Stabilization of live mollicute bacteria in liquid compositions.

Abstract

The present invention relates to a liquid composition of live mollicutes bacteria and a stabilizer, whereby the stabilizer is a natural deep eutectic solvent (NADES), in which the live mollicutes are stabilized without the need for freezing or lyophilization, allowing for a variety of advantageous uses in humans or non-human animals, including diagnostics and pharmaceuticals, particularly liquid vaccines used against infections or diseases caused by mollicutes bacteria.

Description

[Technical field]

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

[0002] Viable bacteria are used in a wide variety of applications, some of the more well-known being traditional and modern biotechnological processes, diagnostics and vaccines. Important in all these applications is the way to stabilize such bacteria and maintain their viability during storage and use. A common technique relies on storage in deep-frozen conditions using cryoprotectants such as glycerol or rich media for stabilization. Alternatively, viable bacteria can be freeze-dried and maintained in lyophilized form under refrigerated or frozen conditions. However, all these stabilization methods have many drawbacks. Rich storage media introduce components of undefined and variable quality such as serum, hydrolysates and extracts. Such undefined components can have unpredictable effects on stability. Furthermore, frozen products require a laborious cold chain for storage and distribution. However, even with these processes, complete survival of viable bacterial cells is not achieved, as the freezing step itself induces a significant drop in viable cell count. Also, freeze-drying is a very costly and time-consuming process. Therefore, improved methods to effectively stabilize viable cell count of bacteria during storage and use are highly desirable.

[0003] A group of agriculturally and medically important bacteria is the class Mollicutes. They are taxonomically classified in the phylum Tenericutes (Ludwig et al., 2009, Bergey's Manual of Systematic Bacteriology, 2nd edn, vol. 3. Springer: New York, USA, pp 1-13). This phylum contains bacteria that are derived from Gram-positive bacteria but have lost their cell wall. They are typically parasites or commensals of eukaryotic hosts.

[0004] Infection of animals with Mycoplasma bacteria as a primary or secondary pathogen often causes respiratory disease and / or a variety of other symptoms that together can cause mild to severe illness and cause a lot of discomfort and economic loss to the affected host. A reference for the impact of Mycoplasma infections in agriculture is "The Merck veterinary manual" (11th ed., 2016, ISBN-10:9780911910612).

[0005] Mycoplasma infections can be treated therapeutically with certain types of antibiotics, but vaccines are commonly used prophylactically. Such vaccines can be based on live attenuated strains or inactivated bacterins (killed bacteria). A bacterin vaccine against M. bovis is MpB Guard™ (Agrilabs). The most used commercial mycoplasma vaccines are for poultry. For example, live attenuated vaccines for M. gallisepticum: Vaxsafe® MG (Bioproperties) (frozen), and Nobilis MG 6 / 85 (MSD Animal Health) (lyophilized, cooled); bacterin vaccines for M. gallisepticum: MG-Bac® (Zoetis) and Nobilis® MG inac (MSD Animal Health), both of which are emulsions with oil adjuvants. Live attenuated vaccines for M. synoviae are Vaxsafe® MS (Bioproperties) (frozen) and Nobilis® MS Live (MSD Animal Health) (lyophilized, cooled). Several other live attenuated mycoplasma vaccines are known and used on a more limited scale, such as Contavax®, a freeze-dried vaccine against M. mycoides in ruminants, which is used in some African countries. Also, in China, a live attenuated M. hyopneumoniae vaccine is used in pigs. A general overview of mycoplasma vaccination is as follows: 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 into host cells, Mollicutes are known contaminants of in vitro cell cultures of a variety of eukaryotic cells. Therefore, several species of live Mollicutes bacteria are used for diagnostic and quality control purposes. It is also a formal requirement in biological production under government regulations such as the European Pharmacopoeia and the US 9-CFR. [Prior art documents] [Patent documents]

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

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

[0009] Mollicutes are the smallest self-replicating organisms known. Bacteria from some species are only 0.2 micrometers in diameter, and some species have genomes of only 580 kbp. The very small number of genes makes Mollicutes highly dependent on resources from the environment, so they typically require very rich media when cultured in vitro. Also, the lack of a cell wall exposes the cell membrane to physical and chemical environmental influences, reducing stability during storage and use.

[0010] Therefore, a key problem in the vaccine industry is how to preserve the viability of such live Mollicutes bacteria as bacterial stocks during the shelf life of the final product and when used, for example, in diagnostic tools or vaccinations. One solution is to produce and use non-live bacterin-type vaccines. However, such vaccines are relatively expensive due to the need for relatively high doses, repeated administration, and the use of adjuvants.

[0011] Vaccines based on live Mollicutes bacteria are deep-frozen or lyophilized and stored in a rich, stabilizing medium. As noted above, such media are less than desirable for a number of reasons. The freezing process itself also results in a significant reduction in viable cell numbers, which can cause up to 10 deaths within a few days. 3 This results in a loss of colony forming units (CFU). Consequently, methods and materials for improving the stabilization of live mollicutes bacteria are highly desirable.

[0012] Deep eutectic solvents (DES) are known liquid compositions that combine many remarkable properties. In general, they are mixtures of compounds that are liquid at ambient temperature but have a low water content. Furthermore, the small amount of water present is tightly bound, which limits its availability for chemical or biological processes. This is expressed by a very low water activity value. DES are described, for example, in Abbott et al., 2003, Chem. Commun., vol1, p.70-71, and WO2009 / 120839.

[0013] DES are highly ionic but at the same time have very low water activity, making them very effective as solvents for polar hydrophobic compounds. Examples range from the extraction of gases, minerals or industrial bulk products to the solubilization of pharmaceuticals. DES have also been used to extract and stabilize biomolecules such as enzymes and RNA, see US8,247,198 and WO2011 / 155829, respectively.

[0014] Since about 2011, special forms of DES composed of natural substances have been investigated. These so-called "natural DES" or NADES are described, for example, in 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 of great interest as "green" solvents that can be used for the extraction of natural products and as solvents for use in, for example, natural pesticides.

[0015] WO2011 / 155829 describes the use of NADES for the extraction of biological materials, one of the options listed being the extraction of proteinaceous material such as subunit vaccines.

[0016] Similarly, WO2014 / 131906 describes a fixative for viruses in blood or tissue, with a particular focus on the extraction and stabilization of viral RNA for clinical diagnosis. The fixative is a DES, and WO2014 / 131906 lists many possible DES compositions, with only compositions containing urea or trifluoroacetamide as the proton acceptor and choline or betaine as the proton donor being used in practice. In WO2014 / 131906, the most preferred DES consists of choline chloride and trifluoroacetamide, and thus it is not a NADES, and trifluoroacetamide is not an acceptable pharmaceutical excipient.

[0017] WO2016 / 108083 describes the use of NADES for their bactericidal activity, and it has been found that some NADES can effectively kill or inactivate fungal cells of Gram-positive and Gram-negative bacteria, as well as Candida.

[0018] WO2016 / 162703 describes the use of NADES, e.g. consisting of betaine and organic acids or polyhydric alcohols, for the extraction of biomolecules, in particular from plant material.

[0019] WO2017 / 222377 describes the use of NADES gel in a mixture with animal feed to provide a source of water that will not dry out under heat. The specific use described is for newly hatched chicks.

[0020] WO2015 / 121463 describes a liquid stable poultry vaccine, specifically of a poultry virus, in a composition of 5-40% sugar alcohol and 0.15-0.75M of an amino acid selected from arginine, glutamic acid or glycine. However, most importantly, WO2015 / 121463 and similar publications such as WO2014 / 029702 and WO2014 / 140239 do not disclose NADES, which contain at least 60% (weight / volume) water and have a water activity (a) of more than 0.85. w ) is described. Comparative data is provided in the Examples section.

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

[0022] Qiao et al. (2018, Appl. Microbiol. Biotechnol., vol. 102, p. 5695-5705) investigated lactic acid bacteria stabilization using NADES-derived compositions. These bacteria are Gram-positive and contain a cell wall. The viability of two species from the Lactobacillus genus was not stabilized or was damaged by the use of NADES in cryopreservation. Streptococcus thermophilus could be stabilized in frozen form using NADES with a 1:3 molar ratio of proline:glycerol. However, by applying a 1+1 volumetric mix of NADES and bacterial culture, Qiao et al. diluted the NADES until the network of hydrogen bonds was not compromised. This is evident from the differential scanning calorimetry (DSC) profile (Qiao et al., Figure 4), which shows the melting and growth of ice crystals. Although these are properties of aqueous compositions, intact NADES would exhibit a glass transition temperature.

