abstract

The present invention relates to a liquid composition of live Mollicutes bacteria and a stabilizer, wherein the stabilizer is a natural deep eutectic solvent (NADES). In such a liquid composition, live Mollicutes can be stabilized without freezing or freeze drying. This allows for various advantageous uses in diagnosis and medicine, in particular as a liquid vaccine for combating infections or diseases caused by Mollicutes bacteria in humans or non-human animals.

Description

[0001] The present invention relates to the fields of bacteriology and vaccinology. In particular, the present invention relates to a liquid composition of live Mollicutes bacteria and a stabilizer, the preparation of the liquid composition, the use of NADES as a stabilizer in the composition, and various medical uses of the composition.

[0002] Live bacteria are used in a wide variety of applications, well known to be classical or modern biotechnological processes, diagnostics and vaccines. For all these applications, methods to stabilize these bacteria to maintain their viability during storage and use are important. Common techniques rely on storage under deep freezing conditions, stabilization using cryoprotectants such as glycerol or rich culture media. Alternatively, live bacteria can be lyophilized and stored in lyophilized form under refrigerated or frozen conditions. However, all these stabilization methods have many disadvantages: enriching the storage medium introduces components of uncertain and variable quality, such as serum, hydrolysates and extracts. This undefined component may have an unpredictable effect on stability. In addition, frozen products require a laborious cold chain for storage and transportation. But even these processes cannot keep live bacterial cells completely alive, because the freezing step itself leads to a significant reduction in the viable cell count. In addition, lyophilization is a very expensive and time-consuming process. Therefore, there is a great need for improved methods that effectively stabilize the viable cell count of bacteria during storage and use.

[0003] A group of bacteria of agricultural and medical interest is the class Mollicutes. These are taxonomically classified into the phylum Tenericutes (Ludwig et al., 2009, in: Bergey's Manual of Systematic Bacteriology, 2nd edn, vol. 3. Springer: New York, USA, pp1–13.). This phylum contains bacteria that are derived from Gram-positive bacteria but have lost their cell wall. They are usually parasites or symbionts of eukaryotic hosts.

[0004] Infection of animals by mycoplasma bacteria, either as primary or secondary pathogens, typically results in respiratory disease and / or a variety of other symptoms that together result in mild to severe illness, great discomfort to the affected host, and economic losses. A reference on the impact of mycoplasma infection in agriculture is: The Merck Veterinary Manual (11th edition, 2016, ISBN-10: 9780911910612).

[0005] Although mycoplasma bacterial infections can be treated with certain types of antibiotics, they are usually prevented using vaccines. Such vaccines can be based on live attenuated strains or killed bacterins (inactivated bacteria). The bacterin vaccine against Mycoplasma bovis (M. bovis) is MpB Guard TM (Agrilabs). Most commercial mycoplasma vaccines in use are for poultry; for example: Live attenuated vaccine for Mycoplasma gallisepticum: MG (Bioproperties) (frozen) and NobilisMG 6 / 85 (MSD Animal Health) (lyophilized, cooled); and bacterin vaccines for Mycoplasma gallisepticum: (Zoetis) and MG inac (MSD Animal Health), both emulsions contain an oily adjuvant. The live attenuated vaccines used for Mycoplasma synoviae are: MS(Bioproperties)(Frozen) and MS Live (MSD Animal Health) (lyophilized, cooled). Several other live attenuated mycoplasma vaccines are known and are being used on a more limited scale, such as Contavax TM , a freeze-dried vaccine against ruminant Mycoplasma mycoides, is used in some African countries. In addition, in China, a live attenuated M. hyopneumoniae vaccine is used in pigs. Review articles on mycoplasma vaccination are: Whithear (1996, Rev. Sci. Tech. Off. Int. 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 cell membranes and even their invasion of host cells, Mollicutes are well-known contaminants of various eukaryotic in vitro cell cultures. Therefore, several live Mollicutes bacteria are used for diagnostic and quality control purposes. This is also a formal requirement in bioproduction according to government regulations such as the European Pharmacopoeia and the US 9CFR.

[0007] Mollicutes are the smallest known self-replicating organisms: some species are only 0.2 micrometers in diameter, and some species have genomes as small as 580 kbp. With so few genes, Mollicutes bacteria are heavily dependent on resources in their environment, so they usually require very rich culture media when cultured in vitro. In addition, the lack of a cell wall exposes their cell membranes to the effects of the physical and chemical environment, resulting in poor stability during storage and use.

[0008] Therefore, an important issue in the vaccine industry is how to maintain the viability of such live Mollicutes bacteria as bacterial stock during the shelf life of the final product and during, for example, diagnosis or vaccination. One solution is to produce and use non-live bacterium-type vaccines. However, such vaccines are relatively expensive because they require relatively high doses, repeated administrations and the use of adjuvants.

[0009] Vaccines based on live Mollicutes bacteria are deep frozen or lyophilized and stored in a rich stabilizing culture medium. As mentioned above, this culture medium is less than ideal for a number of reasons. In addition, the freezing process itself still results in a significant loss of viable cell counts, resulting in a loss of up to 10^3 colony forming units (CFU) within a few days. Therefore, methods and materials for improving the stabilization of live Mollicutes bacteria are highly desirable.

[0010] Deep eutectic solvents (DES) are well-known liquid compositions that combine many remarkable properties. Typically, these are mixtures of compounds that are liquid at ambient temperature but have a low water content. In addition, the small amount of water present is tightly bound, limiting its availability in chemical or biological processes. This manifests itself as very low water activity values. DES are described, for example, by Abbott et al., 2003, Chem. Commun., vol. 1, p. 70-71 and in WO 2009 / 120839.

[0011] DES has high ionicity but at the same time very low water activity and is very effective as a solvent for polar hydrophobic compounds. Examples range from the extraction of gases, minerals or industrial bulk products to the dissolution of drugs. DES is also used for the extraction and stabilization of biomolecules such as enzymes and RNA, see US 8,247,198 and WO 2011 / 155829, respectively.

[0012] Since about 2011, a special form of DES consisting of naturally occurring substances has been considered. These so-called "natural DES" or NADES are described in, for example, Choi et al., 2011, Plant Physiol., vol. 156, p. 1701-1705; and Dai et al. 2013, Anal. Chim. Acta, vol. 766, p. 61-68. NADES are currently attracting attention as "green" solvents that can be used to extract natural products and as solvents for, for example, natural pesticides.

[0013] WO 2011 / 155829 describes the use of NADES for the extraction of biological material. One of the options listed is the extraction of proteinaceous material, such as subunit vaccines.

[0014] Similarly, WO 2014 / 131906 describes a fixative for viruses in blood or tissues, with particular focus on the extraction and stabilization of viral RNA for clinical diagnosis. The fixative is DES, and although WO 2014 / 131906 lists a large number of possible DES compositions, only compositions that include urea or trifluoroacetamide as proton acceptors and choline or betaine as proton donors are actually used. In WO 2014 / 131906, the most preferred DES consists of choline chloride and trifluoroacetamide, so it is not NADES, and trifluoroacetamide is not an acceptable pharmaceutical excipient.

[0015] WO 2016 / 108083 describes the use of NADES for their bactericidal activity: Several NADES were found to be able to effectively kill or inactivate Gram-positive and Gram-negative bacteria, as well as fungal cells of Candida species.

[0016] WO 2016 / 162703 describes the use of NADES made of, for example, betaine and organic acids or polyols for the extraction of biomolecules, in particular from plant material.

[0017] WO 2017 / 222377 describes the use of NADES gel in admixture with animal feed to provide a water source that does not dry out under heating. The specific use described is for newly hatched chicks.

[0018] WO 2015 / 121463 describes a liquid stable poultry vaccine, in particular poultry virus, in a composition of 5-40% sugar alcohol and 0.15-0.75 M of an amino acid selected from arginine, glutamic acid or glycine. However, most importantly, WO 2015 / 121463 and similar publications such as WO 2014 / 029702 and WO 2014 / 140239 do not disclose NADES, but describe a liquid stable poultry vaccine containing at least 60% w / v water and having a water activity (a w ) above 0.85 in aqueous solution. Comparative data are provided in the Examples section.

[0019] Gutierrez et al. (2010, Angew. Chem. Int. Ed., vol. 49, p. 2158-2162) describe cryopreservation of the Gram-negative bacterium Escherichia coli using a NADES diluent made from choline chloride and glycerol in a 1:2 molar ratio, with a solute of 20% w / v. To form NADES, the mixture of bacteria and the NADES diluent was freeze-dried.

[0020] Qiao et al. (2018, Appl. Microbiol. Biotechnol., vol. 102, p. 5695-5705) tested the stabilization of lactic acid bacteria using a composition derived from NADES. These bacteria are Gram-positive and contain cell walls. Due to the use of NADES in frozen storage, the vitality of two species from the genus Lactobacillus was unstable or even destroyed. Streptococcus thermophilus can be stabilized in frozen form using NADES with a 1:3 molar ratio of proline: glycerol. However, by applying a 1+1 volume of NADES and bacterial culture mixture, Qiao et al. diluted their NADES to the extent that the hydrogen bond grid was no longer complete. From the differential scanning calorimetry (DSC) curve showing ice crystal melting and growth (Qiao et al., Figure 4 These are characteristic of aqueous compositions, whereas intact NADES will display a glass transition temperature.

[0021] Qiao et al. also described that bacteria with cell walls can resist the effects of NADES toxicity, namely dehydration and cell membrane damage.

[0022] An object of the present invention is to overcome the disadvantages of the prior art and to meet the needs of the art by providing a composition comprising live Mollicutes bacteria which can be prepared without freeze-drying and does not require frozen storage, but provides adequate stabilization during storage and handling at temperatures above 0°C.

