STABILISATION OF LIVE MOLLICUTES BACTERIA IN A LIQUID COMPOSITION

Abstract

The present invention relates to a liquid composition of live Mollicutes bacteria and a stabiliser, whereby the stabiliser is a natural deep-eutectic solvent (NADES). In this liquid composition the live Mollicutes are stabilised without need for freezing or freeze-drying. This allows various advantageous uses in diagnostics and medicine, specifically as a liquid vaccine for use against infection or disease caused by Mollicutes bacteria, for human- or non-human animals.

Claims

1. A liquid composition comprising live Mollicutes bacteria and a stabiliser, wherein the stabiliser is a natural deep-eutectic solvent (NADES).

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

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

4. The liquid composition of claim 1, wherein the NADES comprises an organic salt and a polyol.

5. The liquid composition of claim 1, wherein the NADES comprises two or more compounds that are all polyols.

6. The liquid composition of claim 4, wherein the polyol is a sugar or a sugar-alcohol.

7. The liquid composition of claim 6, wherein the sugar is selected from sucrose and trehalose, or in that the sugar-alcohol is one or more selected from: erythritol, xylitol, and sorbitol.

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

9. The liquid composition of claim 1, wherein the Mollicutes bacteria are attenuated in respect of their pathogenic properties.

10. A method for manufacturing the liquid composition of claim 1, comprising the steps of; a. preparing the NADES, and b. admixing said NADES with live Mollicutes bacteria.

11. (canceled)

12. A liquid composition obtainable by the method of claim 10.

13. A liquid vaccine for use against infection or disease caused by Mollicutes bacteria, comprising the liquid composition of claim 1.

14. (canceled)

15. (canceled)

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

17. (canceled)

18. A method of vaccinating a human or non-human animal target against infection or disease caused by Mollicutes bacteria, comprising administering to said target the liquid vaccine of claim 13.

19. A method of vaccinating a human or non-human animal target against infection or disease caused by Mollicutes bacteria, comprising mixing from the kit of parts of claim 16, the contents of the container comprising the liquid vaccine with the contents of the container comprising a liquid diluent to form a mixture and then administering the mixture to the human or non-human animal target.

20. A liquid vaccine for use against infection or disease caused by Mollicutes bacteria, comprising the liquid composition of claim 12.

21. A method of vaccinating a human or non-human animal target against infection or disease caused by Mollicutes bacteria, comprising administering to said target the liquid vaccine of claim 20.

Description

EXAMPLES

Example 1: Methods and Materials

1.1 Preparing the NADES Formulations

[0225] NADES can be prepared by mixing of the desired components, which can be accelerated by heating and/or addition of a small amount of water. The samples of NADES as further tested and described herein below were prepared by dissolving the relevant compounds in water via sonification in a waterbath. The required quantities of compounds were added to a flask. The flask was briefly shaken or vortexed, to mix the powders. Next the required amount of water was added. This mixture was placed in a sonication-waterbath, set at 45° C., with sonication up to several hours, until a NADES was formed. During sonication temperatures of up to 80° C. could be reached.

1.2 Measurements of Water Activity

Samples

[0226] For biological containment purposes the water activities were measured of samples of NADES preparations, so without live bacteria. However, when knowing the amount of aqueous bacterial composition that will be added to the NADES to form the liquid vaccine composition according to the invention, the resulting water activity of the combination of NADES and bacterial composition can be calculated according to Dai et al. (2015, Food Chem., vol. 187, p. 14-19).

Sample Preparation

[0227] An amount of NADES sample was transferred to a 10 ml glass vial in order to reach a fill height of approx. ¼ of the vial. For each sample coded vials were filled in duplicate. The vials were closed using an aluminium crimp cap with PTFE stopper, and were placed at room temperature for 12 hours to reach an equilibrium between sample and headspace.

Equipment

[0228] The moisture vapour pressure in the headspace of the samples was determined using the Lighthouse® FMS-1400 Moisture/Pressure analyser. The samples were inserted into the sample chamber as such. Before sample measurement the system was calibrated using calibration headspace pressure standards in the range of 0-0.2 a.sub.w. Also, saturated salt standards were used to calibrate and recalculate the non linear range from 0.2-1 a.sub.w.

