Low-energy electron irradiation-inactivated mollicutes

Abstract

The present invention relates to the field of veterinary vaccines. More specifically, the invention relates to a composition and to vaccines comprising Mollicutes bacteria inactivated by low- energy electron irradiation (LEEI), to methods for producing said composition or said vaccines, and to use of said composition, and of said vaccines for the protection of swine against infection or disease caused by Mollicutes bacteria.

Description

[0001] LOW-ENERGY ELECTRON IRRADIATION-INACTIVATED MOLLICUTES

[0002] TECHNICAL FIELD

[0003] The present invention relates to the field of vaccines. More specifically, the invention relates to a composition and to vaccines comprising Mollicutes bacteria inactivated by low-energy electron irradiation (LEEI), to methods for producing said composition or said vaccines, and to use of said composition and of said vaccines for the protection of a target against infection or disease caused by Mollicutes bacteria.

[0004] BACKGROUND

[0005] A group of bacteria with agricultural and medical significance is the class of Mollicutes.

[0006] Mollicutes derive from Gram positive bacteria but have lost their cell wall. They are typically parasites or commensals of eukaryotic hosts, both human and animal.

[0007] The Mollicutes are the smallest self-replicating organisms presently known: bacteria from some species are only 0.2 pm in diameter, and some have a genome of only 580 kilo-base pairs (kbp). Having so few genes, Mollicutes bacteria heavily rely on resources from their environment, so when cultured in vitro they typically require very rich and complex media.

[0008] Several Mollicutes are known as causes of human- or animal diseases. The best-known genus in the Mollicutes class is Mycoplasma. Infections of animals, such as ruminants, poultry or swine, by Mycoplasma bacteria, either as primary- or as secondary pathogen, often cause respiratory disease and / or a variety of other symptoms. In humans M. pneumoniae, M. hominis, and M. genitalium are well known causes of disease.

[0009] Mycoplasma hyopneumoniae (Mhyo) is a widespread swine respiratory pathogen, causing chronic respiratory disease leading to poor growth and vulnerability to secondary infections. Especially young pigs are affected by this, non-fatal, disease. The disease is highly contagious, and transmission is usually through direct contact with infected respiratory tract secretions, e.g. in the form of infected droplets after coughing / sneezing. The most problematic consequence of this disease is that it predisposes for all kinds of secondary infections of the respiratory system, which may exacerbate disease. It is estimated that e.g. in the USA, 99 % of all pig farms are infected. Yearly losses are estimated to be between 100 and 300 million dollars.

[0010] Without a cell-wall, several members of the class of Mollicutes are naturally resistant to antibiotics that target cell wall synthesis (like the beta-lactam antibiotics). Therefore, treatment is commonly prophylactically, by way of vaccination. Consequently, most pigs worldwide are being vaccinated against Mhyo, typically at young age.

[0011] Vaccines against infections with Mollicutes bacteria can be based on live attenuated strains, or on inactivated bacterins (killed bacteria). Most commercial Mycoplasma vaccines are used for poultry. Examples of live attenuated vaccines for M. gallisepticum are Vaxsafe® MG (Bioproperties) (frozen), and Nobilis® MG 6 / 85 (MSD Animal Health) (freeze-dried, cooled). Live attenuated vaccines for M. synoviae are: Vaxsafe® MS (Bioproperties) (frozen), and Nobilis® MS Live (MSD Animal Health) (freeze-dried, cooled).

[0012] Examples of bacterin type Mycoplasma vaccines are: for M. gallisepticum: MG-Bac® (Zoetis) and Nobilis® MG inac (MSD Animal Health).

[0013] As attenuation is a complex and uncertain process, inactivation is an important step in vaccine development against micro-organisms that would otherwise be too virulent to administer. The inactivation process must efficiently kill the micro-organism, but without damaging the antigens needed to achieve an efficient immune response. Therefore, following inactivation, extensive testing of both complete inactivation and of remaining efficacy, is required before the product can be released for further processing.

[0014] A wide variety of methods is known to inactivate bacteria for the preparation of an inactivated bacterial vaccine. Such methods typically aim to damage the structure, the proteins, or the genome of the bacteria, to render it non-infectious or even non-proliferative. The methods typically employ chemical- or physical inactivation. Examples of physical inactivation are: heating, high shear, high pressure, or exposure to ionizing radiation, e.g. UV light, X-rays or gamma rays. Examples of chemical inactivation are exposure to high- or low pH, or addition of an enzyme, a detergent, an organic solvent, or a chaotropic agent. Typical chemical inactivation agents include formaldehyde (formalin), a lactone, such as beta propiolactone (BPL), or an aziridine such as binary ethylenimine (BEI).

[0015] Chemical inactivation, however, is a time-consuming process, the inactivation efficiency can display variability, and extensive downstream procedures are often required. Even with such downstream procedures, traces of the inactivation agent may still be present in the final product, which can be harmful for the vaccinated target or for other antigens that are to be combined.

[0016] Further, chemical inactivation agents, such as formalin, BEI, and BPL, are highly toxic, potential carcinogens, and require elaborate safety precautions when used, especially at large scale.

[0017] The production and inactivation of mycobacterial cell cultures is more challenging than average bacterial culturing, since the main immunoprotective antigen is not known, and culturing requires rich cell culture media. Vaccines are therefore typically prepared from whole inactivated Mycoplasma cell-cultures. Also, because the product used for vaccine production is the concentrated whole culture, thus including cells and supernatant, the product used for inactivation, formulation, and testing is effectively a crude and viscous composition, which contains high amounts of proteins and fats. This makes effective inactivation a more demanding process, which may require e.g. long incubation times, and large amounts of such harmful chemicals.

[0018] The presence of residual traces of chemical inactivation agents may further be problematic in case of combining the vaccine based on inactivated Mollicutes with antigens from other pathogens, such as, in the case of swine vaccines: Lawsonia intracellularis, and the viruses Porcine reproductive and respiratory syndrome virus (PRRSV), and porcine circovirus type 2 (PCV2). Such a combination is commonly used for swine vaccination for reasons of cost and economy, but also to reduce stress to the piglets resulting from the vaccination. However, the combination of inactivated Mollicutes bacteria and live attenuated PRRSV is a challenge, since trace amounts of chemical inactivation agents may affect or inactivate the PRRSV antigen.

[0019] Currently used physical inactivation processes, such as irradiation technologies (mostly gamma-rays and high energy electrons) require large and complex shielding constructions to protect the environment and staff from radioactivity or X-rays generated during the process. Thus, physical inactivation processes are typically rather cumbersome for large-scale production of vaccines.

[0020] LEEI is a process mainly used in the art for sterilization of food or surfaces. The process has also recently been established as an alternative for inactivation of pathogens in liquid solutions (Fertey et al., 2016, Viruses, 8:319). LEEI uses electrons of <500 keV, which results in a much lower amount of secondary irradiation, such as X-rays, generated. Therefore, LEEI does not require complex shielding. The LEEI process has been proposed for the inactivation of pathogenic viruses or bacteria, e.g. in processes of vaccine manufacturing or other applications such as decontamination and sterilisation. However, it has been described in the art mainly for small scale laboratory procedures using highly diluted- or purified liquid compositions, and for batchwise irradiation (see Fertey et al., 2022, In: Bidmos et al., (eds): Bacterial Vaccines. Methods in Molecular Biology, vol. 2414. Humana, New York, NY). In contrast, the production of vaccines for protection against pathogenic Mollicutes bacteria requires inactivation of whole cell cultures present in rich and complex media.

