WO2025262124A1

WO2025262124A1 - Adjuvanted veterinary vaccines against enteric diseases

Info

Publication number: WO2025262124A1
Application number: PCT/EP2025/067074
Authority: WO
Inventors: Markus Hendrikus ROOSMALEN VAN
Original assignee: Intervet International BV; Intervet Inc
Current assignee: Intervet International BV; Intervet Inc
Priority date: 2024-06-19
Filing date: 2025-06-18
Publication date: 2025-12-26
Anticipated expiration: 2026-12-19

Abstract

The present invention relates to a vaccine composition comprising non live antigens from: a) E.coli; b) bovine rotavirus; c) bovine coronavirus; wherein the antigens are formulated in an adjuvant having a continuous phase that is aqueous. In addition, the invention relates to methods for the preparation of the vaccine composition and to methods for vaccinating ruminants against infection by bovine rotavirus, bovine coronavirus and E. coli.

Description

ADJUVANTED VETERINARY VACCINES AGAINST ENTERIC DISEASES

TECHNICAL FIELD

The invention pertains to adjuvanted vaccine compositions comprising antigens from E.coli, bovine rotavirus and bovine coronavirus. Methods of making and using the vaccine compositions are also provided.

BACKGROUND

Bovine enteric disease is the result of an enteropathogenic intestinal infection in bovines that often manifests itself in some form of diarrhea. One form of diarrhea is neonatal calf diarrhea. Neonatal calf diarrhea remains the most important cause of death in calves under one month of age and thus is responsible for substantial economic loss in the farming industry. Developing a strategy to prevent or treat bovine enteric disease has been very difficult since while it is known that multiple enteric pathogens (also referred to as “enteropathogens”) are present during the infection, it is not known which pathogen or combination of pathogens causes the disease. The most prevalent infections are with enteric pathogens Escherichia coli, rotavirus, coronavirus, and Cryptosporidium parvum (Brunauer M. et al. Animals (Basel). 2021 Apr 3; 11 (4): 1014. doi: 10.3390/ani11041014), as was for example found in a study by Bartels et al. (2010) (Bartels CJ et al. Prev Vet Med. 2010 Feb 1 ;93(2-3): 162-9. doi: 10.1016/j.prevetmed.2009.09.020. Epub 2009 Oct 12). Cases of neonatal calf diarrhea are commonly associated with one or more of these pathogens.

Immunization of bovines and newborn calves to the enteric pathogens rotavirus, coronavirus and E. coli today may be done by administering the commercially available Bovilis® Rotavec® corona vaccine (MSD Animal Health) to a pregnant bovine. This vaccine comprises non live antigens of each of the corresponding pathogens, in particular, the vaccine comprises inactivated rotavirus, inactivated corona virus and fimbrial adhesins of E. coli. Immunization of bovines and newborn calves to the enteric pathogen Cryptosporidium parvum today may done by administering the commercially available Bovilis® Cryptium® vaccine (MSD Animal Health) to a pregnant bovine. These vaccines have been shown to induce protection against enteric disease, such as neonatal calf diarrhea, in the calves of the pregnant bovine.

Both above-mentioned veterinary vaccines, and other commercially available vaccines, are adjuvanted with aluminium-based adjuvants in mineral oil. To maximize the effectiveness of vaccines, especially those containing poorly antigenic components or highly purified antigens, it is standard procedure in the industry to formulate the antigen(s) with an adjuvant, in particular a strong adjuvant that is oil based. Adjuvants can potentially, when given in combination with an antigen, increase the induced immune response to that antigen. Most adjuvants used in veterinary vaccines comprise mineral-based adjuvants (usually aluminium-based), saponins, oils or emulsions, or a combination thereof. A handbook on adjuvants and their uses and effects is: “ Vaccine adjuvants" (Methods in molecular medicine, vol. 42, D. O’Hagan ed., 2000, Humana press, NJ, ISBN: 0896037355).

Typically, emulsions used as adjuvants can be water-in-oil emulsions (w/o) or oil-in- water emulsions (o/w). Upon administration, for example by subcutaneous injection, the first-mentioned cause the most intense local reactions but generally induces a stronger immune response than the oil-in-water emulsions (o/w) which tend to be milder in local reactions and induced immune response (Guidance on the use of adjuvanted veterinary vaccines of the European Medicines Agency (1998)). In other words, the use of (increased amounts of) water as an adjuvant in a vaccine composition works as a two-edged sword in that it generally mildens the severity of local reactions caused by adjuvanted vaccines, but also reduces immune responses induced by the vaccine. Consequently, when aiming at reducing severity of adverse reactions one must be careful in formulating vaccines with adjuvants comprising water as not to risk losing the protective efficacy of the vaccine.

According to the Note for Guidance on the use of adjuvanted veterinary vaccines of the European Medicines Agency (1998) “the ideal adjuvanted vaccine is safe for the treated animal, does not cause clinical or local reactions or allergic reactions and is also safe for consumers of food produced from vaccinated animals. As such an ideal adjuvanted vaccine currently is not available some limitations have to be tolerated, but without compromising consumer safety." Thus, for such “ideal” adjuvants, a balance between safety, efficacy and consumer safety is desirable.

Yet, current adjuvants for veterinary vaccines still comprise limitations, such as the occurrence of adverse reactions to adjuvanted vaccines causing discomfort to vaccinated animals, and thus are far from considered “ideal”.

Nowadays, veterinary vaccine development requires that “adjuvants should be chosen based on the type of immune response desired and [the adjuvant] should be formulated [...] in such a way that the optimal type of immune response with minimal adverse effects is obtained’ (Draft Updated Guideline on the use of adjuvanted veterinary vaccines of the European Medicines Agency (2018)). Yet, many veterinary vaccines, including widely used vaccines such as Bovilis Rotavec corona or Bovilis Cryptium, still remain adjuvanted with aluminium-salts. Whilst effective in inducing immune responses to the antigen(s) of the vaccines, vaccinated animals suffer from adverse effects such as local reactions caused by adjuvants.

Altogether, despite that adjuvants for veterinary vaccines are well-described and broadly used for commercial vaccines, there are limitations to available adjuvanted veterinary vaccines that require a solution. This is easier said than done because “one active substance(s) - adjuvant combination cannot, as a rule, be extrapolated to other active substance(s) - adjuvant combination" (Draft Updated Guideline on the use of adjuvanted veterinary vaccines of the European Medicines Agency (2018)). In other words, an adjuvant that has a desirable efficacy and/or safety profile for one veterinary antigen cannot be used in the formulation of another veterinary antigen without extensive safety and efficacy testing meeting regulatory and legal requirements and pharmacopoeia! guidelines such as the Directive 2001/82/EC, the Guideline on requirements for the production and control of immunological veterinary medicinal products (EMA/CVMP/IWP/206555/2010- Rev.1 ), the Commission Regulation (EU) No 37/2010 of 22 December 2009 on pharmacologically active substances and their classification regarding maximum residue limits in foodstuffs of animal origin Guideline on user safety for immunological veterinary medicinal products (EMEA/CVMP/IWP/54533/2006) and the Ph. Eur. 0062: ‘Vaccines for veterinary use’. Whilst the need is high to move to improved, preferably “ideal”, adjuvanted veterinary vaccines that improve animal wellbeing whilst maintaining a desirable efficacy in protection animals against infection, there are major challenges before arriving at an improved adjuvanted veterinary vaccine.

It is an object of the current invention to solve any one or more of the above challenges in the field and to provide for improved adjuvanted veterinary vaccines.

SUMMARY OF INVENTION

Surprisingly, it was found that this object can be met, and consequently one or more disadvantages of the prior art can be overcome by providing a veterinary vaccine composition wherein the non live antigens of the here pathogens as mentioned here above are formulated in an adjuvant having a continuous phase that is aqueous. This discovery opens up a number of favourable options for vaccine formulation. For example, some vaccine compositions can now be formulated in an adjuvant having a continuous phase that is aqueous, wherein the vaccines are stable whilst at the same time comprise a favourable safety and efficacy profile. It was unexpected that these vaccine compositions, as described and exemplified herein, exhibited adequate antibody titers that are illustrative for immune responses associated with desirable protection profiles in bovines. Moreover, it was unexpected that such vaccine compositions were also well-tolerated by bovines and were found suitable for parenteral administration to bovines. This is achieved for a vaccine composition comprising non live antigens from E.coli, bovine rotavirus and bovine coronavirus, wherein the antigens are formulated in an adjuvant having a continuous phase that is aqueous. Therefore, in one aspect the invention relates to a vaccine composition to aid in the protection against infection with an enteric pathogen in a bovine, the vaccine composition comprising non live antigens from: a) E.colr', b) bovine rotavirus; c) bovine coronavirus; wherein the antigens are formulated in an adjuvant having a continuous phase that is aqueous. Preferably, the vaccine is for subcutaneous administration. DESCRIPTION OF DRAWINGS

Figure 1 : Fig. 1 A - D show an overview of average antibody responses in the serum samples for, respectively, bovine rotavirus (BRV), bovine coronavirus (BCV), E. coli F5/F41 and C. parvum gp40 exhibited by the different adjuvated vaccines and determined by ELISA.

Figure 2: Fig. 2 A - D show lymphocyte (T-cell) stimulation tests against, respectively, BRV VP8, BCV S1 , E. coli F5, and C. parvum gp40.

DEFINITIONS

For purposes of the present invention, the following terms are defined below.

The term “adjuvant” is used here in its common meaning of a substance, compound or composition capable of stimulating an immune response in a target animal in a nonspecific manner.

An “antigen" for the invention refers to a substance that can -in the right circumstances- induce a protective immunological response in a target animal against a pathogen. Typically an antigen is a non pathogenic form of the pathogen (such as killed or live attenuated), or a subunit of this pathogen, either extracted from this pathogen or recombinantly expressed.

Non live antigen of a wild type pathogen is any substance or compound, other than the live pathogen as such, against which an immunological response is to be elicited, such that the corresponding virulent pathogen or one or more of its virulence factors will be recognized by the host’s immune system as a result of this immune response, and are at least partly neutralized. Typical examples of non live antigen of a wild type pathogens are killed whole cell bacteria or viruses, subunits of these pathogens such as surface expressed, secreted (such as toxins) or other proteins. Subunits may or may not be recombinantly expressed. Non live antigens typically induce a humoral immune response as opposed to live antigens which in addition trigger cellular responses. Non live antigens are typically less effective than live antigens since they do not replicate, and hence need an adjuvant for inducing an adequate immune response.

As used herein, by the term "aqueous" in relation to an adjuvant or continuous phase of the adjuvant (e.g., a reference to an "continuous aqueous phase"), it is meant that the adjuvant or continuous phase of the adjuvant is a liquid formulation comprising water and that water constitutes at least 50%, typically at least 51 %,, 55%, 60%, 65%, 70%, 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% by weight of the total amount of the liquid, i.e. water plus any co-solvent or co-solvents freely miscible with water at room temperature present in the adjuvant or continuous phase of the adjuvant.

The terms "aqueous phase" or "oil phase", for example when referring to “an O/W emulsion comprising a continuous aqueous phase and a discontinuous oil phase” according to the present invention should be understood that the main part of said phase is respectively derived from, respectively, a water (when referring to the “aqueous phase”) or an oil (when referring to the “oil phase”).