Calibration

[0229] To calibrate the machine over the full range from a.sub.w 0-1, and to create a reference line of water activity values, a number of standard samples were measured: next to pure water 5 samples of specific saturated salt-solutions were used, and for each of the 6 standards duplo samples were measured in 3 fold. By setting the measured value for pure water to 1.000, the other standard values were determined at a.sub.w of: 0.8511, 0.7547, 0.5438, 0.3307, and 0.1131. Also, a series of certified vapour pressure standards were measured.

[0230] Results of a.sub.w measurements are included in Table 1.

1.3 Testing the DSC Profile of a NADES

[0231] Differential scanning calorimetry (DSC) analysis was done on a Q2000 DSC apparatus from TA Instruments using Thermal Analysis software. For a full DSC profile, samples were first equilibrated at 40° C. and cooled to −90° C. at 5° C./min, followed by equilibration for 5 min at −90° C. after which samples were heated again to 40° C. at 5° C./min. This cycle of cooling and heating was repeated a second time on the same sample to investigate phase hysteresis. The Tg of a sample was determined by the software from the heating curve by the midpoint of the stepwise shift in the baseline, indicating a change in thermal conductivity due to increased mobility during the glass transition.

[0232] A representative DSC profile of a NADES for the invention is presented in FIG. 1, for a NADES formed at a molar ratio for proline, sorbitol, and water of 1:1:2.5.

[0233] FIG. 1 shows the steep shift of the base line, here between −41 and −45° C., that indicates a glass-transition, which corresponds with the properties of an effective NADES for the invention. This profile is essentially different from the one as presented by Qiao et al. (supra), in their FIG. 4, of a NADES that was overly diluted and became an aqueous composition; that shows an essentially even baseline, with raise and drop of the heat flow upon the forming and melting of water-ice crystals; so-called ‘cold-crystallisation’.

1.4 Mixing NADES and Bacterial Compositions

[0234] NADES preparations were blended 9+1 v/v with bacterial compositions in a laminar flow cabinet. Samples were either gently mixed using a roller bank; vortexed; or stirred briefly using a sterile inoculation loop, depending on sample size. From these samples 1 ml aliquots were filled into 3 ml glass vials that were capped with rubber stopper and metal cap.

1.5 Stability Assays

[0235] For each test sample a unique vial was stored at refrigerated conditions (2-8° C.), frozen conditions (−20° C.) or at room temperature (20° C.). After a defined period, the vial was opened and a serial tenfold dilution in PBS was prepared from 10{circumflex over ( )}−1 to 10{circumflex over ( )}−10 dilution. For at least three dilutions a 50 μl sample was plated on agar plates supporting the growth of the specific bacterium, in duplo. Next, plates were incubated at 37° C. and 5% CO.sub.2 for 5-8 days until colonies were of sufficient size to be counted. From this the titre of the Mollicutes was calculated, whereby between 30 and 300 colonies were counted. The titre was expressed as colony forming units per ml sample (CFU/ml). From the data points of the different vials that are tested at the different time points, a graph of the stability data was created, as is represented in FIGS. 2 and 3.

Example 2: Composition of NADES for Use in the Invention

[0236] A large number of different NADES compositions were prepared for use in the invention, with different compounds, with different molar ratios, and with different water contents.

[0237] Table 1 below lists a number of the most effective NADES formulations; these yielded clear liquid solutions, that were fluid at room temperature (about 20° C.). Details on their composition and water content are provided. For several of the samples prepared, the water activity was determined, and the results are included.

TABLE-US-00001 TABLE 1 Compositions and properties of a representative number of NADES for the invention Molar ratios % water (w/w) a.sub.w (21° C.) Tg (° C.) Proline Sorbitol — WFI 1:1:2.5 13.1 0.39 −42 Betaine Sorbitol — WFI 1:1:2.5 13.1 0.32 −50 Arginine Sorbitol — WFI 1:2:6 15.9 0.60 −48 Choline-Cl Xylitol — WFI 2:1:2.5 9.5 0.18 −75 Xylitol Sorbitol Trehalose WFI 2:2:1:10 14.7 0.59 −49

Example 3: Results of Bacterial Stability Assays

[0238] For M. synoviae and M. gallisepticum bacteria, comprised in liquid compositions according to the invention, stability assays were performed over an extended period of time, and temperatures of 2-8° C. and at room temperature (about 20° C.).

[0239] The incubation at room temperature for 8 weeks can serve as an expedited stability test, indicative of the effect of incubation for 1-2 years at 4° C. This is thus a very severe attack on the bacteria's viability, and not all samples were expected to survive under those conditions.