[0021] Further, ionizing radiation (including low-energy electrons), alters the integrity of structural- and functional components of the pathogens treated at the molecular level. On the one hand this is an intended effect, as that results in their inactivation. However, such modifications may also affect the antigenic structures required to induce the immuno-protective effect. This is particularly problematic in the manufacture of vaccines from cultures of Mollicutes, since the immunogenic components of the Mollicutes are not known. Thus, even though structural modifications in the pathogenic cell may be desirable to achieve complete inactivation, it may lead to destruction or partial denaturation of the pathogen, and thus to an impairment of the immunogenic capacity of the vaccine antigen.

[0022] Despite extensive research, the mechanisms of radiation sensitivity are not yet completely understood and probably vary in the effect on different taxa and species of pathogenic microorganisms. Further, it is theorized in the art that radiation susceptibility of microorganisms is additionally affected by further factors, such as medium composition. For example, for Bacillus subtilis, complete inactivation could not be reproducibly achieved by low dose (<6.5 kGy) LEEI (Schopf et al., 2002, Front. Immunol., 13:814767).

[0023] In consequence, it cannot be predicted whether a certain LEEI treatment can provide effective inactivation of Mollicutes bacteria, but if such LEEI-inactivated Mollicutes can still induce an efficient immunogenic reaction, and thus are suitable for use as an antigen in a vaccine.

[0024] It is therefore an object of the present invention to overcome the disadvantages associated with the inactivation processes used in the prior art, and to accommodate to this need in the field by providing immunologically effective inactivated Mollicutes bacteria. SUMMARY OF INVENTION

[0025] It has surprisingly been found in the present invention that the prior art problems can be overcome by performing LEEI to inactivate Mollicutes bacteria. It has further been surprisingly found in the present invention that such LEEI-inactivated Mollicutes bacteria are suitable for use as an antigen in a vaccine for the protection of a target against infection or disease caused by Mollicutes bacteria.

[0026] Thus, in a first aspect is herewith provided a composition comprising inactivated Mollicutes bacteria, such as Mycoplasma, characterised in that said inactivation was performed by LEEI. This is also termed in the following as the “inventive composition” or the “composition according to the invention”.

[0027] In a second aspect is herewith provided a method for the production of a composition comprising inactivated Mollicutes bacteria, the method comprising the steps of:

[0028] (a) providing a liquid composition comprising live Mollicutes bacteria, and

[0029] (b) inactivating the Mollicutes bacteria in said liquid composition by LEEI.

[0030] In a third aspect is provided a composition comprising LEEI-inactivated Mollicutes bacteria for use as a vaccine.

[0031] In a fourth aspect, the invention described herein is directed to the use of the inventive composition for the manufacture of a vaccine, in particular of a vaccine for the protection of swine against infection or disease caused by Mollicutes bacteria.

[0032] Other aspects of the invention described herein are directed to a vaccine comprising the inventive composition, its use in the protection of swine against infection or disease caused by Mollicutes bacteria, and a combination vaccine comprising the inventive composition comprising at least a first antigen, and a second composition comprising at least a second antigen, wherein the first composition is a composition according to the invention, and the second composition, for example, comprises PRRSV. DESCRIPTION OF DRAWINGS

[0033] Figure 1 : Schematic representation of a continuous LEEI inactivation process of a composition comprising biological material using a roller for transporting a liquid sample through an electron beam, for inactivation of the biological material.

[0034] Figure 2: Lung lesion score (LLS)-data resulting from a vaccination-challenge trial (described in Example 1) in swine using the vaccine of the invention compared with a vaccine prepared by chemical inactivation.

[0035] Figure 3: Titration of PRRSV titer following combination with Mhyo antigen concentrate irradiated with 10 kGy LEEI. Samples were incubated for 0 h, 1 h, or 3 h at room temperature (RT), with different starting titers (predilutions) of PRRSV (described in Example 2).

[0036] Figure 4: Titration of PRRSV (10E6.5 TCID50 / mL) combined with Mhyo antigen concentrate samples inactivated with different methods and incubated for 0 h and 3 h at RT (described in Example 2).

[0037] Figure 5: Titration of PRRSV (10E6.5 TCID50 / mL) combined with Mhyo antigen concentrate samples inactivated with different methods and incubated for 0 h or 5 h at RT (described in Example 2).

[0038] Figure 6: Titration of PRRSV (10E4.5 TCID50 / mL) combined with LEEI-inactivated Mhyo concentrated culture (13.6 PCVU / mL), or with BEI-inactivated Mhyo (15.0 PCVU / mL), for 0 h or 3 h at RT (described in Example 2).

[0039] DEFINITIONS

[0040] A “culture medium” (also referred to herein only as “medium”) refers to any solid- (e.g. agar plates), or liquid composition, that can be used for culturing, i.e. cultivation, growth, amplification, of Mollicutes bacteria. Such compositions will be known to the skilled person and are commercially available, for example as ready-to-use media or as powder compositions to be supplemented with a diluent, e.g. water, buffers, and / or nutritional enrichments, before use. For example, classical Mollicutes culture mediums were described by N. Friis (1971 , Acta Vet. Scand., vol. 12, p. 454-456), and variations are: inter alia, the well-known FRIIS medium, Frey medium, and Hayflick medium. The exact composition of the culture medium and the respective culturing conditions may depend on the Mollicutes, e.g. Mycoplasma, type or strain to be cultured, and can be optimised using common general knowledge. Similarly, the required culturing conditions using such media will be known to the skilled person.

[0041] A “vaccine” is a pharmaceutical composition that is safe to administer to a target, and is able to induce protective immunity in that target against a pathogenic microorganism, e.g. to induce a successful prophylactic treatment as defined here below. A vaccine as described in the context of the present invention typically relates to a composition comprising LEEI-inactivated Mollicutes bacteria, which composition or bacteria are suitable to induce protective immunity in a target, e.g. a human, or an animal such as a ruminant, poultry, or swine, against an infection or against disease caused by pathogenic Mollicutes bacteria, such as Mycoplasma bacteria, e.g. such as Mhyo.

[0042] “Non-replicating immunogen” of a pathogen is any substance or compound corresponding to or deriving from the pathogen, other than the live replicating pathogen as a whole (either in wild type or in attenuated form), against which pathogen an immunological response is to be elicited, such that the corresponding pathogen or an immunogen thereof will be recognized by the host's immune system as a result of this immune response, and are ultimately -at least partly- neutralized. Typical examples of non-replicating immunogens are killed bacteria, also termed “bacterin”. Non-replicating immunogen as described in the context of the present invention typically relates to LEEI-inactivated Mollicutes bacteria, but may also relate to immunogens from other pathogenic micro-organisms used in a combination vaccine comprising LEEI-inactivated Mollicutes bacteria. These include for example, bacterial- or viral immunogens, such as nonreplicating immunogens from PCV2 and / or from Lawsonia intracellularis, and / or live PRRSV.

[0043] A pathogen, such as a Mollicutes bacteria, is “inactivated” for the invention when it can no longer infect, or even replicate, in vitro or in vivo, under conditions wherein such pathogen would otherwise be able to proliferate.

[0044] Whether a Mollicutes bacterium has indeed been “inactivated” can readily be detected using common techniques. One way is to test using one of the published cell-lines that allow replication of Mollicutes bacteria. Signs of infection can then be observed based on cytopathogenic effect (cpe) by microscopic inspection, or based on detection of bacterial replication by e.g. RT-qPCR (reverse transcriptase quantitative real-time PCR), Enzyme-linked Immunosorbent Assay (ELISA), immunofluorescence, or flow cytometry. Alternatively, such an inactivation-control test can be performed in vivo by inoculation of target animals, including appropriate positive- and negative controls, and detection of any infection by histo-pathologic screening.

[0045] A “live attenuated pathogen” is a viable, replication-competent form of the pathogen, but displaying a reduced virulence in vitro or in vivo. The process of attenuation takes an infectious pathogen and alters it so that it becomes harmless or less virulent, typically by either multiple passaging of the pathogen in vivo or in vitro, or by genetically modifying the pathogen. A live attenuated pathogen as described in the context of the present invention typically relates to live attenuated PRRSV, which may be used in a combination vaccine including LEEI-inactivated Mollicutes bacteria.