A “bovine" for the invention is taurine cattle (Bos taurus), zebu cattle (Bos indicus), buffalo, bison, yak, or wisent. The bovine can be of any type: dairy or beef, or parental stock for dairy- or beef type.

As is well-known, “colostrum" is the milk that is secreted by the mammary glands of a mammal in the period around delivery.

The term "comprises" (as well as variations such as "comprise", "comprising", and "comprised") as used herein, intends to refer to all elements, and in any possible combination conceivable for the invention, that are covered by or included in the text section, paragraph, claim, etc., in which this term is used, even if such elements or combinations are not explicitly recited; and not to the exclusion of any of such element(s) or combinations. Therefore, any such text section, paragraph, claim, etc., can therefore also relate to one or more embodiment(s) wherein the term "comprises" (or its variants) is replaced by terms such as "consist of", "consisting of", or "consist essentially of”. The term “formulation” in context of the current invention refers to the combining of substances, herein e.g., antigens and adjuvants, to create a vaccine composition in accordance with the invention.

The term “inactivated” when referring to an “inactivated antigen”, such as “inactivated bovine coronavirus" or “inactivated bovine rotavirus” means that the antigen, e.g., an infectious organism or pathogen, is no longer capable of replication or growth and/or cannot cause disease, whilst retaining its immunogenicity (i.e., remaining an immunologically active component). The term comprises dead or killed forms of antigens (e.g., viruses, bacteria, parasites, other). Inactivation may be accomplished by a variety of through methods known in the art, for example those described in by Sanders B et al. Inactivated Viral Vaccines. Vaccine Analysis: Strategies, Principles, and Control. 2014 Nov 28:45-80. doi: 10.1007/978-3-662- 45024-6_2 and in van den Biggelaar, A.H.J., Poolman, J.T. (2015). Live-Attenuated and Inactivated Whole-Cell Bacterial Vaccines. In: Nunnally, B., Turula, V., Sitrin, R. (eds) Vaccine Analysis: Strategies, Principles, and Control. Springer, Berlin, Heidelberg, doi.org/10.1007/978-3-662-45024-6_5).

An “oil" is used here in its common meaning and refers to a nonpolar chemical substance with a relatively high hydro-carbon content that is typically a relatively viscous liquid, has a density lighter than water, and is hydrophobic and lipophilic. An oil can be of mineral origin, or of “non-mineral” origin such as of synthetic-, semi- synthetic-, animal- or vegetable origin. Some oils are metabolizable.

A “pharmaceutically acceptable carried’ refers to a biocompatible medium, viz. a medium that after administration does not induce significant adverse reactions in the treated subject, preferably an animal, capable of presenting the antigen to the immune system of the animal after administration of the composition comprising the carrier. Such a pharmaceutically acceptable carrier may for example be a liquid containing water and/or any other biocompatible solvent or a solid carrier such as commonly used to obtain freeze-dried vaccines (based on sugars and/or proteins), optionally comprising an adjuvant. As used herein “prior to parturition” is equivalent to “prior to calving”. Similarly, “after parturition” is equivalent to “after calving”.

The terms “protection” or “protect’ in the context of protection (or protect) against a pathogenic infection with an infectious agent means arriving at protective immunity in an animal, i.e. , reducing disease symptoms, reducing an infection and/or aiding in preventing, ameliorating, or curing (an) adverse effect(s) caused by the infection with that agent, for example, by inhibiting the replication and/or persistence of a pathogen and/or by reducing the virulence of a virulent factor that is known to contribute to the pathogenicity of an infection in an animal by a microorganism.

The term “ready-to-use" as used herein means: “not requiring the combining of (part of) the content of 2 or more containers in order to be ready for administration” e.g., to a target animal. For example: not requiring a dissolution or an admixing step and being available to the end-user as a suspension in a single bottle. For a ready-to- use vaccine, the necessary combining of compounds has been performed by the manufacturer of the vaccine, in a controlled environment. This has distinct advantages over field-side mixing, mainly in the ease of use for the end-user, especially when vaccinating large numbers of animals. Other advantages are that the manufacturer can perform the combining under aseptic conditions and can apply various quality assurance tests on the final mixture, to guarantee its correct composition and its quality. However, this does not exclude that a ready-to-use vaccine may require some sort of simple pre-treatment, such as brief shaking by hand to remove sedimentation or creaming of an emulsion, or such as warming before administration when the vaccine had been stored refrigerated, or such as the extracting a volume, e.g., by using a suitable applicator such as a syringe, from a bottle or flask containing the vaccine so that it can be administered to the animal. However, the vaccine may also be provided in a substantially sealed, preferably aseptic and/or sterile suitable applicator, such as a syringe. Thus, “ready-to-use” as meant herein may also refer to “ready-to-administer”.

As meant herein, the term "ruminant' for the invention is an animal assigned to the suborder Ruminantia, and/or an animal applying the process of rumination to digest its feed.

The term “vaccine" is herein used to refer to a composition suitable for administration to a mammal, comprising immunologically active components in an immunologically effective amount, typically combined with a pharmaceutically acceptable carrier, which upon administration to the animal induces an immune response that protects the animal against a pathogenic infection with the infectious agent. The “immunologically active component’’, may be one or more antigenic molecule(s) that is recognized by the immune system of a subject, preferably an animal, to which the vaccine is administered and that induce a protective immunological response. The response may originate from the subjects', preferably the animals’, innate- and/or from the acquired immune system and may be of the cellular- and/or of the humoral type.

DESCRIPTION OF EMBODIMENTS

It is contemplated that any product, method, use or composition described herein can be implemented with respect to any other product, method, use or composition described herein. Embodiments disclosed in the context of products, methods, uses or compositions of the invention may be employed with respect to any other product, method, use, or composition described herein. Thus, an embodiment pertaining to one product, method, use or composition may be applied to other products, methods, uses or compositions of the invention as well.

Surprisingly, the inventors found that the formulation of a vaccine comprising non live antigens from E.coli, bovine rotavirus and bovine coronavirus in an adjuvant, wherein said adjuvant has a continuous phase that is aqueous, exhibits a desirable efficacy and/or safety profile. In addition, the inventors found that the formulation of vaccines comprising an antigen from E.coli, an antigen from bovine rotavirus and an antigen from bovine coronavirus with an adjuvant that has a continuous phase that is aqueous resulted in stable adjuvanted vaccines. As previously mentioned, adjuvants that are commonly used for vaccines of animals, and especially for bovines, (see, for example, Bovilis Cryptium, Bovilis Rotavec Corona) comprise mineral-salts, such as aluminium hydroxide in an oil phase, such as light paraffin or other mineral oil. Whilst able to enhance the magnitude of protective immunity provided by an antigen, it is also contemplated that oil-based adjuvants, such as Freund's adjuvant or MONTANIDE™ ISA (Seppic), play a role in the occurrence of side-effects, for example in the form of local reactions, of vaccination. The inventors found that, through the current invention, alternative, improved adjuvants to oil-based adjuvants could be used in the formulation of veterinary vaccines provided herein. The inventors found that the newly developed vaccine compositions comprising an adjuvant that has a continuous phase that is aqueous exhibit efficacy in protecting against infection by an enteric pathogen in bovines. This was surprising as generally for vaccines the use of aqueous adjuvants or increasing the fraction of aqueous adjuvants and/or water as compared to other adjuvant constituents such as oily constituents, leads to a reduced efficacy of the adjuvanted vaccine composition. Accordingly, adjuvanted vaccines are provided that comprise an enhancement of the magnitude of protective immunity provided by an antigen, for example compared to when said antigen is administered by a bovine without said adjuvant and/or compared to commercially available vaccines, such as Bovilis Rotavec Corona.

It was further unexpected that the doses of one or more of the antigens formulated in a vaccine composition according to the invention were comparable, or even similar, to commercial vaccine compositions comprising mineral and/or oil-based adjuvants. Indeed, it was expected that doses of one or more of the antigens formulated in a vaccine composition according to the invention would need to be increased compared to commercial vaccines comprising mineral and/or oil-based adjuvants due to, and without being bound by theory, the immune enhancing effect of the oil-based adjuvants of commercial vaccines and which the newly developed vaccine compositions comprising an adjuvant that has a continuous phase that is aqueous (are considered to) lack. However, vaccine compositions comprising antigens and formulated in an adjuvant having a continuous phase that is aqueous still proved to exhibit desirable safety and efficacy. Therefore, in a first aspect there is provided for a vaccine composition to aid in the protection against infection with an enteric pathogen in a bovine, the vaccine composition comprising the non live antigens from: a) E.coli; b) bovine rotavirus; c) bovine coronavirus; wherein the antigens are formulated in an adjuvant having a continuous phase that is aqueous. As an example, the non live antigens of E. coli are one or more fimbrial adhesins of E. coli (as commonly used in veterinary vaccines), inactivated bovine rota virus and inactivated bovine coronavirus.

Veterinary vaccine compositions in the art generally comprise an antigen and an adjuvant. Veterinary vaccines may further comprise additional constituents such as, but not limited to, preservatives, emulsifiers, stabilizers, and residuals (e.g., cell culture media traces, agents used for inactivating viruses and the like). The vaccine composition as provided herein comprises at least an antigen from E.coli, an antigen from bovine rotavirus, an antigen from bovine coronavirus and an adjuvant having a continuous phase that is aqueous. The adjuvant provided herein as constituent of the vaccine composition according to the invention comprises a continuous phase that is aqueous. The antigens of the vaccine composition according to the invention are formulated in said adjuvant having a continuous phase that is aqueous, or in other words a continuous aqueous phase. Such an adjuvant is, for example, in the form of an aqueous liquid, such as an aqueous suspension, aqueous solution or aqueous dispersion. In general, phases of a matter are known to be continuous if the matter occupies a continually connected region of space (as opposed to dispersed if the phase occupies disconnected regions of space). Thus, it is understood herein that a continuous phase that is aqueous comprises a continually connected region of space wherein the continually connected region of space comprises an aqueous liquid, such as water. In preferred embodiments, the water is of a pharmaceutical acceptable quality.

The vaccine composition according to the invention comprises that the antigens are combined (e.g., admixed) in an adjuvant having a continuous phase that is aqueous. It is preferred that the antigens are combined, e.g., admixed, with the continuous aqueous phase of the adjuvant. It is contemplated that, in preferred embodiments, upon admixing the adjuvant with the antigens that the antigens will be combined with, e.g., suspended or dispersed in, the continuous aqueous phase of the adjuvant. Thus, the continuous aqueous phase of the vaccine composition comprises at least the antigens from E.coli, bovine rotavirus and bovine coronavirus.

As described herein, the vaccine composition according to the invention is suitable for the protection against infection with an enteric pathogen in a bovine and known in the field known to be causative for infections in bovines. The enteric pathogens to which the vaccine composition according to the invention at least provides protection against infection by said pathogen are Escherichia coli, rotavirus and coronavirus. In other embodiments, the enteric pathogens can further comprise Cryptosporidium parvum (Brunauer M. et al. Animals (Basel). 2021 Apr 3; 11 (4): 1014. doi: 10.3390/ani11041014) and/or C.perfringens.