[0023] Surprisingly it has been found that this object can be achieved and one or more disadvantages of the prior art can be overcome by providing a liquid composition capable of stabilizing live Mollicutes bacteria without freezing or freeze drying. This is achieved by using NADES as a stabilizer.

[0024] This discovery opens up many very advantageous applications: live Mollicutes bacteria can now be stored without freezing or freeze-drying, a process which itself would impair viability. Moreover, such processes are laborious, time-consuming and expensive. In addition, the liquid composition of the invention does not use components of uncertain or variable quality, such as hydrolysates for stabilization, but, on the contrary, uses compounds of natural origin and chemical definition. In addition, the liquid form can be conveniently used in various applications, such as diagnostics, quality control, and can also be used as a live attenuated vaccine. This is because NADES is an acceptable pharmaceutical carrier. Liquid compositions containing live Mollicutes bacteria can now be used directly or after dilution for such vaccinations.

[0025] Surprisingly, NADES was able to stabilize live Mollicutes bacteria: as a powerful ionic solvent, NADES is expected to dissolve fragile and exposed bacterial cell membranes and extract their cytoplasmic proteins and nucleic acids. In fact, Qiao et al. (supra) warned that bacteria without cell walls are sensitive to the toxicity of NADES. Moreover, WO 2016 / 108083 (supra) actually used NADES to kill bacteria.

[0026] Thus, the inventors surprisingly found that NADES formulations can provide remarkable stability of viability of live Mollicutes bacteria without the need for freezing or freeze drying. The stability is significantly better than that observed in aqueous liquid compositions. For example, Mycoplasma synoviae can be stored in a liquid composition according to the invention for up to 24 weeks at 4°C with a loss of less than 3 Log10 CFU, and in certain compositions, even less than 1 Log10 CFU. Even at room temperature, remarkable stability was found for up to 8 weeks, showing a loss of only 4 Log10 CFU, while cultures in phosphate buffered solution were completely lost within a week at this temperature.

[0027] It is not clear how or why NADES is such an effective stabilizer for live Mollicutes bacteria. Although the inventors do not want to be bound by any theory or model that might explain these findings, they speculate that the low amount of available water in NADES helps maintain bacterial viability. In addition, Mollicutes bacteria apparently interact with NADES differently than bacteria with cell walls. Because there is no prior art for stabilizing Mollicutes bacteria in NADES, this effect could not be predicted.

[0028] Thus, in one aspect, the present invention provides a liquid composition comprising live Mollicutes bacteria and a stabilizer, characterized in that the stabilizer is a natural deep eutectic solvent (NADES).

[0029] The term "liquid" is used in its ordinary sense to refer to a composition that can flow at a specific temperature and for a specific period of time. One method of determining the characteristics of a liquid is a tilt test, in which a container containing a composition at a specific temperature is tilted, and when any change in the form of the composition is detected after a certain period of time, the composition is classified as a liquid. For the present invention, a composition is a liquid if it changes shape within 15 minutes of tilting the container at 90° at room temperature.

[0030] As used herein, the term "comprising" (and variations such as "comprise", "comprises" and "comprised") is intended to refer to all elements and any possible combinations conceivable of the present invention, which are covered or included in the text part, paragraph, claims, etc., in which the term is used, even if these elements or combinations are not explicitly recited; and does not exclude any such elements or combinations.

[0031] Therefore, any such text part, paragraph, claim etc. may therefore also refer to one or more embodiments, in which the term "comprising" (or variations thereof) is replaced by terms such as "consist of", "consisting of" or "consist essentially of".

[0032] The term "live" is used herein in its ordinary sense in relation to bacteria, referring to their ability to grow, move, absorb and ferment nutrients and / or replicate under appropriate conditions. Methods for determining whether bacteria are dead or alive are well known in the art. This can be determined in vivo, but is more convenient in vitro, for example by culturing in a suitable medium and detecting an increase in the optical density of the culture, or by plating on a suitable agar plate and counting CFU. Another method is to apply live / dead selective staining and counting.

[0033] The term "Milkytophytes" refers to bacteria that have characteristics characteristic of their taxonomic class, such as morphological, genomic and biochemical features, as well as biological features such as physiological, immunological or pathological behavior. The same applies to references to bacterial orders, families, genera or the names of individual bacterial species.

[0034] The class Mollicutes includes the orders Acholeplasmatales, Anaeroplasmatales, Entomoplasmatales, and Mycoplasmatales. The order Acholeplasmatales includes known Mollicutes plant pests of the tentative genus Phytoplasma. The order Entomoplasmatales includes insect symbionts and spirochetal plant pathogens.

[0035] The best known is the order Mycoplasmalles, as it contains the well-known human or animal pathogenic bacteria of the genus Mycoplasma: for example, M. pneumoniae, M. bovis, M. hyopneumoniae, M. meleagridis and M. gallisepticum, which (mainly) infect: humans, ruminants, pigs, turkeys and chickens, respectively.

[0036] As is known in the art, the classification of microorganisms in a particular taxonomic group is based on a combination of these characteristics. Thus, the present invention also includes species of Mollicutes bacteria that are assigned to the order, family, genus and species of Mollicutes. Similarly, this refers to bacteria that have been subclassified therefrom in any manner, such as subspecies, strains, isolates, genotypes, variants, subtypes or subgroups, etc.

[0037] Furthermore, it will be apparent to those skilled in the art that while a particular order, family, genus or species of the class of the class of the present invention may currently be assigned to that group, this is a taxonomic classification that may change over time as new insights may lead to reclassification into new or different taxonomic groups. However, since this does not change the bacterium itself or its antigenic repertoire, but only its scientific name or classification, such reclassified bacteria remain within the scope of the present invention. For example, Gupta et al. (2018, Antonie van Leeuwenhoek, vol. 111, p. 1583-1630) proposed a new framework for the phylum of the class ...

[0038] The Mollicutes bacterial samples used in the present invention may be obtained from various sources, for example as field isolates from humans, or from non-human animals in the wild or on farms, or from various laboratories, (depository) institutions, or (veterinary) universities.

[0039] The "stabilizer" of the present invention is a composition capable of reducing the loss of viable cell count of the Mollicutes bacteria of the present invention due to handling or use and / or storage at a specific temperature for a specific period of time.

[0040] "Stabilization" according to the present invention is expressed as the effect on the difference in CFU before and after a particular treatment of a composition comprising live Mollicutes bacteria, such as incubation at a particular temperature for a particular period of time. Survival in different liquid compositions according to the present invention is thus compared with survival in a standard PBS buffer (10 mM Na2PO4, 1.8 mM KH2PO4, 2.7 mM KCl and 137 mM NaCl in water, pH 7.4). Any survival above that observed in PBS under similar storage conditions is then considered to be an effect on survival provided by the liquid composition according to the present invention.

[0041] A "deep eutectic solvent" (DES) is an ionic liquid known in the art which comprises a mixture of at least two compounds in a molar ratio that forms a deep eutectic mixture, such that the eutectic point of the mixture is significantly lower than the melting points of the individual compounds.

[0042] This lowered melting point of the mixture is caused by the interaction of the compounds, one acting as a hydrogen bond donor and the other as a hydrogen bond acceptor, providing a stable hydrogen bond network. Although the network does contain a small amount of water, it will not crystallize when frozen. This allows the mixture to be liquid at temperatures significantly below the melting points of the components. Usually "eutectic" means: easily meltable.

[0043] The NADES used in the present invention has a complete hydrogen bond network. This can be easily verified by thermal analysis of its phase transition curve using DSC. Therefore, the NADES used in the present invention has a DSC curve showing a glass transition temperature.

[0044] The determination of DSC curves is well known and can be accomplished using standard equipment. Figure 1 An example curve is shown.

[0045] The hydrogen bonding network can tolerate a limited level of dilution. However, when too much dilution is applied, the "DES" will become a regular aqueous solution. In this case, the DSC curve will show the melting enthalpy and crystallization enthalpy, as shown in Qiao et al., supra. Figure 4 middle.

[0046] The individual compounds (excluding water) used to form the NADES of the present invention have a melting point above about 80° 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, while the NADES formed with a molar ratio of betaine, sorbitol and water of 1:1:2.5 has a glass transition temperature of 50° C. and does not show ice crystals and / or ice melting points in its DSC curve.

[0047] The term "natural" is used to indicate that the compounds used to form the natural DES (NADES) of the present invention are organic compounds that can be found under normal conditions in materials from biological sources such as plants or animals, in amounts far above trace amounts. Typically, such natural compounds are or are derived from primary metabolites present in specific materials of plant or animal origin. Those skilled in the art will understand that the term natural is used herein only to characterize the initial source of the compounds used for the NADES of the present invention, and not to characterize the actual source of the compounds used for the present invention. Therefore, natural compounds can also be used in the present invention when derived by (semi-)synthetic production.

[0048] Examples of natural compounds that can be used to form the NADES of the present invention are organic acids, amines, sugars, sugar alcohols, amino acids and quaternary ammonium salts, such as choline derivatives.

[0049] It will be apparent to one skilled in the art that, in the embodiments disclosed herein for the present invention, there will be combinations of certain types of NADES and one type of Mollicutes bacteria that are more favorable than other combinations with respect to bacterial stability. However, a skilled artisan will be able to readily select, optimize, and fine-tune combinations and compositions according to the present invention, using only conventional methods and materials, and the information disclosed herein.

[0050] Further details of preferred embodiments and further aspects of the invention are described below.

