Stabilized liquid vaccine of live virus

Abstract

The present invention relates to a liquid vaccine composition comprising a live virus and a natural deep eutectic solvent (NADES) as a carrier, which further comprises an additive selected from methionine and (hydroxy)ectoine, which additive is capable of reducing the loss of virus titer over time upon storage in NADES-based liquid vaccine compositions containing up to 50% water by weight. Such compositions have a relatively low viscosity which is favorable for the manufacture of the carrier and for the formulation and use of liquid vaccines.

Description

[Technical field]

[0001] The present invention relates to the field of vaccinology, in particular to live virus vaccines. In particular, the present invention relates to a liquid vaccine composition comprising a live virus and a natural deep eutectic solvent (NADES) as a carrier. Furthermore, the present invention relates to a method for producing said liquid vaccine composition, a method for stabilizing a live virus in such a liquid composition, and medical uses of such a liquid vaccine composition. [Background technology]

[0002] Deep eutectic solvents (DES) are liquid compositions that combine many remarkable properties, which stem from the fact that they are ionic but contain very little free water. Essentially they are mixtures of two or more salts, whereby the melting point of the mixture is significantly reduced compared to the melting points of the individual components. As a result, such mixtures, even if composed primarily of salts, are liquid at ambient temperature or even below zero degrees Celsius.

[0003] DESs have been reported, for example, by Abbott et al. (2003, Chem. Commun., vol. 1, p.70-71) and WO2009 / 120839. Smith et al. (2014, Chem. Rev., vol.114, p.11060-11082) have proposed a classification of DESs into four classes. A recent review of the properties and microstructure of DESs is given by Kaur et al. (2020)(J. of Phys. Chem. B, vol.124, p.10601-10616).

[0004] Being ionic liquids, DESs are highly effective as solvents for a variety of chemical and industrial processes. Examples range from the extraction of gases, minerals and industrial bulk products, to metals and pharmaceutical solvents. DESs have also been used to extract biomolecules such as enzymes and RNA, see US8,247,198 and WO2011 / 155829, respectively.

[0005] A special form of DES is one whose components are natural substances. Such so-called "natural DES" or NADES are described 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 used as "green" solvents for various biological compounds such as cellulose and vanillin.

[0006] As is known in the art, the key characteristics that determine the immunological effectiveness of a live virus vaccine are the amount and characteristics of the virus used. Therefore, for a particular vaccine strain, it is paramount to maintain a minimum effective live virus titer (i.e., potency) until the end of the registered shelf life. The traditional method of preserving live viruses is freeze-drying, which is a laborious, energy-intensive, and expensive process.

[0007] In WO2019 / 122329, a liquid vaccine composition was disclosed that overcomes the need for freeze-drying and allows the development of liquid vaccines of sensitive live enveloped viruses with excellent stability even at high temperatures. This was achieved by using NADES as vaccine carriers and virus additives, since even the small amount of water present is not readily available for chemical or biological processes. In the '329 patent application, the NADES are preferably composed of organic salts and polyhydric alcohols, and the vaccines typically contain less than 20% water by weight.

[0008] Several publications, such as WO2011 / 121306, WO2014 / 029702, WO2014 / 140239, WO2015 / 121463, and US2014 / 0271710, describe the stabilization of viruses in liquid vaccine compositions, using compounds that at first glance appear similar to those described in '329. However, these liquid vaccines are not based on NADES, but on conventional aqueous compositions. Such aqueous compositions are essentially different from NADES-based compositions in their physical properties and their ability to stabilize susceptible viruses. In the following Example 1, some of these published aqueous compositions are analyzed with respect to their salt concentration, amount of water, and water activity. It is immediately clear that these compositions are aqueous compositions and use water as a pharma- ceutically acceptable carrier, but are not based on NADES.

[0009] WO2020 / 201048 describes a NADES-based liquid vaccine for Mollicutes.

[0010] DES consists of a hydrogen bond network of intermolecular interactions, in which the water present in the composition is tightly bound. This also determines the maximum amount of water that the DES network can contain. There is agreement in the art that DES can contain up to about 50% water by weight before the network structure of DES is completely disrupted, at which point the composition changes from DES to an aqueous composition. See, for example, Dai et al., 2015 (Food Chem., vol.187, p.14-19);Hammond et al., 2017 (Angew. Chem., Int. Ed., vol.56, p.9782-9785);and Liu et al., 2018 (J. of Nat. Prod., vol.81, p.679-690).

[0011] Although the NADES-based liquid vaccines of WO2019 / 122329 can be administered by injection, some of the compositions can be quite viscous, which can be a disadvantage for large-scale manufacturing and processing, and for certain methods of administration of such vaccines. [Prior art documents] [Patent documents]

[0012] [Patent Document 1] WO2009 / 120839 [Patent Document 2] US8,247,198 [Patent Document 3] WO2011 / 155829 [Patent Document 4] WO2019 / 122329 [Patent Document 5] WO2011 / 121306 [Patent Document 6] WO2014 / 029702 [Patent Document 7] WO2014 / 140239 [Patent Document 8] WO2015 / 121463 [Patent Document 9] US2014 / 0271710 [Patent Document 10] WO2020 / 201048 [Patent Document 11] WO2019 / 122329 [Non-patent literature]

[0013] [Non-Patent Document 1] Abbott et al, 2003, Chem. Commun., vol. 1, p.70-71. [Non-Patent Document 2] Smith et al, 2014, Chem. Rev., vol.114, p.11060-11082. [Non-Patent Document 3] Kaur et al, (2020)(J. of Phys. Chem. B, vol.124, p.10601-10616). [Non-Patent Document 4] Choi et al, 2011(Plant Physiol., vol.156, p.1701-1705). [Non-Patent Document 5] Dai et al, 2013(Anal. Chim. Acta, vol.766, p.61-68) [Non-Patent Document 6] Dai et al., 2015 (Food Chem., vol.187, p.14-19). [Non-Patent Document 7] Hammond et al., 2017 (Angew. Chem., Int. Ed., vol.56, p.9782-9785). [Non-Patent Document 8] Liu et al., 2018 (J. of Nat. Prod., vol.81, p.679-690. Summary of the Invention [Problem to be solved by the invention]

[0014] It is therefore an object of the present invention to overcome the shortcomings of the prior art and address the need in the art by providing a liquid live virus vaccine based on NADES as a pharma- ceutically acceptable carrier, which has low viscosity and can accommodate relatively large amounts of water while reducing loss of viral titer. [Means for solving the problem]

[0015] Surprisingly, it has been found that this objective can be achieved, thereby overcoming one or more of the disadvantages of the prior art, by providing a NADES-based live virus liquid vaccine further comprising methionine, ectoine or hydroxyectoine.

[0016] This discovery allows the development of NADES-based liquid vaccine compositions that have a lower viscosity compared to compositions described, for example, in WO2019 / 122329 and can contain water in amounts of up to 50% by weight, while still providing good stabilization of the live virus. These liquid vaccines will be easier to manufacture and formulate, especially on a large scale, and easier to handle and administer by different routes. The relatively large amount of water also allows for greater flexibility in including viruses and other antigens. For example, this allows the inclusion of live viruses from a lower concentration aqueous formulation and / or the inclusion of several other antigens in addition, to constitute a multi-antigen mixed vaccine.

[0017] Importantly, they still offer the advantages of NADES-based liquid vaccines in that the live virus is highly stable even at temperatures above 0°C, eliminating the need for lyophilization during production, eliminating the need for reconstitution prior to administration to a subject, and allowing for convenient storage at temperatures below 0°C without freezing.

[0018] In an attempt to produce a less viscous NADES-based vaccine, the inventors learned that simply increasing the amount of water was only a partial solution, indeed lowering the viscosity, but unfortunately also reducing the stabilizing ability. A decrease in virus titer was clearly observed in the stability assays already when the amount of water was increased beyond 5 wt%. Inclusion of 20-40 wt% water resulted in moderate to severe losses in live virus titer, respectively, and above 40 wt% a very rapid loss of live virus titer was observed. When water was included above 50 wt%, the composition lost its NADES properties, as indicated by the change in the differential scanning calorimetry (DSC) profile from a glass transition profile characteristic of an aqueous solution to an ice crystallization profile, as described herein.