E. coli is a well-known and extensively described bacterium in the art. E. coli is an important bacterial pathogen associated with neonatal diarrhea in calves during the first week of life. Bovine coronaviruses (BCV) are betacoronaviruses associated with neonatal calf diarrhea. Rotaviruses are the most common cause of neonatal diarrhoea in calves.

As is provided and exemplified herein, by administration of the vaccine composition according to the invention to bovines an immune response (as exemplified herein by antibody titers illustrative for protection against) against antigens from Escherichia coli, bovine rotavirus and bovine coronavirus can be induced. Thus, the vaccine composition according to the invention is able to induce protective antibody titers in a bovine and, as a result of induced protective antibody titers, is suitable for protection against infection with E. coli, bovine rotavirus and bovine coronavirus.

The bovine to which the vaccine composition is administered preferably is an adult cow, pregnant heifer, pregnant cow or calf. In some embodiments, the vaccine composition is administered parenterally as injection, e.g., as a subcutaneous injection. In some embodiments, the vaccine composition is administered orally in the form of colostrum which may be supplemented with the vaccine composition provided herein or, preferably, is colostrum derived from a heifer or cow after parturition and comprising maternally derived antigens (MDA) to an enteric pathogen, preferably comprising at least one, preferably all, of the antigens. Said colostrum preferably is administered to a calf.

In embodiments, the vaccine composition in accordance with the invention is provided as a ready-to-use composition, which preferably is in the form of a liquid. As broadly described herein, a ready-to-use composition comprising the vaccine composition of the current invention comprises that the combining of compounds, e.g., the combining of antigens with a desirable pharmaceutically acceptable carrier, has been performed by the manufacturer of the vaccine. Thus, when providing the vaccine composition in accordance with the invention, the vaccine composition comprises a pharmaceutically acceptable carrier, which at least comprises an adjuvant having a continuous phase that is aqueous. Such a pharmaceutically acceptable carrier may for example be a liquid comprising water and/or any other biocompatible solvents. In embodiments, the ready-to-use liquid composition may be an emulsion, solution, aerosol, gel, dispersion, or suspension e.g., a suspension suitable for injection. In preferred embodiments, the liquid composition comprising the vaccine composition of the invention is suitable for administration to an animal by parenteral injection.

It is preferred that further to the continuous phase that is aqueous, the adjuvant further comprises an oil as a discontinuous phase. Thus, in some embodiments there is provided for a vaccine composition comprising non live antigens from: a) E.coli; b) bovine rotavirus; c) bovine coronavirus; wherein the antigens are formulated in an adjuvant having a continuous phase that is aqueous and comprising an oil as a discontinuous phase. A discontinuous phase may also be referred to as a “dispersed phase” and indicates that the phase occupies disconnected regions of space. A discontinuous phase thus, as illustrative example, may have the form of oil droplets in a body of a continuous phase, e.g., of water. The oil may be a mineral oil or non-mineral oil, or combinations thereof. A mineral oil is an oil that originates from a mineral source, typically from petroleum. A non-mineral oil can be selected from synthetic oils, semi-synthetic oils, animal oils, and vegetable oils. As provided herein, a semi-synthetic oil is an oil that is non- mineral in origin such as an animal or vegetable oil, but which was modified in structure and/or composition by a chemical or physical process.

The adjuvant of the vaccine composition according to the invention comprises a continuous phase that is aqueous and further comprises an oil as a discontinuous phase. Such a discontinuous phase comprises a dispersed phase comprising an oil that typically is randomly distributed throughout the continuous phase that is aqueous. In preferred embodiments, when the adjuvant comprises continuous phase and a discontinuous phase, all of the antigens from E.coli, from bovine rotavirus and from bovine coronavirus are comprised in the continuous aqueous phase and the discontinuous oil phase does not comprise any of said antigens. It may be that the discontinuous oil phase comprises emulsifiers, stabilizers, preservatives and/or other vaccine constituents that have hydrophobic properties and may be soluble in an oily substance.

Adjuvants formulated as oil-in-water (O/W) emulsions can be advantageously used. An “emulsion” is a mixture of at least two immiscible liquids, whereby one is dispersed in another. Fundamentals of emulsion formation and stability are, for example, described by Silva et al. (2022), Developments in Clay Science, Elsevier, Volume 10, Pages 37-59, https://doi.org/10.1016/B978-0-323-91858-9.00009-4. Typically, the droplets of the dispersed phase are very small, in the range of micrometers or less. For the invention the emulsion comprises at least two phases that are liquid, wherein one phase is an oil phase and another phase is an aqueous phase. In emulsions, there commonly is a boundary between the phases, separating these phases, commonly referred to as the "interface". Procedures and equipment for the preparation of an emulsion at any scale are well-known in the art, and are for instance described in handbooks such as: “Remington: the science and practice of pharmacy” (2000, Lippincot, USA, ISBN: 683306472), and: “Veterinary vaccinology” (P. Pastoret et al. ed., 1997, Elsevier, Amsterdam, ISBN 0444819681 ). In preferred embodiments of the invention, the adjuvant is an emulsion that comprises at least one dispersed and one continuous phase. For example, oil and water can form an oil-in-water emulsion, in which the oil is the dispersed (or discontinuous) phase, and the aqueous liquid, e.g., water is the continuous phase. When the adjuvant comprises an oil-in-water (O/W) emulsion, the continuous outer phase is aqueous, and the dispersed internal phase comprises oil. Thus, in other words, an oil-in-water emulsion typically comprises an outer aqueous phase and a dispersed internal oil phase.

Preferably, the adjuvant is formulated as an oil-in-water (O/W) adjuvant. When the adjuvant comprises an O/W emulsion, it is preferred that the antigens are comprised in the aqueous phase.

Thus, there is also provided for a vaccine composition comprising non live antigens from: a) E.coli; b) bovine rotavirus; c) bovine coronavirus; wherein the antigens are formulated in an adjuvant having a continuous phase that is aqueous and comprising an oil as a discontinuous phase, wherein the adjuvant is formulated as an oil-in-water (O/W) emulsion.

In some preferred embodiments the adjuvant is selected from the group consisting of:

- an emulsion of a mineral oil and a tocopherol in water;

- an emulsion of a non-mineral oil and a tocopherol in water.

Therefore, in one embodiment, there is provided for a vaccine composition comprising non live antigens from: a) E.colr', b) bovine rotavirus; c) bovine coronavirus; wherein the antigens are formulated in an adjuvant, wherein the adjuvant is an emulsion of a mineral oil and a tocopherol in water. In another embodiment, there is provided for a vaccine composition comprising non live antigens from: a) E.coli; b) bovine rotavirus; c) bovine coronavirus; wherein the antigens are formulated in an adjuvant, wherein the adjuvant is an emulsion of a non-mineral oil and a tocopherol in water. It is understood that when an emulsion of a mineral oil or non-mineral oil, and a tocopherol in water is provided this is an oil-in-water (O/W) adjuvant. In an emulsion of a mineral oil and a tocopherol in water, the discontinuous phase typically comprises the mineral oil and the tocopherol, and the continuous phase typically comprises water or another aqueous liquid. In an emulsion of a non-mineral oil and a tocopherol in water the discontinuous phase typically comprises the non-mineral oil and the tocopherol, and the continuous phase typically comprises water or another aqueous liquid. It was found that an O/W emulsion of any one of a mineral oil or a non-mineral oil and a tocopherol in water can advantageously be used in the formulation of the vaccine composition according to the invention.

Emulsions comprising more than two phases (e.g., "water-in-oil-in-water" or “oil-in- water-in-oil”) are also envisioned herein as emulsions that may comprise the vaccine constituents, such as the herein provided antigens, of the invention. Such an emulsion comprising more than two phases may be, for example, a "water-in-oil- in-water" (W/O/W) emulsion. A W/O/W emulsion has two oil-water interfaces and comprises one internal water phase, which is dispersed in oil, and one external water phase, wherein the oil phase is dispersed. Without being bound by theory it is considered that W/O/W emulsions are particularly suitable for poorly soluble materials, as the internal water phase can optionally change the solvent conditions to meet the requirement of high solubility while the internal oil phase can provide a natural barrier to outside influences, such as light, oxygen and ions. It is considered that the antigens of the vaccine composition according to the invention can be comprised in the internal, the external or in both water phases of a W/O/W emulsion. In some embodiments, the outer water phase comprises a different aqueous solution than the inner water phase. In other embodiments, when the adjuvant comprises a W/O/W emulsion the outer water phase and inner water phase comprise the same or a similar aqueous solution. In one non-limiting example, two of the antigens, e.g., an antigen from E.coli and an antigen from bovine rotavirus may be comprised in the internal water phase, and another antigens, e.g., an antigen from bovine coronavirus may be comprised in the outer water phase.

In embodiments, when the adjuvant comprises a W/O/W emulsion, all antigens are comprised in the outer water phase, i.e. , in the continuous water phase. Another emulsion comprising more than two phases may be, for example, an "oil-in-water- in-oil" (O/W/O) emulsion. An O/W/O emulsion has two water-oil interfaces and comprises one internal oil phase, which is dispersed in water, and one external oil phase, wherein the water phase is dispersed. In preferred embodiments, when the adjuvant comprises an O/W/O emulsion wherein the water phase is a continuous phase, all antigens are comprised in the water phase.

The inventors found desirable antibody responses (as exemplified in, but not limited to, Example 1 wherein as shown by determining average antibody titer levels in bovines subsequent to a first and/or second (booster) vaccination), when administering to bovines vaccine compositions comprising an adjuvant selected from an emulsion having a continuous phase that is aqueous (e.g., water) and a discontinuous phase that is an oil. Preferably, the oil is a non-mineral oil. Preferably, the adjuvant further comprises a tocopherol. It can further be envisioned in light of the current invention that the adjuvant comprises an emulsion comprising a mixture of a mineral oil, a non-mineral oil and a tocopherol, wherein said mixture of oils is dispersed in a continuous water phase.

A mineral oil is an oil that originates from a mineral source, typically from petroleum. In preferred embodiments the mineral oil preferably is a liquid paraffin oil. A liquid paraffin oil, is a type of mineral oil, also named a white (mineral) oil, or light liquid paraffin oil, and has CAS number: 8042-47-5. It is generally available, also in pharmaceutical grade quality. Examples are: Drakeol® 6VR (Penreco), Marcol® 52 (Exxon Mobile), and Klearol® (Sonneborn). A non-mineral oil can be selected from synthetic oils, semi-synthetic oils, animal oils, and vegetable oils. As provided herein, a semi-synthetic oil is an oil that is nonmineral in origin such as an animal or vegetable oil, but which was modified in structure and/or composition by a chemical or physical process. Preferably, the nonmineral oil is selected from a squalane, a squalene, and a vegetable oil, wherein, preferably, said vegetable oil is an oleate, more preferably ethyl-oleate. In one preferred embodiment, the non-mineral oil is squalane. Squalane is a chemical compound with CAS number 111-01 -3. Some alternate names are: hydrogenated shark liver oil, hexamethyltetracosane, or perhydrosqualene. This is not to be confused with squalene (CAS nr. 111-02-4) which is a poly-unsaturated C30 oil and is metabolisable as a compound of the cholesterol pathway. Originally the precursor to squalane was obtained from shark livers, but over environmental concerns this has shifted to other natural sources, such as olive oil, or to chemical synthesis. Therefore, squalane may be provided in its natural, synthetic or semi-synthetic form, or mixtures thereof. Squalane is commercially available in a variety of purities, for example: from vegetable source, from Worlee (Squalane, vegetable), or Croda (Pripure Squalane); or synthetic, e.g., from Kuraray (Squalane-PE). For the invention, a high purity of the squalane is preferred: preferably over 75 % purity, more preferably over 80, 90, or even over 95 % purity, in that order of preference. Other examples of synthetic oils suitable for use in the adjuvant of the vaccine composition according to the invention include, for example, a Shell Ondina® oil, e.g. Shell Ondina X420 or Shell Ondina X409.