[0051] The water present in the NADES used in the present invention is tightly bound in the NADES hydrogen bond grid structure, which greatly reduces the availability of water to chemical or biological processes that may affect the stability of Mollicutes bacteria. This characteristic is usually expressed by indicating the total water and the amount of free water. The availability of this water (free water) is represented by the water activity value, symbolized by: w. Water activity varies between an upper limit of 1.0 for pure water and a lower limit of 0. Water activity is usually measured by comparing (at the same temperature) the vapor pressure of a test composition relative to the vapor pressure of pure water and a number of saturated salt solutions of known water activity. This is described in various manuals, reviews and handbooks, for example in FAO Agricultural Service Bulletin No. 149 (Cánovas et al. FAO, Rome, 2003, ISBN 92-5-104861-4), and in a review article ('Fundamentals of water activity', Decagon Devices Inc., Washington, 2015 (http: / / pdf.directindustry.com / pdf / decagon-devices-inc / fundamentals-water-activity / 64142-634433.html)).

[0052] When the water activity is below about 0.8, most bacteria stop growing; below 0.7, most yeasts and molds stop growing; and when the water activity is below 0.4, most enzyme activities are effectively stopped.

[0053] Equipment and procedures for measuring water activity are well known and available, for example by using headspace pressure analysis.

[0054] It was found that effective stabilization of Mollicutes bacteria in the liquid composition according to the invention can be achieved when the composition has a water activity below about 0.85.

[0055] Therefore, in one embodiment of the liquid composition according to the invention, the composition has a water activity of 0.85 or less.

[0056] For the present invention, the indicated water activity refers to the water activity of the liquid composition according to the present invention in the final product form, such as provided by a commercial manufacturer, and in this form can be stored for a long time. When using the composition, it can be diluted or mixed with other components, which may then change the water activity value.

[0057] In one embodiment of the liquid composition according to the present invention, the water activity of the composition is less than about 0.8; more preferably, less than about 0.7, about 0.6, about 0.5, about 0.4, about 0.3 or even less than about 0.2, in the following order of preference.

[0058] Preferably, the water activity of the liquid composition according to the invention is between 0.8 and 0.1. More preferably, in the following order of preference, between 0.7 and 0.1, between 0.6 and 0.1, between 0.5 and 0.1, between 0.4 and 0.1, between 0.3 and 0.1, or even between 0.2 and 0.1.

[0059] For the present invention, indications of ranges are intended to include the stated endpoints.

[0060] For the present invention, "about" means that a number can vary between ±10% of its indicated value. Preferably, "about" means ±9% around its value, and more preferably, "about" means, in the following preferred order, ±8, 7, 6, 5, 4, 3, 2, 1 or even 0% around its value.

[0061] The water used in the liquid composition according to the present invention is preferably high quality and high purity water, such as (doubly) distilled water or reverse osmosis water. More preferably, the water is of pharmaceutical quality and suitable for parenteral injection; such water is usually sterile and substantially pyrogen-free, for example: water for injection (WFI).

[0062] The NADES used in the liquid composition according to the present invention is prepared with a limited amount of water. By varying the water content of the liquid composition, its properties, such as the viscosity of the liquid composition, can be adjusted and optimized. Effective NADES-containing compositions with low viscosity can be produced with a total water content of up to 50% w / w (Dai et al., 2015, vol. 187, p. 14-19). Large amounts of water may disrupt the hydrogen bonding network of the NADES.

[0063] Therefore, in one embodiment of the liquid composition according to the invention, the composition has a water content of 50% w / w or less.

[0064] The water content of the liquid composition according to the invention is expressed as a weight percentage "w / w", which is the weight of the total amount of water in the liquid composition according to the invention, as a part of the weight of the liquid composition. The water content can be measured using different procedures, for example by Karl Fischer titration, which is well known in the art.

[0065] With respect to water activity, a liquid composition herein is a composition in final product form before any mixing or dilution is applied.

[0066] Preferably, the liquid composition according to the invention has a water content of 40% w / w or less, more preferably in the following order of preference, 30, 25, 20, 15, 10, 8, 7, 6 or even 5% w / w or less.

[0067] The lower limit of water content in the liquid composition according to the present invention will be determined by practical factors such as how much water is sufficient to dissolve the NADES forming compound and how much water is needed to achieve an acceptable viscosity level for the NADES so that it can be conveniently processed or used.

[0068] Therefore, in one embodiment of the liquid composition according to the invention, the water content in the liquid composition is, in the following preferred order, between 50 and 0.5% w / w; between 40 and 1% w / w; between 30 and 1.5% w / w; between 20 and 2% w / w; or even between 10% and 3% w / w.

[0069] Furthermore, by mixing NADES with a live Mollicutes bacterial sample, a certain amount of water is introduced into the mixture to prepare a liquid composition according to the present invention. However, the liquid composition of the present invention should not be excessively diluted.

[0070] Therefore, in one embodiment of the liquid composition according to the present invention, the ratio of the volume of NADES to the volume of the bacterial sample to be combined will preferably be at least 3:1 v / v NADES: bacterial sample; more preferably at least 5:1 v / v NADES: bacterial sample in the following preferred order; even at least 10:1 v / v NADES: bacterial sample. Note: A ratio of 10:1 means a combination of 9 parts of NADES to 1 part of bacterial sample. For example: NADES with a molar ratio of proline, sorbitol and water of 1:1:2.5 has a total water content of 13.1% w / w. After mixing with the mycoplasma bacterial sample used in the present invention at a ratio of 9 parts NADES + 1 part bacteria, the total water content of the liquid composition obtained according to the present invention is 21.8% w / w.

[0071] In order to limit over-dilution of the NADES of the present invention into aqueous compositions, it may be necessary to concentrate the bacterial samples to be combined.

[0072] It has been found that NADES effective as stabilizer for live Mollicutes bacteria in liquid compositions according to the invention are formed from different combinations and ratios of organic salts and / or polyols, or from several polyols.

[0073] Thus, in one embodiment of the liquid composition according to the invention, the NADES comprises an organic salt and a polyol.

[0074] In an alternative embodiment of the liquid composition according to the invention, the NADES comprises two or more compounds which are both polyols.

[0075] In some of these embodiments, the hydrogen bond grid is formed by an organic salt acting as a hydrogen bond donor as an ionic species and a polyol as a hydrogen bond acceptor. Alternatively, when the NADES comprises only a polyol, the grid is formed by hydrogen bonds between these molecules.

[0076] For purposes of the present invention, a "polyol" is an organic compound containing two or more hydroxyl groups. However, very large polymers, such as cellulose, which fall under the definition of a polyol, are not very effective in forming NADES as defined herein and therefore should not be preferably used in the present invention. Therefore, the polyols used in the present invention preferably have a molecular weight of less than about 10,000 grams per mole.

[0077] In a further preferred embodiment, the polyols used in the present invention have a molecular weight of less than 5000, or even less than 1000 g / mol.

[0078] The compounds used in the NADES of the present invention are easily obtained from various commercial suppliers with different purity and quality. Preferably, the compound is used with pharmaceutical grade quality. Such excipients are described, for example, in government regulations such as the European Pharmacopoeia and the U.S. 9CFR and manuals such as The Handbook of Pharmaceutical Excipients (R. Rowe et al., Pharmaceutical press 2012, ISBN 0857110276); Remington: the science and practice of pharmacy (2000, Lippincot, USA, ISBN: 683306472), and: "Veterinary vaccinology" (P. Pastoret et al. ed., 1997, Elsevier, Amsterdam, ISBN 0444819681)).

[0079] Preferred polyols for use in the present invention are sugars or sugar alcohols, as they have been shown to be versatile components that allow for the production of a variety of effective NADES for use in the present invention.

[0080] In a preferred embodiment, the sugar is a non-reducing sugar.

[0081] In one embodiment of the non-reducing sugar used in the liquid composition according to the present invention, the non-reducing sugar is selected from sucrose and trehalose.

[0082] Preferably, the non-reducing sugar is trehalose. Preferably, the trehalose is CAS nr. 6138-23-4.

[0083] For the purposes of the present invention, a "sugar alcohol" is a hydrogenated sugar which contains 3 or more carbon atoms and may be based on a monosaccharide, a disaccharide or a polysaccharide.

[0084] In one embodiment of the sugar alcohol used in the liquid composition of the present invention, the sugar alcohol is one or more selected from erythritol, xylitol and sorbitol.

[0085] Erythritol is preferably CAS nr. 149-32-6. Xylitol is preferably CAS nr. 87-99-0. Sorbitol is preferably D-sorbitol, CAS nr. 50-70-4.

[0086] In one embodiment of the sugar alcohol used in the liquid composition of the present invention, the sugar alcohol is one or more selected from xylitol and sorbitol.

[0087] In a more preferred embodiment, the sugar alcohol is sorbitol.

[0088] The polyols used in the liquid composition according to the invention may be used as different salts, isomeric forms, hydrates or anhydrous forms etc. The skilled person is fully capable of selecting and testing suitable forms of polyols for use in the present invention.

[0089] For the present invention, an "organic salt" is a salt of any organic acid or base, including zwitterions, which is within the definition of a natural compound as set out above and is capable of forming the deep eutectic solvents of the present invention as described herein. The skilled person is fully capable of selecting an organic salt for the present invention and applying it to form a NADES.

[0090] In one embodiment of the liquid composition according to the present invention, the organic salt is one or more selected from the following salts: betaine, proline, choline, methionine and arginine.

[0091] For the present invention, "betaine" is preferably the compound N,N,N-trimethylglycine, CAS nr. 107-43-7, also known as glycine-betaine. Proline is preferably CAS nr. 609-36-9. Choline is preferably CAS nr. 62-49-7. Methionine is preferably L-methionine, CAS nr. 63-68-3.

[0092] In a preferred embodiment, the choline is choline chloride, CAS nr. 67-48-1.

[0093] For the above polyols, the organic salts may be used in different isomeric forms, hydrates or anhydrous forms, etc. The skilled person is fully capable of selecting and testing suitable forms of organic salts for use in the present invention.

[0094] In one embodiment of the liquid composition according to the invention, when the organic salt is arginine, arginine is used as a mixture of L-arginine (CAS nr. 74-79-3) and L-arginine-HCl (CAS nr 1119-34-2), wherein L-arginine-HCl is the main component of the mixture.