[0019] Next, the inventors had to find a way to increase the survival time of the virus in these reduced viscosity NADES-based vaccines. One option considered was to add additional components. However, many different compounds were tested, most of which did not sufficiently reduce the loss of viral titer. In fact, only a few compounds, methionine and (hydroxy)ectoine, were able to actually reduce the loss of viral titer over time when the live virus was stored in a NADES-based liquid vaccine composition according to the invention containing up to 50% water by weight.

[0020] This was unexpected and clearly indicates that the virus stabilizing properties of compounds in typical aqueous compositions are unlikely to occur in very different physical conditions such as NADES-based compositions, where the free water levels are very low. As a result, previous publications on the stabilizing effects of compounds used in aqueous compositions cannot be used to predict, generalize, or extrapolate that such effects will also occur in the context of NADES-based compositions.

[0021] It is not known exactly how and why methionine and (hydroxy)ectoine are able to extend the viability of live virus in NADES-based liquid vaccine compositions containing up to 50% water by weight. This was particularly surprising since some of the compounds used to form the NADES are thought to have stabilizing and / or antioxidant effects themselves, and these compounds are present in large excess compared to the added methionine or (hydroxy)ectoine.

[0022] While the inventors do not wish to be bound by any theory or model that may explain these findings, they speculate that methionine, ectoine and hydroxyectoine may exert this favorable effect by somehow avoiding the detrimental effect that the presence of water has on the titer of live virus over time.

[0023] Thus, in one aspect the present invention relates to a liquid vaccine composition comprising a live virus and a pharma- ceutically acceptable carrier, wherein the carrier is a natural deep eutectic solvent (NADES), and wherein the vaccine has a water activity of up to 0.8, the water content of the liquid vaccine composition is up to 50% by weight, and wherein the vaccine also comprises an excipient selected from methionine, ectoine and hydroxyectoine. [Brief description of the drawings]

[0024] [Figure 1] Figure 1 from the literature, reproduced here, originally from WO2020 / 201048, shows the differential scanning calorimetry (DSC) glass transition profile of the complete NADES. Specifically, this is the DSC profile of the NADES with a molar ratio of proline, sorbitol, and water of 1:1:2.5. [Diagram 2] Figure 4 from the literature, reproduced here originally from Qiao et al., (2018, Appl. Microbiol. Biotechnol., vol. 102, p. 5695-5705), shows DSC profiles indicating the enthalpy of fusion and crystallization of overdiluted NADES in aqueous compositions. [Diagram 3] Results of incubation of CPI in compositions containing different additives. Incubation was carried out at 20° C. Details are given in Example 2. [Figure 4A] Figure 4 shows the results of incubation of BCV in compositions containing different additives. Incubation was carried out at 28°C (Figure 4A) or 4°C (Figure 4B). Details are given in Example 2. [Figure 4B] Figure 4 shows the results of incubation of BCV in compositions containing different additives. Incubation was carried out at 28°C (Figure 4A) or 4°C (Figure 4B). Details are given in Example 2. [Diagram 5]FIG. 1 shows the results of incubation of IBV in a NADES-based composition containing methionine as an additive. Incubation was carried out at 4° C. Details are given in Example 3. [Figure 6A] 6A-6C are results of incubation of CDV in NADES-based liquid compositions containing different amounts of water. Stability studies were performed at 20° C. (FIG. 6A) or 4° C. (FIG. 6B) in compositions containing 27% water by weight; alternatively, incubations were performed at 4° C. (FIG. 6C) in compositions containing 40% water by weight. Details are provided in Example 3. [Figure 6B] 6A-6C are results of incubation of CDV in NADES-based liquid compositions containing different amounts of water. Stability studies were performed at 20° C. (FIG. 6A) or 4° C. (FIG. 6B) in compositions containing 27% water by weight; alternatively, incubations were performed at 4° C. (FIG. 6C) in compositions containing 40% water by weight. Details are provided in Example 3. [Figure 6C] 6A-6C are results of incubation of CDV in NADES-based liquid compositions containing different amounts of water. Stability studies were performed at 20° C. (FIG. 6A) or 4° C. (FIG. 6B) in compositions containing 27% water by weight; alternatively, incubations were performed at 4° C. (FIG. 6C) in compositions containing 40% water by weight. Details are provided in Example 3. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] The term "liquid" is used in its general sense and refers to a composition that can flow at a particular temperature and within a particular time period. To determine liquid properties, a tilt test is performed in which a container containing the composition at a particular temperature (e.g., ambient temperature) is tilted, and if a change in the morphology of the composition is detected after a particular time (e.g., 30 minutes), the composition is classified as being liquid.

[0026] It is well known that a "vaccine" is a composition having a medical effect. A vaccine comprises an immunologically active ingredient and a pharma- ceutically acceptable carrier. In this case, the "immunologically active ingredient" comprises a live virus. After administration of the vaccine, the immunogen is recognized by the target human or animal immune system, which induces a protective immune response. This response may arise from the target's innate and / or adaptive immune system and may be of the cellular and / or humoral type.

[0027] Vaccines are generally effective in reducing the severity of infection by reducing the number of pathogens or the period of time that the pathogen can replicate within the host.

[0028] Also, or perhaps as a consequence, vaccines are generally effective in reducing or ameliorating the (clinical) symptoms of disease that may be caused by such infection or replication of a pathogen, or a target's response to that infection or replication.

[0029] 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.

[0030] 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."

[0031] Strictly speaking, it is biologically inaccurate to call a virus "live", however, this is a common way of referring to a virus that has not been inactivated, and therefore, for purposes of the present invention, the term "live" refers to a virus that is capable of replicating (i.e., "replicative") under appropriate conditions, e.g., in a suitable host cell.

[0032] In practice, the live virus contained in the liquid vaccine composition according to the invention is a virus suitable for vaccination of a human or animal target. Typically, this means that the vaccine virus is less pathogenic for that target. This phenotype may result from natural properties of the virus itself, e.g., less pathogenic when used in a particular target species. Alternatively, the virus may be an attenuated virus, also called a "modified live (virus)".

[0033] "Attenuated" in the present invention is defined as causing a lower level of disease symptoms, e.g., having a reduced rate of infection or replication, compared to the native, non-attenuated, or "wild-type" form of such a virus. Viral attenuation can be obtained in a variety of ways, e.g., by passage and selection through laboratory animals, or in cell culture, or by random or targeted mutation, e.g., via chemicals, radiation, or via recombinant DNA techniques, all of which are known in the art.

[0034] Viruses are well-known microorganisms, many of which are pathogenic for humans and animals. A large variety of virus types and families are described in handbooks such as Fields Virology (4th ed. 2001, Lippincott Williams & Wilkins, ISBN-10:0781718325).

[0035] In the present invention, the "titer" of a composition comprising a live virus refers to the amount of infectious virus in the composition. The reduction in titer occurs due to the virus losing its ability to infect host cells and / or to replicate once in the host cell. This can be caused, for example, by damage to the viral envelope, structural proteins, and / or nucleic acid. Consequently, the stability of a live virus can be effectively indicated in terms of its effect on its titer. This can be determined in vivo, but more conveniently, in vitro, for example using fertilized eggs or cell cultures of suitable host cells. The viral titer can then be compared, for example, before and after storage, and can be expressed, for example, in tissue culture infectious doses (TCID50), cell infectious doses (CID), plaque forming units (pfu), or egg infectious doses (EID).

[0036] For the present invention, a "pharmaceutically acceptable carrier" is a liquid with a high grade of purity and suitable for medical purposes. In this case, the carrier is a NADES. The carrier may contain additional excipients.