In an embodiment the tocopherol is an alpha-tocopherol; more preferably, the alphatocopherol is selected from Vitamin E and Vitamin E-acetate. Vitamin E-acetate is a chemical compound with CAS number: 58-95-7. Some alternate names are: tocopheryl acetate, or alpha-tocopherol-acetate. Vitamin E-acetate is an acetateester of vitamin E (tocopherol) and can be derived from vegetable materials such as seeds, nuts, fruits or leaves, or from fatty meats, but may also be produced synthetically. Vitamin E-acetate may be provided in a natural, synthetic or semisynthetic form, or mixtures thereof. Vitamin E-acetate is commercially available, in different degrees of purity. In non-limiting examples, O/W emulsions suitable for use as adjuvant in the vaccine composition of the invention may be:

• A combination of two O/W emulsion adjuvant components, one which is based on vitamin E acetate (e.g., the emulsion disclosed in EP0382271 ), and one which is based on liquid paraffin oil (e.g., the emulsion disclosed in WO 2009/144,088);

• An O/W emulsion comprising light liquid paraffin oil and Vitamin E-acetate;

• An O/W emulsion comprising squalane and Vitamin E-acetate;

• An O/W emulsion comprising a synthetic non-mineral oil and Vitamin E- acetate.

The adjuvants can comprise further additives, such as stabilizers, preservatives or emulsifiers. Emulsions, such as O/W emulsions, can be formed and stably maintained by selecting an appropriate kind and concentration of emulsifier(s). An emulsifier for the adjuvant, when said adjuvant comprises an O/W emulsion, in a vaccine composition according to the invention is a molecule with amphiphilic properties, having both a hydrophobic- and a hydrophilic side. An emulsifier typically takes position at the interphase between water and oil and stabilizes the droplets of the internal, dispersed phase. Many different emulsifiers are known and are suitable for pharmaceutical use, such as in vaccines. One example is the emulsifier polysorbate 80, also known as polyoxyethylene sorbitan mono-oleate, and commercially available as Tween® 80. In some preferred embodiments, the adjuvant comprises an emulsifier. One class of hydrophilic non-ionic emulsifiers that may be used in the invention may be polyoxyethylene cetostearyl ether, which is an ether of cetostearyl alcohol.

In some preferred embodiments, the adjuvant comprises a polyethoxyethylene cetostearyl ether, preferably polyethoxyethylene 12 cetostearyl ether, a tocopherol, preferably alpha-tocopheryl acetate, and a non-mineral oil, preferably squalane or a synthetic oil, such as a Shell Ondina® oil, for example Shell Ondina X GTL- based medicinal white oil. In some preferred embodiments, the adjuvant comprises a polyethoxyethylene cetostearyl ether polyethoxyethylene 12 cetostearyl ether, a tocopherol, preferably alpha-tocopheryl acetate, and a mineral oil, preferably light (or white) liquid paraffin, such as Marcol® (Exxon Mobile) or Drakeol® (Penreco).

In some preferred embodiments, the vaccine composition of the invention comprises an adjuvant which is an O/W emulsion comprising:

• about 2 to about 3 % w/w of an emulsifier, preferably a polyethoxyethylene cetostearyl ether, more preferably polyethoxyethylene 12 cetostearyl ether,

• about 2 to about 3 % w/w tocopherol, preferably alpha-tocopheryl acetate; and

• about 30 to about 40 % w/v; preferably about 35 % of a mineral oil, preferably light (or white) liquid paraffin.

This adjuvant composition is described in the art (e.g., in WO2023118553A1 ) as Xsolve2.0. Xsolve2.0 adjuvant is a nano emulsion.

• about 6 to about 7 % w/w of an emulsifier, preferably a polyethoxyethylene cetostearyl ether, more preferably polyethoxyethylene 12 cetostearyl ether;

• about 15 to about 16 % w/w of tocopherol, preferably alpha-tocopheryl acetate;

• about 13 to about 14 % w/w of a non-mineral oil, preferably squalane.

This adjuvant composition is known in the art (e.g., in WO2023118553A1 )as SVEA- E. SVEA-E adjuvant is a nano emulsion.

In some embodiments, the vaccine composition according to the invention comprises an adjuvant having a continuous phase that is aqueous, wherein said adjuvant comprises matrix-like particles. Such matrix-like particles are comprised in the continuous aqueous phase. Preferably, the matrix-like particles comprise saponins. In more preferred embodiments, the adjuvant is a dispersion of saponin matrix particles in water. Saponins are natural glycosides of steroid or triterpene. Saponins have a diverse range of properties and may act as emulsifiers, surfactants or foam-forming agents and are typically used in soaps, shampoos, but also in drinks, cosmetics, fire extinguishers etc. Further, saponins have been used as vaccine adjuvants and have been explored as such for several vaccines, such as in infectious diseases and cancer therapy (see, Dalsgaard et al. Acta Vet Scand. 1977;18(3):367-73. doi: 10.1186/BF03548434; Skene et al. Methods, Volume 40, Issue 1 , 2006, Pages 53-59, ISSN 1046-2023, doi.org/10.1016/j.ymeth.2006.05.019; Sanders et al. 2005. Immunol Cell Biol 83:119-128, doi.org/10.1111/j.1440-1711 ,2005.01319.x). Saponins may be provided in their natural, synthetic, or semi-synthetic form, or mixtures thereof. Matrices comprising saponins, specifically the saponin Quil A, extracted from the bark of Quillaja Saponaria Molina, have been first described by Morein et al. as immune stimulating complexes (“iscom”), self-assembling particles of approximately 40nm - 60nm in diameter in which virus membrane proteins were presented in a multimeric form (Morein et al. Nature 308, 457-460 (1984). doi.org/10.1038/308457a0). Saponins, e.g., Quil A, form such matrices (ISCOMs) when mixed with polar lipids, such as phospholipids and cholesterol (Kersten et al., Biochimica et Biophysica Acta (BBA) - Biomembranes, Volume 1062, Issue 2, 1991 , Pages 165-171 , doi.org/10.1016/0005-2736(91 )90388-0). One commercially available saponin-based adjuvant for vaccines is Matrix-M™ (Novavax Inc.). The inventors found desirable antibody responses in bovines upon administration of the vaccine composition according to the invention and adjuvanted with a dispersion of saponin matrix particles in water, preferably wherein the saponin is Quil A, more preferably wherein Quil A saponins are formulated as immune stimulating complexes (ISCOMs). Further, and of relevance to some embodiments, the inventors found that by using a dispersion of saponin matrix particles in water as adjuvant for a vaccine composition according to the invention a lesser dose of C. parvum gp40 antigen and/or bovine coronavirus antigen (inactivated bovine coronavirus) could be used to achieve a response comparable or improved response compared to higher doses. In other words, the adjuvant was found to have a stimulatory effect on the response of the immune system, as determined by average antibody titers, to a vaccine composition comprising the antigens of C. parvum gp40 antigen and/or bovine coronavirus. Further, the vaccine composition combined with an adjuvant comprising a dispersion of saponin matrix particles in water was shown to exhibit acceptable safety, in some non-limiting examples shown by the absence of clinical signs due to vaccination and/or absence of morbidity and/or adverse effects. Hence, in one embodiment the invention provides for a vaccine composition comprising non live antigens from: a) E.coli; b) bovine rotavirus; c) bovine coronavirus; wherein the antigens are formulated in an adjuvant having a continuous phase that is aqueous, wherein the adjuvant is a dispersion of saponin matrix particles in water.

It is preferred that the antigen from E.coli comprises inactivated E. coli or an extract thereof. For example, the E. coli may be inactivated by formalin treatment, but other methods known to a skilled person and are likewise encompassed herein. The extract preferably is a cell-free extract.

Enteropathogenic E coli contains several virulence factors associated with production of diarrhea. Common virulence factors are fimbrial antigens, which enable (enteropathogenic) E.coli to attach to and colonize the villi of the small intestine of neonatal calves in the first days of life. Therefore, the current invention preferably comprises E.coli fimbrial adhesins as antigens. These E. coli fimbrial adhesins and may also be referred to in the art as pili or fimbriae.

Provided herein, the E.coli fimbrial adhesins can be selected from enterotoxins fimbrial adhesins and therefore comprise any one or more from the group consisting of F4, F5, F6, F18, and F41. (Dubreuil JD, et al. Animal enterotoxigenic Escherichia coli. EcoSal Plus 2016;7(1 ): 10.1128/ecosalplus. ESP-0006-2016). As also described by Dubreuil et al. E.coli strains that are responsible for enterotoxicity in calves most commonly possess F5 (K99), F17a and/or F41 fimbrial antigens. These enterotoxic E.coli strains thus are one of the most common causes of E. co//-caused diarrhoea in bovines and account for a large portion of all calf mortality on dairy farms. Accordingly, the invention provides for a vaccine composition comprising non live antigens from: a) E.colr', b) bovine rotavirus; c) bovine coronavirus; wherein the antigen is a cell-free extract comprising one or more E.coli fimbrial adhesins selected from F4, F5, F6, F18, and F41.

In more preferred embodiments, the invention provides for a vaccine composition as described herein and comprising non live antigens from: a) E.colr', b) bovine rotavirus; c) bovine coronavirus; wherein the antigen from E.coli comprises the E.coli fimbrial adhesins F5 and F41 . As described above, these fimbrial adhesins are preferably derived from inactivated E.coli and thus the antigens from E.coli as provided herein, as a result, comprise inactivated E.coli fimbrial adhesins F5 and F41. The E. coli strain providing said fimbrial adhesins may be, for example, Strain CN7985. Other strains may be used likewise. Preferably, the E. coli antigens are antigens, preferably fimbrial adhesins, isolated or derived from E. coli that has been inactivated.

Preferably, the antigen from bovine rotavirus is inactivated bovine rotavirus.

Thus, the invention may provide for a vaccine composition as described herein and comprising: a) a non live antigen from E.colr', b) inactivated bovine rotavirus; c) a non live antigen from bovine coronavirus.