[0095] This mixture of arginine compounds helps to bring the pH of the liquid composition according to the invention closer to the physiological value when the composition is diluted, for example before use as a vaccine, or when used for counting.

[0096] Preferably, the molar ratio of L-arginine to L-arginine-HCl is between 1:1 and 1:100; more preferably, the molar ratio of L-arginine to L-arginine hydrochloride is between 1:1 and 1:50, more preferably the molar ratio is about 1:19.

[0097] In one embodiment of the liquid composition according to the invention, the NADES comprises three polyols.

[0098] In a preferred embodiment, the three polyols are sorbitol, xylitol and trehalose.

[0099] In one embodiment of the NADES for use in the liquid composition according to the invention, the organic salt is selected from: salts of betaine, proline, choline and arginine; the polyol is selected from: xylitol, sorbitol.

[0100] For the present invention, organic salts and polyols or polyols can be used in different molar ratios to each other to provide NADES with different properties. These can be optimized by the skilled person to achieve the best stabilization of live Mollicutes bacteria in the liquid composition according to the invention.

[0101] Thus, in one embodiment of the liquid composition according to the invention, the molar ratio between the organic salt and the polyol, both as defined herein, is between 1 :5 and 5:1.

[0102] In a preferred embodiment of the liquid composition according to the invention, the molar ratio between the organic salt and the polyol, both as defined herein, is between 1:4 and 4:1, between 1:3 and 3:1, or even between 1:2 and 2:1, in the following order of preference.

[0103] In one embodiment of the liquid composition according to the present invention, the molar ratio between any of the three polyols xylitol, trehalose and sorbitol is as defined herein and may be between 1 and 5. Thus, the molar ratio of sorbitol:xylitol:trehalose is between 1:1:1 and one of the following: 1:1:5, 1:5:1, 5:1:1, 1:5:5, 5:1:5, 5:5:1 and 5:5:5.

[0104] One advantageous use of the liquid composition according to the invention is as a vaccine. In such applications, Mollicutes bacteria are used as active ingredients of live bacterial vaccines, which require them to have attenuated properties in order not to cause unacceptable vaccination reactions in the target animals.

[0105] Thus, in one embodiment of the liquid composition according to the invention, the Mollicutes bacteria are attenuated with respect to their pathogenic properties.

[0106] The term "attenuated" of the present invention is used in its ordinary sense to refer to bacteria that have a lower level of pathogenicity due to a reduced infection and / or replication rate in a host compared to an unmodified, non-attenuated or "wild-type" form of a Mollicutes bacterium. Such attenuated live bacteria are also referred to as "modified live" bacteria.

[0107] Attenuation of Mollicutes bacteria can be obtained in different ways, for example by passaging wild-type bacteria through experimental animals or in cell culture, by using mutagens, or by recombinant DNA techniques, all of which are well known in the art.

[0108] The liquid composition according to the present invention can effectively stabilize the activity of various bacteria from the class Mollicutes.

[0109] Thus, in one embodiment, the Mollicutes bacteria are selected from one or more of the following bacterial orders: Acholeplasmalles, Anaeromycoplasmalles, Entomoplasmalles and Mycoplasmalles.

[0110] In one embodiment, the Mollicutes bacteria used in the present invention are selected from one or more of the following bacterial families: Acholeplasmataceae, Anaeroplasmataceae, Entomoplasmataceae, Spiroplasmataceae and Mycoplasmataceae.

[0111] In one embodiment, the Mollicutes bacteria of the present invention are selected from one or more of the following bacterial genera: Acholeplasma, Phytoplasma, Anaeroplasma, Asteroleplasma, Entomoplasma, Mesoplasma, Spiroplasma, Hepatoplasma, Marinoplasma, Moeniiplasma, Mycoplasma and Ureaplasma.

[0112] Preferably, the Mollicutes bacteria used in the present invention are one or more selected from the following bacterial genera: Asterococcus, Spirochete and Mycoplasma.

[0113] More preferably, the Mollicutes bacterium used in the present invention is from the bacterial genus Mycoplasma.

[0114] In one embodiment of the liquid composition according to the present invention, the Mollicutes bacteria is one or more selected from Mycoplasma pneumoniae, Mycoplasma bovis, Mycoplasma hyopneumoniae, Mycoplasma meleagridis, Mycoplasma gallisepticum and Mycoplasma synoviae.

[0115] In a preferred embodiment, the Mollicutes bacterium is Mycoplasma gallisepticum and / or Mycoplasma synoviae.

[0116] In one embodiment of the liquid composition according to the invention, the Mollicutes bacteria are Mycoplasma gallisepticum and Mycoplasma synoviae.

[0117] Experiments have shown that Mycoplasma gallisepticum and Mycoplasma synoviae can be stored in combination in a liquid composition according to the invention, whereby no significant effect of one on the stability of the other was detected when tested at 2-8°C for up to 23 weeks. In this mixture, the two bacteria can be detected and counted when the same mixed sample is plated on two different types of agar plates: a plate containing NAD (nicotinamide adenine dinucleotide) and a plate without NAD, and the number of colonies counted is compared. As described in the literature (Olson & Kish Meadows, 1972, Avian Dis., vol. 16, p. 387-396), the growth of Mycoplasma synoviae requires NAD, so on the plate without NAD, all colonies observed are from Mycoplasma gallisepticum.

[0118] In a preferred embodiment, the liquid composition according to the present invention is capable of providing stabilization of live Mollicutes bacteria, whereby the number of CFU of Mollicutes is reduced by no more than 3 Log10 after incubation for 12 weeks at 4° C. Even more preferably, the number of CFU of Mollicutes is reduced by no more than 7 Log10 after incubation for 8 weeks at room temperature.

[0119] Room temperature is a temperature of 20±5°C; 4°C is 2 to 8°C.

[0120] In one embodiment of the liquid composition according to the invention, one or more or all of the conditions apply which are selected from:

[0121] - the water activity of the liquid composition is 0.85 or less;

[0122] - the water activity of the liquid composition is less than about 0.8; more preferably, in the following order of preference, less than about 0.7, about 0.6, about 0.5, about 0.4, about 0.3 or even less than about 0.2;

[0123] - the water activity of the liquid composition is between 0.8 and 0.1; more preferably between 0.7 and 0.1, 0.6 and 0.1, 0.5 and 0.1, 0.4 and 0.1, 0.3 and 0.1 or even between 0.2 and 0.1 in the following order of preference;

[0124] - the liquid composition has a water content of 50% w / w or less;

[0125] - The liquid composition has a water content of 40% w / w or less, more preferably in the following order of preference 30, 25, 20, 15, 10, 8, 7, 6 or even 5% w / w or less.

[0126] - the water content of the liquid composition is, in the following order of preference, between 50 and 0.5% w / w; between 40 and 1% w / w; between 30 and 1.5% w / w; between 20 and 2% w / w; or even between 10% and 3% w / w;

[0127] - the ratio of the volume of NADES to the volume of the bacterial sample to be combined is preferably at least 3:1 v / v NADES: bacterial sample; more preferably at least 5:1 v / v NADES: bacterial sample in the following preferred order; or even at least 10:1 v / v NADES: bacterial sample;

[0128] -NADES contains organic salts and polyols;

[0129] -NADES comprises two or more compounds which are all polyols;

[0130] - the molecular weight of the polyol used in the present invention is less than 5000, or even less than 1000 g / mol;

[0131] - the polyol is a sugar or a sugar alcohol;

[0132] - the sugar is a non-reducing sugar;

[0133] - the non-reducing sugar is selected from sucrose and trehalose; preferably, the non-reducing sugar is trehalose; trehalose is preferably CAS nr. 6138-23-4;

[0134] - the sugar alcohol is one or more selected from erythritol, xylitol and sorbitol;

[0135] - Erythritol is preferably CAS nr. 149-32-6; xylitol is preferably CAS nr.

[0136] 87-99-0; sorbitol is preferably D-sorbitol, CAS nr.50-70-4; - the sugar alcohol is one or more selected from xylitol and sorbitol; in a more preferred embodiment, the sugar alcohol is sorbitol;

[0137] - the organic salt is one or more selected from the group consisting of betaine, proline, choline, methionine and arginine salts;

[0138] - betaine is preferably the compound N,N,N-trimethylglycine, CAS nr. 107-43-7, also known as glycine-betaine; proline is preferably CAS nr. 609-36-9; choline is preferably CAS nr. 62-49-7; methionine is preferably L-methionine, CAS nr. 63-68-3; arginine is preferably L-arginine, CAS nr. 74-79-3;

[0139] - the choline is choline chloride, CAS nr. 67-48-1;

[0140] - when the organic salt is arginine, arginine is used in the form of a mixture of L-arginine (CAS nr. 74-79-3) and L-arginine hydrochloride (CAS nr 1119-34-2);

[0141] - the molar ratio of L-arginine to L-arginine-HCl is between 1:1 and 1:100; more preferably, the molar ratio of L-arginine to L-arginine hydrochloride is between 1:1 and 1:50, more preferably, the molar ratio is about 1:19;

[0142] - the organic salt is selected from the group consisting of betaine, proline, choline and arginine salts; the polyol is selected from the group consisting of xylitol and sorbitol;

[0143] - the molar ratio of the organic salt to the polyol is between 1:5 and 5:1;

[0144] - the molar ratio of the organic salt to the polyol is, in the following order of preference, between 1:4 and 4:1, between 1:3 and 3:1, or even between 1:2 and 2:1;

[0145] -NADES comprises three polyols; in a preferred embodiment, the three polyols are sorbitol, xylitol and trehalose;