[0037] "Deep-eutectic solvents" (DES) are well known in the art as ionic liquids that contain a mixture of at least two compounds in a molar ratio that forms a eutectic mixture, whereby the eutectic point of the resulting mixture is significantly lower than the melting points of the individual compounds. This reduction in the melting point of the mixture is caused by molecular interactions of the compounds, one acting as a proton donor and the other as a proton acceptor. This allows the development of stable hydrogen bonds without crystallization, allowing the mixture to be in liquid form at much lower temperatures compared to its constituent components. In general, "eutectic" means easy melting.

[0038] In the present invention, the individual compounds used to form the DES for the present invention have melting points above about 80° C., while the DES has a melting point below about 40° C. For example, the melting points of proline and sorbitol are 228° C. and 112° C., respectively, while a NADES formed with a molar ratio of proline, sorbitol, and water of 1:1:2.5 formed a clear liquid at ambient temperature and remained fluid even at −20° C.

[0039] Thus, DESs consist primarily of liquefied salts, optionally with small amounts of water. Thus, DESs are essentially different from aqueous compositions that consist primarily of water with a certain amount of salt in it. The difference between these two types of liquids is the water activity (a w This is also evident by comparing parameters such as the amount of water (wt%) and the amount of ice (wt%), and is reflected in the different DSC profiles. The DSC profile of the aqueous composition shows ice crystal formation and melting, whereas the NADES have a glass transition profile, as described in more detail herein.

[0040] "About" indicates that the value may vary by ±10%, preferably, about means ±9% around the value, more preferably, about means ±8, 7, 6, 5, 4, 3, 2% around the value, or even about means ±1% around the value, in that order of preference.

[0041] In the terms "natural deep eutectic solvents" and "NADES", the feature "natural" serves to indicate that the compounds used to form this type of DES are organic compounds present in materials from biological sources such as plants or animals in amounts well above trace amounts. Typically, such natural compounds are or are derived from primary metabolites present in certain materials of plant or animal origin. As the skilled person will understand, the term natural is used herein only to characterize the initial origin of the compound, and not the form in which the compound used in the NADES of the present invention is actually provided. Thus, the natural compounds of the present invention can be obtained from (semi)synthetic production.

[0042] Examples of naturally occurring compounds that can be used to form NADES are organic acids, amines, sugars, sugar alcohols, and amino acids.

[0043] A further advantageous feature of the liquid vaccine composition according to the invention is that the water present in the vaccine is tightly bound to the structure of the NADES, which results in a very limited amount of water available for chemical or biological processes that could affect, for example, the stability of the live virus. This feature is usually represented by the symbol: w Water activity is expressed by a value of "water activity" indicated by the formula: 1.0 (high limit for pure water) to 0 (low limit). Water activity can vary between 1.0 for pure water and 0 for pure water. Water activity is usually measured by comparing the vapor pressure of the test composition (at the same temperature) with pure water and a number of saturated salt solutions with known water activity. This is described in various handbooks, compendia and manuals, such as, for example, the FAO agricultural service bulletin no. 149 (Canovas et al, FAO, Rome, 2003, ISBN 92-5-104861-4) on fruit and vegetable preservation, and the compendia published in 'Fundamentals of water activity', Decagon Devices Inc., Washington, 2015 (http: / / pdf.directindustry.com / pdf / decagon-devices-inc / fundamentals-water-activity / 64142-634433.html).

[0044] A water activity below 0.8 stops the growth of most bacteria, a water activity above 0.7 stops the growth of most yeasts and molds, and a water activity below 0.4 effectively stops the activity of most enzymes.

[0045] Instruments and procedures for measuring water activity are known and available, for example, by using headspace pressure analysis.

[0046] In the present invention, the indicated water activity refers to the water activity of the liquid vaccine composition according to the invention in the form of the final product, e.g. as provided by a commercial producer and in a form that can be stored for a long period of time before administration to a target. The indicated water activity therefore does not relate to the dilution of the liquid vaccine composition according to the invention, which is carried out, e.g. immediately before administration to a target, e.g. upon admixture with another vaccine, or e.g. when mixing with another vaccine, or when diluting for administration, e.g. by spray or via drinking water.

[0047] Liquid vaccines according to the invention contain components other than NADES, and these other components may contain water, such as, for example, a stock solution of live virus in water, and therefore the water activity of the NADES carrier will typically be lower than the water activity of the final liquid vaccine composition, to allow for the addition of such other components without exceeding the maximum water activity of the liquid vaccine of the invention.

[0048] In the present invention, "water content" refers to the amount of water present in the composition of a liquid vaccine according to the invention, which is calculated by dividing the total weight of water in the liquid vaccine by the total weight of the vaccine, expressed as a percentage.

[0049] Water content can be measured using a variety of different procedures, such as Karl Fischer titration, which are well known in the art.

[0050] In the experiments leading to the present invention, samples of NADES-based vaccines containing live virus were prepared and stored at specific temperatures, e.g., -20, 4, 20 or 37°C, for extended periods of time. The residual virus titer after storage was then measured. As disclosed in detail in the Examples below, it is believed that the additives described in the present invention, to increase the amount of water, can reduce the loss of virus titer over time when compared to similar compositions without the additive. Thus, the additives defined in the present invention can be included in NADES-based compositions containing up to 50% water by weight to reduce the loss of live virus titer during storage.

[0051] Obviously, the advantages of the liquid vaccine according to the invention are lost if the composition is diluted too much and loses its NADES properties to become an aqueous composition, i.e., if the amount of water is increased from about 50% by weight. As the skilled person will appreciate, the exact boundary at which NADES integrity is lost depends on the details of the composition under consideration. Thus, the amount of water in the liquid vaccine according to the invention is limited to an amount of water up to a maximum of 50% by weight.

[0052] Like the water activity, the water content of a liquid vaccine according to the invention relates to the liquid vaccine in its final product form before any dilution has taken place, for example immediately prior to administration to a target.

[0053] By limiting the amount of water to 50% by weight or less, the use of NADES as carriers in the liquid vaccine composition according to the invention provides the vaccine with a complete hydrogen bond network typical of DES. This can be easily confirmed by thermal analysis of the phase transition profile of the vaccine using differential scanning calorimetry (DSC). As a result, the liquid vaccine according to the invention has a DSC profile that exhibits a glass transition temperature and no ice crystallization profile.

[0054] Determination of DSC profiles is well known and can be performed using standard equipment. An example of a glass transition profile for a complete NADES is shown in Figure 1 of WO2020 / 201048 and represented herein as Figure 1. Specifically, this is the DSC profile for a NADES with a molar ratio of proline, sorbitol, and water of 1:1:2.5.

[0055] An example of a DSC profile showing the enthalpy of fusion and enthalpy of crystallization of an aqueous composition resulting from overdilution of a NADES is shown in Figure 4 of Qiao et al. (2018, Appl. Microbiol. Biotechnol., vol. 102, p. 5695-5705) and is shown herein as Figure 2.

[0056] The term "additive" is used in the present invention in its general sense and refers to an added compound, e.g. an excipient, which can reduce the loss of infectivity of live virus in a liquid vaccine composition according to the present invention, e.g. during storage.

[0057] The results in the Examples section show that some titer loss may still occur in the compositions of the present invention. However, as one skilled in the art can appreciate, even a reduction in the rate of titer loss compared to the control represents a valuable effect in virus stabilization, primarily because it overcomes the need for lyophilization. Moreover, it may allow, for example, to assign a two-year shelf life to liquid vaccine products, which would not be possible without the present invention. Thus, although clearly desirable, the criterion for a beneficial effect is not zero titer loss, but a significantly reduced rate or total level of titer loss compared to a similar composition that does not contain the additive of the present invention or is not NADES-based.

[0058] "Methionine" is a known amino acid, and "ectoine" and its derivative "hydroxyectoine" are known compounds of bacterial origin.

[0059] Details of embodiments and further aspects of the invention are described below.

[0060] In the present invention, the positive effect of the presence of an additive in the liquid vaccine composition according to the invention is manifested by a reduced loss of viral titer compared to the titer remaining after a control incubation. The control incubation is preferably carried out with a similar composition, which is preferably maintained under similar storage conditions, such as temperature and duration. Preferably, the reduction in titer loss caused by the presence of the additive is at least 5% after incubation for a certain period of time. This means that the viral titer in the composition containing the additive is preferably at least 5% higher than in the control incubation composition after the incubation period.