Bovine rotavirus is a non-enveloped RNA virus that belongs to the family Reoviridae. Based on the group specific epitopes localized in an immunodominant site of VP6 between amino acid residue 48 and 75, rotaviruses have been divided into five serological species (A-E) and two additional tentative species (F and G) according to the International Committee on Taxonomy of Viruses (ICTV). Most commonly, Bovine rotaviruses of Bovine rotavirus group A are associated with neonatal diarrhea in young calves. Bovine rotavirus group A strains can be classified into VP4 or P types (for protease-sensitive) and VP7 or G types (for glycoprotein) (Estes & Kapikian, Fields Virology, Vol. 2, Lippincott Williams & Wilkins/Wolters Kluwer, Philadelphia (2007), pp. 1917-1974). Bovine RVA strains belonging to G6, G8, and G10, in association with P1 , P5, and P11 , are commonly found in cattle. It has been described by Papp et al. that the predominant genotype combination among bovine RVA strains was found to be G6P5 (Papp et al., Veterinary Microbiology, Volume 165, Issues 3-4, 2013, Pages 190-199, ISSN 0378-1135, doi.org/10.1016/j.vetmic.2O13.03.020). Papp et al. found that the predominance of strains comprising this genotype combination was seen across continents over time. The prevalence of G6 was followed by G10 in Americas, Europe, Asia, and Australia, and G8 in Africa. Hence, it is preferred that the bovine rotavirus as provided herein at least comprises strain UK-compton (serotype G6P5). Inactivated bovine rotavirus strain UK-compton (serotype G6P5) is commercially available in, for example, the product Bovilis Rotavec Corona (MSD Animal Health), and two inactivated bovine rotavirus strains, i.e., NCDV G6P1 and B233(G10P[11 ]), are commercially available in the product Bovilis® Guardian® (Merck Animal Health). There are other commercially available veterinary rotavirus vaccines licensed for use in other geographic regions (e.g., Scourguard® 4KC (Zoetis), or Trivacton® 6 (Boehringer Ingelheim). Further, exemplary rotavirus strains that may be include in the vaccine composition according to the current invention include strain G10 and G8. Multiple strains of rotavirus can be included, such as a combination of rotavirus strains G10 and G6 either alone or in combination with other rotavirus strains such as strain G8, combinations of G10 or G8 with other rotavirus strains, and combinations of rotavirus strains that do not include rotavirus strains G8 or G10. Said strains G6, G10 and G8 may be in association with any one of P 1 , P5, or P11 . Accordingly, in some embodiments, the vaccine composition may comprise two inactivated bovine rotavirus strains, i.e., G10P11 and G6P5. Other sets of inactivated bovine rotavirus strains can be envisioned by a skilled person and are also encompassed by the current invention.

Preferably, the antigen from bovine coronavirus provided herein is inactivated bovine coronavirus. Thus, the invention may provide for a vaccine composition as described herein and comprising: a) a non live antigen from E.coli; b) a non live antigen from bovine rotavirus; c) inactivated bovine coronavirus. Bovine coronavirus is a single-stranded positive-sense RNA virus with a lipid envelope belonging to the order Nidovirales. Coronaviruses are causative of calf enteritis. The inactivated bovine coronavirus, strain Mebus is commercially available in, for example, the product Bovilis Rotavec Corona (MSD Animal Health). Thus, preferably the inactivated bovine coronavirus of the vaccine composition according to the invention is of strain Mebus. The skilled person is aware of other suitable inactivated bovine coronavirus strains that may be used as alternative in the vaccine composition of the current invention. In line with the above, the invention in preferred embodiments provides for a vaccine composition that comprises: a) E. co// fimbrial adhesins F5 and F41 , for example inactivated E. co// fimbrial adhesins F5 and F41 ; b) inactivated bovine rotavirus; and c) inactivated bovine coronavirus wherein the antigens are formulated in an adjuvant having a continuous phase that is aqueous.

Other causes for bovine enteric disease have been described in the art. The pathogens that may cause bovine enteric disease comprise viruses, parasites, and bacteria. Examples of these pathogens are described in the art, such as in Cho et al. (2014), Foster et al. (2009) and Gillhuber et al. (2014) (Cho et al. J Vet Sci. 2014;15(1 ):1 -17. doi: 10.4142/jvs.2014.15.1 .1 . Epub 2013 Dec 27.; Foster DM et al. Vet Clin North Am Food Anim Pract. 2009 Mar;25(1 ): 13-36, xi. doi: 10.1016/j.cvfa.2008.10.013.; Gillhuber J et al. BMC Res Notes. 2014 Feb 26;7:112. doi: 10.1186/1756-0500-7-112).

It is contemplated that, in certain embodiments, the vaccine composition of the invention may comprise a further enteric pathogen that is an antigen derived from a virus, a parasite, or a bacterium, preferably wherein said virus is selected from bovine viral diarrhoea virus, bovine torovirus, bovine norovirus and bovine nebovirus, preferably wherein said parasite is selected from Giardia spp., such as, G. bovis or G. duodenalis, Cryptosporidium spp., such as Cryptosporidium parvum or Cryptosporidium bovis, and Eimeria spp, such as E. bovis and E. zuernii, and preferably said bacterium is selected from Salmonella spp. , such as S. typhimurium, S. Dublin and one from Clostridium spp., such as C. perfringens, C chauvoei, C septicum, C novyi, and C sordellii.

One exemplary pathogen that is considered a cause of bovine enteric disease comprises the parasite genus Cryptosporidium. The term “Cryptosporidium" refers to a genus of parasites of the phylum Apicomplexa, and the subclass Coccidia. These micro-organisms have the characterizing features of their taxonomic class, such as the morphologic, genomic, and biochemical characteristics, as well as the biological characteristics such as physiologic, immunologic, or pathologic behaviour. A large number of species of Cryptosporidium parasites are known. These can infect a wide variety of non-human animals as well as humans. Well known species of Cryptosporidium parasite is C. parvum, which appears in two genotypes: genotype I which is considered to be infectious for humans only, and genotype II that is a proven zoonotic agent. Both genotypes of C. parvum cause Cryptosporidiosis, especially in vulnerable targets. A reference for the characteristics and the effects of C. parvum in veterinary medicine is: "The Merck veterinary manual" (11th ed., 2016, ISBN-10: 9780911910612). The glycoprotein Cryptosporidium parvum gp 40 is a protein that occurs on the surface of the motile parasite stages and is heavily glycosylated. It must be noted that protein naming for Cryptosporidia is confusing, and gp40 (or its encoding gene or gene-product) is also called Cpgp40/15 (Cevallos et al., 2000, Inf. & Imm., vol. 68, p. 4108-4116); gp15/45/60 (Strong et al., 2000, Inf. & Imm., vol. 68, p. 4117-4134); or S60 (Winter et al., 2000, Funct. Integr. Genomics, vol. 1 , p. 207-217). Related is also the Cp17 protein (Priest et al., 2000, Mol. Biochem. Parasit., vol. 106, p. 261 -271 ). The differences in indicated molecular weight reflect variability in sequence and glycosylation level. Of note is also that the antigen named ‘Cp15/60’ is a different protein (Jenkins et al., 1993, Inf. and Imm., vol. 61 , p. 2377-2382; GenBank acc.nr. U22892). The same applies to the antigens named ‘CP15’ (GenBank acc.nr. L34568), or ‘cp41 ’ (WO 01/040439). The expression of gp40 in a recombinant expression system and use for (passive) vaccination has been suggested since many years, e.g., in: WO 93/024649, WO 01/040248, WO 01/077293, and US 2002/0081312. However, only until recently, a functional subunit vaccine is available, e.g., in the form of the commercially available Bovilis Cryptium (MSD Animal Health). WO2021122896A1 describes methods of preparing safe and effective vaccines comprising the Cryptosporidium parvum gp40 protein or immunogenic part thereof by incubating the gp40 protein with an aziridine before its use as a vaccine in humans and non-humans. It is encompassed herein that the antigen Cryptosporidium parvum gp40 provided in the vaccine composition of this invention is prepared in accordance with methods of WO2021122896A1 . Hence, the Cryptosporidium parvum gp40 may be inactivated, for example as a result of alkylation of gp40 by a method of WO2021122896A1 .

Accordingly, in some preferred embodiments, the present invention may comprise a vaccine composition comprising: a) a non live antigen from E.coli; b) a non live antigen from bovine rotavirus; c) a non live antigen from bovine coronavirus; and d) an antigen from Cryptosporidium parvum, preferably a non live antigen.

Preferably, the antigen from Cryptosporidium parvum is the glycoprotein Cryptosporidium parvum gp40.

In some preferred instances, as exemplified herein, the addition of an antigen from C. parvum, preferably C. parvum gp40, to the vaccine composition in accordance with the invention even resulted in a synergetic effect on serology for some of the antigens from E.coli, preferably on E.coli fimbrial adhesins F5/41 , from bovine rotavirus, preferably inactivated bovine rotavirus, and from bovine coronavirus, preferably bovine coronavirus.

Other exemplary pathogen that is considered a cause of bovine enteric disease comprises is a bacterium from Clostridium spp., for example, C. perfringens. Therefore, in particular embodiments, the antigen may be from Clostridium spp. For example, Clostridium spp. may comprise a Clostridium spp. bacterin. In some embodiments, the Clostridium spp. bacterin is inactivated. Examples of such inactivation methods include, but are not limited to, heat, formaldehyde, formalin, binary ethylenimine (13E1 ), radiation, and beta-propiolactone treatment. In additional embodiments, the antigen may be isolated or derived from Clostridium spp. bacteria that have been inactivated by any suitable method available to one of ordinary skill in the art. In certain embodiments the vaccine composition according to the invention comprises antigens that elicit an immune response to Clostridium spp. In some embodiments, the further antigen comprises a bacterin toxoid from C. perfringens types C and/or D (and/or C. perfringens types A and/or B). Accordingly, in some embodiments, there is provided for a vaccine composition comprising non live antigens from: a) E.coli b) bovine rotavirus; c) bovine coronavirus; a) Clostridium spp.

Preferably, the antigen from Clostridium spp is an inactivated antigen. Preferably the antigen from Clostridium spp is from C. perfringens, and more preferably is inactivated C. perfringens Types C and/or D toxoid.

In some embodiments, the vaccine composition of the current invention may be provided in a volume comprising between 0.2 mL - 5 mL per dose. The volume may be any volume between 0.2 mL and 5 mL, preferably about 0.2 mL, 0.5 mL, about

1 mL, about 1 ,5 mL, about 2 mL, about 2,5 mL, about 3 mL, about 3,5 mL, about 4 mL, about 4,5 mL and/or about 5 mL. Preferably, the vaccine is provided commercially in a container suitable for containing at least one dose of vaccine, such as a vial, bottle, flask and/or suitable applicators, such as a syringe. Also encompassed are containers suitable for containing multiple doses of the vaccine composition. In one illustrative and non-limiting example, the vaccine composition according to the invention can be provided in a container comprising 10 vials of 2 ml (10 x 1 dose), or comprising 1 vial of 10 ml (5 doses) or Cardboard box with 1 vial of 40 ml (20 doses), or Cardboard box with 1 vial of 100 ml (50 doses). In embodiments, the vaccine composition of the current invention may be given as single dose or in two (e.g., a booster vaccine) or more doses. In certain embodiments, the primary vaccine is with a single 0.5 mL - 5 mL, preferably about

2 mL, dose of vaccine. In certain embodiments, the primary vaccine and booster vaccine are with two 0.5 mL - 5 mL, preferably about 2 mL, doses of vaccine. In some embodiments, the vaccine composition comprises per dose an amount of:

• between 500 - 10.000 AU of inactivated bovine rotavirus;

• between 25 - 500 CVU of inactivated bovine coronavirus; and/or

• between 500 - 10.000 AU of E. coli fimbrial adhesins F5 and F41 . Thus, a vaccine composition having a volume of about 2 mL may comprise, for example, 2000 All/2 mL of the antigen inactivated bovine rotavirus, 110 CVll/2 mL of inactivated bovine coronavirus and 2200 AU/2 mL of E. co// fimbrial adhesins F5 and F41 . Preferably, the vaccine composition comprises effective amounts of the antigens inactivated bovine rotavirus, inactivated bovine coronavirus and E. coli fimbrial adhesins F5 and F41 . It is contemplated that the effective amounts may vary depending upon the strain or strains of E. coli, bovine rotavirus and/or bovine coronavirus, and optionally Cryptosporidium parvum, used to generate the vaccine. The effective amount preferably is an amount that is sufficient (and/or more than sufficient) to evoke a protective immune response to the corresponding pathogen in the bovine, pregnant heifer and/or the pregnant cow and/or transfer of protective passive immunity to the new-born calf.