[0023] Qiao et al. also describe that the presence of a cell wall allows bacteria to resist the effects of NADES toxicity, namely dehydration and damage to the cell membrane.

[0024] The object of the present invention is to overcome the shortcomings of the prior art and address the need in the art by providing a composition comprising live Mollicutes bacteria that can be prepared without lyophilization, does not require maintenance in a frozen state, but provides sufficient stabilization during storage and handling at temperatures above 0°C. [Means for solving the problem]

[0025] Surprisingly, it has been found that this objective can be achieved by providing a liquid composition capable of stabilizing live mollicutes without freezing or lyophilization, thereby overcoming one or more of the drawbacks of the prior art. This is achieved by employing NADES as a stabilizing agent.

[0026] This discovery opens up many highly favorable fields of application. Live Mollicutes bacteria can now be stored without freezing or lyophilization, which are laborious, time-consuming and expensive processes. Furthermore, the liquid composition of the present invention does not use components of uncertain or variable quality such as hydrolysates for stabilization, but on the contrary uses chemically defined compounds of natural origin. Furthermore, the liquid form facilitates the use in various applications such as diagnostics, quality control, etc., but also allows the use as live attenuated vaccines, since NADES are acceptable pharmaceutical carriers. Such vaccines can now be administered by administering the liquid composition containing live Mollicutes bacteria, either directly or after dilution.

[0027] That NADES can stabilize mollicute bacteria is unexpected: as strong ionic solvents, NADES would be expected to dissolve the delicate exposed bacterial cell membrane and extract its cytoplasmic proteins and nucleic acids. Indeed, Qiao et al. (Qiao et al., supra) warn that bacteria without cell walls are susceptible to the toxicity of NADES. Also, WO2016 / 108083 (supra) employs NADES to actually kill bacteria.

[0028] Thus, the inventors were surprised to find that NADES formulations can provide significant stabilization of viability of live Mollicutes bacteria without freezing or lyophilization, which stabilization was significantly better than that observed in aqueous-based liquid compositions. For example, M. synoviae was able to grow at a 3 Log 10 It can be maintained with less than 1 Log CFU loss, with some compositions losing less than 1 Log 10 Even at room temperature, significant stabilization was observed for up to 8 weeks, with only 4 Log 10 Although there was a loss of CFU, cultures in phosphate buffered saline were completely lost within 1 week at that temperature.

[0029] It is not known exactly how or why NADES are such effective stabilizers for live mollicutes. Although the inventors do not wish to be bound by any theory or model that may explain these findings, they speculate that the low amount of available water in NADES helps maintain the viability of the bacteria. Also, mollicutes appear to interact with NADES in a different way than do bacteria with cell walls. This effect was not predictable, as there is no prior art on the stabilization of mollicutes in NADES.

[0030] Thus, in one aspect, the present invention provides a liquid composition comprising a promollicute bacterium and a stabilizer, characterized in that the stabilizer is a natural deep eutectic solvent (NADES). [Brief description of the drawings]

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

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

[0033] As used herein, the term "comprising" (and variations such as "comprise", "comprises", and "comprised") refers to all elements and all possible combinations contemplated herein that are covered or contained in the statement, paragraph, claim, etc. in which the term is used, even if the element or combination is not explicitly recited, and does not exclude any of such elements or combinations.

[0034] Thus, such statements, paragraphs, claims, etc. may be directed to one or more embodiments in which the term "comprising" (or variations thereof) is replaced with terms such as "consist of," "consisting of," or "consist essentially of."

[0035] The term "live" is used herein in a general sense with respect to bacteria and refers to their ability to grow, move, take up and ferment nutrients, and / or replicate under appropriate conditions. Methods for determining whether bacteria are dead or alive are known in the art. This can be determined in vivo, but more conveniently in vitro, for example by culturing in a suitable medium and detecting an increase in the optical density of the culture, or plating on suitable agar plates and counting CFU. Another method uses live / dead selective staining and counting.

[0036] The term "mollicutes" refers to bacteria that have characteristic features of a taxonomic classification, such as morphological, genomic, and biochemical characteristics, as well as biological characteristics, such as physiological, immunological, or pathological behavior. The same applies to references to bacterial orders, families, genera, or names of individual bacterial species.

[0037] The class Mollicutes includes the orders Acholeplasmales, Anaeloplasmales, Entomoplasmales, and Mycoplasmales. Acholeplasmales includes known Mollicutes plant pests of the genus Phytoplasma (Candidatus). Entomoplasmales includes insect symbionts and plant pathogens of the genus Spiroplasma.

[0038] The best known are the Mycoplasmales, which include known human or animal pathogenic bacteria of the genus Mycoplasma, such as M. pneumoniae, M. bovis, M. hyopneumoniae, M. meleagridis, and M. gallisepticum, which (mainly) infect humans, ruminants, pigs, turkeys, and chickens, respectively.

[0039] As is known in the art, the classification of microorganisms in particular taxonomic groups is based on a combination of such features. Thus, the present invention also includes species of Mollicute bacteria from the orders, families, genera, and species classified in the Mollicute class. Similarly, this refers to bacteria that are subclassified therein in any way, such as, for example, subspecies, strains, isolates, genotypes, variants, subtypes, or subgroups.

[0040] Furthermore, it will be apparent to those skilled in the art that, although a particular order, family, genus or species of Mollicutes bacteria in the present invention may currently be assigned to that group, it is a taxonomic classification that may change over time as new insights may be gained that may lead to reclassification into new or different taxonomic groups. However, such reclassified bacteria remain within the scope of the present invention, as this does not change the bacterium itself or its antigenic repertoire, but only its scientific name or classification. For example, Gupta et al. (Gupta et al., 2018, Antonie van Leeuwenhoek, vol. 111, p. 1583-1630) have proposed a new framework for the Tenericutes phylum based on the latest genomic data.

[0041] Samples of Mollicutes bacteria for use in the present invention can be obtained from various sources, for example from humans, or from wild or farm non-human animals, or as wild isolates from various laboratories, (repository) institutions or (veterinary) universities.

[0042] A "stabilizer" in the present invention is a composition that can reduce the loss of viable cell count of the Mollicutes bacterium of the present invention due to handling or use and / or due to storage at a particular temperature for a particular period of time.

[0043] "Stabilization" in the present invention is expressed as the effect on the difference in CFU before and after a specific treatment of a composition containing live Mollicutes bacteria, for example from incubation at a specific temperature for a specific period of time. Thereby, the survival rate in different liquid compositions according to the present invention is compared with the survival rate in a standard PBS buffer (10 mM Na2PO4, 1.8 mM KH2PO4, 2.7 mM KCl, and 137 mM NaCl in water at pH 7.4). A survival rate that exceeds that observed in PBS under similar storage conditions is then considered as the survival effect provided by the liquid composition according to the present invention.

[0044] "Deep eutectic solvents" (DES) are known in the art as ionic liquids comprising a mixture of at least two compounds in a molar ratio that forms a eutectic mixture, whereby the eutectic point of the mixture is significantly lower than the melting points of the individual compounds.

[0045] This reduction in the 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, which provides a stable hydrogen bond network. The network incorporates small amounts of water, which cannot crystallize upon freezing. This allows the mixture to be in liquid form at temperatures well below the melting points of the constituent components. In general, "eutectic" means easily soluble.

[0046] NADES for use in the present invention have an intact hydrogen bond network, which can be easily confirmed by thermal analysis of the phase transition profile using DSC. As a result, NADES for use in the present invention have a DSC profile that exhibits a glass transition temperature.

[0047] Determination of DSC profiles is known and can be performed using standard equipment, an example of a profile is shown in Figure 1.

[0048] The hydrogen bond network allows a limited level of dilution. However, if it is diluted too much, the "DES" turns into a normal aqueous solution. The DSC profile then displays the enthalpy of fusion and the enthalpy of crystallization, as in Qiao et al., supra, Figure 4.