[0046] “Prophylactic treatment” against an infection with a pathogen aims at inducing or assisting with preventing or ameliorating an infection with that pathogen or a disease arising from that infection post treatment, and / or to reduce the pathogen’s load in the host after such infection, and optionally to aid in preventing or ameliorating one or more clinical manifestations resulting from the post treatment infection with the pathogen.

[0047] DESCRIPTION OF EMBODIMENTS

[0048] Composition

[0049] In the first aspect, the present invention provides a composition comprising LEEI-inactivated Mollicutes bacteria. This class of bacteria was found in the present invention to be sensitive to LEEI inactivation. Hence, the invention is generally applicable to members within the class of Mollicutes bacteria.

[0050] Most well-known in the Mollicutes class is the Mycoplasmatales order, as it contains well known human- and animal pathogenic bacteria of the genus Mycoplasma: e.g. M. pneumoniae, M. bovis, M. hyopneumoniae, M. meleagridis, M. synoviae, and M. gallisepticum, which (mainly) infect respectively: humans, ruminants, swine, turkeys, and chickens.

[0051] Mycoplasma hyopneumoniae (Mhyo) is a species of Mycoplasma bacteria known to cause the disease porcine enzootic pneumonia, a highly contagious and chronic disease affecting pigs. Mhyo is difficult to grow in vitro due to its complex nutritional requirements and the high chances of contamination during such culture in rich medium. Thus, in a preferred embodiment, the Mycoplasma bacteria used in the present invention are Mhyo. The most relevant Mhyo strains include strain 98, strain 232, strain J, strain 7448, strain 168, strain 168L, strain 7422, and strain 11, as described in Kamminga et al. (2020, Front Microbiol., 11 :1679). Preferred Mhyo strains include strain 11 and / or strain J, most preferably strain 11.

[0052] The composition used for inactivation by LEEI is typically a crude culture used for growth and cultivation of the Mollicutes bacteria, such as Mycoplasma, in particular Mhyo. Thus, in a preferred embodiment, the composition used for inactivation by LEEI comprises or consists, preferably consists, of culture medium including said bacteria.

[0053] It has surprisingly been found for the first time in the present invention that a complete and efficient inactivation of Mollicutes bacteria can be achieved by LEEI inactivation, although the bacteria are present in a complex, or even in a concentrated, medium, i.e. , in the presence of high amounts of medium components, such as fats, lipids, proteins, carbohydrates and / or salts. In theory, all these components, especially when present in high amounts, may shield the bacteria from the radiation and / or lead to the generation of structural changes, modification or degradation products of medium components, which may impact inactivation efficiency and may further impact the immunological efficacy of the inactivated bacteria for use as an inactivated antigen in a vaccine.

[0054] Moreover, high amounts of medium components may considerably impair the performance of LEEI inactivation, since it may become difficult to achieve thin liquid films typically required for efficient LEEI inactivation.

[0055] Typically, the medium used for culturing the Mollicutes bacteria is directly applied to LEEI inactivation. In this embodiment, the “crude” culture medium is used, i.e., no further processing steps are performed on the Mollicutes culture before performing LEEI inactivation.

[0056] In an alternative embodiment, the culture medium comprising the Mollicutes bacteria may be processed by one or more processing steps before LEEI inactivation is performed. Such processing steps include, inter alia, one or more of concentration, dilution, (dia- and / or ultra-) filtration, dialysis, or centrifugation. Preferably, the culture medium applied to the LEEI inactivation process is processed by a concentration step. In an alternative embodiment, such processing steps may be performed after LEEI inactivation.

[0057] Filtration or centrifugation may be advantageous to clear the culture medium from solid materials, contaminants or precipitates resulting from or appearing during culturing of the Mollicutes bacteria. The presence of such solid materials may be detrimental to the inactivation efficiency or may lead to difficulties in performing LEEI inactivation, such as the formation of thin films typically required for the inactivation process, as described herein below. Of course the immunologically relevant fraction itself needs to be retained.

[0058] Since vaccination against infection caused by pathogenic Mollicutes typically requires a certain amount of immunogenic material in order to achieve an immunogenic reaction by the vaccinated target, it may be desirable to provide a concentrate of the inventive composition. Concentrating of culture medium can be performed, for example, by gravitation, centrifugation, filtration, such as diafiltration and / or ultrafiltration, for example using filters with a cut-off size of 100-1000 kDa, preferably with a cut-off size of about 500 kDa.

[0059] Thus, the composition used for LEEI-inactivation may be a concentrate of said Mollicutes culture, i.e. the composition comprises, or consists of, a culture concentrate, such as a 5x to 15x concentrate of the culture, preferably a 6x to 12x concentrate, e.g., a 7x, 8x, 9x, 10x or 11x concentrate of the culture.

[0060] The culture medium suitable for growth and cultivation of Mollicutes bacteria, such as Mycoplasma, is typically a rich and complex medium, i.e. it typically comprises one or more of: a protein hydrolysate, vegetable extract, yeast extract, and animal serum.

[0061] An “animal serum”, as used herein, refers to any fluid and solute component of blood derived from an animal source without the clotting factors, or as blood from which all cells and clotting factors were removed. For the culture of Mycoplasma, well-known animal serum is one or more of: bovine serum, porcine serum, and equine serum.

[0062] The base media may be supplemented with one or more additional components, such as carbohydrates, nutritional supplements, Mycoplasma supplement, or Mycoplasma enrichment as needed, depending on the culture medium used, and the Mycoplasma to be cultured. All this is well-known to the skilled person. Mycoplasma supplement typically contains yeast extract and animal serum. Yeast extract is typically used to provide the nucleic acid precursors that are required by Mycoplasma spp. Animal serum is typically used to supply cholesterol and several other growth factors. Mycoplasma enrichment is a selective enrichment containing the inhibitor thallium acetate, to which a beta-lactam antibiotic of choice (e.g. bacitracin, penicillin G, or a broad-spectrum semi-synthetic penicillin) can be added at the time of use, to make it selective against gram-positive and gram-negative bacteria, and to inhibit the synthesis of the cell wall of potentially contaminating gram-positive bacteria during culture of Mycoplasma bacteria.

[0063] The most widely used liquid medium for culture of Mhyo was developed by Niels Friis (N. Friis, 1971, supra), and is thus commonly referred to as “FRIIS medium” in the art. FRIIS medium may be supplemented with animal serum, e.g., horse serum or swine serum, to provide useable cholesterol and fatty acids. Meat digests, peptones, beef extract, and yeast extract typically provide the nitrogen, vitamins, amino acids, and carbon sources in these media. Sodium chloride is commonly used to maintain the osmotic balance of these formulations.

[0064] The pH value of Mycoplasma medium is typically in the range of 7.0 to 8.0, preferably from 7.2 to 7.6.

[0065] Therefore, the composition used for LEEI-inactivation may contain one or more of the components typically used in Mycoplasma medium, including one or more of brain heart infusion, meat digest, peptone, animal serum, e.g. porcine serum, horse serum; yeast extract, NaCI, mineral supplements, and beta-lactam antibiotics, as listed above. Whereby the skilled person will readily appreciate that the exact composition of a spent culture medium, i.e. after the proliferation of the Mollicutes bacteria, will not be the same as that of the original fresh medium at the start of the culture.

[0066] The composition used for LEEI-inactivation is typically provided in liquid (fluid) form, since inactivation is preferably performed by transporting a thin liquid film through an electron beam. Subsequently, the inventive, i.e., irradiation-inactivated liquid composition may be further processed, such as by lyophilization, freeze-drying, or spray-drying, to result in a solid composition, e.g., in a solid- or powder form.