[0061] The incubation (storage) period is preferably at least one week at 20° C., or at least one month at 4° C. More preferably, it is at least 2, 3, 4, 6, 12, 18, or 24 months at 4° C.

[0062] Preferably, the reduction in virus titer loss caused by the presence of the additive is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or at least 75%, in that order of preference.

[0063] In one embodiment of the liquid vaccine composition according to the invention, the NADES is formed from an organic salt, a polyol, and water. The organic salt is preferably one or more selected from the salts of betaine, proline, carnitine, and choline. The polyol is preferably a sugar or sugar alcohol, the sugar is preferably one or more selected from the salts of fructose, maltose, sucrose, glucose, and trehalose, and the sugar alcohol is preferably one or more selected from the salts of glycerol, xylitol, mannitol, and sorbitol.

[0064] In a preferred embodiment of the liquid vaccine composition according to the invention, the NADES is formed from proline, sorbitol and water.

[0065] "Proline" is a known amino acid, and "sorbitol" is a known sugar alcohol.

[0066] In a preferred embodiment, proline has the CAS number 609-36-9.

[0067] In a preferred embodiment, the sorbitol is D-sorbitol, more preferably the D-sorbitol has the CAS number 50-70-4.

[0068] In a preferred embodiment, proline, sorbitol and water are present in the NADES in a molar ratio of proline:sorbitol:water from 1:1:1 to 1:1:16.

[0069] For NADES with a molar ratio of proline, sorbitol and water of 1:1:16, the amount of water is 49.2% by weight and the water concentration is 32.3 M. However, since there is little room to include an aqueous virus preparation while keeping the amount of water in the vaccine below 50% by weight, the molar ratio of proline:sorbitol:water in NADES is preferably 1:1:1 to 1:1:15, more preferably 1:1:1 to 1:1:12, or 1:1:1 to 1:1:10.

[0070] Particularly sensitive, and therefore requiring appropriate stabilization in liquid formulations, are live viruses that are relatively large, have an RNA genome, and / or have a viral envelope (i.e., are surrounded by an envelope). Most sensitive are live viruses that are large, have an RNA genome, and have a viral envelope, as these viruses are sensitive to both physical and chemical influences on their infectivity and ability to replicate.

[0071] Thus, in one embodiment of a liquid vaccine composition according to the invention, the live viruses are large, i.e. greater than 50 nanometers in diameter. More preferably, the large viruses of the invention are greater than 75, 100, 150 or 200 nanometers in diameter, in that order of preference.

[0072] In one embodiment of the liquid vaccine composition according to the invention, the live virus has an RNA genome.

[0073] The RNA genome can have a positive or negative translational orientation, also called the plus or minus strand, or sense or antisense, respectively. As is well known, the positive, plus or sense orientation allows direct translation. The RNA genome can be single-stranded or double-stranded. The RNA genome can be segmented or non-segmented, all of which are well known to those skilled in the art.

[0074] In one embodiment of the liquid vaccine composition according to the invention, the live virus is a live enveloped virus.

[0075] "Enveloped viruses" are a well-known type of virus that has a phospholipid envelope. Examples include the Asfar-, Baculo-, Hepadna-, Herpes-, and Pox virus families; the Corona-, Flavi-, and Toga virus families; the Arena-, Bunya-, Filo-, Orthomyxo-, Paramyxo-, Pneumo-, and Rhabdo virus families; and the Reo- and Retro virus families.

[0076] The viral envelope is a lipid bilayer that surrounds such viruses and is obtained by the virus budding from a eukaryotic host cell. The envelope, which comprises phospholipids and proteins, is highly sensitive to damage and is important for the ability of the enveloped virus to further infect host cells. Damage or degradation of the viral envelope, such as by storage for a long period of time and / or storage at high temperatures, significantly reduces the infectious ability of the enveloped virus, i.e., its infectious titer. As for other types of viruses, the stability of a live enveloped virus can be effectively demonstrated in terms of its effect on its infectious titer, i.e., the amount of infectious virus.

[0077] In a preferred embodiment of the liquid vaccine composition according to the invention, the live virus is large, has an RNA genome and is enveloped.

[0078] In one embodiment of the liquid vaccine composition according to the invention, the large live enveloped RNA virus is selected from a paramyxovirus and a coronavirus.

[0079] These virus families have members that are relatively large viruses with an envelope and an RNA genome, making them particularly susceptible to biological degradation and loss of potency during storage, especially when in a liquid environment.

[0080] For the purposes of the present invention, a viral family as referred to herein refers to viruses that have characteristic features of the taxonomic group members, such as morphological, genomic and biochemical properties, as well as biological properties such as physiological, immunological or pathological behavior, etc. The same applies to references to names of viral genuses or individual viral species.

[0081] As is known in the art, the classification of microorganisms into particular taxa is based on a combination of such characteristics. Thus, the present invention also includes virus species from a designated family or designated species name, which are further classified therefrom in any way, such as into subspecies, strains, isolates, genotypes, variants, subtypes, or subgroups.

[0082] Furthermore, it will be apparent to one of ordinary skill in the art that, although a particular family, subfamily, genus or species of a virus of the invention may currently be assigned to a group, it is a taxonomic classification that may change over time as new knowledge may result in reclassification into a new or different taxonomic group. However, such reclassified viruses remain within the scope of the invention, as this does not change the virus itself or its antigenic repertoire, only its scientific name or classification.

[0083] In a preferred embodiment, the paramyxovirus is selected from canine parainfluenza virus (CPI) and canine distemper virus (CDV).

[0084] In preferred embodiments, the coronavirus is a bovine coronavirus (BCV) or an infectious bronchitis virus (IBV).

[0085] All these viruses, CPI, CDV, BCV and IBV, are known and attenuated vaccine strains of each are publicly available, or alternatively, attenuated viruses can be developed using standard procedures.

[0086] These virus samples can come from a variety of sources, for example from field isolates from humans, from wild or farm animals, or from various research laboratories, (repository) institutions and (veterinary) universities.

[0087] The relevance of these viruses in veterinary medicine is documented in well-known handbooks such as the “Merck Veterinary Manual” (11th edition, 2016, ISBN-10:9780911910612).

[0088] In a preferred embodiment of the liquid vaccine composition according to the invention, the methionine is L-methionine, more preferably the L-methionine has the CAS number 63-68-3.

[0089] In a preferred embodiment of the liquid vaccine composition according to the invention, the ectoine has the CAS number 96702-03-3.

[0090] In a preferred embodiment of the liquid vaccine composition according to the invention, the hydroxyectoine is 5-hydroxyectoine, more preferably the 5-hydroxyectoine has the CAS number 165542-15-4.

[0091] The additives of the compositions of the present invention are carefully added to reach a substantial and effective amount. Thus, they are not compounds present in trace amounts, e.g., resulting from the cultivation of a virus or the production of another antigen, contained in the liquid vaccine of the present invention. For example, a typical culture medium for mammalian cells, such as RPMI1640 or DMEM, contains 15-30 mg / L methionine, i.e., 0.0015-0.003% by weight. After cultivation, harvesting and formulation into a vaccine, only very small traces of that methionine are carried over to the final vaccine product, and therefore are not considered to be present as additives in the present invention.

[0092] Therefore, in one embodiment of the liquid vaccine composition of the present invention, the additive is contained at 0.05 to 1% by weight, preferably 0.05 to 0.5% by weight, or 0.1% by weight.

[0093] In the present invention, the amounts of additives are preferably expressed relative to the weight of the NADES support and the ranges expressed include the recited endpoints.

[0094] In a preferred embodiment of the liquid vaccine composition according to the invention, the additive is methionine.

[0095] The compounds for use in the NADES of the present invention are readily available in different purity and quality 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, 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, Lippincott, USA, ISBN: 683306472), and "Veterinary vaccinology" (P. Pastoret et al. ed., 1997, Elsevier, Amsterdam, ISBN 0444819681).