The units “AU” and “CVU” as used herein to indicate the dose of the antigens comprised in the vaccine composition are known to a skilled person in the art and are commonly used to indicate the amount of viral, parasitic and/or bacterial antigens present in a composition such as those disclosed herein. As is used in some examples provided herein a 100% dose corresponds to the doses of the available commercial products Bovilis Rotavec Corona and/or Bovilis Cryptium. Thus, for example, 4000 AU/dose inactivated bovine rotavirus, 220 CVU/dose inactivated bovine coronavirus and 4400 AU/dose E. coli fimbrial adhesins F5 and F41 corresponds to the amounts/dose of said antigens in the available commercial product Bovilis Rotavec Corona., and for example 1.5 AU/dose Cryptosporidium parvum gp40 corresponds to the amount/dose of said antigen in the available commercial product Bovilis Cryptium.

The amounts of the antigens comprised in the vaccine composition of the invention may be determined by using methods known in the art, such as a BRV potency ELISA, BCV potency ELISA and/or E. coli F5 (K99) and F41 potency ELISA, and optionally Cryptosporidium parvum GP40 potency ELISA.

As described herein the vaccine composition preferably is provided as a ready-to- use liquid composition. The vaccine composition may be provided in a suitable container, such as a vial, bottle, flask and/or suitable applicators, such as a syringe. When provided in a suitable applicator, the vaccine composition may be provided in a form that is “ready-to-use" such that the vaccine can be administered, e.g., via a syringe, to an animal, preferably a bovine. The vaccine composition in the form of a ready-to-use liquid composition, e.g., when provided in a suitable container or suitable applicator, is provided in a volume that is sufficient to administer as a single dose. In some embodiments, the vaccine composition may be provided in forms, volumes, and/or suitable containers, allowing the recent development vaccines of which the dosing is flexible were developed (“flex dosing”), for instance as 2 times 1 ml, or as once 2 ml.

Further provided herein are methods for the preparation of the vaccine composition according to the current invention. The method may comprise the steps of 1 ) providing the antigens, comprising at least an antigen from bovine rotavirus, preferably inactivated bovine rotavirus, an antigen from bovine coronavirus, preferably inactivated bovine coronavirus, and an antigen from E. coli, preferably E.coli fimbrial adhesins F5 and F41 ; 2) admixing the antigens with an adjuvant having a continuous phase that is aqueous. Preferably the adjuvant is an O/W emulsion comprising a non-mineral oil and a tocopherol or a dispersion of saponin matrix particles in water.

It may be that the method for preparing the vaccine composition comprises the inactivation of any one or more of the antigens.

It is preferred that the method for the preparation of the vaccine composition comprises the step of admixing the antigens and an adjuvant to obtain the vaccine composition of the invention. In even more preferred embodiments, said method comprises the step of admixing the antigens and any one adjuvant selected from the group consisting of: an O/W emulsion of a mineral oil and a tocopherol in water, an O/W emulsion of a non-mineral oil and a tocopherol in water and a dispersion of saponin matrix particles in water. It will be appreciated by the skilled person that the admixing can be performed by using arrangements, tools and/or methods known and generally used in the art for the preparation of vaccine compositions. As previously described, the vaccine composition provided herein preferably is “ready- to-use". Therefore, it is contemplated that the methods for the preparation of the vaccine composition are performed by a manufacturer and/or supplier of said vaccine. In one further aspect, the invention provides for methods for immunization and/or a vaccine composition for use in the immunization of a bovine against enteric disease due to an infection with an enteric pathogen. Preferably, the methods provide for the effective immunizing of a bovine, thus comprising that a protective immune response to the corresponding pathogen is evoked in the bovine, pregnant heifer and/or the pregnant cow and/or comprising that there is a transfer of protective passive immunity to a new-born calf.

In one embodiment, the methods for immunization and/or comprises administering to the bovine the vaccine composition according to the invention. The administering may comprise the parental e.g., intramuscular, subcutaneous, intraperitoneal, or intradermal, preferably subcutaneous, administering of the vaccine composition to a bovine. Preferably, the vaccine composition is administered in effective amounts. Thus, methods provided by the present invention also can be used to provide active immunity against enteric disease in a bovine.

In some illustrative examples provided herein, to which the invention is not limited, after administration of the vaccine composition to bovine serum-antibody titers were achieved that are illustrative for immune protection by the vaccine composition against at least one, two, or all the enteric pathogens for which antigens are comprised in the vaccine composition, in the vaccinated cow.

In some illustrative examples, serum-antibody titers were obtained for the antigens comprised in the vaccine composition of the invention, that are illustrative for improved immune protection compared to positive and/or negative controls and/or compared to commercial vaccine compositions available in the art. In related embodiments, a cow may be administered a primary and a booster (secondary) vaccination with the vaccine composition provided herein. In further related embodiments, said administration is to a pregnant cow and prior to parturition. In some illustrative examples, to which the invention is not limited, the booster (secondary) vaccination with the vaccine composition improved antibody titers, against at least one, two, three, four or all the enteric pathogens for which antigens are comprised in the vaccine composition, to a level illustrative for improved immune protection by the vaccine composition in the vaccinated cow.

In one further illustrative example, non-limiting for the invention, it was shown that the vaccine composition according to the invention had a desirable effect on the average parasite load (C. parvum) of serum and colostrum, i.e. , vaccination reduced parasite load compared to a control.

In another embodiment the immunization comprises administering to a bovine calf colostrum comprising maternally derived antigens (MDA) to an enteric pathogen, wherein the colostrum is derived from a bovine to which the vaccine composition according to the invention has been administered. Thus, a variant of a vaccine composition according to the invention is the colostrum containing antibodies against bovine rotavirus, bovine coronavirus and E. coli that is generated by a pregnant heifer or pregnant cow that was immunized with the vaccine composition according to the invention. In related embodiments, a pregnant cow or pregnant heifer may be administered a primary and a booster (secondary) vaccine of the vaccine composition provided herein prior to parturition.

The colostrum can be fed to or drunk by the calf. Alternatively, antibodies derived from such colostrum can be administered. The calf receives passive immunity through ingestion of colostrum, which may be by allowing the calf to nurse from a vaccinated cow or vaccinated heifer after giving birth to a calf and/or may be by bottle-feeding colostrum obtained from a vaccinated cow or vaccinated heifer to the calf. Thus, methods provided by the present invention also can be used to provide passive immunity against enteric disease, preferably neonatal calf diarrhea, in calves.

In some embodiments, the vaccine composition according to the invention is administered as a single vaccine dose to a pregnant cow or heifer between about 20 - 2 weeks, e.g., 15, 14, 13, 12, 11 , 10, 9, 8, 7, 6, 5, 4, 3, 2 weeks before parturition. In some embodiments, the methods for preventing enteric disease due to an infection with an enteric pathogen in a bovine may comprise the administering of a primary and a booster vaccine. The booster vaccine comprises the vaccine composition as provided herein and is preferably administered after between about 2 - 18 weeks, e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, and 18 weeks, preferably between 4, 8, 12, 14 or 17 weeks after administering the primary vaccine. Preferably, in case the vaccine composition is administered to a pregnant heifer or pregnant cow, both primary and booster vaccines are administered prior to parturition.

Further, the invention provides for methods for preventing enteric disease due to an infection with an enteric pathogen in a bovine, wherein the method comprises administering vaccine composition according to the invention to the bovine. Accordingly, some embodiments of the invention provide methods for the prevention and/or amelioration of E. coli, bovine rotavirus and/or bovine coronavirus infection (and optionally C. parvum and/or C. perfringens infection) in a bovine that comprises administering parentally, preferably subcutaneously, to a bovine a vaccine composition of the present invention. Alternatively, there is provided for methods for preventing enteric disease due to an infection with an enteric pathogen in a bovine, wherein the method comprises administering colostrum of a vaccinated bovine, wherein said bovine has been vaccinated with the vaccine composition according to the invention, to a calf. Accordingly, some embodiments of the invention also provide methods for the prevention and/or amelioration of E. coli, bovine rotavirus and/or bovine coronavirus infection (and optionally C. parvum and/or C. perfringens infection) in a bovine calf that comprises administering to a heifer and/or cow pregnant with the calf a vaccine composition of the present invention and/or administering to said calf, after birth, the colostrum obtained from a heifer and/or cow vaccinated with the vaccine composition according to the invention.

The vaccine composition according to the invention wherein the enteric pathogen is selected from the group consisting of: bovine rotavirus, bovine coronavirus, E. coli and combinations thereof. The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art (including the contents of the references cited herein), readily modify and/or adapt for various applications, such as specific embodiments, without undue experimentation, without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein.

All references cited herein, including journal articles or abstract, published, or corresponding patent applications, patents, or any other references, are incorporated by reference herein in its entirety, including all data, tables, figures, and text presented in the cited references. Additionally, the entire contents of the references cited within the references cited herein are also entirely incorporated by reference.

It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance presented herein, in combination with the knowledge of one of ordinary skill in the art.

Having now generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration and are not intended to be limiting to the present invention. Further aspects and embodiments will be apparent to those skilled in the art. EXAMPLE

- Example 1 is a safety & efficacy study of several vaccine compositions comprising the antigens provided herein.

- Example 2 is a serology study in serum and colostrum of ISCOM-adjuvanted vaccine compositions with the antigens provided herein.

Example 1: Serology study of vaccine compositions containing bovine rotavirus, bovine coronavirus, Escherichia Col i and Cryptosporidium parvum antigens

Study Design

The study was performed in 64 animals (Friesian Holstein or cross breed) of which 48 were calves between 4 and 9 months old and the remaining 16 animals were cows between 13 and 21 months old. The animals were allocated to eight groups of eight animals each (see Table 1 ) in such way that each group consisted of six calves and two cows.

Adjuvants tested herein are:

- GNE (proprietary product of MSD Animal Health): a non-bacterial oil/water adjuvant;

- Alhydrogel® (Croda Pharma): an aluminum-based wet gel suspension;

- SVEA-E® (MSD Animal Health) (see, e.g., WO2023118553A1 ): an emulsion of a non-mineral oil and a tocopherol in water;

- X-Solve® 2.0 (MSD Animal Health): (see, e.g., WO2023118553A1 ) an emulsion of a (synthetic) non-mineral oil and a tocopherol in water;

- Emunade® (proprietary product of MSD Animal Health): emulsion of a mineral oil in water, and containing alhydrogel;

- ISCOM: a dispersion of saponin matrix particles in water;

- Alu-oil: oil (Montanide™ ISA 70 (Seppic) (water-in-oil formulation) + aluminum (Alhydrogel). Table 1 : Study design

Treatment

All animals received a 2 mL SC injection of the described vaccine preparations according to Table 2, in the right side of the neck. A second administration of 2 mL of the described vaccine preparations according to Table 8 was given SC in the left side of the neck to half of the animals grouped in the groups 3, 5 and 7, respectively 14 (for the calves) and 17 weeks (for the cows) after the first administration. Table 2: Vaccine preparation

Procedures Daily observations

Animals were inspected on a daily basis for general wellbeing and signs of disease.