[0146] - the molar ratio of sorbitol:xylitol:trehalose is between 1:1:1 and one of the following: 1:1:5, 1:5:1, 5:1:1, 1:5:5, 5:1:5, 5:5:1 and 5:5:5;

[0147] - the pathogenic properties of Mollicutes bacteria are attenuated;

[0148] - the Mollicutes bacteria is selected from one or more of the following bacterial orders: Acholeplasmalles, Anaeromycoplasmalles, Entomoplasmalles and Mycoplasmalles;

[0149] - the Mollicutes bacterium is selected from one or more of the following bacterial families: Acholeplasmataceae, Anaplasmataceae, Entomoplasmataceae, Spiroplasmataceae and Mycoplasmataceae;

[0150] - the Mollicutes bacterium is one or more selected from the following bacterial genera: Asteroclasts, Phytoplasmas, Anaplasma, Astrophytoma, Entomoplasma, Centroplasma, Spirochete, Hepatoplasma, Algal Plasma, Moniplasma, Mycoplasma and Ureaplasma;

[0151] - the Mollicutes bacterium is selected from one or more of the following bacterial genera: Asterococcus, Spirochete and Mycoplasma;

[0152] - Mollicutes bacteria from the bacterial genus Mycoplasma;

[0153] - the Mollicutes bacteria is one or more selected from Mycoplasma pneumoniae, Mycoplasma bovis, Mycoplasma hyopneumoniae, Mycoplasma melitensis, Mycoplasma gallisepticum and Mycoplasma synoviae;

[0154] - the Mollicutes bacterium is Mycoplasma gallisepticum and / or Mycoplasma synoviae; and

[0155] -Bacteria from the class Mollicutes are Mycoplasma gallisepticum and Mycoplasma synoviae.

[0156] In one embodiment of the liquid composition according to the present invention, the liquid composition has a water activity of 0.85 or less and a water content of 50% w / w or less; the NADES comprises an organic salt and a polyol, wherein the polyol is xylitol or sorbitol, and the organic salt is selected from betaine, proline, choline and arginine; the Mollicutes bacteria are attenuated in their pathogenic properties, and the Mollicutes bacteria are one or more selected from Mycoplasma pneumoniae, Mycoplasma bovis, Mycoplasma hyopneumoniae, Mycoplasma meleagridis, Mycoplasma gallisepticum and Mycoplasma synoviae.

[0157] In a preferred embodiment, the Mollicutes bacterium is Mycoplasma gallisepticum and / or Mycoplasma synoviae, and the NADES comprises betaine and sorbitol.

[0158] In one embodiment of the liquid composition according to the present invention, the liquid composition has a water activity of 0.85 or less and a water content of 50% w / w or less; the NADES comprises three polyols of sorbitol, xylitol and trehalose; the Mollicutes bacteria are attenuated in their pathogenic properties, and the Mollicutes bacteria are one or more selected from Mycoplasma pneumoniae, Mycoplasma bovis, Mycoplasma hyopneumoniae, Mycoplasma meleagridis, Mycoplasma gallisepticum and Mycoplasma synoviae.

[0159] As mentioned above, the liquid composition according to the present invention can be produced using common techniques and materials. The details and examples of such methods, uses or processes for preparing the liquid composition according to the present invention are described herein, and such procedures can be easily applied by those skilled in the art using conventional materials and methods.

[0160] For example, the NADES of the present invention can be produced on an industrial scale and then prepared into a liquid composition according to the present invention by mixing with a live preparation of Mollicutes bacteria. The mixture is then filled into containers of appropriate size. The various stages of the preparation process can be monitored by appropriate tests, such as biochemical or immunological tests to detect the quality and quantity of the bacteria.

[0161] Therefore, in another aspect, the present invention provides a method for preparing a liquid composition according to the present invention, the method comprising the steps of:

[0162] a. Preparation of NADES, and

[0163] b. Mixing the NADES with live Mollicutes bacteria.

[0164] Conveniently, the two steps of the method according to the invention can be carried out separately in time and / or place, as well as intermediate storage, transport or even further steps. This will allow operational flexibility in terms of planning and logistics.

[0165] Conventional tools and methods can be used to make NADES for use in the methods according to the invention. One convenient method is to heat the combined compounds to a temperature that allows them to mix and dissolve, such as in autoclaving. However, some NADES compounds or compositions for use in the invention may not tolerate temperatures that are too high; the skilled person can optimize and fine-tune such a process. Heating can be combined, for example, with stirring or ultrasonic treatment.

[0166] Furthermore, one of the components forming the NADES can initially be dissolved in water in a more or less concentrated form, after which it can be mixed with (a) the other component. The NADES structure is then formed by removing excess water. Preferably, this removal is not done by freeze drying. An alternative method is, for example, rotary evaporation under reduced pressure in a water bath at 70°C for several hours.

[0167] The composition comprising live Mollicutes bacteria used in the method according to the invention may be conveniently derived from in vivo cultures in different ways, but is preferably derived from in vitro cultures of the bacteria in a suitable medium. Such bacterial cultures may be produced in any desired size or volume, up to several thousand liters in large-scale industrial-scale fermenters applying automatic control and monitoring technology.

[0168] The bacteria are then harvested from the culture in a manner that depends on the characteristics of the bacteria: as a supernatant, a cell pellet, or as the entire culture. The bacteria are then mixed with the NADES, either directly or indirectly by first mixing the bacteria with a liquid, such as a buffer or medium.

[0169] The bacterial composition used in the method of the invention can be prepared using further process steps such as concentration (e.g. using concentration through an ultrafiltration membrane). The bacterial composition is then checked for viability and quantity. All of this is well known to the skilled person.

[0170] In the method according to the invention, the mixing of NADES and the bacterial composition is preferably performed using aseptic techniques.

[0171] The order and manner of mixing is not important, as the NADES can be added to the bacterial composition, or vice versa. Preferably, any mechanical mixing used will be low speed and low impact to prevent unnecessary shear forces on the bacteria. In addition, mixing and / or filling can be performed in a protective atmosphere of an inert gas (e.g., nitrogen) to reduce oxygen exposure.

[0172] Convenient equipment for mixing and filling relatively viscous liquids is available from a variety of process equipment suppliers.

[0173] In another aspect, the present invention provides the use of a NADES according to the present invention as a stabilizer in a liquid composition comprising live Mollicutes bacteria.

[0174] The “stabilizer”, “liquid composition” and “live Mollicutes bacteria” are all as defined herein in any one or more embodiments described herein.

[0175] As mentioned above, an advantageous use provided by the present invention is the medical use of the liquid composition according to the present invention as a bacterial vaccine.

[0176] Therefore, in another aspect, the present invention provides a liquid composition according to the present invention, or as obtainable by a method of preparation according to the present invention, for use as a vaccine against an infection or disease caused by bacteria of the class Mollicutes.

[0177] Similarly, in another aspect, the present invention provides a liquid vaccine for combating an infection or disease caused by bacteria of the class Mollicutes, said vaccine comprising a liquid composition according to the present invention, or being obtainable by a method according to the present invention.

[0178] And, in another aspect, the present invention provides use of the NADES according to the present invention as a pharmaceutically acceptable carrier in the liquid vaccine according to the present invention.

[0179] As is well known, a "vaccine" is a composition with a medical effect. A vaccine comprises an immunologically active component and a pharmaceutically acceptable carrier. In this case, the "immunologically active component" is represented by live Mollicutes bacteria. The vaccine is recognized by the immune system of the target animal and induces a protective immune response. The response can originate from the innate immune system and / or the acquired immune system of the target and can be of cellular and / or humoral type.

[0180] Vaccines are often effective in reducing the severity of an infection, for example by reducing the amount of pathogen present or shortening the duration that the pathogen can replicate in the host animal.

[0181] Additionally, or possibly as a result thereof, the vaccine may generally be effective in reducing or ameliorating (clinical) symptoms of disease that may be caused by such infection or replication or the target's response to the infection or replication.

[0182] The "pharmaceutically acceptable carrier" of the present invention is a liquid of high purity and preferably sterile. In the present case, the carrier is NADES as defined herein. The carrier may contain other additives, such as stabilizers, antioxidants or preservatives.

[0183] Details of "infections or diseases caused by Mollicutes bacteria" are well known in the art and described, for example, in manuals such as The Merck Veterinary Manual (supra) and Diseases of Poultry, 12th edition, 2008, Wiley-Blackwell, ISBN:0813807182.

[0184] The liquid vaccine according to the present invention comprises an amount of live, immunologically effective Mollicutes bacteria.

[0185] Those skilled in the art of the present invention will not only be able to determine and optimize the immunologically effective amount of such live Mollicutes bacteria in a liquid vaccine according to the present invention, for example by monitoring the immune response following vaccination. In addition, where non-human animals are the target, a challenge infection can be performed and then the target can be monitored for signs of disease, clinical scores, and spread and transmission of the pathogen, and these results can be compared to the vaccination challenge response observed in mock vaccinated animals.

[0186] Methods for determining the amount of live bacteria in a vaccine dose or animal sample are well known in the art and are generally performed using bacterial culture and counting techniques. Conveniently, such bacterial amounts may be expressed as a numerical value, such as CFU.

[0187] The liquid vaccine according to the invention is targeted at humans or non-human animals in need of vaccination against infections or diseases caused by the pathogenic forms of specific live Mollicutes bacteria contained in the vaccine. The age, weight, sex, immune status and other parameters of the target to be vaccinated are not critical, although it is advantageous to vaccinate healthy, uninfected targets and to vaccinate as early as possible to prevent any field infection.

[0188] The liquid vaccine according to the invention may additionally comprise other compounds, such as additional antigens or microorganisms, cytokines or immunostimulatory nucleic acids comprising unmethylated CpG, adjuvants, etc. Alternatively, the liquid vaccine according to the invention itself may be added to a vaccine.