[0067] Since Mollicutes inactivation is performed in the present invention by LEEI, which achieves a complete inactivation of the Mollicutes bacteria contained in the composition, the inventive composition does not require any other- or further inactivation methods to be performed additionally.

[0068] Therefore, the compositions of the invention are preferably substantially free of chemical inactivation agents conventionally used in the art, such as formalin, BEI, and BPL. For the invention, chemical inactivation agents include the corresponding neutralisation agent(s), e.g. thiosulphate. Therefore, the inventive composition typically does not contain any chemical inactivation agents.

[0069] “Substantially free” in the context of the present invention means that the amount of chemical inactivation agent is typically below 1000 ppm, preferably below 100 ppm, and most preferably is below the analytical detection limit.

[0070] In an embodiment of the composition according to the invention, the composition is obtainable by the method according to the invention, as defined herein below.

[0071] Low-energy Electron-Irradiation (LEEI)

[0072] In the second aspect, the present invention provides a method for the production of the abovedescribed inventive composition comprising LEEI-inactivated Mollicutes bacteria. The method comprises the steps of (a) providing a liquid composition comprising live Mollicutes bacteria, and (b) inactivating the Mollicutes bacteria in said liquid composition by LEEI.

[0073] “LEEI” as used in the context of the present invention refers to the use of electron beams in which electrons are accelerated with low-energy to inactivate the Mollicutes bacteria contained in the compositions as described herein.

[0074] As is well-known, in the technical production of low-energy electrons -after power is switched on- the acceleration voltage applied between cathode and anode during their emission determines their energy content in electron volts (eV). The higher the energy content of the electrons, the deeper those electrons can penetrate into the irradiated material.

[0075] The radiation power is the product of beam current (amount of electrons generated), and acceleration voltage, specified in kilowatts (kW).

[0076] Low-energy in the context of LEEI typically refers to electrons accelerated using an acceleration energy of between 50 keV and 500 keV, more preferably accelerated by between 100 keV and 400 keV, even more preferably between 150 keV and 300 keV, or even at about 200 keV.

[0077] The use of low-energy electrons for inactivation and sterilization of biological material is described in the art and commonly known to the skilled person. The use of low-energy electrons for irradiation of biological material (in short referred to herein as “LEEI process”) is for example described in WO 2015 / 011265 and WO 2018 / 041953, which are incorporated herein in their entirety, and in Fertey et al. (2016, supra).

[0078] LEEI according to the invention is preferably carried out using a device for generating electron beams which operates continuously or in rapid pulses. Preferably, the irradiation process is performed continuously. A continuous LEEI process suitable for use in the methods of the present invention is described, for example, in WO 2018 / 041953. In a further preferred embodiment of the LEEI process, the compositions and vaccines according to the invention are transported continuously through the electron beam.

[0079] In the LEEI process of the invention, the dose rate (beam current per unit of time) can typically be adjusted in relation to the desired final, applied dose, evidently a high beam current requires a short irradiation time, and a low beam current requires a longer irradiation time. The dose rate can be adjusted by the skilled person in consideration of e.g. the flow rate of the liquid medium, and the type of radiator, which is as defined in WO 2015 / 011265 and WO 2018 / 041953.

[0080] For example, in the method according to the invention, a radiation dose can be applied in the range of 5 to 50 kGy, particularly 10 to 30 kGy, over a time period of up to 1000 seconds; preferably over a time period of 1 to 200 seconds, most preferably between 10 to 120 seconds.

[0081] In a further preferred embodiment of the method according to the invention, the irradiation time is therefore in the range of 1 second to 1000 seconds, preferably between 2 seconds and 200 seconds, most preferably between 10 seconds and 120 seconds.

[0082] During the irradiation, a temperature increase may occur, which depends on the applied dose rate. To avoid denaturing processes, it is therefore advantageous if the temperature rise is controlled or limited. The device may thus preferably be equipped with a cooling device for cooling of the material before, during and / or after the irradiation process. Alternatively or additionally, the temperature of the composition to be irradiated has a temperature between 1 °C and 40 °C, preferably between 5 °C and 37 °C, more preferably between 10 °C and 32 °C, even more preferably between 15 °C and 30 °C.

[0083] The LEEI process typically requires that the compositions as defined herein are irradiated in the form of liquid films. The film thickness may affect the inactivation efficiency, since low-energy electrons have a limited penetration depth, especially in complex and / or concentrated liquids. To ensure an efficient and complete inactivation, the film thickness is typically within a range of 0.05 to 0.5 mm, preferably between 0.1 and 0.4 mm. A lower film thickness may be economically undesirably, since then only low throughput volumes are achieved. On the other hand, when the thickness of the liquid film is higher than the upper limit, the full radiation dose cannot reach the bottom of the layer, leading to incomplete inactivation. Thus, at higher film thicknesses, a higher electron energy and / or higher dose rate may be necessary to achieve complete inactivation. Such optimisation and fine-tuning is well within the routine capabilities of the skilled person. In case a roller is used for continuously transporting a film of the liquid composition through the electron beam, the film thickness can be adjusted by the roller speed, and / or by the surface properties of the roller. Alternatively, the film thickness may be adjusted by altering the temperature and / or the viscosity of the composition.

[0084] In a preferred embodiment, the method according to the invention is characterized in that the compositions described herein and containing the Mollicutes bacteria, are irradiated with an electron beam dose of at least 5 kGy, and preferably a beam dose of between 10 kGy and 50 kGy, preferably between 10 and 30 kGy.

[0085] Higher electron beam doses can lead to faster inactivation, but may be economically undesirable due to higher energy consumption. Moreover, higher electron beam doses may lead to excessive and undesirable degradation or structural changes and modifications in antigenic components of the Mollicutes bacteria. Thus, higher electron beam doses may be detrimental for the immunogenic properties of the irradiated composition. Electron beam doses of lower than 5 kGy may lead to incomplete or inefficient inactivation, and may require lower film thicknesses, which may be difficult to process in particular when using a concentrate of the culture medium, which may have an increased viscosity due to high concentration of culture components.

[0086] LEEI inactivation is specifically suitable for continuously supplying the compositions described herein for the purpose of dose-controlled irradiation. In a particularly preferred embodiment, the composition is provided in liquid form, most preferably as a liquid film, for continuously inactivating the Mollicutes contained therein.

[0087] The continuous method generally comprises the steps of: (a) supplying the composition to the irradiation device; (b) rotating a roller in the device such that a continuous liquid film of determinable thickness forms on the revolving roller surface; (c) subjecting the liquid film on the roller surface to LEEI; and (d) collecting the LEE irradiated liquid.

[0088] A typical continuous method comprises at least the following steps: in step (a) the liquid composition, which contains the live Mollicutes, is supplied to a roller. In step (b) a continuous fluid film of a predeterminable thickness is formed from the supplied liquid composition or vaccine on the revolving roller surface. In step (c), the liquid film formed and exposed on the roller surface is irradiated with low-energy electrons at an inactivating dose. The radiation dose is determined by the radiation intensity of the radiation source, for the radiation-exposed volume of the liquid film, which is determined by the radiation window and the thickness of the film, and also preferably by the flow velocity or flow rate of the liquid film, which can be determined by the rotation speed of the roller. In step (d), the liquid containing the irradiation-inactivated Mollicutes after passing through the radiation window, can be collected from the roller surface.

[0089] Preferably, the LEEI dose is determined by the radiation source and the rotational speed. Since a radiation energy dose gradient arises within the liquid composition, it is particularly desirable in many cases for the layer of the liquid on the roll to have the least possible height. This advantageously results in minimal differences in the radiation dose within the transported liquid. The dose may be determined and regulated by the rotation speed of the roller, so that the rotation speed effectively determines the residence time of a specific volume of the liquid composition in the irradiated area. The rotational speed also optionally determines the thickness of the fluid film. In addition, the dose and penetration depth can be determined by direct control of the radiation source.