[0049] The individual compounds (except water) used to form the NADES of the present invention have melting points greater than about 80° C., and the resulting NADES have melting points less than about 40° C. For example, the melting points of betaine and sorbitol are 310° C. and 95° C., respectively, but a NADES formed with a 1:1:2.5 molar ratio of betaine, sorbitol, and water had a glass transition temperature of −50° C. and did not exhibit ice crystallization and / or ice melting in its DSC profile.

[0050] The term "natural" is intended to indicate that the compounds used to form the natural DES (NADES) of the present invention are organic compounds that can be found in materials from biological sources, such as plants or animals, in amounts well above trace under normal conditions. Typically, such natural compounds are or are derived from primary metabolites present in a particular material of plant or animal origin. As the skilled artisan will appreciate, the term natural is used herein only to characterize the initial origin of the compounds used in the NADES of the present invention, and not the actual source of the compounds to be used in the present invention. Thus, natural compounds can also be used in the present invention when derived by (semi)synthetic production.

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

[0052] It will be apparent to those skilled in the art that within the embodiments disclosed herein of the present invention, there will be combinations of certain types of NADES and certain types of Mollicutes bacteria that are more favorable than other combinations in terms of bacterial stability. Nevertheless, those skilled in the art will be able to readily select, optimize and fine-tune combinations and compositions according to the present invention using only routine methods and materials with the information disclosed herein.

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

[0054] The water present in the NADES used in the present invention is tightly bound to the NADES hydrogen bond network structure, strongly reducing the availability of water for chemical or biological processes that may affect the stability of the mollicute bacteria. This characteristic is generally represented by showing the amount of total water and the amount of free water. The availability of the water (free water) is represented by the symbol a w Water activity is indicated by a value of water activity indicated by: Water activity varies between an upper limit of 1.0 for pure water and a lower limit of 0. Water activity is generally measured by comparing the vapor pressure of the test composition to that of pure water and to saturated salt solutions with a number of known water activities (at the same temperature). This is described, for example, in various handbooks, compendia and manuals on fruit and vegetable preservation in FAO Agricultural Service Bulletin No. 149 (Canovas et al., FAO, Rome, 2003, ISBN 92-5-104861-4), and the compendia "Fundamentals of water activity", Decagon Devices Inc., Washington, 2015 (http: / / pdf.directindustry.com / pdf / decagon-devices-inc / fundamentals-water-activity / 64142-634433.html).

[0055] Below a water activity of about 0.8, most bacterial growth ceases, below 0.7, most yeast and mold growth ceases, and below 0.4, most enzyme activity is effectively stopped.

[0056] Instruments and procedures for measuring water activity are known and available, for example, by using head space pressure analysis.

[0057] It has been found that effective stabilization of mollicute bacteria in liquid compositions according to the present invention can be achieved when the compositions have a water activity of less than about 0.85.

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

[0059] In the present case, the indicated water activity refers to the water activity of the liquid composition according to the invention in the form of the final product, for example as provided by a commercial manufacturer, in which form it can be stored for a long time. When using this composition, it can be diluted or mixed with other ingredients, which can change the water activity value.

[0060] In one embodiment of a 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 that order of increasing preference.

[0061] The water activity of the liquid composition according to the present invention is preferably 0.8 to 0.1, more preferably 0.7 to 0.1, 0.6 to 0.1, 0.5 to 0.1, 0.4 to 0.1, 0.3 to 0.1, or even 0.2 to 0.1, in that order of preference.

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

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

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

[0065] NADES for use in the liquid composition according to the invention are prepared with a limited amount of water. By varying the water content of the liquid composition, its properties can be adjusted and optimized, for example with respect to the viscosity of the liquid composition. Effective NADES-containing compositions with little viscosity can be made with a total water content of up to 50% by weight (Dai et al., 2015, vol. 187, p. 14-19). Relatively large amounts of water can disrupt the hydrogen bond network of NADES.

[0066] Thus, in one embodiment of the liquid composition according to the invention, the composition has a water content of less than or equal to 50% by weight.

[0067] The water content of the liquid compositions according to the present invention is expressed as a percentage of weight per weight "w / w", which is the weight of the total amount of water in the liquid composition according to the present invention as a fraction of the weight of the liquid composition. The water content can be measured using various procedures, for example by Karl Fischer titration, as known in the art.

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

[0069] Preferably, liquid compositions according to the present invention have a water content of less than or equal to 40% by weight, more preferably less than or equal to 30, 25, 20, 15, 10, 8, 7, 6, or 5% by weight, in that order of increasing preference.

[0070] The lower limit of water content in a liquid composition according to the present invention is determined by practical factors such as, for example, how little water is sufficient to solubilize the compounds that form the NADES, and also the amount of water required to reach an acceptable level of viscosity of the NADES so that it can be conveniently processed or used.

[0071] Therefore, in one embodiment of the liquid composition according to the present invention, the water content in the liquid composition is 50 to 0.5% by weight; 40 to 1% by weight; 30 to 1.5% by weight; 20 to 2% by weight; or even 10 to 3% by weight, in this order of increasing preference.

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

[0073] Thus, in one embodiment of the liquid composition according to the invention, the ratio of the volume of the NADES to the volume of the bacterial sample to be combined is preferably at least 3:1 volume ratio NADES:bacterial sample; more preferably at least 5:1 volume ratio NADES:bacterial sample; or even at least 10:1 volume ratio NADES:bacterial sample, in that order of increasing preference. Note: a ratio of 10:1 means a combination of 9 parts NADES and 1 part bacterial sample. For example, a NADES of proline, sorbitol and water in a molar ratio of 1:1:2.5 has a total water content of 13.1% by weight. After mixing with a sample of mycoplasma bacteria according to the invention in a ratio of 9 parts NADES + 1 part bacteria, the total water content of the resulting liquid composition according to the invention is 21.8% by weight.

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

[0075] It has been found that NADES effective as stabilizers of live mollicute bacteria in liquid compositions according to the present invention are formed from various combinations and ratios of organic salts and / or polyhydric alcohols, or are formed from several polyhydric alcohols.

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

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

[0078] In some of these embodiments, the hydrogen bond network is formed by the organic salt as the ionic species acting as the hydrogen bond donor and the polyhydric alcohol as the hydrogen bond acceptor, or, if the NADES comprises only polyhydric alcohol, the network is formed by hydrogen bonding between these molecules.

[0079] In the present invention, a "polyhydric alcohol" is an organic compound containing two or more hydroxyl groups. However, very large polymers under the definition of polyhydric alcohol, such as cellulose, are less effective in forming NADES as defined herein and should preferably not be used in the present invention. As a result, the polyhydric alcohols used in the present invention preferably have a molecular weight of less than about 10,000 grams / mole.

[0080] In a further preferred embodiment, the polyhydric alcohols used in the present invention have a molecular weight of less than 5000 / mole, or even less than 1000 grams / mole.

[0081] Compounds for use in the NADES used in the present invention are readily available in different purities and qualities from various commercial suppliers. Preferably, the compounds are used in pharmaceutical grade quality. Such excipients are described, for example, in government regulations such as the European Pharmacopoeia and the US 9 CFR, and in handbooks 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).

[0082] Preferred polyhydric alcohols for use in the present invention are sugars or sugar alcohols, as these have proven to be versatile components that allow for the production of a variety of effective NADEEs for use in the present invention.

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

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

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

[0086] In the present invention, a "sugar alcohol" is a hydrogenated sugar containing three or more carbon atoms and can be based on a monosaccharide, disaccharide or polysaccharide.

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

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

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

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

[0091] The polyhydric alcohols used in the liquid compositions according to the invention can be used as different salts, isomers, hydrates or anhydrous forms etc. The skilled person is fully capable of selecting and testing suitable forms of the polyhydric alcohols to be used in the present invention.

[0092] In the present invention, an "organic salt" is any salt of an organic acid or base, including zwitterions, that falls within the definition of being a naturally occurring compound presented herein above and has the ability to form a deep eutectic solvent for the invention described herein. A person skilled in the art is fully capable of selecting an organic salt for the present invention and applying it to form a NADES.