[0189] Liquid vaccines according to the present invention can advantageously be combined with one or more other antigens or microorganisms, for example derived from microorganisms that are pathogenic to the intended human or non-human animal target. Such additional antigens themselves can be infectious microorganisms, live, weakened or inactivated, or can be subunits, extracts or fractions, etc. Other antigens can be composed of biological or synthetic molecules, such as proteins, carbohydrates, lipopolysaccharides, lipids or nucleic acid molecules.

[0190] The liquid vaccine according to the invention may contain a preservative, such as thimerosal, thimerosal, phenolic compounds and / or gentamicin. Preferably no preservative is used.

[0191] The liquid vaccine of the present invention may contain a stabilizer, but preferably such a stabilizer is not a variable or undefined compound as described herein.

[0192] The liquid vaccine according to the invention may contain an antioxidant, such as ascorbic acid, citrate, methionine or tocopherol.

[0193] The liquid vaccine according to the present invention may be used as a preventive treatment or a therapeutic treatment, or both.

[0194] The liquid vaccine according to the present invention can be used as an effective priming vaccination, which can be followed by one or more booster vaccinations using the same or different vaccines.

[0195] Liquid vaccines according to the present invention may be prepared using well known techniques which will be combined with the methods for preparing the liquid compositions according to the present invention.

[0196] Therefore, in another aspect, the present invention provides the use of a NADES according to the present invention for the preparation of a liquid vaccine according to the present invention.

[0197] This preparation will be combined with microbiological testing for sterility and the absence of foreign agents; and ultimately vaccination studies in animals to confirm efficacy and safety. Vaccine products can only be marketed after quality, quantity, sterility, safety, and efficacy testing have been completed.

[0198] All of this is well known to the skilled person, and the general techniques and considerations applicable to the preparation of vaccines under well-known pharmaceutical production standards are described, for example, in government directives and regulations ((Pharmacopoeia, 9 CFR)) and in well-known manuals (Pastoret, Lippincot, both supra).

[0199] The liquid vaccine according to the present invention can be administered to humans or non-human animals in a form provided, for example, by a pharmaceutical manufacturer. However, depending on the specific composition of a particular embodiment of the liquid vaccine according to the present invention, it may be advantageous to dilute the liquid vaccine. This may facilitate actual administration to the target, for example by reducing viscosity, or may reduce local reactions after vaccination of the target.

[0200] The diluent used for such dilution is typically liquid and may be provided separately from the liquid vaccine composition according to the invention.Preferably, the diluent is provided together with the liquid vaccine according to the invention, for example as a kit of parts.

[0201] Thus, in another aspect, the invention relates to a kit of parts comprising at least two containers: one container comprising a liquid vaccine according to the invention, and one container comprising a liquid diluent.

[0202] A "liquid diluent" for diluting a liquid vaccine according to the present invention, such as a "liquid diluent" for inclusion in a kit according to the present invention, is a liquid suitable for diluting a liquid vaccine according to the present invention. The liquid diluent may therefore be aqueous, such as water or a buffer. However, due to the low water content of the liquid vaccine according to the present invention, the diluent may also be oily, or may be an emulsion of oil and water, such as oil-in-water, water-in-oil or a double emulsion.

[0203] In one embodiment, the liquid diluent may itself be a liquid vaccine, such as a reconstituted lyophilized live vaccine, or an emulsion vaccine.

[0204] The choice of liquid diluent for diluting a liquid vaccine according to the invention, e.g. comprised in a kit of parts according to the invention, will depend on several factors, such as: the intended use of the vaccine after dilution; the Mollicutes bacteria it contains; the intended human or non-human animal target for vaccination, etc. The skilled person is well within the ability to select, prepare and use such diluents.

[0205] In one embodiment of the kit of parts according to the invention, the kit includes instructions for use of the kit and / or its components. In a preferred embodiment, the instructions are provided on or with one or more components of the kit; or by reference to instructions in electronic form, such as information that can be viewed or downloaded on an Internet website (e.g., from a manufacturer or distributor of the kit).

[0206] In one embodiment, the kit of parts is a box comprising at least two containers and comprising instructions for use presented on an information carrier (eg, a card or leaflet) contained on, with, or within the box.

[0207] In one embodiment of the kit of parts, the kit may also be an offer (in connection with commercial sales), for example on an internet website, of the components for use in the vaccination method according to the invention.

[0208] The liquid vaccine according to the invention, or a liquid vaccine that can be manufactured by the method or use according to the invention, or a liquid vaccine that can be prepared in diluted form, for example using a kit of parts according to the invention, is intended for administration to a human or non-human animal target in need of such vaccination. Vaccination induces active immunity to reduce infection and / or disease symptoms that may be caused by infection with a non-attenuated counterpart of the live Mollicutes bacteria of the invention. In turn, this will result in a reduction in the number, intensity or severity of lesions caused by bacterial infection and the attendant clinical symptoms of disease in the target human or non-human animal. Such vaccines are colloquially referred to as vaccines "against" specific Mollicutes bacteria, or as "Molicutes vaccines".

[0209] Thus, in another aspect, the present invention provides a liquid vaccine according to the present invention for use in a method of protecting a human or non-human animal target against infection and / or disease caused by a pathogenic form of a live Mollicutes bacterium contained in said liquid vaccine.

[0210] Such a "pathogenic form of a live Mollicutes bacterium contained in the liquid vaccine" is, for example, an unattenuated parent Mollicutes bacterium from which the attenuated form of the Mollicutes bacterium used in the present invention is produced. Alternatively, such a pathogenic form of the bacterium can be easily derived from a variety of sources, as described herein. A person skilled in the art of the present invention is fully capable of determining whether a Mollicutes bacterium is a pathogenic form, for example by an in vitro or in vivo comparative test as described herein.

[0211] A "method of protection" involves administering a liquid vaccine according to the invention to said human or non-human animal target such that the vaccine is able to "succeed" (ie establish a protective infection) and demonstrate its beneficial immunological effects in the target.

[0212] In another aspect, the invention provides a method of vaccinating a human or non-human animal target against infection or disease caused by bacteria of the class Mollicutes, the method comprising administering to the target a liquid vaccine according to the invention or a kit comprising the use of a component according to the invention.

[0213] In the vaccination method according to the invention, the liquid vaccine according to the invention can be used for direct administration to a human or non-human animal target. Alternatively, the liquid vaccine according to the invention can be administered to a human or non-human animal target in a diluted form. According to the latter embodiment of the vaccination method according to the invention, handling and administration by injection can be facilitated due to the reduced viscosity. Alternatively, or additionally, administration in a diluted form can reduce local reactions in the human or non-human animal target, such as caused by tissue irritation at the vaccination site.

[0214] Vaccination according to the method of the invention may be performed by a parenteral route, i.e. through the skin, for example: intramuscularly, intraperitoneally, intradermally, submucosally or subcutaneously. The preferred parenteral route of use of the liquid vaccine composition according to the invention is by intramuscular or subcutaneous injection.

[0215] Furthermore, the liquid vaccine according to the present invention can be administered by enteral or mucosal routes, i.e., by eye drops, nasal drops, oral, enteral, oronasal drops, spray, aerosol, etc.

[0216] Alternatively, the liquid vaccine according to the invention may be administered to the target by mass administration methods, such as by drinking water, coarse spray, atomization, feeding, etc. Another alternative is administration to embryonic birds by the in ovo route.

[0217] It goes without saying that the best route of administration of the liquid vaccine according to the invention will depend on the properties of the vaccine used and the specific characteristics of the target. The skilled person is fully capable of selecting and optimizing such routes and methods of administration.

[0218] Liquid vaccines according to the present invention can be administered parenterally in a volume acceptable to a target human or non-human animal, and can be, for example, in a volume between about 0.01 ml and about 10 ml. Preferably, the volume of one dose is between about 0.05 ml and about 5 ml, more preferably, the volume of one dose is between 0.1 ml and 3 ml.

[0219] When administered by the intramuscular route, the volume of a dose is preferably between about 0.5 ml and about 3 ml, more preferably between about 1 ml and about 2 ml.

[0220] When administered by the intradermal route, the volume of a dose is preferably between about 0.1 ml and about 0.5 ml, more preferably about 0.2 ml.

[0221] When administered in ovo, the volume of a dose is preferably between about 0.01 ml and about 0.5 ml, more preferably about 0.05 ml.

[0222] The administration method, time and volume of the liquid vaccine according to the present invention are preferably integrated into the existing vaccination plan of other vaccines that the target human or non-human animal may need to reduce stress on the target and reduce labor costs. These other vaccines can be administered in a simultaneous, parallel or sequential manner and in a manner compatible with their registered use.

[0223] The technician can further optimize the liquid vaccine according to the present invention. Generally, this involves fine-tuning the vaccine efficacy to further improve the immune protection it provides. This can be achieved by, for example, adjusting the dosage, volume or antigen content of the vaccine, or by different approaches, methods or regimen applications. All of these are within the scope of the present invention.

[0224] The invention will now be further described by way of the following non-limiting examples. Example

[0225] Example 1: Methods and Materials

[0226] 1.1 Preparation of NADES preparation

[0227] NADES can be prepared by mixing the desired ingredients, which can be accelerated by heating and / or adding a small amount of water. As further tested and described below, NADES samples are prepared by dissolving the relevant compounds in water via ultrasonic treatment in a water bath. The desired amount of compound is added to a flask. The flask is briefly shaken or vortexed to mix the powder. The desired amount of water is then added. The mixture is placed in an ultrasonic treatment water bath set at 45°C and ultrasonicated for up to several hours until NADES is formed. Temperatures as high as 80°C can be reached during the ultrasonic treatment.