[0096] Similarly, the water used in the liquid vaccine compositions according to the invention is preferably water of high purity, of pharmaceutical grade and suitable for parenteral injection. The nature of such water is typically sterile and essentially pyrogen-free, such as (multiple) distilled water, reverse osmosis water or water for injection (WFI).

[0097] Further optimization of the liquid vaccine composition 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 it provides. This can be done 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.

[0098] In one embodiment of the liquid vaccine composition according to the invention, the water content of the liquid vaccine composition is 50% by weight or less, more preferably the water content is 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 35, 30, 25, 20 or 15% by weight or less, in that order of preference.

[0099] The additives in liquid vaccine compositions according to the invention have a favorable effect on maintaining the titer of the live virus contained in such compositions. This effect is already evident from a water amount of about 5% by weight. Thus, in one embodiment, the liquid vaccine composition according to the invention has a minimum water amount of 5% by weight, preferably a minimum water amount of 7, 10, 12, 15, 20, 30% by weight, in that order of preference.

[0100] Therefore, a liquid vaccine composition according to the present invention preferably has a water content in a range that is a combination of the preferred upper and lower limits indicated, for example, 5 to 50, 7 to 45, 10 to 40% by weight, etc. In a preferred embodiment, the vaccine composition according to the present invention has a water content in the range of 20 to 50% by weight, more preferably 30 to 50% by weight, or in some cases 35 to 50% by weight.

[0101] As explained, liquid vaccine compositions according to the invention can contain more water and still maintain live virus titers compared to excipient-free NADES-based compositions, resulting in reduced viscosity, which has several advantages in administration and formulation.

[0102] In one embodiment of a liquid vaccine composition according to the invention, the viscosity of the composition is 1000 mPa·s or less, more preferably 750, 500, 400, 300, 250, 200, 150, or 100 mPa·s or less. Viscosity can be measured by routine methods and using standard equipment as described herein.

[0103] The liquid vaccine compositions according to the present invention contain an immunologically effective amount of live virus.

[0104] One skilled in the art of the invention can more than determine and optimize such immunologically effective amounts of live virus in a liquid vaccine composition according to the invention, for example by monitoring the immunological response after vaccination or (in the case of an animal target) after a challenge infection, e.g., by monitoring the target's signs of disease, clinical scores, or by re-isolation of the pathogen, and comparing these results with the vaccination-challenge response seen in sham-vaccinated animals.

[0105] Methods for determining the amount of live virus in a vaccine dose are well known in the art and typically use techniques of virus titration, such as plaque assays, titration in eggs, animals or microtitration plates. Thus, such virus infectious titers can be expressed, for example, in TCID50, EID50, CID50 or plaque forming units (pfu).

[0106] The liquid vaccine composition according to the invention may further comprise other compounds such as additional antigens or microorganisms, cytokines, or immunostimulatory nucleic acids containing unmethylated CpG, or alternatively the liquid vaccine composition according to the invention may be added to the vaccine itself.

[0107] The liquid vaccine composition according to the invention can advantageously be combined with one or more further antigens, for example derived from a microorganism pathogenic to the intended human or animal target. Such further antigens can be the infectious microorganism itself, or inactivated or subunits. The further antigens can be biological or synthetic molecules (e.g. proteins, carbohydrates, lipopolysaccharides, lipids, or nucleic acid molecules).

[0108] In one embodiment of the liquid vaccine composition according to the invention, the vaccine comprises an additional antigen.

[0109] In a preferred embodiment, the additional antigen is a bacterial antigen, more preferably the bacterial antigen is one or more selected from the genera Leptospira, Bordetella, Borrelia, Ehrlichia, Mycoplasma, Porphyromonas and Bacteroides.

[0110] In a preferred embodiment, the additional antigen is an antigen from Leishmania or Microsporum.

[0111] The relevance of these pathogens in veterinary medicine is known and described, for example, in "The Merck Manual" (supra). Suitable strains of these bacteria are readily available from a variety of sources. The bacterial antigens can be included as live attenuated bacteria, as inactivated bacteria (bacterins), or as parts thereof (lysates, homogenates, extracts, fractions, etc.).

[0112] In a preferred embodiment, the Leptospira bacterium is one or more selected from the following:

[0113] - L. interrogans serogroup Canicola, more preferably serovar Portland-vere or serovar Canicola. - L. interrogans serovar Icterohaemorrhagiae, more preferably serovar Copenhageni or serovar Icterohaemorrhagiae, - L. interrogans serogroup Pomona, more preferably serovar Pomona, - L. interrogans serogroup Australis, more preferably serovar Bratislava or serovar Australis, and - L. kirschneri serogroup Grippotyphosa, more preferably serovar Dadas, serovar Grippotyphosa, or serovar Bananal / Lianguang.

[0114] In a preferred embodiment, the Bordetella bacterium is B. bronchiseptica.

[0115] In a preferred embodiment of the liquid vaccine composition according to the invention comprising an additional antigen, the additional antigen is a further virus.

[0116] In a more preferred embodiment, the additional virus is one or more selected from rabies virus, canine adenovirus type 1 (CAV-1), canine adenovirus type 2 (CAV-2), canine parvovirus (CPV), canine coronavirus (CCV), canine distemper virus (CDV), and canine herpesvirus.

[0117] The additional viruses may be included as live attenuated viruses or as inactivated viruses. The relevance of these additional viruses in veterinary medicine is known and is described, for example, in "The Merck Manual" (supra).

[0118] In one embodiment of the liquid vaccine composition according to the invention, one or more conditions selected from the group consisting of the following apply:

[0119] -NADES are formed from an organic salt, a polyol, and water; the organic salt is preferably one or more selected from salts of betaine, proline, carnitine, and choline; the polyol is preferably one or more selected from sugars or sugar alcohols; the sugar is preferably one or more selected from fructose, maltose, sucrose, glucose, and trehalose; the sugar alcohol is preferably one or more selected from glycerol, xylitol, mannitol, and sorbitol; -NADES are formed from proline, sorbitol and water; - proline preferably has CAS number 609-36-9; - sorbitol is D-sorbitol, preferably D-sorbitol has the CAS number 50-70-4; proline, sorbitol and water are present in the NADES such that the molar ratio of proline:sorbitol:water is from 1:1:1 to 1:1:16, preferably from 1:1:1 to 1:1:15, more preferably from 1:1:1 to 1:1:12, or from 1:1:1 to 1:1:10; -Live viruses are large, i.e., greater than 50 nanometers in diameter; -Live viruses have an RNA genome; -Live viruses are live enveloped viruses; -Live viruses have an RNA genome and are also enveloped; - the live enveloped RNA virus is selected from paramyxoviruses and coronaviruses; - the paramyxovirus is selected from canine parainfluenza virus (CPI) and canine distemper virus (CDV); - the coronavirus is bovine coronavirus (BCV) or infectious bronchitis virus (IBV); - ectoine preferably has CAS number 96702-03-3; - hydroxyectoine is 5-hydroxyectoine, preferably 5-hydroxyectoine has CAS number 165542-15-4; the additive is present in an amount of 0.05 to 1% by weight, preferably the additive is present in an amount of 0.05 to 0.5% by weight, or even 0.1% by weight; -The additive is methionine; - methionine is L-methionine, preferably L-methionine has the CAS number 63-68-3; the liquid vaccine composition has a water content of less than or equal to 50% by weight, more preferably less than or equal to 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 35, 30, 25, 20 or 15% by weight; - the liquid vaccine composition has a minimum water content of 5% by weight, preferably, in order of preference, a minimum water content of 7, 10, 12, 15, 20% by weight; the liquid vaccine composition has a viscosity of less than 1000 mPa·s, more preferably less than or equal to 750, 500, 400, 300, 250, 200, 150, or 100 mPa·s; - the liquid vaccine composition comprises an additional antigen, preferably the additional antigen is a bacterial antigen, more preferably the bacterial antigen is one or more selected from Leptospira and Bordetella bacteria: The Leptospira bacterium is one or more selected from the following: L. interrogans serogroup Canicola, more preferably serovar Portland-vere or serovar Canicola; L. interrogans serogroup Icterohaemorrhagiae, more preferably serovar Copenhageni; L. interrogans serogroup Pomona, more preferably serovar Pomona; L. interrogans serogroup Australis, more preferably serovar Bratislava; and L. kirschneri serogroup Grippotyphosa, more preferably serovar Dadas, serovar Grippotyphosa, or serovar Bananal / Lianguang; - The Bordetella species is B. bronchiseptica; - in a liquid vaccine composition comprising an additional antigen, the additional antigen is a further virus; said further virus is one or more selected from rabies virus, canine adenovirus (CAV), canine parvovirus (CPV), and canine coronavirus (CCV).