[0090] In a further aspect, the present invention relates to a composition comprising LEEI-inactivated Mollicutes bacteria as described above (first aspect) which is obtainable by the method described above (second aspect).

[0091] Therefore, in an embodiment, the invention regards a method for the production of a composition comprising inactivated Mollicutes bacteria, the method comprising the steps of: providing a liquid composition comprising live Mollicutes bacteria, and inactivating the Mollicutes bacteria in said liquid composition by LEEI.

[0092] In a preferred embodiment of the method according to the invention, the step of inactivating is done by way of a continuous process.

[0093] In a preferred, and in a more preferred, embodiment of the method according to the invention, the liquid composition is irradiated in the form of a liquid film having a film thickness from 0.05 mm to 0.4 mm.

[0094] In a preferred, and in a more preferred, embodiment of the method according to the invention, electron irradiation is performed at doses from 5 to 30 kGY.

[0095] Vaccine

[0096] The composition as described in the first aspect is preferably used as a vaccine. Thus, in a third aspect, provided herein is a composition comprising LEEI-inactivated Mollicutes bacteria for use as a vaccine (in the following also referred to as: the vaccine according to the present invention).

[0097] In an embodiment of the composition according to the invention for use as a vaccine, the composition is obtainable by the method according to the invention.

[0098] The vaccine may be used for the protection of a human or animal target against infection or disease caused by Mollicutes bacteria, such as Mycoplasma bacteria, e.g. infections of swine caused by Mhyo. Thus, in a further preferred embodiment, the present invention relates to a vaccine for use in the prophylaxis of an infection with Mhyo in swine.

[0099] In an embodiment of the vaccine according to the invention, the vaccine comprises a composition as is obtainable by the method according to the invention.

[0100] In a fourth aspect, the invention is directed to the use of the composition as described in the first aspect for the manufacture of a vaccine, in particular for the protection of a target against infection or disease caused by Mollicutes bacteria.

[0101] In an embodiment of the use of the composition for the manufacture of a vaccine according to the invention, the composition is a composition as obtainable by the method according to the invention.

[0102] In a preferred embodiment, the vaccine is used for the prophylaxis of an infection with Mhyo in swine.

[0103] In another aspect, the present invention relates to a method for the prophylaxis of an infection with Mhyo in swine comprising administering an immunologically effective amount of the vaccine.

[0104] In yet another aspect, the present invention relates to the use of the vaccine of the present invention for the manufacture of a medicament for the prophylaxis of an infection with Mhyo in swine.

[0105] The vaccine of the invention may contain one or more pharmaceutically acceptable excipients, such as a pharmaceutically acceptable carrier, and may comprise an adjuvant. A “pharmaceutically acceptable carrier” for the invention is an aqueous liquid of a high grade of purity and preferably sterile, for example: water, a physiological salt solution, or a phosphate buffered saline solution. The carrier can comprise further additives, such as stabilizers or preservatives, all well-known in the art.

[0106] An “adjuvant” is typically used in vaccines comprising non-replicating antigen to provide an immune-stimulation for the non-replicating antigen, which would otherwise not be (sufficiently) immunogenic. The adjuvant may trigger different routes of the immune system, but the basic mechanisms are not well understood. Adjuvants in general can be classified according to the immunological events they induce. The first class, comprising i.a. ISCOM’s (immunostimulating complexes), saponins (or fractions and derivatives thereof such as Quil A), aluminum hydroxide, liposomes, cochleates, polylactic / glycolic acid, facilitates the antigen uptake, transport and presentation by APC’s (antigen presenting cells). The second class, comprising i.a. oil emulsions, gels, polymer microspheres, non-ionic block copolymers and most probably also aluminum hydroxide, provide for a depot effect. The third class, comprising i.a. CpG-rich motifs, monophosphoryl lipid A, mycobacteria (muramyl dipeptide), yeast extracts, cholera toxin, is based on the recognition of conserved microbial structures, so-called pathogen associated microbial patterns (PAMPs), defined as signal 0. The fourth class, comprising i.a. oil emulsion, surface active agents, aluminium hydroxide, hypoxia, is based on stimulating the distinguishing capacity of the immune system between dangerous and harmless (which need not be the same as self and non-self). The fifth class, comprising i.a. cytokines, is based on the upregulation of costimulatory molecules, signal 2, on APCs. An adjuvant helps in providing an adequate immune response. Although the present nanoparticles cannot be clearly identified to belong to one of these classes, it appears that in some particular constellations an adjuvating effect may still be obtained.

[0107] Preferably, the adjuvant is based on emulsions of water and oil. A preferred adjuvant composition for use in a vaccine of the invention comprises an emulsion of water, a tocopherol or a pharmaceutically acceptable ester thereof, and a polyethoxy ethylene cetostearyl ether. In said composition, the tocopherol or the pharmaceutically acceptable ester thereof acts as an oily adjuvant, and the polyethoxy ethylene cetostearyl ether acts as an emulsifier. An example of such adjuvant is described in WO 2023 / 118553. Such an adjuvant composition was found to solve the occurrence of emulsion instability, even in complex mixtures, and even when using relatively crude preparations of antigens, such as in the vaccines described herein comprising crude compositions comprising LEEI-inactivated Mollicutes bacterial cultures. As used herein, the term “polyethoxy ethylene cetostearyl ether" refers to a class of hydrophilic, non-ionic emulsifiers that are used in the manufacturing of various O / W emulsions. This term is used interchangeably with the term “polyoxyethylene cetostearyl ether”. Polyoxyethylene cetostearyl ether is an ether of cetostearyl alcohol. Cetostearyl alcohol, cetearyl alcohol or cetylstearyl alcohol is a mixture of fatty alcohols, consisting predominantly of cetyl (16 C) and stearyl alcohols (18 C) and is classified as a fatty alcohol. In the present invention, the term “polyethoxy ethylene cetostearyl ether” has the same meaning as “a mixture comprising a poly ethoxy ethylene cetyl ether and a poly ethoxy ethylene stearyl ether”.

[0108] Particular examples include polyethoxyethylene 12 cetostearyl ether (INCI: Ceteareth-12, CAS number: 68439-49-6, Ph. Eur.: Macrogol cetostearyl ether 12), polyethoxyethylene 20 cetostearyl ether (Ceteareth-20, Macrogol cetostearyl ether 20, CAS number: 68439-49-6) and polyethoxyethylene 30 cetostearyl ether (Ceteareth-30, CAS number: 68439-49-6).

[0109] “Tocopherol", as used herein, refers to a class of organic chemical compounds having vitamin E activity. Tocopherol includes alpha-tocopherol (CAS number: 10191-41-0), beta-Tocopherol (CAS number: 148-03-8), gamma-Tocopherol (CAS number: 54-28-4), and delta-Tocopherol (CAS number: 119-13-1).

[0110] Pharmaceutically acceptable esters of tocopherol (vitamin E) particularly include alpha- tocopheryl-acetate which is also termed tocopheryl acetate or vitamin E-acetate having CAS number 58-95-7. Alpha-tocopheryl-acetate can be derived from vegetable materials such as seeds, nuts, fruits or leaves, or from fatty meats, but may also be produced synthetically. Thus, included in the definition of vitamin E-acetate are natural, synthetic or semi-synthetic forms, or mixtures thereof. Vitamin E-acetate is commercially available, in different degrees of purity.

[0111] An alpha-tocopheryl-acetate for the adjuvant composition according to the invention is preferably DL-alpha-tocopherol -acetate, which is the racemate of the chemical with CAS number: 7695-91-2.