[0228] 1.2 Measurement of water activity

[0229] sample

[0230] For biocontrol purposes, the water activity of the NADES formulation samples was measured without live bacteria. However, when the amount of aqueous bacterial composition to be added to the NADES to form the liquid vaccine composition according to the present invention is known, the final water activity of the combination of NADES and bacterial composition can be calculated according to the method of Dai et al. (2015, Food Chem., vol. 187, p. 14-19).

[0231] Sample preparation

[0232] A certain amount of NADES sample was transferred to a 10 ml glass vial to reach approximately 1 / 4 of the vial fill height. For each sample, the coded vial was filled in duplicate. The vial was closed using an aluminum crimp cap with a PTFE stopper and left at room temperature for 12 hours to achieve equilibrium between the sample and the headspace.

[0233] equipment

[0234] Using Lighthouse TM The FMS-1400 moisture / pressure analyzer measures the moisture pressure in the sample headspace. The sample is inserted into the sample chamber. Before the sample is measured, use a 0-0.2a w The headspace pressure standard calibration system is used for calibration within the range. In addition, saturated salt standards are used for calibration and recalculation of 0.2-1a w non-linear range.

[0235] calibration

[0236] In order to w To calibrate the machine over the entire range of 0-1 and create a reference line for the water activity values, a number of standard samples were measured: in addition to pure water, 5 samples of specific saturated salt solutions were used and each of the 6 standard samples was measured 3 times. By setting the measured value of pure water to 1.000, the a values ​​of the other standard values ​​were w They were determined to be: 0.8511, 0.7547, 0.5438, 0.3307 and 0.1131. In addition, a series of certified vapor pressure standards were measured.

[0237] a w The results of the measurements are contained in Table 1.

[0238] 1.3 Testing the DSC curve of NADES

[0239] Differential Scanning Calorimetry (DSC) analysis was performed on a Q2000DSC instrument from TA Instruments using thermal analysis software. For a complete DSC curve, the sample was first equilibrated at 40°C and cooled to -90°C at 5°C / min, then equilibrated at -90°C for 5 minutes, and then the sample was heated to 40°C again at 5°C / min. This cooling and heating cycle was repeated again for the same sample to study the phase lag. The Tg of the sample was determined by the software from the midpoint of the stepwise shift of the baseline based on the heating curve, indicating a change in thermal conductivity due to increased fluidity during the glass transition.

[0240] A representative DSC curve of the NADES of the present invention is shown in Figure 1 , which is for NADES formed with a molar ratio of proline, sorbitol and water of 1:1:2.5.

[0241] Figure 1 shows a sharp shift in the baseline, here between -41 and -45°C, indicating a glass transition, which corresponds to the characteristics of an effective NADES of the present invention. This curve is consistent with that of Qiao et al. (supra) in their Figure 4 (NADES is over-diluted and becomes an aqueous composition); this shows a substantially uniform baseline with a rise and fall in heat flow as water ice crystals (so-called "cold crystals") form and melt.

[0242] 1.4 Mixed NADES and bacterial composition

[0243] NADES preparations were blended with the bacterial composition at 9+1 v / v in a laminar flow cabinet. The samples were gently mixed using a roller set; vortexed; or briefly stirred using a sterile inoculating loop, depending on the sample size. From these samples, 1 ml aliquots were filled into 3 ml glass vials, which were capped with rubber stoppers and metal caps.

[0244] 1.5 Stability determination

[0245] For each test sample, a unique vial was stored under refrigerated conditions (2-8°C), frozen conditions (-20°C), or room temperature (20°C). After the specified time, the vial was opened and ten-fold serial dilutions from 10^-1 to 10^-10 dilutions were prepared in PBS. For at least three dilutions, 50 μl of the sample was plated in duplicate on agar plates that support the growth of the specific bacteria. Next, the plates were incubated at 37°C and 5% CO2 for 5-8 days until the colonies were large enough to be counted. The titer for the class Mollicutes was calculated from this, from which 30 to 300 colonies were counted. The titer is expressed as colony forming units per milliliter of sample (CFU / ml). Based on the data points from different vials tested at different time points, stability data graphs were created, such as Figure 2 and 3 shown.

[0246] Example 2: Composition of NADES for use in the present invention

[0247] A number of different NADES compositions were prepared for use in the present invention, having different compounds, different molar ratios, and different water contents.

[0248] Table 1 below lists some of the most effective NADES formulations; these produced clear liquid solutions that were fluid at room temperature (about 20°C). Detailed information on their composition and water content is provided. For several samples prepared, the water activity was determined and the results are included.

[0249] Table 1: Composition and properties of representative quantities of NADES of the present invention

[0250]

[0251]

[0252] Example 3: Results of bacterial stability assay

[0253] For the M. synoviae and M. gallisepticum bacteria comprised in the liquid composition according to the invention, stability tests were performed at temperatures between 2 and 8°C and at room temperature (about 20°C) over extended periods of time.

[0254] Incubation for 8 weeks at room temperature serves as an accelerated stability test, demonstrating the effects of incubation for 1-2 years at 4°C. Therefore, this is a very severe attack on bacterial viability and not all samples will survive under these conditions.

[0255] Conclusions from stability results

[0256] The control (i.e., a 1+9 v / v mixture in standard phosphate buffered saline (PBS)) demonstrated that M. synoviae and M. gallisepticum bacteria have inherently poor stability during storage. Figure 2 and Figure 3 See for exemplary details. In this control liquid composition, at the first tested time point, no bacterial colonies could be detected any more at any of the studied dilutions. This applies to storage conditions of 2-8°C and room temperature.

[0257] It was concluded from these control samples that there was no detectable survival of M. synoviae and M. gallisepticum in PBS.

[0258] Surprisingly, in stark contrast to the PBS control, both M. synoviae and M. gallisepticum showed impressive survival when combined with NADES in the liquid composition according to the invention. When mixed with different NADES at 1+9 v / v, measurable titers were obtained after storage at 20°C for up to 8 weeks.

[0259] For M. synoviae, NADES at a molar ratio of 1:1:2.5 for betaine:sorbitol:WFI and 2:2:1:10 for sorbitol:xylitol:trehalose:WFI showed the strongest stabilization effect at 20°C. Impressive survival titers were found after storage at 2-8°C for up to 6 months, with several NADES showing a loss of less than 3Log10CFU / ml.

[0260] For M. gallisepticum, NADES with a molar ratio of 1:2:6 of arginine:sorbitol:WFI and 1:1:2.5 of proline:sorbitol:WFI showed the strongest stabilization effect at 20°C. Impressive survival titers were found after storage at 2-8°C for up to 3 months, with several NADES showing a loss of less than 3Log10 CFU / ml.

[0261] Example 4: Administration of NADES to experimental animals

[0262] To evaluate the ease and safety of administration of the NADES used in the present invention in target animals, examples of the NADES were administered to experimental animals and post-mortem materials.

[0263] To this end, two variants of proline:sorbitol-based NADES were prepared and tested: one with high viscosity (NADES 3A) and the other with low viscosity (NADES 3B). These were used to compare the application characteristics of the NADES of the invention at the two extremes of the viscosity spectrum. The characteristics of NADES 3A are described in Table 1. In addition, their viscosity was measured using standard conditions: at about 20°C, using a Brookfield TM DV-I+ Viscometer, using a No. 62 spindle, at 60 rpm for 30 seconds in a standard measuring cup with a volume of approximately 80 ml.

[0264] deal with

[0265] High viscosity NADES 3A can be passed through a hypodermic needle size of 21G or smaller, i.e. a needle with an outer diameter of 0.8mm or larger.

[0266] Table 3: NADES formulations tested by administration to animals

[0267]

[0268] Subcutaneous administration in vivo

[0269] In the safety study in dogs, NADES formulations 3B and 3C were administered by subcutaneous injection. Particular attention was paid to any adverse reactions that might occur after local or systemic administration of the stabilizer formulations.

[0270] Both formulations were prepared as described above, by heating and sonicating proline, sorbitol and water for injection in closed vials to obtain clear, uniform liquids. In addition, they were sterilized by filtration, and their bioburden and endotoxin levels were determined. It was found that the scores of both formulations were below the minimum dilution standard of 1 unit / ml, meeting the general acceptance criteria for sterile formulations. The NADES 3C formulation was prepared by diluting 1 volume of NADES 3A with 9 volumes of WFI to obtain a formulation with a 1:1:5 ratio of proline:sorbitol:WFI and a viscosity of 289mPa.s. The viscosity of NADES 3B was 33.4mPa.s.

[0271] Six healthy beagle dogs aged 7 months were divided into two groups of 3 dogs each. Each group was assigned to a NADES formulation and received two doses of 1 ml of the designated formulation, two weeks apart, via subcutaneous inoculation using a 1 ml syringe and 21G 5 / 8 needle. Dogs in Group 1 received the low-viscosity NADES 3B and dogs in Group 2 received the high-viscosity NADES 3C. All dogs were clinically monitored daily throughout the study, including for local reactions at the inoculation site.

[0272] Vaccinations were administered at the nape of the neck; the first vaccination was administered on the left side of the neck and the second vaccination was administered on the right side of the neck. The vaccination site was shaved before each administration and the injection site was circled with a marker. No discomfort was observed during any of the injections. Four hours after the injection, the injection site was palpated and little or no material was felt at the injection site, and no swelling or redness was observed.

[0273] Measure rectal temperature 3, 2, and 1 days before each vaccination; on the day of vaccination: just before and 4 hours after vaccination; and daily for 7 days starting on the day after vaccination.

[0274] Throughout the study, no significant increase in body temperature was observed in any dog, and no clinical adverse effects were observed.

[0275] Conclusion: Subcutaneous administration of NADES formulations 3C or 3B to dogs did not induce any adverse vaccination reactions, either local or systemic.