[0120] In one embodiment of the liquid vaccine composition according to the present invention, the NADES is formed from proline, sorbitol and water, which are present in the NADES in a molar ratio of proline:sorbitol:water of 1:1:1 to 1:1:10, the additive is present at 0.1% by weight, the additive is methionine, the live virus is one or both selected from CPI and CDV, the liquid vaccine composition comprises an additional antigen which is one or more bacterial antigens selected from Leptospira and Bordetella, and the liquid vaccine composition comprises a further additional antigen which is one or more additional viruses selected from canine adenovirus (CAV), canine parvovirus (CPV), and canine coronavirus (CCV).

[0121] The liquid vaccine composition according to the invention can be produced using conventional techniques and materials. Details and examples of methods, uses or processes for producing the liquid vaccine composition according to the invention are described herein, and such procedures can be easily adapted by a person skilled in the art using conventional materials and methods.

[0122] For example, the NADES of the invention can be produced on an industrial scale and have a low viscosity, making this part of the production easier. The additives defined in the invention are then added. This can be added at different stages, for example during the formation of the NADES, to the NADES after formation, to the live virus preparation before it is mixed with the NADES, or to the mixture of NADES + virus.

[0123] The mixture is then filled into containers of appropriate size. The various stages of the manufacturing process are monitored by sufficient testing, for example, by immunological tests to check the quality and quantity of virus; by microbiological tests to ensure sterility and freedom from foreign matter; and finally by vaccination studies in animals to check efficacy and safety. Once quality, quantity and sterility tests are complete, the vaccine product is available for sale.

[0124] All of this is well known to those of skill in the art, and the general techniques and considerations that apply to the manufacture of vaccines under known standards of pharmaceutical manufacturing are described, for example, in government directives and regulations (Pharmacopeia, 9 CFR, supra) and known handbooks (Pastoret, Remington, supra).

[0125] Therefore, in a further aspect, the present invention relates to a method for producing a liquid vaccine composition according to the present invention, said method comprising the steps of: - providing a NADES as defined in the present invention, - mixing said NADES with an additive as defined in the present invention, and - mixing said NADES and said additive with a composition comprising a live virus. The present invention relates to a method comprising the steps of:

[0126] Conveniently, these steps of the method of the invention can be carried out separated in time and / or location, with intervening storage, transport or further steps, allowing flexibility in planning and arranging operations.

[0127] The preparation of the NADES used in the process according to the invention can be carried out using conventional means and methods. One convenient method is to heat the mixed compounds to a temperature at which they mix and dissolve easily without causing damage, for example about 80° C. The heating can be carried out, for example, in combination with stirring or ultrasonication.

[0128] Additionally, one of the components of the NADES may first be dissolved in water in a somewhat concentrated form and then mixed with additional components to form the NADES of the present invention by evaporating the excess water.

[0129] The additives defined in the present invention can be mixed with the NADES after it has been formed or can be added to the compound before the NADES is formed. The additives can be added as dry salts, which allows the total amount of water in the liquid vaccine according to the present invention to be controlled. Alternatively, the additives can be added from an aqueous stock solution. Obviously, when preparing the liquid vaccine composition according to the present invention, the amount of water thus added to the composition must be taken into account.

[0130] The compositions comprising live virus used in the methods according to the invention can be conveniently derived in a variety of ways, advantageously from in vitro culture of the virus in a suitable cell line. Virus cell culture can be carried out in any desired size or volume, up to several thousand liters, in large industrial size fermenters applying automated control and monitoring techniques.

[0131] The virus is then harvested from the culture in an appropriate manner, for example as supernatant, cell pellet or whole culture. This can be fine-tuned to the characteristics of the virus and the culture system used. The virus preparation used in the method according to the invention can be further processed, if necessary, using further processing steps, such as concentration by ultrafiltration. The virus composition is then checked for quality and quantity. All this is known to the skilled person.

[0132] Mixing of the NADES components, additives, and virus in the methods of the present invention is preferably performed using aseptic techniques.

[0133] The order and method of mixing is not important, and the NADES can be added to the virus composition or vice versa, and additives can be added at any stage of the process. Preferably, any mechanical mixing used is slow and low impact so as to be gentle on the live virus. Additionally, mixing and / or filling can be performed in a protective atmosphere, for example with nitrogen or argon gas, to reduce exposure to oxygen.

[0134] Convenient equipment for mixing and filling liquid vaccine compositions is available from a variety of process-equipment suppliers.

[0135] In one embodiment of a method for producing a liquid vaccine composition according to the invention, the steps of preparing the NADES and adding an additive are combined to combine the NADES components with the additive, after which the NADES is formed as described, for example, proline, sorbitol, and methionine are combined as dry salts, with a small amount of water, and upon heating, optionally with sonication, the NADES+additive is formed.

[0136] In a further aspect, the present invention relates to the use of an additive as defined in the present invention for stabilising a live virus in a liquid vaccine composition according to the invention.

[0137] In one embodiment of the use of the additive according to the present invention, the additive is contained in the liquid vaccine composition at 0.05 to 1% by weight, preferably 0.05 to 0.5% by weight, or 0.1% by weight.

[0138] In the present invention, "stabilization" refers to the reduction in loss of viral titer over time, as defined above.

[0139] In a further aspect, the present invention relates to a method for stabilising a live virus in a liquid vaccine composition according to the invention, comprising the step of including an additive as defined in the present invention in said liquid vaccine composition.

[0140] In one embodiment of the stabilization method according to the present invention, the additive is contained in the liquid vaccine composition at 0.05 to 1% by weight, preferably 0.05 to 0.5% by weight, or 0.1% by weight.

[0141] As mentioned above, the present invention allows for a liquid live virus vaccine composition that has low viscosity but retains good virus stability, and therefore can be advantageously used as a vaccine against a variety of targets and diseases.

[0142] Therefore, in a further aspect, the present invention relates to a liquid vaccine composition according to the invention for use in a method for protecting a human or animal target against infection and / or disease caused by a pathogenic form of a virus contained in said liquid vaccine composition.

[0143] The "method of protection" comprises the step of administering a liquid vaccine composition according to the invention to a subject in need of such protection. As explained, this administration can be carried out in a variety of ways, particularly due to the reduced viscosity of NADES-based vaccines.

[0144] Thus, in one embodiment, the use in a method of protection according to the invention comprises administration by a mucosal route, preferably involving a route selected from the ocular, nasal and oral routes. The mucosal route can also be a combination, such as, for example, ocular-nasal or nasal-oral.

[0145] Obviously, the method of protection according to the invention can also be carried out by administration via the parenteral route, i.e. via the skin, for example via the intramuscular, intraperitoneal, intradermal, submucosal or subcutaneous route. The preferred route of parenteral administration of the liquid vaccine composition according to the invention is by intramuscular or subcutaneous injection.

[0146] Furthermore, the method of protection according to the invention may involve administering a liquid vaccine composition according to the invention via the enteral route.

[0147] Alternatively, liquid vaccines can be administered by mass dosing methods such as via drinking water, coarse spray, atomization, feeding, and the like.

[0148] The method of protection according to the present invention can be applied as either a prophylactic or therapeutic treatment, or both.

[0149] The liquid vaccine composition according to the present invention may serve as an effective priming vaccine, which can then be followed and amplified by booster vaccinations with the same or a different vaccine.