Conclusions from the Stability Results

[0240] The inherent poor stability of both M. synoviae and M. gallisepticum bacteria during storage was demonstrated by the control, being the 1+9 v/v admixture in standard Phosphate Buffered Saline (PBS). Please refer to FIGS. 2 and 3 for exemplary details. In this control liquid composition, already at the first timepoint tested no bacterial colonies could be detected anymore, at any of the investigated dilutions. This applied both to storage conditions at 2-8° C. and at RT.

[0241] From these control samples, it was concluded that there was no detectable survival of either M. synoviae or M. gallisepticum in PBS.

[0242] Surprisingly, and in sharp contrast with the PBS controls, both M. synoviae and M. gallisepticum showed impressive survival when combined with NADES in a liquid composition according to the invention. When admixed 1+9 v/v with different NADES, measurable titres were obtained upon storage at 20° C. for up to 8 weeks.

[0243] For M. synoviae the stabilizing effect at 20° C. was strongest with the NADES' Betaine:Sorbitol:WFI at 1:1:2.5 molar ratio, and Sorbitol:Xylitol:Trehalose:WFI at 2:2:1:10 molar ratio. Upon storage at 2-8° C. for up to 6 months impressive survival titres were found, with losses of less than 3 Log 10 CFU/ml for several NADES.

[0244] For M. gallisepticum the stabilizing effect at 20° C. was strongest with the NADES' Arginine:Sorbitol:WFI at 1:2:6 molar ratio, and Proline:Sorbitol:WFI at 1:1:2.5 molar ratio. Upon storage at 2-8° C. for up to 3 months, impressive survival titres were found, with losses of less than 3 Log 10 CFU/ml for several NADES.

Example 4: Administration of NADES to Experimental Animals

[0245] To assess the ease of administration, and the safety of a NADES for use in the invention in target animals, examples of a NADES were administered to experimental animals and to post mortem materials.

[0246] For this purpose, two variants of a Proline:Sorbitol based NADES, were made and tested: one with high (NADES 3A) and one with low (NADES 3B) viscosity. These served to compare the administration-properties of NADES for the invention at two extremes of the viscosity spectrum. The characteristics of NADES 3A are described in Table 1. Further, their viscosities were measured using standard conditions: at about 20° C., using a Brookfield® DV-I+ viscometer, utilising spindle type No. 62, for 30 seconds at 60 r.p.m., in a standard measuring cup of about 80 ml volume.

Handling

[0247] The high viscosity NADES 3A, could be pushed through a hypodermic needle of size 21 G or less, i.e. a needle with an outer diameter of 0.8 mm or more.

TABLE-US-00002 TABLE 3 NADES formulations tested by administration to animals Ionic molar ratio Viscosity # species Polyol Pro:Sor:WFI (mPa .Math. s) 3A Proline Sorbitol 1.0 1.0 2.5 5197 3B Proline Sorbitol 1.0 1.0 10.0 33.4 3C Proline Sorbitol 1.0 1.0 5.0 289

Subcutaneous Administration In Vivo

[0248] The NADES formulations 3B and 3C were administered by subcutaneous injection, in a safety study in dogs. Special attention was given to any adverse reactions that could occur after the administration of the stabilizer formulations, either locally or systemically.

[0249] Both formulations were prepared as described before, by heating and sonification of proline, sorbitol, and water for injection, in a closed vial, resulting in a clear and homogeneous liquid. Additionally, they were filter-sterilised, and their bioburden and endotoxin levels were determined. Both formulations were found to score below the lowest dilution standard of 1 unit/ml, meeting the general acceptance criteria for sterile formulations. The NADES 3C formulation was prepared by diluting 1 volume part NADES 3A with 9 volume parts WFI, resulting in a formulation with Proline:Sorbitol:WFI in 1:1:5 ratio, and a viscosity of 289 mPa.Math.s. NADES 3B has a viscosity of 33.4 mPa.Math.s.

[0250] Six healthy beagle dogs of 7 months old, were divided into two groups of 3 dogs. Each group was assigned to a NADES formulation, and received two doses of 1 ml of the assigned formulation, using a 1 ml syringe and a 21 G ⅝ needle by subcutaneous inoculation, with a two-week interval. Group 1 dogs were given the low viscosity NADES 3B, and the Group 2 dogs received the high viscosity NADES 3C. All dogs were clinically monitored daily throughout the study, including for local reactions at the site of the inoculations.