[0112] The use of an adjuvant composition comprising an emulsion of water, a tocopherol or a pharmaceutically acceptable ester thereof, and a polyethoxy ethylene cetostearyl ether, as described above, is particularly suitable for use in a combination vaccine comprising LEEI- inactivated Mollicutes bacteria, and one or more other antigens used in the protection against important other swine pathogens, e.g. PCV2, PRRSV, and Lawsonia intracellularis, as described further below. The vaccine may optionally contain auxiliary substances, such as pH buffering agents, viscosity enhancing additives, and / or preservatives. Suitable auxiliary substances are for example disclosed in “Pharmaceutical Preformulation and Formulation”, by Mark Gibson, Taylor & Francis Inc. (Publ.); vol. 199, 2ndedition, 2009.

[0113] It is to be understood that the vaccine according to the invention contains the inactivated bacteria in an immunologically effective amount. Such an amount can be easily established based on common general knowledge, and could for example be the amount of Mhyo bacterin as present in the commercial product Porcilis® Mhyo ID ONCE (MSD Animal Health), or any of the other Mycoplasma bacterin products available in the market.

[0114] In an embodiment, the vaccine is administered to a target in a volume of between 0.1 and 3 ml per dose, preferably between 0.2 and 2 ml per dose.

[0115] In an embodiment for swine, the vaccine for use is administered between week 1 and week 5 of age, preferably between week 2 and week 4 of age.

[0116] In another embodiment, the vaccine according to the invention is administered systemically. In contrast to a local administration (such as for example intranasally or orally), systemic administration means that the vaccine is delivered into the circulatory system of the target and thus inherently affects its whole body. Examples of systemic administration include: intramuscular (IM), intravenous (IV), intradermal (ID), transdermal (TD) and sub-cutaneous (SC).

[0117] In an embodiment, the vaccine is administered intradermally. It is preferred that the vaccine is administered by needle-free injection by using an IDAL™ (intradermal application of liquids) injection system. IDAL is a pressure-based application system (from MSD Animal Health).

[0118] In an embodiment, the vaccine according to the invention is administered as a single dose. Thus, the vaccine is administered as a one-shot administration in contrast to being administered multiple times (e.g., a two-shot application comprising a prime- and a boost vaccination).

[0119] In a preferred embodiment, the vaccine is administered to a target intradermally as a single dose. Typically, the vaccine is used for the reduction of lung lesions in swine due to the infection with Mhyo. It was found that such lung lesions are reduced by 10 to 100%, and even by 50 to 100% compared to mock-vaccinated - challenged control pigs.

[0120] A lung lesion score (LLS) using a common scoring system, may be reduced by 10 to 100%, typically by 50 to 100% compared to untreated control pigs.

[0121] The LLS is measured at three weeks after challenge, as described in more detail in Example 1. The reduction of the lung lesions and the LLS refers to the comparison of pigs treated with the vaccine according to the invention, in comparison to non-vaccinated pigs. The LLS is a helpful indicator regarding the effectiveness of immunization by the vaccine. The LLS can be determined by measuring the percentage of lung lesions and transforming this into a Goodwin & Whittlestone score (Goodwin & Whittlestone, 1973, British Vet. Journ., vol. 129, p. 456 -464), resulting in the LLS.

[0122] Combination vaccine

[0123] The vaccine according to the invention is particularly suitable for use as a combination vaccine. Since the vaccine contains LEEI-inactivated Mollicutes bacteria, it typically does not contain any chemical inactivation agents, which may be harmful for, or which may interfere with, any replicating antigens to be combined with the vaccine of the invention. The vaccine according to the fourth aspect is thus preferably used as part of a combination vaccine.

[0124] The vaccine of the invention is particularly suitable for combination with one or more antigens used in the protection against important other swine pathogens, e.g. PCV2, PRRSV, and / or Lawsonia intracellularis. The vaccine is particularly suitable for use in a combination vaccine comprising live attenuated PRRSV, as described in WO 2007 / 116032, which is hereby incorporated in its entirety. The vaccine is further particularly suitable for use in a combination vaccine comprising non-replicating immunogen of PCV2, as described in WO 2017 / 162741 , which is hereby incorporated in its entirety.

[0125] In an embodiment, the present invention relates to a combination vaccine comprising a mixture of a first composition comprising at least a first antigen and a second composition comprising at least a second antigen, wherein the first composition is a composition according to the present invention. In a preferred embodiment the second composition comprises live attenuated PRRSV as the second antigen. In the inventive combination vaccine, a pharmaceutically or veterinary acceptable adjuvant may be provided together with the first composition and / or with the second composition. In a particularly preferred embodiment, the adjuvant is provided in the second composition, such as a composition comprising PRRSV antigen, which is combined with the second composition not comprising an adjuvant. For example, LEEI-inactivated Mhyo antigen may be combined with live attenuated PRRSV antigen, for example as reconstituted from a freeze-dried preparation.

[0126] In a further preferred embodiment, the combination vaccine comprises a first composition according to the invention comprising at least LEEI-inactivated Mollicutes bacteria as a first antigen, and a second composition comprising at least a second antigen, such as PRSSV, wherein the first composition further comprises a non-replicating antigen from PCV2 and / or a non-replicating antigen from Lawsonia intracellularis.

[0127] The combination vaccine is preferably provided in the form of an emulsion vaccine, most preferably an O / Wtype emulsion vaccine comprising an oily adjuvant, more preferably a composition comprising an emulsion of a tocopherol or a pharmaceutically acceptable ester thereof, and a polyethoxy ethylene cetostearyl ether as an emulsifier, and the LEEI-inactivated composition according to the invention as the aqueous phase. Such an adjuvant composition has been found to achieve increased emulsion stability, which is often a problem in combination vaccines comprising relatively crude antigens.

[0128] In an embodiment of the combination vaccine according to the invention, the combination vaccine comprises a composition as is obtainable by the method according to the invention.

[0129] The present invention also relates to a kit of parts comprising a first vaccine, and a second vaccine, wherein the first vaccine is a vaccine according to the present invention, and the second vaccine comprises live attenuated PRRSV.

[0130] In yet another aspect, the present invention relates to a combination vaccine comprising in a vial a mixture of a first vaccine and a second vaccine, wherein the first vaccine is a vaccine according to the present invention, and the second vaccine comprises live attenuated PRRSV. The combination vaccine may be a so-called RTU (ready-to-use) vaccine, thus delivered to an end user in a pre-mixed form, as produced by the manufacturer, or may for example be a vaccine that is mixed field-side, just prior to administration to a subject animal, for example by dissolving the PRRS virus in the Mhyo vaccine at the farm. EXAMPLES

[0131] Example 1 : Efficacy of LEEI inactivation compared to chemical inactivation in Mhyo vaccine

[0132] 1.1 Objective

[0133] Mhyo antigens that were inactivated using LEEI were tested for their efficacy as vaccine antigens against Mhyo challenge infection. Lung lesion scores of vaccinated animals were compared to animals that were not vaccinated.

[0134] 1.2 Cultivation and Inactivation procedure

[0135] Mhyo strain 11 was batchwise cultivated in FRIIS medium with 10 % porcine serum, at a temperature of 37 °C and pH 7.4 in a single-use bioreactor.

[0136] 1.2.1 LEEI inactivation

[0137] Mhyo strain 11 antigen used for LEEI irradiation was stored at 4 °C or at -80 °C until use. To elevate infectivity titer of the sample prior to irradiation 1 mL of spike antigen was added to the 600 mL Mhyo culture, and the Mhyo concentrate, right before irradiation. This resulted in a starting titer of the Mhyo material of ~5x10E6 CCU / mL.

[0138] LEEI inactivation was performed in a continuous system as described by Fertey et al. (2020, Sci. Rep., vol. 10, 12786) using a custom-built electron beam device, equipped with an electron emitter of maximally 300 keV (type EBA 300 / 270 / 4, ebeam™ Technologies). The distance between electron exit window and substrate was 35 mm. The acceleration energy was set to 200 keV. Adjustments of irradiation doses were made by regulation of the beam current at a constant sample speed.