[0150] The target of the liquid vaccine composition for use according to the invention is a human or animal in need of vaccination against an infection and / or disease caused by the pathogenic form of the live virus contained in the vaccine. The age, weight, sex, immune status and other parameters of the target to be vaccinated are not critical, although it is preferred to vaccinate healthy, uninfected targets and to vaccinate as soon as possible to prevent field infections.

[0151] In one embodiment of the method of protection according to the invention, the target is a mammal or bird, preferably the mammal is a bovine or canine.

[0152] In an embodiment of a liquid vaccine composition for use according to the present invention, the characteristics of the liquid vaccine and the live virus are all as disclosed herein for the invention.

[0153] In a further aspect, the present invention relates to a method for vaccinating a human or animal target, comprising administering to said target a liquid vaccine composition according to the invention.

[0154] In an embodiment of the vaccination method according to the invention, the administration and characteristics of the liquid vaccine are all as disclosed herein for the invention.

[0155] The invention is described herein in various aspects and embodiments, it being understood that any combination of these is considered within the scope of the invention, although for the sake of brevity, not all possible combinations have been exhaustively described herein.

[0156] The invention will now be further illustrated by the following non-limiting examples. EXAMPLES

[0157] Example 1: Comparison of properties of different compositions In the field of live virus vaccines, most compositions described are lyophilized cakes. Although some publications describe liquid formulations, these are typically aqueous compositions, i.e. based on water as a carrier, and are not NADES. These aqueous compositions of live viruses differ from NADES-based liquid vaccines mainly in their DSC profiles, and in the amount, concentration and availability of water present. As some of these aqueous compositions may be superficially similar to the compositions of the present invention, they are analyzed and compared in more detail here, thereby clearly showing that they are in fact essentially different.

[0158] Aqueous compositions from the literature WO2014 / 029702 and related publications, such as WO2014 / 140239, WO2015 / 121463, US2014 / 0271710, describe liquid stable preparations of many different viruses in compositions that include a sugar alcohol and one or more amino acids. One exemplary composition taken from '702 includes 30% by weight sorbitol and 0.6M arginine in water. A more detailed analysis of this composition reveals the following: If the (estimated) density of the composition is 1.1 g / mL, there will be 1100 grams per liter. Sorbitol is 300 g / g, which gives 330 g / L, and Mw is 182 g / mol, which gives 1.8M sorbitol. Similarly, arginine is 0.6M, and Mw is 174.2 g / mol, which corresponds to 105 g / L of arginine. In this case, the water content is 1100-330-105=665 grams / liter, or 18 g / mol, so this is 37M water. This corresponds to 60.5% water by weight. The water activity of this composition was also measured, and found to be Aw=0.89.

[0159] In conclusion, the liquid compositions described in WO2014 / 029702 and related publications have water contents of 60%, water concentrations of 37M, and water activities of 0.89, all of which are significantly above the limiting and preferred values ​​for the NADES-based compositions of the present invention.

[0160] NADES characteristics When comparing the aqueous compositions described above to the NADES-based compositions of the present invention, it is immediately apparent that they have substantially different compositions and properties. For example, NADES with a molar ratio of proline:sorbitol:water of 1:1:2.5 has a water activity Aw = 0.39 and a water content of only 13.2 wt%.

[0161] Even in the case of NADES with a molar ratio of proline:sorbitol:water of 1:1:10, which further contains water, Aw=0.76 and the water content is 37.7 wt% or less.

[0162] For more information, see Table 1, which also outlines the glass transition temperatures (Tg) of the two extreme NADESs shown, as well as the decrease in viscosity that is obtained when the NADES is prepared using more water.

[0163] The Tg of the samples was measured by differential scanning calorimetry (DSC) using thermal analysis software on a TA Instruments Q2000 DSC instrument. For a complete DSC profile, the sample was first equilibrated at 40°C, then 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. This cooling and heating cycle was repeated once more on the same sample to investigate phase hysteresis. The Tg of the sample was determined by the software based on the heating curve at the midpoint of the stepwise shift in baseline, indicating a change in thermal conductivity from an increase in mobility at the glass transition.

[0164] Viscosity was measured at 20° C. using standard conditions: Brookfield™ DV-I+ viscometer, spindle type No. 62, 60 rpm for 30 seconds in a standard measuring cup (80 mL capacity).

[0165] Table 1: Characteristics of NADES with increasing water volume [Table 1]

[0166] NADES-Based Compositions According to the Invention In the preparation of a liquid vaccine composition according to the invention, several parts by volume of NADES are mixed with one part by volume of an aqueous preparation of live virus. Typically, 9 parts by volume of NADES are mixed with one part by volume of virus suspension, but within the spirit of the invention, a lower relative amount of NADES can also be used, and vice versa, and a higher relative amount of virus suspension (and other antigens) can be added to further dilute the NADES. This reduces the viscosity, but may also affect the stability of the virus, which can be largely prevented by adding the additives defined in the present invention.

[0167] Mixing the antigen suspension slightly increases the water content in the final liquid vaccine composition, but this is not a linear effect. The higher the initial water content in the NADES, the less the relative increase in water content due to such dilution with the virus sample than when the starting water content is lower. This was analyzed, for example, by adding 1 part pure water to 9 parts NADES consisting of proline:sorbitol:water (P:S:W) in a molar ratio of 1:1:2.5. This increased the water content from 13.2% by weight of the NADES to 25.8% by weight of the NADES base composition. For other P:S:W NADES mixed 9+1 with water, the increase in water content was as follows: 1:1:5 went from 23.3% to 37.0% by weight, and 1:1:10 went from 37.7% to 50% by weight. In this last case, Aw increased from 0.76 to 0.80.

[0168] Example 2: Different additive compounds tested A number of compounds were tested for their potential to compensate for the negative impact on virus stability of increased water content in liquid vaccine compositions according to the invention. Different virus types and different incubation temperatures were used to obtain the broadest possible effect of additive effects in the context of NADES-based virus compositions.

[0169] 2.1 Canine parainfluenza virus CPI is a paramyxovirus that was mixed into a NADES carrier containing water and various additives and incubated at various temperatures for increasing periods of time. The NADES used was a molar ratio of 1:1:10 proline:sorbitol:water (P:S:W). The components of the NADES were mixed with 0.1 wt% of one of the different additives and dissolved by sonication in a heated water bath. The same NADES but without additives was used as a control.

[0170] After cooling, 0.9 mL of NADES+additive or control NADES was filled into a number of 3 mL glass vials. At the start of the stability experiment, 0.1 mL of 10^9 TCID50 / mL CPI solution was added to each vial, which was then closed and mixed thoroughly. The amount of water was 38% by weight before mixing with the additive and virus, and 50% by weight after mixing.

[0171] The t=0 vial was immediately titered on Vero cells. Standard 96-well plates were prepared the day before with Vero cells at 1x10^5 cells / mL in 100 μL of standard medium and incubated overnight at 37°C in 5% CO2.

[0172] At each time point tested, serial 1:10 dilutions were prepared by dissolving a vial of the virus+NADES composition in 1 mL of medium and adding 0.2 mL to 1.8 mL of medium for seven additional consecutive cycles. 100 μL of each dilution was then added to each well of a 96-well plate containing Vero cells and incubated for 7 days. The cpe was then read and the titer was calculated as TCID50 expressed as Log10 / mL.

[0173] Except for the t=0 sample, the other samples were stored at room temperature (i.e., 20° C.) for at least 24 weeks. At appropriate time points after the start of the experiment: 4, 8 and 24 weeks, vials were removed and their contents titrated according to the method described.

[0174] The various additives tested here were citric acid monohydrate, ectoine, EDTA, methionine and lecithin, all used at 0.1% by weight relative to the weight of the NADES. An additional control series was also included, in which CPI was placed in phosphate buffered saline (PBS) and treated and stored in the same way.