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

[0094] In the present invention, "betaine" is preferably the compound N,N,N-trimethylglycine, CAS number 107-43-7, which is also known as glycine betaine. Proline is preferably CAS number 609-36-9. Choline is preferably CAS number 62-49-7. Methionine is preferably L-methionine, CAS number 63-68-3.

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

[0096] As with the polyhydric alcohols mentioned above, the organic salts can be used in different isomeric forms, hydrated or anhydrous forms, etc. Those skilled in the art are fully capable of selecting and testing the appropriate forms of the organic salts for use in the present invention.

[0097] In one embodiment of the liquid composition according to the invention, when the organic salt is arginine, the arginine is used as a mixture of L-arginine (CAS No. 74-79-3) and L-arginine-HCl (CAS No. 1119-34-2), whereby L-arginine-HCl constitutes the majority of the mixture.

[0098] Such a mixture of arginine compounds serves to bring the pH value of the liquid composition according to the invention closer to physiological values, for example when diluting the composition before use as a vaccine or when depositing for counting.

[0099] Preferably, the molar ratio of L-arginine:L-arginine-HCl is from 1:1 to 1:100; more preferably, the molar ratio of L-arginine:L-arginine hydrochloride is from 1:1 to 1:50, and more preferably, the molar ratio is about 1:19.

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

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

[0102] In one embodiment of the NADES used in the liquid composition according to the invention, the organic salt is selected from salts of betaine, proline, choline and arginine, and the polyhydric alcohol is selected from xylitol and sorbitol.

[0103] In the present invention, the organic salt and the polyhydric alcohol or polyhydric alcohols can be applied in different molar ratios to each other to provide NADES with different properties, which can be optimized by the skilled person to reach the optimal stabilization of the live Mollicutes bacteria in the liquid composition according to the present invention.

[0104] Thus, in one embodiment of the liquid composition according to the present invention, the molar ratio between the organic salt and the polyhydric alcohol is from 1:5 to 5:1, all as defined herein.

[0105] In preferred embodiments of the liquid composition according to the invention, the molar ratio between the organic salt and the polyhydric alcohol is from 1:4 to 4:1, from 1:3 to 3:1, or even from 1:2 to 2:1, in that order of increasing preference, all as defined herein.

[0106] In one embodiment of the liquid composition according to the invention, the molar ratio between any of the three polyhydric alcohols xylitol, trehalose and sorbitol, all as defined herein, may be from 1 to 5. Consequently, the molar ratio of sorbitol:xylitol:trehalose is one of 1:1:1 and 1:1:5, 1:5:1, 5:1:1, 1:5:5, 5:1:5, 5:5:1 and 5:5:5.

[0107] One type of advantageous use of the liquid composition according to the invention is as a vaccine, in which the mollicute bacterium is used as the active ingredient of a live bacterial vaccine and must be of an attenuated nature so as not to induce an unacceptable vaccination response in the target animal.

[0108] Thus, in one embodiment of the liquid composition according to the invention, the Mollicutes bacteria are attenuated with respect to their pathogenicity.

[0109] The term "attenuated" in the present invention is used in its general sense to refer to a bacterium that causes a lower level of pathogenic effects by reducing the rate of infection and / or replication in a host compared to the unmodified, non-attenuated or "wild type" of such Mollicutes bacterium. Such live attenuated bacteria are also referred to as "modified live bacteria".

[0110] Attenuation of Mollicutes bacteria can be achieved in a variety of ways, for example, by passaging wild-type bacteria in laboratory animals or cell culture, by the use of mutagens, or via recombinant DNA techniques, all as known in the art.

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

[0112] Thus, in one embodiment, the Mollicutes bacterium is one or more selected from the bacterial orders: Acholeplasmales, Anaeroplasmales, Entomoplasmales, and Mycoplasmales.

[0113] In one embodiment, the Mollicutes bacterium of the invention is one or more selected from the bacterial families Acholeplasmataceae, Anaeroplasmataceae, Entomoplasmataceae, Spiroplasmataceae, and Mycoplasmataceae.

[0114] In one embodiment, the Mollicutes bacterium of the present invention is one or more selected from the bacterial genera Acholeplasma, Phytoplasma, Anaeroplasma, Asterolplasma, Entomoplasma, Mesoplasma, Spiroplasma, Hepatoplasma, Marinoplasma, Moeniplasma, Mycoplasma, and Ureaplasma.

[0115] Preferably, the Mollicutes bacterium of the present invention is one or more selected from the bacterial genera Acholeplasma, Spiroplasma and Mycoplasma.

[0116] More preferably, the Mollicutes bacterium according to the present invention is from the genus Mycoplasma.

[0117] In one embodiment of the liquid composition according to the present invention, the Mollicutes bacterium is one or more selected from M. pneumoniae, M. bovis, M. hyopneumoniae, M. meleagridis, M. gallisepticum, and M. synoviae.

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

[0119] In an embodiment of the liquid composition according to the present invention, the Mollicutes bacteria are M. gallisepticum and M. synoviae.

[0120] Experiments have shown that M. gallisepticum and M. synoviae can be stored in combination in the liquid composition according to the present invention, and no significant effect of one on the stability of the other can be detected when tested at 2-8°C for up to 23 weeks. In such a mixture, the two types of bacteria can be detected and counted when the same mixed sample is plated on two different types of agar plates (one with NAD (nicotinamide adenine dinucleotide) and the other without NAD) and the colony numbers are compared. As described in the literature (Olson & Kish Meadows, 1972, Avian Dis., vol. 16, p. 387-396), M. synoviae requires NAD for its growth, so on the plate without NAD, all the colonies observed are from M. gallisepticum.

[0121] In a preferred embodiment, the liquid composition according to the present invention can provide stabilization of live mollicutes bacteria, such that the number of mollicutes CFUs is reduced by 3 Log after 12 weeks of incubation at 4° C. 10 Even more preferably, the number of Mollicutes CFU does not decrease by more than 7 Log 10 The reduction should not exceed 10%.

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

[0123] In embodiments of the liquid composition according to the invention, one or more or all of the following conditions apply:

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

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

[0126] The water activity of the liquid composition is from 0.8 to 0.1; more preferably from 0.7 to 0.1, 0.6 to 0.1, 0.5 to 0.1, 0.4 to 0.1, 0.3 to 0.1, or even from 0.2 to 0.1, in this order of increasing preference.

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

[0128] The liquid composition has a water content of less than or equal to 40% by weight, more preferably less than or equal to 30, 25, 20, 15, 10, 8, 7, 6 or 5% by weight (in that order of increasing preference).

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

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

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

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

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

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

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

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

[0137] The sugar alcohol is one or more selected from erythritol, xylitol and sorbitol.

[0138] -erythritol is preferably CAS number 149-32-6; xylitol is preferably CAS number 87-99-0; sorbitol is preferably D-sorbitol, CAS number 50-70-4.

[0139] the sugar alcohol is one or more selected from the group of xylitol and sorbitol; in a more preferred embodiment the sugar alcohol is sorbitol.

[0140] the organic salt is one or more selected from the salts of betaine, proline, choline, methionine and arginine;

[0141] -betaine is preferably the compound N,N,N-trimethylglycine, CAS number 107-43-7, also known as glycine betaine;proline is preferably CAS number 609-36-9;choline is preferably CAS number 62-49-7;methionine is preferably L-methionine, CAS number 63-68-3;arginine is preferably L-arginine, CAS number 74-79-3.

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

[0143] When the organic salt is arginine, the arginine is used as a mixture of L-arginine (CAS number 74-79-3) and L-arginine hydrochloride (CAS number 1119-34-2).

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

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

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

[0147] The molar ratio of the organic salt to the polyhydric alcohol is preferably 1:4 to 4:1, more preferably 1:3 to 3:1, and even more preferably 1:2 to 2:1, in this order.

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

[0149] The molar ratio of sorbitol:xylitol:trehalose is 1:1:1, 1:1:5, 1:5:1, 5:1:1, 1:5:5, 5:1:5, 5:5:1, and 5:5:5.

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

[0151] The Mollicutes bacteria is one or more selected from the bacterial orders Acholeplasmales, Anaeroplasmales, Entomoplasmales and Mycoplasmales.