[0139] Irradiation was performed in the continuous system with the parameters as displayed in Table 1. Table 1: LEEI irradiation parameters

[0140] Directly after irradiation each sample was transferred to sterile 50 mL tubes in 30 mL aliquots, and stored at 4 °C until use.

[0141] 1.2.2 Chemical inactivation

[0142] BEI was used for chemical inactivation of Mhyo antigen in a final concentration of 20 mM and was incubated for 18-31 h at 37 °C and a pH of 7.4. The combined mixture with BEI was transferred to a new vessel, and after inactivation was neutralized with sodium thiosulfate at a final concentration of 60 mM for at least 2 hours at 20 - 37 °C.

[0143] 1.2.3 Inactivation control test

[0144] A titration and inactivation control (IC) test was performed. For the titration, a 10Log dilution range was made from 10E-2 to 10E-9 in tubes, where 0.5 mL sample was added to 4.5 mL medium. The tubes were incubated for 20 days in total at 37 °C, whereby after 1 week, 2 weeks and 20 days, the tubes were checked for a color change. The starting color was red, and if active Mhyo was still present, an orange or yellow color would indicate the presence of live cell replication, and be counted as positive. Results of inactivation control tests showed that all samples 1, 2 and 3 had been successfully LEEI inactivated. Samples 1 and 3 were chosen for vaccination challenge experiments as described in the following.

[0145] 1.3 Study design

[0146] Pigs with no antibody titers for Mhyo were used for this study. Groups of 12 animals each were vaccinated at the age of 3 weeks (+ / - three days). One group was not vaccinated for Mhyo and served as challenge control.

[0147] Five weeks post vaccination, all animals were infected with a virulent Mhyo strain. Three weeks post challenge infection, all animals were investigated for lung lesion scores. Blood samples were taken prior to vaccination, just before challenge infection and at necropsy. 1.4 Test, control article(s), and challenge material

[0148] All vaccines used are shown in the Table 2 below. The vaccines were ready-to-use emulsions for intradermal injection. Vaccines were stored at 2-8 °C and transported at ambient temperature. Mhyo one-shot vaccines were formulated at 5 PCVU / mL.

[0149] Table 2: Vaccination study design

[0150] 1.5 Preparation of challenge materials

[0151] Mhyo strain 98 was diluted 2000 times in FRIIS+ 20 % SPF swine serum. This culture was incubated at 37 °C (50 RPM) for four to five days. After four and five days of incubation samples of this culture were taken for challenge. Viable count was performed before and after challenge by inoculating 4.5 mL FRIIS + 20 % SPF swine serum with 0.5 mL culture, mixing and transferring 0.5 mL of culture from this tube to the next, until 10E-10. CCU tubes were incubated at 37 °C for three weeks.

[0152] 1.6 Assignment of animals to treatment groups and blinding procedures

[0153] Piglets were allotted to 4 groups of 12 animals each as they come to hand. The first animal of a sow was assigned to group 1, the second animal to group 2 etc. When all suitable animals were assigned to a group, the procedure was continued with the next sow. The pigs were treated as described in the table 3 below in the right side of the neck.

[0154] Table 3: Treatment groups 1.7 Results

[0155] Blood samples were taken (vena jugularis, 2-8 mL) just before first vaccination (T=0, three weeks of age), prior to challenge (T=5, 8 weeks of age), and at post-mortem (T=8, 11 weeks of age). Serology of blood samples was performed by ELISA; results are shown in Table 4 below:

[0156] Table 4: Results of Serology

[0157] Table 4 shows that serology was significantly improved by the use of LEEI-inactivated samples, as compared to use of a chemically inactivated sample.

[0158] In addition, LLS were determined at 3 weeks post challenge infection at post mortem. The results of LLS are presented in Figure 2 (columns 1-4 in Figure 2 correspond to the study groups 1-4 of Tables 4 and 5). Median values are shown in Table 5 below:

[0159] Table 5: Results of Lung Lesion Scores (LLS)

[0160] Median score reduction of LLS by 50 % is indicative of good efficacy. Thus, Table 5 shows performance of LEEI-inactivated antigen that is comparable to that of BEI-inactivated antigen.

[0161] The above Example thus demonstrates the successful generation of fully inactivated Mhyo strain 11 antigen concentrate, by subjecting the samples to LEEI inactivation, based on a continuous LEEI inactivation process. Irrespective of the irradiation dose tested, the inactivated antigen samples retained their immunogenicity, as is demonstrated by their capacity to induce seroconversion in the target animal, and induce a protection against challenge that is comparable to that of BEI-inactivated Mhyo vaccine formulation, being applied as a single-shot intradermal vaccine.

[0162] Example 2: Impact of LEEI-inactivated Mhyo antigen on vaccine formulation and stability

[0163] 2.1 Objective

[0164] In view of a desired combination vaccine for commercial use, Mhyo antigen is intended for combination with antigens from PCV2, Lawsonia, and / or PRRSV. However, currently when the PRRSV antigen is combined with chemically-inactivated Mhyo, such as by using BEI, the viability of the live PRRSV antigen greatly decreases. It is hypothesized that this may be due to the impact of thiosulfate present in the BEI-inactivated Mhyo antigen, which was needed to neutralize the BEI.

[0165] The objective of this experiment was thus to study the impact of LEEI-inactivated Mhyo antigen on vaccine formulation stability, specifically on PRRSV infectivity titer, in a combination vaccine with live-attenuated PRRSV antigen. Several titrations were performed wherein the effect of coincubation with Mhyo antigen on PRRSV infectivity titer was evaluated.

[0166] 2.2 Materials & Methods

[0167] 2.2.1 Preparation of samples and LEEI treatment

[0168] Under frozen conditions, 600 mL Mhyo strain 11 culture, and 600 mL Mhyo strain 11 10x concentrate were provided together with 2x 1 mL Mhyo spike (strain 11) with a concentration of 10E9 CCU / mL.

[0169] Both the spiked culture and concentrate were subsequently LEEI treated as described in Example 1.

[0170] The antigenic mass of LEEI-inactivated Mhyo culture was measured in packed cell volume units (PCVU); for the 600 mL of undiluted concentrate, this was 13.6 PCVU.

[0171] For all titrations of PRRSV, MA-104 cells were used.

[0172] 2.2.2 First titration with LEEI-inactivated Mhyo combined with PRRSV

[0173] The titer of Porcilis® PRRSV in a 1 mL vial was 10E6.5 TCID50 / mL. Different concentrations of PRRSV were tested in combination with Mhyo concentrate LEE irradiated with 10 kGy, to see if this could cause differences in stability. Furthermore, several incubation times at room temperature were tested. A PRRSV sample dissolved in PBS with phenol red, was used as a positive control, to check the titer at the start of the incubation.

[0174] Table 6: Schedule of the first titration performed with LEEI-inactivated Mhyo combined with PRRSV

[0175] 2.2.3 Second titration with inactivated Mhyo combined with PRRSV

[0176] For the second titration, Mhyo inactivated with LEEI technology (10 and 25 kGy) was tested in combination with PRRSV. Mhyo inactivated with BEI was used as reference.

[0177] Table 7: Schedule of the second titration performed with LEEI-inactivated and BEI-inactivated Mhyo combined with PRRSV

[0178] 2.2.4 Third titration with inactivated Mhyo combined with PRRSV

[0179] In the third study the impact of a longer incubation time at room temperature on the stability of PRRSV in combination with LEEI-inactivated Mhyo antigen was investigated.