[0276] Intranasal administration

[0277] Two NADES formulations: 3A and 3B were tested for their spreading pattern on the nasal mucosal surface when administered intranasally. Both NADES formulations were tested without the addition of water. Thus, the viscosity of NADES 3A was 5197 mPa.s and that of NADES 3B was 33.4 mPa.s. A spatula-tip amount of "Patent Blue" colorant was added to 30 ml of each NADES formulation to obtain a dark blue color. PBS containing the colorant was used as a control.

[0278] Calf heads were obtained from necropsy of calves approximately 2-4 weeks of age. Administration was performed with the head held in an upright position, mimicking the position of calves during field inoculation, with the nostrils tilted slightly upward.

[0279] The fluid was then administered according to standard procedures for intranasal inoculation, whereby a 2.5 ml needleless syringe containing 1.5 ml of fluid was placed into the nostril and the fluid was then ejected at a steady stream. The head was next dissected in half, the septum removed and the nasal passages examined for the presence of a blue color.

[0280] For all samples, the blue liquid was observed to pass easily through the nasal passages. The low viscosity of the PBS sample made it easy to inject, but all liquids passed quickly. The blue color was observed throughout the nasal cavity including the beginning of the trachea.

[0281] The injection of NADES 3A, which has a very high viscosity, required a little force to eject from the syringe. In addition, as expected, the blue liquid appeared longer below the head. After dissecting the head, it was observed that this viscous liquid also spread evenly into the nasal passages, where it appeared to form a thick layer, leaving more material in the nasal passages. This suggests that when administered to a calf during intranasal vaccination, improved retention and less progression to the upper respiratory tract occurs.

[0282] The effect of administration of the low viscosity NADES 3B formulation was very similar to that of the PBS sample, with the liquid flushing through the nasal cavity.

[0283] Conclusions: The distribution of coloured formulations of different viscosities can be monitored after intranasal injection into the calf head. The liquid distribution was comparable for both NADES formulations and PBS as judged by the blue colour, with some differences in ease of application and the amount of residual material, such that the NADES 3A formulation was a little more difficult to expel but produced a thicker residual layer in the nasal passages. This suggests that delayed release of antigen is possible from this viscous NADES formulation.

[0284] Example 5: Comparative Measurements of Aqueous Stabilizers

[0285] An aqueous stabilizer composition as described in WO 2015 / 121463 was prepared having: 30% w / w sorbitol + 0.6M arginine in 0.04M PBS. The properties of the composition, such as water activity and DSC curve, were tested to demonstrate that it did not have the characteristics that make the composition a NADES as defined and outlined above.

[0286] water activity

[0287] As described in WO 2019 / 122329, Example 1, a was determined by headspace vapor pressure measurement. w The water activity results were determined twice and the average w Clearly, this high level of available water indicates that this composition is not NADES.

[0288] DSC curve

[0289] The determination of the DSC curves of the aqueous prior art stabilizers was performed as described in Example 1.3 herein.

[0290] The results are shown in Figure 4 Among them (such as Figure 1 The upper horizontal part of the graph (shown) represents a cooling stage from 40°C to -90°C at 5°C / min (from right to left), and the bottom horizontal part represents a heating stage from -90°C to 40°C at 5°C / min (from left to right).

[0291] During cooling, a large amount of free water was observed to suddenly crystallize into ice at a temperature of about -27°C. Then, after reheating, a typical ice crystal melting curve was observed with a peak at -5.5°C.

[0292] In addition, Figure 4 In the heating phase, the horizontal baseline after the ice melts returns to the same level as before melting. This indicates that the heat capacity of this sample has not changed, which is clearly different from the stepwise baseline shift usually observed in the case of glass transition.

[0293] It is also important to Figure 4 Melting of ice occurs at Figure 1 The glass transition temperature (-45°C) is much higher than that (-5°C), again indicating that Figure 4 The samples tested in are not deep eutectic mixtures.

[0294] exist Figure 4 In the graph, the two peaks, the high melting temperature and the return of the baseline to its previous level, all indicate the formation and melting of ice crystals, thus demonstrating the presence of a large amount of free water in the tested composition and therefore not NADES.

[0295] However, on the contrary, Figure 1 In the sample of (NADES according to the invention), no ice crystals formed or melted and a clear glass transition shift of the baseline was found.

[0296] Example 6: Additional Stability Results: Mycoplasma bovis

[0297] Another Mollicutes bacterium, Mycoplasma bovis, was tested for stability in a liquid composition according to the invention. The experiments were performed essentially as described in Examples 1-3 herein for M. synoviae and M. gallisepticum.

[0298] The results of the stability tests in different compositions according to the invention and at room temperature or at 2-8° C. are shown in Figure 5 middle:

[0299] Panel A: room temperature, incubate for up to 10 weeks; and

[0300] Panel B: 2-8°C, incubation up to 26 weeks.

[0301] The starting titer was 9.9 Log10 CFU / ml. In PBS (negative control), some survival was observed in incubations at room temperature and refrigerated for two weeks. However, no viable cells were recovered in samples incubated in PBS for longer periods of time.

[0302] Several NADES compositions achieved good initial stability at room temperature (2-4 weeks of incubation). However, the best results were found when using arginine:sorbitol:water (2:1:6), which was able to stabilize the bacteria at room temperature for at least 10 weeks.

[0303] A similar situation was observed when incubating under refrigeration: good initial stability was observed for 6-13 weeks for several compositions tested. The best long-term stabilizer (26 weeks of incubation) was also arginine:sorbitol:water (2:1:6), although the differences observed between the different compositions tested were not as large as for room temperature incubation.

[0304] In general, the liquid compositions according to the present invention were effective in stabilizing M. bovis at different storage temperatures above freezing without freeze-drying.

[0305] Example 7: Extended stability test results

[0306] The stability test results of Mycoplasma synoviae and Mycoplasma gallisepticum in the liquid composition according to the present invention were obtained by continuous incubation at 2-8°C for up to 1 year. These data are from the same series of samples as described in Example 3 and extend to the present invention. Figure 2 The results are depicted in B and 3B.

[0307] Figure 6 Extended results are provided:

[0308] Panel A: M. synoviae incubated at 2-8°C for up to 54 weeks; and

[0309] Panel B: M. gallisepticum incubated at 2-8°C for up to 52 weeks.

[0310] For Mycoplasma synoviae, the three tested liquid compositions according to the invention can stabilize the viability of these bacteria at 2-8°C for at least one year; among which proline:sorbitol:water (1:1:2.5) and betaine:sorbitol:water (1:1:2.5) show the same final results.

[0311] For Mycoplasma gallisepticum, in betaine:sorbitol:water (1:1:2.5) at 2-8°C, viable bacteria were only retained until 52 weeks of incubation. BRIEF DESCRIPTION OF THE DRAWINGS

[0312] Figure 1 : Differential scanning calorimetry curve of NADES formed with a molar ratio of proline, sorbitol and water of 1:1:2.5.

[0313] Figure 2: Stability results of Mycoplasma synoviae in liquid compositions with different NADES. Panel A: incubated at room temperature; Panel B: incubated at 2-8°C.

[0314] Figure 3 : Stability results of Mycoplasma gallisepticum in liquid compositions with different NADES. Panel A: incubated at room temperature; Panel B: incubated at 2-8°C.

[0315] Figure 4 : DSC curve of an aqueous stabilizer from the prior art, comprising 30% w / w sorbitol and 0.6 M arginine in 0.04 M PBS.

[0316] Figure 5 : Stability data of M. bovis in NADES at room temperature (A) and 2-8°C (B).

[0317] Figure 6 : Long-term stability data of Mycoplasma synoviae (A) and Mycoplasma gallisepticum (B) in NADES at 2-8°C.

Claims

1. A liquid composition comprising live Mollicutes bacteria and a stabilizer, characterized in that The stabilizer is a natural deep eutectic solvent (NADES), the composition has a water activity of 0.85 or less, and the composition has a water content of 50% w / w or less.

2. The liquid composition according to claim 1, characterized in that The NADES comprises an organic salt and a polyol.

3. The liquid composition according to claim 1 or 2, characterized in that The NADES comprises two or more compounds that are both polyols.

4. A liquid composition according to claim 2 or 3, characterised in that The polyol is a sugar or a sugar alcohol.

5. The liquid composition according to claim 4, characterized in that The sugar is selected from sucrose and trehalose, or the sugar alcohol is selected from one or more of erythritol, xylitol and sorbitol.

6. The liquid composition according to claim 2, characterized in that The organic salt is one or more selected from betaine, proline, choline, methionine and arginine.

7. A method for preparing a liquid composition according to any one of claims 1 to 6, comprising the steps of: a. Preparation of NADES, and b. Mixing the NADES with live Mollicutes bacteria.

8. Use of the NADES according to any one of claims 1 to 6 as a stabilizer in a liquid composition comprising live Mollicutes bacteria.

9. The liquid composition according to any one of claims 1 to 6, or as obtainable by the method according to claim 7, for use as a vaccine against infections or diseases caused by bacteria of the class Mollicutes.

10. A liquid vaccine for combating infections or diseases caused by bacteria of the class Mollicutes, said vaccine comprising a liquid composition according to any one of claims 1 to 6 or obtainable by the method of claim 7.

11. Use of the NADES according to any one of claims 1 to 6 as a pharmaceutically acceptable carrier in the liquid vaccine according to claim 10.

12. Use of the NADES according to any one of claims 1 to 6 in the preparation of a liquid vaccine according to claim 10.

13. A kit of parts comprising at least two containers: one container comprising a liquid vaccine according to claim 10, and one container comprising a liquid diluent.

14. The liquid vaccine according to claim 10, for use in a method of protecting a human or non-human animal target against infection and / or disease caused by a pathogenic form of a live Mollicutes bacterium contained in said liquid vaccine.

15. Use of a liquid vaccine according to claim 10 in the preparation of a medicament for vaccinating a human or non-human animal target against an infection or disease caused by bacteria of the class Mollicutes.

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