[0251] The vaccinations were performed in the scruff of the neck; the first vaccination was done on the left side, and the second vaccination was done on the right side of the neck. The vaccination sites were shaved prior to each administration, and the injection site was circled with a marker pen. No discomfort was observed during any of the injections. At 4 hours post-injection, injection sites were palpated, and little or no material was palpable at the injection sites, and no swelling or redness was observed.

[0252] Rectal temperatures were taken on days −3, −2, −1 before each of the vaccinations; at the day of vaccination: just before vaccination and 4 hours after vaccination; and next daily for seven days post vaccination.

[0253] Throughout the whole study no significant increase in body temperature was observed in any of the dogs and no clinical adverse effects were observed.

[0254] In conclusion: the inoculation of NADES formulations 3C or 3B by subcutaneous route to dogs, did not induce any adverse inoculation reaction, neither locally, nor systemically.

Intra-Nasal Administration

[0255] Two NADES formulations: 3A and 3B were tested for their pattern of spread on the mucosal surface of the nose, when administered intranasally. Both NADES formulations were tested as such, without adding water. Consequently, NADES 3A has a viscosity of 5197 mPa.Math.s, and NADES 3B of 33.4 mPa.Math.s. A spatula tip of ‘patent blue’ colourant was added to 30 ml of each of the NADES formulations, to obtain a deep blue colour. As a control PBS with colourant was used.

[0256] Calf heads were obtained post-mortem from calves of about 2-4 weeks old. The administration was done holding the head in an upright position mimicking the position of a calf during i.n. vaccination in the field, with the nostrils tilted slightly upwards.

[0257] The liquids were then administered according to a standard procedure for intranasal vaccination, whereby a 2.5 ml syringe without needle, containing 1.5 ml liquid, was placed into a nostril, and then the liquid was squirted out in one steady flow. Next the head was dissected into two halves and the septum was removed and inspected for presence of blue colour in the intranasal tract.

[0258] For all samples it was observed that the blue liquids had moved easily through the nasal tract. The low viscosity of the PBS sample allowed for easy injection, but all liquid rushed through quickly. A blue colour was observed all through the whole of the nasal cavity including the beginning of the trachea.

[0259] The injection of NADES 3A with the very high viscosity took a little force to inject out of the syringe. Also, as expected, it took longer for the blue liquid to emerge below the head. After dissection of the head, it was observed that also this viscous liquid had spread evenly through the intranasal tract, where it appeared to have formed a thick layer, leaving more material in the nasal tract. This indicates an improved retention and less progression to the upper respiratory tract would occur, when administered to a life calf during i.n. vaccination.

[0260] The effect of the administration of the NADES 3B formulation, with the low viscosity, closely resembled that of the PBS sample, where the liquid rushed through the nasal cavity.

[0261] In conclusion: it was possible to monitor the distribution of coloured preparations with different viscosity after intranasal injection into calve-heads. Distribution of the liquid as judged by the blue colour was comparable for the two NADES preparations and PBS, with some difference with regard to ease of administration and amount of residual material, whereby the NADES 3A formulation was a little more difficult to eject, but resulted in a thicker layer of residue within the nasal tract. This suggests a delayed release of antigen from such viscous NADES formulations may be obtained.

Example 5: Comparative Measurements on an Aqueous Stabiliser

[0262] An aqueous stabiliser composition as described in WO 2015/121463 was prepared having: 30% w/w Sorbitol+0.6 M Arginine in 0.04 M PBS. This composition was tested for its properties such as water activity and DSC profile, to clarify that it does not possess the characterising features which make a composition a NADES as defined and outlined hereinabove.

Water Activity

[0263] Determination of a.sub.w was done by way of headspace vapour pressure measurement, as described in WO 2019/122329, Example 1. Result for water activity was determined in duplo, giving an average a.sub.w of 0.89. Clearly, this high level of available water demonstrates that this composition is not a NADES.

DSC Profile

[0264] Determination of the DSC profile of the aqueous prior art stabiliser was done as described herein in Example 1.3.

[0265] The results are presented in FIG. 4, whereby (as in FIG. 1 herein) the upper horizontal part of the graph presents the cooling phase from 40° C. to −90° C. at 5° C./min (going right to left), and the bottom horizontal part the heating phase from −90° C. to 40° C. at 5° C./min (left to right).

[0266] During the cooling, an extensive and sudden crystallisation of free water into ice is observable, at a temperature of about −27° C. Next, upon re-heating a typical ice-crystal melting profile was observed with a peak at −5.5° C.