[0152] The Mollicutes bacteria is one or more selected from the bacterial families Acholeplasmataceae, Anaeroplasmataceae, Entomoplasmataceae, Spiroplasmataceae and Mycoplasmataceae.

[0153] The Mollicutes bacteria is one or more selected from the bacterial genera Acholeplasma, Phytoplasma, Anaeroplasma, Asterolplasma, Entomoplasma, Mesoplasma, Spiroplasma, Hepatoplasma, Marinoplasma, Moeniplasma, Mycoplasma and Ureaplasma.

[0154] The Mollicutes bacteria is one or more selected from the bacterial genera Acholeplasma, Spiroplasma, and Mycoplasma.

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

[0156] The Mollicutes bacteria is one or more selected from M. pneumoniae, M. bovis, M. hyopneumoniae, M. meleagridis, M. gallisepticum, and M. synoviae.

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

[0158] - Mollicutes bacteria are M. gallisepticum and M. synoviae.

[0159] 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 moisture content of 50% or less by weight; the NADES comprises an organic salt and a polyhydric alcohol, where the polyhydric alcohol is xylitol or sorbitol and the organic salt is selected from betaine, proline, choline and arginine; the Mollicutes bacterium is attenuated with respect to its pathogenicity, and the Mollicutes bacterium is one or more selected from M. pneumoniae, M. bovis, M. hyopneumoniae, M. meleagridis, M. gallisepticum, and M. synoviae.

[0160] In a preferred embodiment, the Mollicutes bacteria is M. gallisepticum and / or M. synoviae and the NADES include betaine and sorbitol.

[0161] 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 moisture content of 50% by weight or less; the NADES comprise three polyhydric alcohols: sorbitol, xylitol and trehalose; and the Mollicutes bacterium is attenuated with respect to its pathogenicity, the Mollicutes bacterium being one or more selected from M. pneumoniae, M. bovis, M. hyopneumoniae, M. meleagridis, M. gallisepticum, and M. synoviae.

[0162] As explained, the liquid compositions according to the present invention can be manufactured using common techniques and materials. Details and examples of such methods, uses or processes for preparing the liquid compositions according to the present invention are described herein, and such procedures are readily accessible to those skilled in the art using routine materials and methods.

[0163] For example, the NADES of the present invention can be manufactured on an industrial scale and then formulated into a liquid composition according to the present invention by mixing with a preparation of live Mollicutes bacteria. The mixture is then filled into containers of suitable size. The various stages of the manufacturing process can be monitored by adequate testing, for example biochemical or immunological testing of the quality and quantity of the bacteria.

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

[0165] Conveniently, the two steps of the method according to the invention can be separated in time and / or location and can be interposed by storage, transportation or even further steps, which allows flexibility in planning and logistics operations.

[0166] The preparation of the NADES used in the method according to the invention can be carried out using routine tools and methods. One convenient method is to heat the combined compounds to a temperature that allows them to be mixed and solubilized, for example by autoclaving. However, some compounds or compositions of the NADES used in the invention may not tolerate excessively high temperatures; a person skilled in the art can optimize and fine-tune such a process. The heating can be carried out, for example, in combination with stirring or ultrasonication.

[0167] Alternatively, one of the components forming the NADES can first be dissolved in water in a somewhat concentrated form and then (a) mixed with the further component. The excess water is then removed to form the NADES structure. Preferably, such removal is not done by freeze-drying. Another method is, for example, rotary evaporation in a water bath at 70° C. under reduced pressure for several hours.

[0168] The compositions comprising live Mollicutes bacteria for use in the methods according to the invention can be conveniently derived in a variety of ways, from in vivo cultures, but preferably from in vitro cultures of the bacteria in suitable media. Such bacterial cultures can be carried out in large industrial size fermenters applying automated control and monitoring techniques, at any desired size or volume, up to several thousand liters.

[0169] The bacteria are then harvested from the culture as a supernatant, cell pellet, or as the whole culture in a manner dependent on the characteristics of the bacteria. 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.

[0170] The bacterial composition used in the method of the invention can be prepared using further process steps, such as concentration, for example by passage through an ultrafiltration membrane. The bacterial composition is then checked for viability and quantity, all of which is known to those skilled in the art.

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

[0172] The order and method of mixing is not important in that the NADES can be added to the bacterial composition or otherwise. Preferably, any mechanical mixing used is low speed and low impact to prevent unnecessary shear forces on the bacteria. Additionally, mixing and / or filling can be performed in a protective atmosphere of inert gas (e.g., nitrogen) to reduce oxygen exposure.

[0173] Convenient equipment for mixing and filling relatively viscous liquids is available from a variety of process equipment suppliers.

[0174] In a further aspect, the present invention provides the use of the NADES described in accordance with the present invention as a stabiliser in a liquid composition comprising live mollicute bacteria.

[0175] "Stabilizer", "liquid composition", "live Mollicutes bacteria" are all as defined in any one or more of the embodiments described herein of the present invention.

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

[0177] Thus, in a further aspect, the present invention provides a liquid composition according to the invention or obtainable by a process according to the invention for use as a vaccine against infection or disease caused by a Mollicutes bacterium.

[0178] Similarly, in a further aspect, the present invention provides a liquid vaccine for use against an infection or disease caused by a Mollicutes bacterium, which vaccine comprises a liquid composition according to the present invention or obtainable by a method according to the present invention.

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

[0180] A "vaccine" is known to be a composition having a medical effect. It comprises an immunologically active ingredient and a pharma- ceutical acceptable carrier. The "immunologically active ingredient" in this case is represented by a live Mollicutes bacterium. The vaccine is recognized by the immune system of the target animal, which induces a protective immune response. The response may be derived from the innate and / or adaptive immune system of the target, and may be of the cellular and / or humoral type.

[0181] Vaccines are generally effective in reducing the severity of an infection, for example by reducing the number of pathogens present or by shortening the period of time for the pathogen to replicate in the host animal.

[0182] Additionally, or perhaps as a consequence thereof, a vaccine is generally effective in reducing or ameliorating the (clinical) symptoms of a disease which may be caused by such infection or replication, or by the target's response to that infection or replication.

[0183] A "pharmaceutically acceptable carrier" in the present invention is a liquid of high purity, preferably sterile. In this case, the carrier is a NADES as defined herein. The carrier may contain further additives such as stabilizers, antioxidants, or preservatives.

[0184] Details regarding "infections or diseases caused by Mollicutes bacteria" are known in the art and can be found, for example, in handbooks such as the Merck Veterinary Manual (supra) and Diseases of Poultry, 12th edition, 2008, Wiley-Blackwell, ISBN: 0813807182.

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

[0186] Those skilled in the art of the invention are well able to determine and optimize such immunologically effective amounts of live Mollicutes bacteria in liquid vaccines according to the invention, for example by monitoring the immune response after vaccination and, in the case of non-human animal targets, administering a challenge infection and then monitoring the targets for disease signs, clinical scores, and pathogen dissemination and spread, and comparing these results with the vaccination challenge response seen in mock-vaccinated animals.

[0187] Methods for determining the amount of live bacteria in a vaccine dose or animal sample are known in the art and typically involve the use of bacterial culture and counting techniques. Conveniently, such amounts of bacteria can be expressed as a numerical value, for example in CFU.

[0188] The target of the liquid vaccine according to the invention is a human or non-human animal in need of vaccination against an infection or disease caused by the pathogenic form of the particular live Mollicutes bacterium contained in the vaccine. The age, weight, sex, immunological status and other parameters of the vaccinated target are not critical, but it is preferred to vaccinate healthy, uninfected targets and to vaccinate as soon as possible to prevent field infections.

[0189] A liquid vaccine according to the invention may further comprise other compounds such as additional antigens or microorganisms, cytokines, or immunostimulatory nucleic acids including unmethylated CpG, adjuvants, etc. Alternatively, a liquid vaccine according to the invention may itself be added to the vaccine.

[0190] The liquid vaccine according to the invention can advantageously be combined with one or more further antigens or microorganisms, for example derived from a microorganism pathogenic to the intended human or non-human animal target. Such further antigens can be live, attenuated or inactivated infectious microorganisms themselves, or they can be subunits, extracts or fractions etc. The further antigens can consist of biological or synthetic molecules, such as proteins, carbohydrates, lipopolysaccharides, lipids or nucleic acid molecules.