Local reactions

Prior vaccination and on day 1 , 3, 7, 14, 21 , 28, 42 and 56 after vaccination, animals of groups 1 -7 were checked for local reactions by visual inspection and palpation only when a local reaction was visible in accordance with standard procedures. In case of a visible local reaction, the findings: estimated size in length x width x height (L x Wx H) [cm], boundary definition, consistency, painful and warm were recorded individually.

Serum samples

Serum samples were tested for specific antibody response against C. parvum gp40 were determined in an antibody ELISA. Serum samples were analyzed with commercial tests from Bio-X Diagnostics for BRV (BIO K 126, Monoscreen AbELISA Bovine rotavirus I competition), BCV (BIO K 392, Monoscreen AbELISA Bovine coronavirus I competition) and E. coli F5 (BIO K 295, Monoscreen AbELISA E. coli F5 (K99) I blocking) antibodies according to the manufacturer’s instructions.

Blood sampling

Plain blood samples for serum preparation were taken in accordance with standard procedures Samples were drawn prior to each vaccination (DO) and two, four, six and eight weeks thereafter.

Two heparin blood samples were taken from all cows plus three calves per group at four and eight weeks after the first vaccination and at two and six weeks after the second vaccination to determine cellular immune reactions using a lymphocyte stimulation test.

Morbidity, veterinary treatment

No animals required treatment by the Responsible Veterinarian for any clinical sign attributable to vaccination. Neutralization assay

Pools of serum samples of group 3, 5 and 7 were analyzed for neutralizing antibodies against C. Parvum, BRV, BCV. Samples for neutralizing antibodies against C. parvum were tested in an in-vitro invasion inhibition test (Ml); samples for neutralizing antibodies against BRV and BCV were tested in a VN test.

Lymphocyte stimulation test

Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood using density gradient centrifugation. The cells were labeled with a proliferation dye and stimulated independently with BRV VP8, BCV S1 , E. coli F5 and C. parvum gp40 for four days. The number of reactive cells after vaccination was determined using flow cytometry.

Results

Overall, the serological responses upon subcutaneous administrations of vaccines adjuvanted with SVEA-E (Group 3), X-Solve 2.0 (Group 5) and ISCOM (Group 7) resulted in desirable antibody responses. An overview of average antibody responses in the serum samples for BRV, BCV, E. coli F5/F41 and C. parvum gp40 exhibited by the different adjuvated vaccines and determined by ELISA is shown in Fig. 1 A - D. Most adjuvanted vaccines were found to be acceptable in terms of safety.

Bovine rotavirus antibody levels

At the start of the study the average BRV antibody level was 58.3% inhibition. After the first vaccination (see, Table 3), the average antibody levels of the groups 2, 4, 5, 6, 7 and 8 increased to maximal 55.4 (4w post V1 ), 65.3 (2w post V1), 73.6 (2w post V1 ), 69.7 (2w post V1 ), 77.0 (2w post V1 ) and 77.5% (2w post V1 ) inhibition for respectively group 2, 4, 5, 6, 7 and 8. No increase was observed in group 1 and 3 after the first vaccination (V1 ). Table 3: Average inactivated bovine rotavirus antibody levels (% inhibition)

Two weeks after the booster vaccination (V2) (see, Table 4) of the animals in group

3 (SVEA-E), 5 (X-Solve 2.0) and 7 (ISCOM), maximum average antibody titers levels of 43.6, 48.8, 49.0% inhibition were found, which were 10-30% lower than the maximum titers of the first part of the study.

Table 4: Average inactivated bovine rotavirus antibody levels (% inhibition) after after V2

Bovine coronavirus antibody levels

At the start of the study the average BCV antibody level was 49.4% inhibition. After the first vaccination, the average antibody levels of all groups increased to maximal 80.8 (8w post V1 ), 85.7 (4w post V1 ), 69.2 (2w post V1 ), 65.5 (2w post V1 ), 65.8 (8w post V1 ), 77.1 (2w post V1 ) and 82.5% (2w post V1 ) inhibition for respectively group 1 , 2, 3, 4, 6, 7 and 8. The titers of group 5 didn’t increase after one vaccination. The highest average increase was found in group 1 (GNE) throughout the first part of the study. Two weeks after the booster vaccination of the animals in group 3 (SVEA-E), 5 (XSolve) and 7 (ISCOM), the average antibody titers increased to maximum levels of 75.4, 73.3 and 89.3% inhibition respectively, after which they decreased. Table 5: Average inactivated bovine coronavirus antibody levels (% inhibition)

Table 6: Average inactivated bovine coronavirus antibody levels (% inhibition) after

V2 E. coli F5 antibody levels

At the start of the study the average E. coli F5 antibody level was 6.8% inhibition.

After the first vaccination (V1 ), the average antibody levels of all groups increased to maximal 96.5 (6w post V1 ), 96.6 (4w post V1 ), 72.8 (8w post V1 ), 68.3 (8w post V1 ), 71.9 (6w post V1 ), 58.0 (6w post V1 ), 64.3 (6w post V1 ) and 95.1 % (8w post V1 ) inhibition for respectively group 1 till 8. The highest average increases were found in group 1 (GNE) and 2 (GNE + alhydrogel) throughout the first part of the study. Two weeks after the booster vaccination of the animals in group 3 (SVEA-E), 5 (X-Solve 2.0) and 7 (ISCOM), the average antibody titers of these groups increased to even higher maximum levels of 97.1 % inhibition, after which they remained stable.

Table 7: Average E. coli F5 (K99) antibody levels (% inhibition)

Table 8: Average E. coli F5 (K99) antibody levels (% inhibition) after booster

C. parvum Gp40 antibody levels

At the start of the study the average IgG antibody titer against C. parvum gp40 10.7 Iog2. Two weeks post first vaccination (V1 ) the antibody responses in all groups increased to 14.9 till 17.0 Iog2. Four/six weeks post vaccination (V1 ) the average antibody levels in all groups decreased roughly 1 Iog2, except for group 1 and 2 where they increased slightly. The highest responses were found in group 7 (ISCOM) at two weeks. Two weeks after the booster vaccination of the animals in group 3 (SVEA-E), 5 (X-Solve 2.0) and 7 (ISCOM), the average antibody titers increased to maximum levels of 19.2, 18.4 and 19.6 Iog2 respectively, after which they decreased 3 Iog2. Table 9: Average C. parvum gp40 antibody antibody levels (Iog2)

Table 10: Average C. parvum gp40 antibody antibody levels (Iog2) after booster BRV neutralization assay

The average neutralizing antibody responses against BRV were analyzed for the groups 3, 5 and 7. In group 3 and 7 the average neutralizing antibody responses decreased after the first vaccination, but after the second vaccination they increased until the same levels compared to the starting level or two weeks post V1 . In group 5 (X-Solve 2.0) the level remains around 8 Iog2 during the study, but 2 weeks after the second vaccination it increases to 9.3 Iog2.

Table 11 : Average BRV neutralizing antibodies (Iog2) BCV neutralization assay

The average neutralizing antibody responses against BCV were analyzed for the groups 3, 5 and 7. During the study the average neutralizing antibody responses against BCV in group 3 (SVEA-E) and 5 (X-Solve 2.0) increased about 1-2 Iog2 after the first vaccination and after the second vaccination it increased further with about 1 Iog2 (around 11 Iog2). In group 7 (ISCOM) the neutralizing antibody level against BCV kept rising after each vaccination from 8.3 Iog2 at the start to 12.5 Iog2 two weeks after the second vaccination.

Table 12: Average BCV neutralizing antibodies (Iog2)

C. parvum I VI I

The average parasite load was tested for the groups 3 (SVEA-E), 5 (X-Solve 2.0), 5 and 7 (ISCOM). At the start of the experiment, the average parasite load for group 5 and 7 were at the same level as the IVI I negative (control consisting of fetal calf serum (FCS)). After both vaccinations (primary and booster) the average parasite loads decreased for all three groups until levels below the IVI I positive control (consisting of polyclonal serum pool): 922, 1149 and 1600 for respectively group 3, 5 and 7.

Table 13: Average of C. parvum IVII results (SQ)

Lymphocyte activation assay Results of the lymphocyte stimulation tests against BRV VP8, BCV S1 , E. coli F5, and C. parvum gp40 are depicted in Figure 2A - D. Although the percentages of proliferated antigen-specific T cells were low (after correction for medium control), increases were observed for all groups. For the BRV VP8- and C. parvum gp40- specific responses, the ISCOM adjuvanted vaccine showed the highest percentage of proliferation after the booster vaccination. In general, the combination vaccine of the invention in any one of the tested adjuvants showed an increase of proliferation after primary- and/or booster vaccination (Fig. 2A - D).

Safety & Adverse reactions

Mild or no clinical signs were observed after vaccination. All tested vaccines caused raised rectal temperatures for 1-2 days after each administration. After the first vaccination, local reactions (see, Table 14). were found in 8, 7, 3, 5, 3, 7, 8 animals per group for respectively groups 1 till 7. The local reactions sizes in the animals of group 1 and 2 increased over time until unacceptable large sizes at eight weeks post V1 .

Animals having received a vaccine composition in accordance with the invention, see e.g., groups 3 and 5, showed reduced occurrence (i.e., local reactions were found less than average number of animals) and a reduced size of the local reactions (see, e.g., group 3 and 5) to the vaccination. Also, local reactions in animals having received a vaccine composition in accordance with the invention, see, e.g., groups 3, 5 and 7, were almost gone in the animals 1w post V1 , and could not be detected 2w post V1 . Also, it was found that groups having received a vaccine composition comprising alu-oil ass adjuvant (e.g., group 2 or 4) showed more extensive local reactions than their counterparts that do not comprise alu-oil (e.g., group 1 or 3).

Table 14: Average local reaction sizes in cm3 (LxWxH) after first vaccination

After the second vaccination (V2) (see, Table 15), local reactions were found in the groups 3, 5 and 7. The local reactions in the individual animals of group 3 resolved after one day, whilst in the other two groups the local reactions resolved between 1 to 14 days. All animals moved easily through the feeding fence and didn’t show abnormal behavior, indicating that the local reactions didn’t cause discomfort to the animals and were found to be acceptable in terms of safety.

Table 15: Average local reaction sizes in cm3 (LxWxH) after booster

Overall, the local reactions sizes in the animals vaccinated with GNE (group 1 ) and GNE + alhydrogel (group 2) showed increases to unacceptable large sizes at after one SC administration. The local reaction sizes in the other groups were clearly smaller and resolved within 1-3 weeks after each vaccination. Overall, the adjuvanted vaccine compositions according to the current invention were found to be acceptable in terms of safety.