[0267] Further, in FIG. 4, during the heating phase, the horizontal base line after the ice-melt returns to the same level at which it was before the melt. This signifies there was no change in the thermal capacity of that sample, which is clearly different from the stepwise baseline shift that is typically observed in the case of a glass transition.

[0268] Also it is significant that the ice-melt in FIG. 4 occurs at a much higher temperature (−5° C.) than the glass-transition in FIG. 1 (−45° C.), again illustrating that the sample tested in FIG. 4 was not a deep-eutectic mixture.

[0269] In FIG. 4, both peaks, the high melting temperature, and the return of the base line to its previous level, are all indicative for the formation and the melting of ice-crystals, and therefore demonstrate that the composition tested had much free water present, and thus was not a NADES.

[0270] On the contrary however, in the sample of FIG. 1 (a NADES according to the invention), no ice crystals were formed or melted, and a clear glass-transition shift of the base line was found.

Example 6: Additional Stability Results: M. bovis

[0271] A further species of Mollicutes bacteria was tested for stabilisation in the liquid compositions according to the invention: Mycoplasma bovis. Experiments were performed essentially as described herein in Examples 1-3 for M. synoviae and M. gallisepticum.

[0272] Results of stability assays in different compositions according to the invention, and either at room-temperature or at 2-8° C. are presented in FIG. 5: [0273] panel A: room temperature, up to 10 weeks; and [0274] panel B: 2-8° C., up to 26 weeks of incubation.

[0275] Starting titre was 9.9 Log 10 CFU/ml. In PBS (the negative control) some survival was observed at two weeks of incubation, both at room temperature and when refrigerated. However no viable cells could be retrieved anymore in samples incubated for longer periods in PBS.

[0276] At room temperature good initial stability (2-4 weeks of incubation) was obtained in several of the NADES compositions. However best results were found when using Arginine:Sorbitol:water (2:1:6), which was able to stabilise this bacterium for at least 10 weeks at room temperature.

[0277] At refrigerated incubation, a similar picture was observed: good initial stabilisation for 6-13 weeks, was observed for several of the compositions tested. Best long-term stabiliser (26 weeks of incubation) was also Arginine:Sorbitol:water (2:1:6), although the differences observed between the different compositions tested were not as big as for room temperature incubation.

[0278] Overall, M. bovis could effectively be stabilised without freeze-drying in the liquid compostions according to the invention, at different storage temperatures above freezing.

Example 7: Extended Stability Test Results

[0279] Stability test results for M. synoviae and M. gallisepticum in liquid compositions according to the invention, were obtained from a continued incubation up to 1 year at 2-8° C. These data result from the same series of samples as described in Example 3, and extend on the results as depicted in FIGS. 2 B and 3 B herein.

[0280] The extended results are provided in FIG. 6: [0281] panel A: M. synoviae at 2-8° C. up to 54 weeks; and [0282] panel B: M. gallisepticum at 2-8° C. up to 52 weeks of incubation.

[0283] For M. synoviae, three of the tested liquid compositions according to the invention can stabilise the viability of these bacteria for at least a year at 2-8° C.; whereby Proline:Sorbitol:water (1:1:2.5) and Betaine:Sorbitol:water (1:1:2.5) showed the same final results.

[0284] For M. gallisepticum, viable bacteria were only retained until 52 weeks of incubation at 2-8° C., in Betaine:Sorbitol:water (1:1:2.5).

LEGEND TO THE FIGURES

[0285] FIG. 1:

[0286] Differential scanning calorimetry profile of a NADES formed at a molar ratio for proline, sorbitol, and water of 1:1:2.5.

[0287] FIG. 2:

[0288] Stability results of Mycoplasma synoviae in liquid compositions with different NADES.

[0289] Panel A: incubated at room temperature; Panel B: incubated at 2-8° C.

[0290] FIG. 3:

[0291] Stability results of Mycoplasma gallisepticum in liquid compositions with different NADES.

[0292] Panel A: incubated at room temperature; Panel B: incubated at 2-8° C.

[0293] FIG. 4:

[0294] DSC profile of an aqueous stabiliser from the prior art, comprising 30% w/w sorbitol and 0.6 M arginine in 0.04 M PBS.

[0295] FIG. 5

[0296] Stability data for M. bovis in NADES, at room temperature (A), and at 2-8° C. (B).

[0297] FIG. 6:

[0298] Long term stability data of M. synoviae (A) and of M. gallisepticum (B) in NADES at 2-8° C.