[0175] The results of the incubation of the CPI virus in the different compositions are shown in the graph in Figure 3. As is quite clear, the CPI was a fairly stable virus and was detectable in the PBS composition even after 8 weeks of incubation at room temperature. However, the PBS sample was negative for live CPI at 24 weeks. In the control NADES composition, a CPI titer of 5.9 was still detectable at 24 weeks. Next, the effect of various additives was compared. Citric acid, EDTA, and lecithin did not provide a clear improvement to the stabilization by the control NADES after 24 weeks. However, samples containing ectoine or methionine maintained titers much better over time.

[0176] The fact that not all samples recorded titers of 10^8 at t=0 is partly due to test variability but also to the effect of early mixing of virus with NADES, which may have led to a slight decrease in the titer of the NADES itself.

[0177] It is clear that even the NADES-based liquid composition according to the present invention is unable to stabilize the CPI at its initial level for long periods at high temperatures. However, it was impressive that the CPI virus could be stored in liquid form at room temperature for at least half a year with only a loss of titer of about 1.5 Log10 / mL. This shows that NADES-based compositions containing a significant amount of water can effectively stabilize live virus without the need for lyophilization.

[0178] 2.2 Bovine coronavirus BCV was used in a similar series of experiments with various additives and was found to be more sensitive than CPI to long-term storage in liquid form, as it showed a significant loss of potency even at 4°C.

[0179] The experimental setup was the same as described for CPI, with BCV used in each vial at a titer of 6.5 Log10 TCID50. The combination with NADES was in a 1+9 volume ratio, and the control NADES was P:S:W 1:1:10, and incubation was at 28°C for up to 2 weeks or at 4°C for up to 13 months. BCV was titrated in Madin Darby bovine kidney cells in microtitration plates. Infection (live virus replication) was detected by IFT. The additives were used at 0.1% by weight and mixed during the preparation of the NADES.

[0180] The results of BCV incubation are shown in Fig. 4 , with panels A and B showing incubations at 28°C and 4°C, respectively.

[0181] Incubation at 28°C served as a simple stability test. Under these conditions, the control NADES failed to stabilize BCV at an adequate level. After one week, 2.5 Log10 of viral titer was lost, and after two weeks, no BCV was detectable at all. This was not improved by the use of citric acid or vitamin C as additives, which instead showed increased titer loss. Surprisingly, however, hydroxyectoine and methionine were able to maintain the BCV titer at approximately 3 Log10 after two weeks of incubation at 28°C in liquid conditions. This is an impressive performance by any standards.

[0182] More practical conditions were focused on incubation at 4°C, which was carried out for a very long period of time. Among the compositions tested, a further control was storage in pure water, which showed a complete loss of BCV titer at 2 months. The control NADES gradually lost titer after the first month, and by 13 months live BCV was no longer detectable. Results using citric acid or iron-citric acid as additives were slightly less, but vitamin C was much worse.

[0183] However, hydroxyectoine and methionine were still able to significantly improve the stabilization capacity of the NADES control, even after storage in liquid state at 4°C for 13 months.

[0184] Example 3: Further test viruses To expand on these positive results, more viruses were tested.

[0185] 3.1 Infectious bronchitis viruses IBV is an avian coronavirus that was used in tests with various NADES compositions by incubation at 4°C for up to 36 weeks.

[0186] The experimental set-up was essentially as described above. IBV strain MA5 was mixed with NADES in a 1:1:5 ratio P:S:W containing 0.1% methionine by weight in a 1+9 volume ratio. IBV was mixed in PBS as a control. IBV samples were titrated on fertilized eggs and after 3 days of incubation, allantoic fluid was harvested and used in an antigen mass ELISA to quantify IBV.

[0187] The results of IBV incubation are shown in FIG.

[0188] Notably, IBV titers were rapidly lost in PBS, with no live virus detected after 36 weeks at 4° C. Surprisingly, however, the 1:1:5 NADES composition containing methionine was able to retain most of the live IB virus even after 36 weeks of incubation at 4° C. in liquid state.

[0189] 3.2 Canine distemper virus CDV is a further paramyxovirus with which stability studies were performed up to 2 months at room temperature, up to 8 months at 4°C and even up to 23 months. The CDV strain Onderstepoort was used with an initial titer of 6.1 Log10 TCID50. The CDV virus was grown and titered on Vero cells. The NADES used were P:S:W 1:1:5 with or without 0.1% methionine by weight. In the first sample series (section 3.2.1 below), CDV was incorporated into the NADES at a 1+9 volume ratio, increasing the water content from 23.3 to 26.2% by weight. In a parallel sample series (section 3.2.2 below), a CDV virus sample was used with further viruses: canine adenovirus, canine parvovirus and canine parainfluenza virus. This larger virus sample increased the water content of the P:S:W 1:1:5 NADES from an initial 23.3 to a final 40.1% by weight.

[0190] As an additional control, a CDV sample was taken in a conventional liquid stabilizer composition called "IDK" which contains NZ amine and sorbitol in an aqueous phosphate buffer. The IDK stabilizer is a typical aqueous composition with a water content of 87.4% by weight.

[0191] The results of CDV incubation are shown in Figure 6, with panels A and B showing incubation in a composition containing 27% water by weight at 20°C and 4°C, respectively, and panel 6C showing incubation in a composition containing 40% water by weight.

[0192] 3.2.1: CDV stability in NADES-based compositions containing 26 wt% water At 20°C, the control NADES slowed the decline in virus titer somewhat but was not effective. The conventional aqueous stabilizer IDK was even worse, with live CDV no longer detectable at all at the 2-month time point. However, NADES + methionine was able to improve the stability seen with the control NADES.

[0193] The same trend was observed at 4 °C, but the effect was more pronounced. The IDK stabilizer was able to maintain CDV titers to some extent even after incubation for up to 8 months at 4 °C in liquid conditions. However, methionine-containing NADESs provided superior stabilization, again much better than the control NADES.

[0194] 3.2.2: Stability of CDV in NADES-based compositions containing 40 wt.% water In FIG. 6C, the aqueous stabilizer was the same IDK as above.

[0195] Samples containing 40% water by weight were incubated at 4° C. CDV in the NADES control gradually lost its titer, and after 23 months of incubation in liquid form at 4° C. no live CDV was detectable. However, the addition of 0.1% methionine by weight delayed this loss of titer by almost another year.

Claims

1. A liquid vaccine composition comprising a live virus and a pharmaceutically acceptable carrier, the carrier is a natural deep eutectic solvent (NADES) and the vaccine has a water activity of up to 0.8; 10. A liquid vaccine composition, characterized in that the liquid vaccine composition has a water content of up to 50% by weight, and that the vaccine also comprises an additive selected from methionine, ectoine, and hydroxyectoine.

2. 2. The liquid vaccine composition according to claim 1, wherein the NADES is formed from proline, sorbitol and water.

3. 3. The liquid vaccine composition of claim 2, wherein the proline, sorbitol and water are present in the NADES in a molar ratio of proline:sorbitol:water of 1:1:1 to 1:1:

10.

4. The liquid vaccine composition according to claim 1, wherein the additive is contained in an amount of 0.05 to 0.5% by weight.

5. 2. The liquid vaccine composition of claim 1, wherein the live virus is a live enveloped virus.

6. 6. The liquid vaccine composition according to claim 5, wherein the live enveloped virus is selected from paramyxoviruses and coronaviruses.

7. 7. The liquid vaccine composition according to claim 6, wherein the paramyxovirus is selected from canine parainfluenza virus (CPI) and canine distemper virus (CDV).

8. 10. A method for producing the liquid vaccine composition of claim 1, comprising: - preparing a NADES as defined in claim 1, - mixing said NADES with an additive as defined in claim 1, and - mixing said NADES and additives with a composition containing said live virus; A method comprising:

9. 10. Use of an additive selected from methionine, ectoine, and hydroxyectoine to stabilize a live virus in a liquid vaccine composition as defined in claim 1.

10. 10. The liquid vaccine composition of claim 1 for use in a method for protecting a human or animal target against infection and / or disease caused by a pathogenic form of the virus contained in said liquid vaccine composition.

11. A method for vaccinating a non-human animal target, the method comprising administering to said target the liquid vaccine composition of claim 1.

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