Conclusion

Vaccines adjuvanted with O/W emulsions (SVEA-E (Group 3), X-Solve 2.0 (Group 5)) and saponins (ISCOM (Group 7)) after primary vaccination resulted in desirable safety and efficacy results. Also, after an additional booster vaccination these vaccines adjuvanted with O/W emulsions (SVEA-E (Group 3), X-Solve 2.0 (Group 5)) and saponins (ISCOM (Group 7)) resulted in desirable safety and efficacy results.

Example 2: Serology study in serum and colostrum upon subcutaneous (SC) administration of ISCOM -adjuvanted vaccine compositions

Study design

The study was performed in pregnant heifers (Friesian Holstein or cross breed). All animals were vaccinated twice (primary + booster) subcutaneous (SC) in the left side of the neck with the vaccines formulated at two dose levels (see Table 16 and 17).

The vaccines were composed of inactivated antigens with no toxic activity. The first dose (primary) was given approximately six weeks before the expected calving date (DO) and the second dose (booster) four weeks later (D28). Table 16: Study design

Table 17: Vaccine preparation

Treatment

All animals received a 2 mL SC injection of the described vaccine preparations according to Table 17, at approximately six (DO) and two (D28) weeks prior to expected calving date, in the left side of the neck. All injections were administered using 3 mL syringes and needles in accordance with standard procedures.

Procedures

Daily observations

Blood- and serum sampling

Plain blood samples without anti-coagulant were taken from all animals just before the vaccinations (DO, D14 and D28) and two (D42) and four (D56) weeks after the last vaccination.

Colostrum sampling

From the animals colostrum from the first and second milking was collected. The udders were hygienical ly cleaned before each colostrum milking. The first milking took place within 6 hours post-partum for the first milking colostrum. The second milking took place within 20 hours post-partum. From each animal two samples of approximately 50 mL of the first two milkings was collected in 50 mL tubes. The collected colostrum samples from a single milking from each individual cow will be labelled individually and stored at 2-8°C until transport to the lab.

Morbidity, veterinary treatment

No animals required treatment by the Responsible Veterinarian for any clinical sign attributable to vaccination.

ELISA Specific antibody responses in colostrum and/or serum samples against C. parvum gp40 were tested using in-house assays. Serum and/or colostrum samples were analysed with commercial tests from Bio-X Diagnostics for BRV (BIO K 126, Monoscreen AbELISA Bovine rotavirus I competition), BCV (BIO K 392, Monoscreen AbELISA Bovine coronavirus I competition) and E. coli F5 (BIO K 295, Monoscreen AbELISA E. coli F5 (K99) I blocking) antibodies according to the manufacturer’s instructions at MSD Animal Health Center for Diagnostic Solutions (R&D Service Lab).

C. parvum in-vitro invasion inhibition (I VI I) test

Pools of serum samples and pools of colostrum samples were tested for neutralizing antibodies against C. parvum in an IVI I test.

Results

Overall, the serological responses upon subcutaneous administrations of vaccines adjuvanted with ISCOM resulted in desirable antibody responses. Acceptable antibody titers could be determined in serum and/or colostrum. Vaccines containing C. parvum gp40 antigens clearly reduced average parasite load in IVI I compared to vaccines without C. parvum gp40 antigen and controls.

Bovine rotavirus antibody levels

At the start of the study the average BRV antibody level was, respectively in group 1 , 2 and 3, 34.8, 32.0 and 41 .5% inhibition. Two weeks after the V1 , a small increase in antibody response was seen for group 2 (40.4%) and 3 (47.5%) whilst in group 1 the responses remained stable. Four weeks after V2 the average antibody responses increased to maximum levels of 53.1 , 65.9 and 49.7% in group 1 till 3 respectively.

The average antibody levels against BRV in the first milking colostrum were 21.1 , 27.6 and 26.9% inhibition respectively for group 1 , 2 and 3. The average antibody titers in the second milking colostrum were 9.6, 12.3, and 13.6% inhibition respectively for group 1 , 2 and 3. The responses in the colostrum of the vaccination groups were comparable. Table 18: Summary of BRV antibody ELISA (% inhibition)

Bovine coronavirus antibody levels

At the start of the study the average BCV antibody level was, respectively in group 1 , 2 and 3, 43.1 , 36.1 and 43.6% inhibition. Two weeks after V1 , the average antibody levels of all RCC vaccination groups increased to maximal 84.8, 83.6 and 84.8% inhibition for respectively group 1 till 3. After this point the average titers decreased slightly until two weeks after V2 after which they remained stable.

The average antibody titers against BCV in the first milking colostrum were 39.2, 26.4 and 30.1 % inhibition respectively for group 1 , 2 and 3. The average antibody titers in the second milking colostrum were 17.3, 8.2 and 11.8% inhibition respectively for group 1 , 2 and 3. The average response in group 1 was slightly higher compared to group 2 and 3 for both (the first and second) milking(s).

Table 19: Summary of BCV antibody ELISA (% inhibition)

E. coli F5 antibody levels

At the start of the study the average E. coli F5 antibody level was, respectively in group 1 , 2 and 3, 9.5, 11.0 and 3.5% inhibition. Two weeks post V1 the antibody responses in the vaccinated groups increased to 69.0, 54.4 and 60.0% inhibition. At the time of V2 (four weeks after V1 ), the average antibody titers decreased roughly 25%. Two weeks after V2, the average antibody titers increased to maximum levels of 95.7, 93.6 and 96.8% inhibition in group 1 , 2 and 3 respectively. No differences were found between groups 1 , 2 and 3.

The average antibody titers against E. coli F5 in the first milking colostrum were 96.8, 85.3 and 95.8% inhibition respectively for group 1 , 2 and 3. The average antibody titers in the second milking colostrum were 96.7, 75.2 and 96.0% inhibition respectively for group 1 , 2 and 3. Similarly high titers were found in groups 1 , 2 and 3 for both (the first and second) milking(s). Between the vaccinated groups a slight difference titer was detected both for first and second milking, with the lowest antibody titer corresponding to the highest vaccine dose. No differences were found between groups 1 and 3.

Table 20: Summary of E. coli F5 antibody ELISA (% inhibition)

C. parvum Gp40 antibody levels

Two weeks post first vaccination (V1 ) the antibody responses in the vaccinated groups increased to 16.9 and 16.4 Iog2. At the time of V2 (four weeks after V1 ), the average antibody titers decreased roughly 1.8 log2. Two weeks after V2, the average antibody titers increased to maximum levels of 17.6 and 17.1 Iog2 in group 1 and 2 respectively, after which they decreased slightly. The highest response was found in group 1 .

The average IgG antibody titers against C. parvum gp40 in the first milking colostrum were 20.1 , 19.5 Iog2 respectively for group 1 and 2. The average antibody titers in the second milking colostrum were 18.6, 17.6 Iog2 respectively for group 1 and 2.

Table 21 : Summary of C. parvum gp40 antibody ELISA (Iog2)

C. parvum I VI I

At the start of the study, the average parasite load was 27684.1 for group 1 and 2. The parasite loads in the C. parvum gp40 vaccinated groups 1 and 2 clearly decreased at two weeks post V1 (2503.9 and 4663.3 SQ) and two weeks post V2 (2617.9 and 1305.2 SQ), A dose effect was seen in the serum from group 1 and 2 at two weeks post V2.

Table 22: Summary of C. parvum IVI I results of serum pools (SQ) In colostrum, the parasite loads in the first milking colostrum pools of the C. parvum gp40 vaccinated groups (1 and 2) were reduced in both milking colostrum pools compared to vaccinated groups not vaccinated with C. parvum gp40. The parasite load in the colostrum pool of the group not vaccinated with C. parvum gp40 was

878.9 SQ.

Table 23: Summary of C. parvum IVI I results of colostrum pools (SQ)

Conclusion

The serological responses in serum and colostrum of two ISCOM adjuvanted vaccine compositions containing C. parvum gp40, containing regular and high antigen levels, and one ISCOM adjuvanted vaccine composition without C. parvum, containing regular antigen levels were investigated.

Average antibody responses against C. parvum gp40, BRV, BCV and E. coli F5 in the serum samples and colostrum samples increased after V1 for all antigens. Antibody titers were present in colostrum in both the first and second milkings for all antigens.

For C. parvum there was a clear effect of average parasite load in the serum and colostrum after vaccination with vaccines containing a C. parvum antigen.

All tested vaccine compositions according to the invention were found to result in a desirable antibody response and were found to be acceptable in terms of safety, thus showing that the ISCOM-based adjuvants as used in the tested vaccine compositions and for the particular antigen combinations are effective and safe.

Claims

1. A vaccine composition to aid in the protection against infection with an enteric pathogen in a bovine, the vaccine composition comprising non live antigens from: a) E.coli; b) bovine rotavirus; c) bovine coronavirus; and wherein the non live antigens are formulated in an adjuvant having a continuous phase that is aqueous.

2. The vaccine composition according to Claim 1 , wherein the adjuvant comprises an oil as a discontinuous phase.

3. The vaccine composition of any one of the previous claims wherein the adjuvant is formulated as an oil-in-water (O/W) emulsion.

4. The vaccine composition of any one of the previous claims wherein the adjuvant is selected from the group consisting of:

- an emulsion of a mineral oil and a tocopherol in water;

- an emulsion of a non-mineral oil and a tocopherol in water.

5. The vaccine composition according to Claim 4, wherein the non-mineral oil is squalane.

6. The vaccine composition according to Claim 4 or 5, wherein the tocopherol is Vitamin-E-acetate.

7. The vaccine composition according to Claim 1 , wherein the adjuvant is a dispersion of saponin matrix particles in water.

8. The vaccine composition of any one of the previous claims, wherein the antigen from E.coli comprises inactivated E. coli or an extract thereof, preferably wherein the extract thereof is a cell-free extract comprising one or more E.coli fimbrial adhesins selected from F4, F5, F6, F18, and F41 , more preferably wherein the antigen from E.coli comprises the E.coli fimbrial adhesins F5 and F41 .

9. The vaccine composition of any one of the previous claims, wherein the antigen from bovine rotavirus comprises inactivated bovine rotavirus and/or the antigen from bovine coronavirus comprises inactivated bovine coronavirus.

10. The vaccine composition of any one of the previous claims comprising one or more further antigens, preferably wherein the one or more further antigens are selected from enteric pathogens of a bovine species.

11 . The vaccine composition of any one of the previous claims, comprising one or more further antigens from Clostridium spp. and/or from Cryptosporidium parvum, preferably wherein the one or more further antigens comprises Cryptosporidium parvum gp40.

12. A vaccine composition for use to aid in the protection against infection with an enteric pathogen in a bovine, the vaccine composition comprising non live antigens from: a) E.coli; b) bovine rotavirus; c) bovine coronavirus; and wherein the non live antigens are formulated in an adjuvant having a continuous phase that is aqueous.

13. A vaccine for use according to claim 12, characterised in that the vaccine is administered subcutaneously.

14. A method of aiding in the protection against infection with an enteric pathogen in a bovine, the method comprising administering a vaccine composition to the bovine, the vaccine composition comprising non live antigens from: a) E.coli; b) bovine rotavirus; c) bovine coronavirus; and wherein the non live antigens are formulated in an adjuvant having a continuous phase that is aqueous.

15. A method according to claim 14, characterised in that the vaccine is administered subcutaneously.