[0180] Table 8: Schedule of the third titration with LEEI-inactivated- and BEI-inactivated Mhyo combined with different concentrations of PRRSV

[0181] 2.2.5 Fourth titration with inactivated Mhyo combined with PRRSV

[0182] In the third titration, undiluted Mhyo antigen concentrate was used, having an antigenic mass of 13.6 PCVU / mL. However, in a commercial type Mhyo vaccine, an antigenic mass of about 5.0

[0183] PCVU / mL is typically used. Therefore, to test whether there is an effect of the Mhyo antigenic mass itself on the stability of PRRSV, a fourth titration was performed. PRRSV diluted in PBS was used as control. Table 9: Schedule of the fourth titration with different concentrations of LEEI-inactivated and BEI-inactivated Mhyo, combined with PRRSV

[0184] 2.3 Results The first PRRSV titration showed that for all the incubation times of 0 h, 1 h, and 3 h, the PRRSV titer remained stable, corresponding to the different predilutions of PRRSV applied (TCID50 / mL of 10E6.5, 10E5.5 and 10E4.5). The positive control, PRRSV diluted in PBS, showed the same titer as the samples dissolved in Mhyo Ag concentrate irradiated with 10 kGy (Figure 3). The results indicate that there is no impact of LEEI-inactivated Mhyo antigen on the infectivity level of PRRSV vaccine over the course of 3 hours.

[0185] In Figure 4 the impact on PRRSV infectivity titers can be seen, following incubation with Mhyo LEEI-inactivated with 10 kGy, 25 kGy, and BEI-inactivated, when combined with PRRSV 10E6.5 TCID50 / mL.

[0186] At 0 h and 3 h, the titer was 10E6.5 TCID50 / mL, showing that PRRSV at this concentration is stable in combination with the LEEI-inactivated Mhyo samples. However, PRRSV in combination with BEI-inactivated Mhyo showed a decrease in titer, to a concentration of (on average) 10E5.25 TCID50 / mL at t=0 h, and to 10E4.85 TCID50 / mL at t=3 h. This means that at the start of incubation, an immediate decrease could already be seen, and this continued over the course of 3 h. The titration of PRRSV at 10E5.5 showed a similar pattern, where the PRRSV combined with LEEI-inactivated Mhyo treatment stayed stable, while the PRRSV combined with BEI-inactivated Mhyo quickly reduced in titer. This indicates that BEI-inactivated Mhyo antigen has an acute negative impact on the viability of live PRRSV, while PRRSV stays stable when combined with LEEI-inactivated Mhyo.

[0187] In Figure 5 the outcome for PRRSV with 10E6.5 TCID50 / mL can be seen. Again, PRRSV diluted with Mhyo antigen following 10 kGy or 25 kGy LEEI-inactivation demonstrated no impact on infectivity titer after 5 hr, opposed to PRRSV diluted with BEI-inactivated Mhyo antigen, which acutely decreased to an average of 10E5.25 at 0 h incubation, further declining to 10E4.85 after 5 h incubation.

[0188] For PRRSV prediluted to a titer of 10E4.5 TCID50 / mL the same pattern could be seen: PRRSV combined with LEEI-inactivated Mhyo antigen remained stable over the course of 5 hours, while PRRSV combined with BEI-inactivated Mhyo antigen showed an immediate decrease in titer directly at 0 h, but did not decrease further over the course of 5 h. This indicates that PRRSV stays stable when combined with LEEI-inactivated Mhyo, even over the course of 5 hours, which is beneficial for use as a combination vaccine.

[0189] Further, it can be seen in Figure 6 that PRRSV with a TCID50 / mL of 10E4.5 stays stable when combined with LEEI-inactivated Mhyo concentrate of 13.6 PCVU / mL (in comparison to the positive control with PBS). PRRSV quickly drops in titer when combined with BEI-inactivated Mhyo with 15.0 PCVU / mL, to an average TCID50 / mL of 10E2.6 at 0 h incubation at RT, and a TCID50 / mL of 10E2.1 at 3 h incubation at RT.

[0190] For the PRRSV combined with LEEI-inactivated Mhyo with concentration 5.0 PCVU / mL, the pattern is the same, as it again stays stable after 3 h when compared to the PBS-control. However, the PRRSV combined with BEI- inactivated Mhyo with concentration of 5.0 PCVU / mL is again not stable, with an average TCID50 / mL of 10E2.35 at 0 h incubation at RT, and a TCID50 / mL of 10E2.25 at 3 h incubation at RT.

[0191] These data suggest that with different antigenic masses of LEEI-inactivated Mhyo antigen PRRSV stays stable, while combining with different concentrations of BEI- inactivated Mhyo the infectivity titer of PRRSV decreases.

[0192] Concluding, the results show that PRRSV stays stable for up to (at least) 5 hours when combined with LEEI-inactivated Mhyo antigen, opposed to PRRSV combined with chemically, i.e. BEI-inactivated Mhyo antigen.

[0193] These data support the hypothesis that circumventing the negative impact of thiosulfate / BEI from Mhyo antigen production by applying an alternative inactivation method may be an attractive strategy towards PRRSV compatibility within a desired combination vaccine, thus decreasing the handling time and number of vaccinations needed to effectively protect pigs.

[0194] Conclusions

[0195] The Examples demonstrate that LEEI completely and efficiently inactivated Mhyo present in crude culture medium, and even when that was concentrated. The LEEI-inactivated Mhyo antigen was suitable for use as a vaccine and achieved protection against Mhyo infection, once applied as a single-shot intradermal vaccine. Thus, it could be demonstrated that the LEEI- inactivated Mhyo is suitable for use in a vaccine against infection or disease caused by Mollicutes bacteria.

[0196] Further, it could be experimentally demonstrated that the LEEI-inactivated Mhyo is suitable for use in a combination vaccine, resulting in a stable combination with live-attenuated PRRSV antigen.

Claims

CLAIMS1. Composition comprising inactivated Mollicutes bacteria, characterised in that said inactivation was performed by low-energy electron-irradiation (LEEI).

2. Composition according to claim 1, wherein the Mollicutes bacteria are Mycoplasma bacteria.

3. Composition according to claim 2, wherein the Mycoplasma bacteria are Mycoplasma hyopneumoniae (Mhyo).

4. Composition according to any one of the preceding claims, wherein the composition comprises a culture of the said bacteria, or a concentrate of said culture.

5. Composition according to claim 4, wherein the culture or concentrate thereof comprises one or more of a: protein hydrolysate, vegetable extract, yeast extract, and animal serum.

6. Composition according to claim 5, wherein the animal serum comprises one or more of: bovine serum, porcine serum, and equine serum.

7. Composition according to any one of claims 4 to 6, wherein the composition is a 5x to 15x concentrate of the culture.

8. Composition according to any one of the preceding claims, wherein the composition is substantially free of chemical inactivation agents.

9. Composition according to any one of claims 1 to 8, for use as a vaccine.

10. Use of the composition according to any one of claims 1 to 8, for the manufacture of a vaccine.

11. Use of the composition according to claim 10, wherein the vaccine is for the protection of swine against infection or disease caused by Mollicutes bacteria.

12. Vaccine comprising the composition according to any one of claims 1 to 8.

13. Vaccine according to claim 12 for use in the protection of swine against infection or disease caused by Mollicutes bacteria.

14. The use according to claims 10 or 11 , and the vaccine according to claims 12 or 13, wherein the Mollicutes bacteria are Mycoplasma bacteria.

15. A combination vaccine comprising a mixture of a first composition comprising at least a first antigen, and a second composition comprising at least a second antigen, wherein the first composition is a composition according to claims 1 to 8, and the second composition comprises live attenuated porcine reproductive and respiratory syndrome virus.

16. The combination vaccine according to claim 15, wherein the first composition further comprises a non-replicating antigen from porcine circovirus type 2, and / or a nonreplicating antigen from Lawsonia intracellularis.

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