[0191] Liquid vaccines according to the invention may contain preservatives such as thimerosal, merthiolate, phenolic compounds, and / or gentamicin. Preferably, no preservatives are used.

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

[0193] Liquid vaccines according to the invention may contain antioxidants such as ascorbic acid, citrate, methionine or tocopherol.

[0194] The liquid vaccine according to the present invention may be used as a prophylactic or therapeutic treatment, or both.

[0195] A liquid vaccine according to the invention can serve as an effective priming vaccination, followed by one or more booster vaccinations with the same or different vaccines.

[0196] Liquid vaccines according to the invention can be prepared using known techniques incorporating methods for producing liquid compositions according to the invention.

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

[0198] Such manufacturing incorporates microbiological testing for sterility and absence of adventitious agents; and ultimately vaccination trials to confirm efficacy and safety. After quality, quantity, sterility, safety and efficacy testing is completed, the vaccine product can be released.

[0199] All of this is known to those of skill in the art, and the general techniques and considerations that apply to vaccine production under known standards of pharmaceutical manufacturing are described, for example, in government directives and regulations (Pharmacopeia, 9 CFR) and known handbooks (Pastoret, Lippincott, both supra).

[0200] The liquid vaccine according to the invention can be administered to humans or non-human animals, for example in the form provided by the pharmaceutical company. However, depending on the specific composition of a particular embodiment of the liquid vaccine according to the invention, it may be preferable to carry out a dilution of the liquid vaccine. This may facilitate the actual administration to the target, for example by reducing the viscosity, or may reduce local reactions upon vaccination of the target.

[0201] The diluent for such dilution is typically a liquid and 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.

[0202] Therefore, in a further aspect, the present invention relates to a kit of parts comprising at least two containers: one container containing a liquid vaccine according to the invention, and one container containing a liquid diluent.

[0203] A "liquid diluent" for diluting a liquid vaccine according to the invention, for example included in a kit of parts according to the invention, is a liquid suitable for diluting a liquid vaccine according to the invention. The liquid diluent can therefore be water or water-based, such as a buffer. However, due to the low water content of liquid vaccines according to the invention, the diluent can also be oil-based or an emulsion of oil and water, such as an oil-in-water emulsion, a water-in-oil emulsion, or a double emulsion.

[0204] In one embodiment, the liquid diluent may itself be a liquid vaccine, for example a reconstituted live lyophilized vaccine or an emulsion vaccine.

[0205] The choice of a liquid diluent for diluting a liquid vaccine according to the invention, e.g. for inclusion in a kit of parts according to the invention, depends on several factors, such as the intended use of the vaccine after dilution, the Mollicute bacterium it contains, the intended human or non-human animal target of vaccination, etc. The skilled person is fully capable of selecting, preparing and applying such a diluent.

[0206] In one embodiment of a kit of parts according to the invention, the kit includes instructions for use of the kit and / or its component parts. In a preferred embodiment, the instructions are provided on or with one or more of the components of the kit, e.g., by a manufacturer or distributor of the kit; or by reference to instructions in electronic form, such as information viewable or downloadable from an internet website.

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

[0208] In one embodiment of the kit of parts, the kit may also be an offering (in association with commercial sale) of the components for use in a vaccination method according to the invention, for example on an internet website.

[0209] The liquid vaccine according to the invention, or the liquid vaccine that can be produced by the method or use according to the invention, or in diluted form that can be prepared, for example, with the kit of parts according to the invention, is intended for administration to a human or non-human animal target in need of such vaccination. The vaccination induces active immunity to reduce infection and / or disease signs that may be caused by infection with a non-attenuated counterpart of a live Mollicutes bacterium of the invention. This then leads to a reduction in the number, intensity or severity of lesions and the resulting clinical signs of disease caused by bacterial infection in the target human or non-human animal. Such a vaccine is colloquially referred to as a vaccine "against" a specific Mollicutes bacterium, or a "Molicutes vaccine".

[0210] Thus, in a further aspect, the present invention provides a liquid vaccine according to the present invention for use in a method for protecting a human or non-human animal target against infection and / or disease caused by a pathogenic type of a live Mollicutes bacterium contained in the liquid vaccine.

[0211] Such a "pathotype live Mollicutes bacterium contained in said liquid vaccine" is, for example, the non-attenuated parent Mollicutes bacterium from which the attenuated form of the Mollicutes bacterium of the present invention was generated. Alternatively, such a pathotype bacterium can be readily derived from a variety of sources, as described herein. One skilled in the art of the present invention is fully capable of determining whether a Mollicute is a pathotype, for example, by comparative in vitro or in vivo testing as described herein.

[0212] A "method of protection" involves administering a liquid vaccine according to the present invention to said human or non-human animal target such that the vaccine "obtains" (i.e. establishes a protective infection) and exhibits its favorable immunological effect in the target.

[0213] In a further aspect, the present invention provides a method for vaccinating a human or non-human animal target against an infection or disease caused by a Mollicutes bacterium, the method comprising administration to said target of a liquid vaccine according to the invention or comprising the use of a kit of parts according to the invention.

[0214] 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. This latter embodiment of the vaccination method according to the invention may facilitate handling and administration by injection due to its reduced viscosity. Alternatively or additionally, administration in a diluted form may reduce local reactions in the human or non-human animal target, for example due to tissue irritation at the site of inoculation.

[0215] Vaccination according to the method of the invention can be carried out by the parenteral route, i.e. through the skin, for example intramuscularly, intraperitoneally, intradermally, submucosally or subcutaneously. The preferred route of parenteral administration of a liquid vaccine composition according to the invention is by intramuscular or subcutaneous injection.

[0216] Furthermore, the liquid vaccine according to the present invention can be administered by enteral or mucosal routes, ie via eye drops, nasal drops, oral, enteral, oronasal drops, spray, aerosol.

[0217] Alternatively, liquid vaccines according to the present invention can be administered to targets such as via mass administration methods via drinking water, coarse spray, atomization, feed addition, etc. A further alternative is administration to embryonic birds via the in ovo route.

[0218] Of course, the optimal route of administration of a liquid vaccine according to the invention will depend on the details of the vaccine used and the specific properties of the target, and the skilled artisan is fully capable of selecting and optimizing such route and method of administration.

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

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

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

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

[0223] The method, timing and volume of administration of the liquid vaccine according to the invention is preferably integrated into existing vaccination schedules of other vaccines that the target human or non-human animal may require, in order to reduce stress on the target and to reduce labor costs. These other vaccines can be administered in a simultaneous, contemporaneous or sequential manner, in a manner compatible with their registered use.

[0224] Further optimization of the liquid vaccine according to the invention is within the reach of a person skilled in the art. In general, this involves fine-tuning the efficacy of the vaccine to further improve the immune protection provided. This can be done, for example, by adjusting the dose, volume or antigen content of the vaccine, or by applying it via a different route, method or administration method. All of this is within the scope of the present invention.

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

[0226] Example 1: Methods and Materials 1.1: Preparation of NADES formulations NADES can be prepared by mixing the desired ingredients, which can be accelerated by heating and / or the addition of small amounts of water. Samples of NADES, further tested and described herein below, were prepared by dissolving the relevant compound in water via sonication in a water bath. The required amount of compound was added to a flask. The flask was briefly shaken or vortexed to mix the powder. The required amount of water was then added. This mixture was placed in a sonication-water bath set at 45° C. and sonicated for up to several hours until the NADES was formed. Temperatures of up to 80° C. could be reached during sonication.

[0227] 1.2: Measurement of water activity sample For biocontainment purposes, the water activity of a sample of the NADES preparation that does not contain viable bacteria was thus measured. However, knowing the amount of aqueous bacterial composition added to the NADES to form a liquid vaccine composition according to the present invention, the resulting water activity of the combination of the NADES and the bacterial composition can be calculated according to Dai et al. (2015, Food Chem., vol. 187, p. 14-19).

[0228] Sample preparation An amount of NADES sample was transferred to a 10 mL glass vial to reach a fill height of approximately ¼ of the vial. For each sample, a coded vial was filled in duplicate. The vials were closed using aluminum crimp caps with PTFE stoppers and left at room temperature for 12 hours to allow equilibrium between the sample and the headspace to be reached.

[0229] Device The water vapor pressure in the headspace of the samples was measured using a Lighthouse™ FMS-1400 moisture / pressure analyzer. The samples were inserted directly into the sample chamber. Prior to sample measurement, the sample was placed in a 0-0.2a wThe system was calibrated using calibration headspace pressure standards ranging from 0.2 to 1a. Saturated salt standards were also used to calibrate the system. w The nonlinear range of was calibrated and recalculated.

[0230] calibration a w To calibrate the machine over the full range of 0-1 and to create a baseline of water activity values, several standard samples were measured: pure water, followed by five samples of a specific saturated salt solution, measured in triplicate for each of the six standard duplicate samples. By setting the pure water reading at 1.000, the a values ​​of 0.8511, 0.7547, 0.5438, 0.3307, and 0.1131 were obtained. w Other standard values ​​were determined using the standard. A series of certified vapor pressure standards were also measured.

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

[0232] 1.3: Examination of DSC profile of NADES Differential scanning calorimetry (DSC) analysis was performed on a Q2000 DSC instrument from TA Instruments using Thermal Analysis software. For a complete DSC profile, the sample was first equilibrated at 40°C, cooled to -90°C at 5°C / min, followed by equilibration at -90°C for 5 min, after which the sample was heated again to 40°C at 5°C / min. This cooling and heating cycle was repeated a second time on the same sample to investigate phase hysteresis. The T of the sample was determined by the software from the heating curve by the midpoint of the stepwise shift in the baseline. g was calculated, which indicates the change in thermal conductivity due to the increased mobility during the glass transition.

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

[0234] 1 shows a sharp shift in the baseline, here from -41 to -45°C, indicating a glass transition, which corresponds to a characteristic of a useful NADES in the present invention. This profile is essentially different from that presented by Qiao et al. (supra) in their FIG. 4 for a NADES that is too diluted into an aqueous composition and shows an essentially flat baseline, with an increase and decrease in heat flow upon the formation and melting of water-ice crystals: the so-called "cold-crystallization".

[0235] 1.4: Mixing of NADES and bacterial compositions The NADES preparation was mixed 9+1 (by volume) with the bacterial composition in a laminar flow cabinet. Samples were mixed gently using a roller bank; vortexed; or briefly stirred using a sterile inoculation loop depending on the sample size. From these samples, 1 mL aliquots were filled into 3 mL glass vials and capped with rubber stoppers and metal caps.

[0236] 1.5: Stability assay 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 a designated period, the vial was opened and a 10-fold dilution in PBS was added to 10 -1 ~10 -10 Dilutions were prepared. For at least three dilutions, in duplicate, 50 μL samples were plated on agar plates that support the growth of the specific bacteria. The plates were then incubated at 37°C and 5% CO2 for 5-8 days until the colonies were large enough to be counted. From this, the titer of Mollicutes bacteria was calculated, whereby 30-300 colonies were counted. The titer was expressed as colony forming units per mL of sample (CFU / mL). Data points from different vials examined at different time points allowed the construction of stability data graphs, as shown in Figures 2 and 3.

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

[0238] Table 1 below lists a number of the most effective NADES formulations; these produced clear liquid solutions that were fluid at room temperature (approximately 20°C). Details of their composition and water content are provided. The water activity of some of the samples prepared was determined and the results are included.

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

[0240] Example 3: Bacterial stability assay results Stability assays were carried out for M. synoviae and M. gallisepticum bacteria contained in liquid compositions according to the present invention over extended periods of time at temperatures between 2-8° C. and at room temperature (approximately 20° C.).

[0241] Incubation at room temperature for 8 weeks could serve as a rapid stability test to show the effect of incubation for 1-2 years at 4° C. Thus, this was a very serious attack on bacterial viability, and not all samples were expected to survive under these conditions.

[0242] Conclusions from stability results The inherent low stability during storage of both M. synoviae and M. gallisepticum was demonstrated by a control, a 1+9 (volume ratio) mixture in standard phosphate buffered saline (PBS). See Figures 2 and 3 for exemplary details. In this control liquid composition, already at the first time point tested, bacterial colonies were no longer detectable in any of the dilutions examined. This was true for both storage conditions at 2-8°C and room temperature.

[0243] From these control samples it was concluded that there was no detectable survival of either M. synoviae or M. gallisepticum in PBS.

[0244] Surprisingly, and in stark contrast to the PBS control, both M. synoviae and M. gallisepticum showed significant survival when combined with NADES in a liquid composition according to the invention, and measurable titers were obtained when mixed with various NADES at a 1+9 (volume ratio) and stored at 20° C. for up to 8 weeks.

[0245] For M. synoviae, the stabilizing effect at 20°C was strongest for NADES with a molar ratio of 1:1:2.5 betaine:sorbitol:WFI and a molar ratio of 2:2:1:10 sorbitol:xylitol:trehalose:WFI. Storage at 2–8°C for up to 6 months resulted in significant viability titers, with some NADES losing over 3 Log 10 There was a loss of less than CFU / mL.

[0246] For M. gallisepticum, the stabilizing effect at 20°C was strongest for NADES with a molar ratio of arginine:sorbitol:WFI of 1:2:6 and a molar ratio of proline:sorbitol:WFI of 1:1:2.5. Storage at 2–8°C for up to 3 months resulted in significant viability titers, with some NADES showing a stabilizing effect of up to 3 Log. 10 There was a loss of less than CFU / mL.

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

[0248] For this purpose, two variants of NADES based on proline:sorbitol were prepared and tested: one with high viscosity (NADES 3A) and one with low viscosity (NADES 3B). These served to compare the dosing characteristics of the NADES of the present invention at the two extremes of the viscosity spectrum. The characteristics of NADES 3A are shown in Table 1. Furthermore, their viscosities were measured using standard conditions at about 20° C. using a Brookfield™ DV-I+ viscometer, spindle type No. 62, at 60 rpm for 30 seconds in a standard measuring cup of about 80 mL.

[0249] handling High viscosity NADES 3A could be forced into hypodermic needles of size 21G or smaller, i.e., needles with an outer diameter of 0.8 mm or greater.

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

[0251] In vivo subcutaneous administration NADES formulations 3B and 3C were administered by subcutaneous injection in a safety study in dogs. Special attention was paid to any possible side effects following local or systemic administration of the stabilizer formulations.

[0252] Both formulations were prepared by heating and sonicating proline, sorbitol, and water for injection in a sealed vial as described above to obtain a clear, homogenous liquid. They were further filter-sterilized and their bioburden and endotoxin levels were determined. Both formulations were found to be below the minimum dilution criterion of 1 unit / mL and to meet the general acceptability criteria for sterile formulations. NADES 3C formulation was prepared by diluting 1 part by volume of NADES 3A with 9 parts by volume of WFI to obtain a formulation containing proline:sorbitol:WFI in a ratio of 1:1:5 with a viscosity of 289 mPa·s. The viscosity of NADES 3B is 33.4 mPa·s.

[0253] Six healthy beagle dogs, 7 months of age, were divided into two groups of three dogs. Each group was assigned to a NADES formulation and administered two doses of 1 mL of the assigned formulation via subcutaneous inoculation, two weeks apart, using a 1 mL syringe and a 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.

[0254] Vaccinations were administered in the nape of the neck: the first vaccination was administered on the left side and the second vaccination on the right side of the neck. Prior to each dose, the vaccination site was shaved and the injection site was circled with a marker pen. No discomfort was noted during any of the injections. Four hours after injection, the injection site was palpated and there was little or no palpable material at the injection site, and no swelling or redness was observed.

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

[0256] No significant increases in body temperature were observed in any of the dogs throughout the study, and no clinical side effects were observed.

[0257] Conclusions: Inoculation of dogs with NADES formulations 3C or 3B via the subcutaneous route did not induce any adverse inoculation reactions, either locally or systemically.

[0258] Intranasal 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 neat, without the addition of water. The resulting viscosity of NADES 3A is 5197 mPa·s and that of NADES 3B is 33.4 mPa·s. A spatula tip of "patent blue" dye was added to 30 mL of each NADES formulation to obtain a deep blue color. PBS containing dye was used as a control.

[0259] Calf heads were obtained postmortem from calves approximately 2-4 weeks of age. Dosing was performed with the head held in an upward position with the nasal passages tilted slightly upward to mimic the position of the calf during nasal dosing on the farm.

[0260] The liquid was then administered according to the standard procedure for intranasal vaccination, whereby a 2.5 mL syringe without a needle containing 1.5 mL of liquid was placed in the nostril and the liquid was then squirted out in one steady stream. The head was then bisected and the septum removed to examine the presence of blue color in the intranasal passages.

[0261] In all samples, blue liquid was observed moving easily through the nasal passages. The low viscosity of the PBS samples made them easier to inject, but all liquid passed rapidly. The blue color was observed throughout the entire nasal cavity, including the beginning of the trachea.

[0262] Injection of the very viscous NADES 3A required little force to inject from the syringe. Also, as expected, it took a relatively long time for the blue liquid to appear under the head. After incision of the head, this viscous liquid was also observed to spread evenly in the intranasal passages and appear to form a thick layer, with more material remaining in the nasal cavity. This indicates that there is likely to be improved retention and less penetration into the upper respiratory tract when administered to live calves during nasal vaccination.

[0263] The effect of administration of the low viscosity NADES 3B formulation was very similar to that of the PBS sample, which allows liquid to pass through the nasal cavity.

[0264] Conclusion: After intranasal injection into the head of a calf, the distribution of colored formulations of different viscosities could be monitored. The distribution of the liquid, as judged by the blue color, was comparable for the two NADES formulations and PBS, but there were some differences regarding the ease of administration and the amount of residue, with the NADES 3A formulation being slightly more difficult to expel but resulting in a thicker layer of residue in the nasal passages. This suggests that a sustained release of antigen from such viscous NADES formulations may be obtained.

[0265] Example 5: Comparative measurements of aqueous stabilizers An aqueous stabilizer composition was prepared as described in WO2015 / 121463 having 30 wt% sorbitol + 0.6M arginine in 0.04M PBS. This composition was characterized, such as for water activity and DSC profile, and revealed that it did not possess the characteristic properties that make it a NADES as defined and outlined above.

[0266] water activity a w The water activity was measured by headspace vapor pressure measurement according to the method described in Example 1 of WO2020 / 122329. The water activity results were measured in duplicate and the average a w was 0.89. Clearly, this high level of available water indicates that this composition is not a NADES.

[0267] DSC Profile Measurement of the DSC profile of the prior art aqueous stabilizer was carried out according to the method described herein in Example 1.3.

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

[0269] During cooling, extensive and sudden crystallization of free water into ice can be observed at a temperature of about −27° C. Then, upon reheating, a typical ice crystal melting profile was observed, with a peak at −5.5° C.

[0270] Moreover, in Figure 4, during the heating period, the horizontal baseline after ice melting returns to the same level as it was before it melted, meaning that there was no change in the heat capacity of the sample, which is clearly different from the gradual baseline shift typically observed in the case of a glass transition.

[0271] It is also important to note that the melting of ice in Figure 4 occurs at a much higher temperature (-5 °C) than the glass transition in Figure 1 (-45 °C), again indicating that the sample examined in Figure 4 was not a deep eutectic mixture.

[0272] In FIG. 4, both peaks, the high melting temperature and the return of the baseline to previous levels are all indicative of ice crystal formation and melting, and therefore indicate the presence of a large amount of free water in the composition studied and therefore that it is not a NADES.

[0273] However, in contrast, the sample of FIG. 1 (the NADES according to the present invention) neither formed nor melted ice crystals, and a clear baseline glass transition shift was observed.

[0274] Example 6: Additional Stability Results: M. bovis A further species of Mollicute bacteria: Mycoplasma bovis was tested for stabilization with the liquid composition according to the invention. The experiments were carried out essentially according to the methods described in Examples 1-3 for M. synoviae and M. gallisepticum.

[0275] Different compositions according to the invention and the results of a stability assay at either room temperature or 2-8° C. are shown in FIG.

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

[0277] The initial titer was 9.9 Log 10 CFU / mL. Some survival was observed in PBS (negative control) at 2 weeks of incubation, both at room temperature and when refrigerated. However, in samples incubated for longer periods in PBS, no viable cells were recoverable any more.

[0278] At room temperature, good initial stability (2-4 weeks of incubation) was obtained with several NADES compositions, however, best results were obtained using arginine:sorbitol:water (2:1:6), which was able to stabilize the bacteria at room temperature for at least 10 weeks.

[0279] A similar picture was observed with refrigerated incubation, with good initial stabilization from 6 to 13 weeks observed for some of the compositions examined. The best long-term stabilizer (26 weeks of incubation) was also arginine:sorbitol:water (2:1:6), but the differences observed between the different compositions examined were not as great as with incubation at room temperature.

[0280] Overall, M. bovis could be effectively stabilized in liquid compositions according to the present invention at various storage temperatures above freezing without lyophilization.

[0281] Example 7: Results of an expanded stability study Stability test results for M. synoviae and M. gallisepticum in liquid compositions according to the invention were obtained from continuous incubation for up to one year at 2-8° C. These data were obtained from the same set of samples as described in Example 3 and extend the results shown in Figures 2B and 3B herein.

[0282] The enlarged results are shown in FIG.

[0283] Panel A: M. synoviae, 2–8°C, up to 54 weeks; and Panel B: M. gallisepticum, incubated at 2–8°C for up to 52 weeks.

[0284] In the case of M. synoviae, three of the liquid compositions tested according to the invention are able to stabilize the viability of these bacteria for at least one year at 2-8°C; thus, proline:sorbitol:water (1:1:2.5) and betaine:sorbitol:water (1:1:2.5) showed the same end result.

[0285] In the case of M. gallisepticum, viable bacteria were only retained in betaine:sorbitol:water (1:1:2.5) at 2–8°C for up to 52 weeks of incubation.

Claims

1. A liquid composition comprising a live Mollicutes bacterium and a stabilizer, the stabilizer being a natural deep eutectic solvent (NADES); wherein the composition has a water activity of 0.85 or less, and wherein the composition has a water content of 50% by weight or less, And here, the NADES is i: an organic salt and a sugar alcohol, or ii) two or more compounds, each of which is a polyhydric alcohol; wherein the sugar alcohol is selected from xylitol and sorbitol; wherein the polyhydric alcohol is selected from trehalose and one or more sugar alcohols; and wherein the organic salt is selected from the group consisting of betaine, proline, choline, methionine and arginine salts. liquid composition.

2. The NADES is i. betaine and sorbitol, ii. proline and sorbitol, iii. choline and sorbitol; iv. arginine and sorbitol, and v. sorbitol, xylitol and trehalose, The liquid composition of claim 1 , wherein the liquid composition is selected from:

3. 3. The liquid composition according to claim 1 or 2, wherein the mollicute bacteria are attenuated with respect to their pathogenicity.

4. A method for producing the liquid composition according to any one of claims 1 to 3, comprising the steps of: - preparing said NADES, and - mixing said NADES with live Mollicutes bacteria The method includes:

5. Use of a NADES according to any one of claims 1 to 3 as a stabilizer in a liquid composition comprising live mollicute bacteria.

6. The liquid composition according to any one of claims 1 to 3 for use as a vaccine against infection or disease caused by Mollicutes bacteria.

7. A liquid vaccine for use against an infection or disease caused by a Mollicutes bacterium, comprising the liquid composition according to any one of claims 1 to 3.

8. Use of a NADES according to any one of claims 1 to 3 as a pharma- ceutically acceptable carrier in a liquid vaccine according to claim 7.

9. Use of the NADES according to any one of claims 1 to 3 for the manufacture of a liquid vaccine according to claim 7.

10. A kit of parts comprising at least two containers, one containing a liquid vaccine according to claim 7, and one containing a liquid diluent.

11. 8. A liquid vaccine according to claim 7 for use in a method for protecting a human or non-human animal target against infection and / or disease caused by a pathogenic type of a live Mollicutes bacterium contained in said liquid vaccine.

12. A liquid vaccine according to claim 7 for vaccination of human or non-human animal targets against infection or disease caused by Mollicutes bacteria.

13. A kit of parts according to claim 10 for vaccination of a human or non-human animal target against an infection or disease caused by a Mollicutes bacterium.

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