Veterinary vaccines against enteric diseases

Abstract

The present invention relates to a combination vaccine comprising the antigens: a) Cryptosporidium parvum gp40; b) inactivated bovine rotavirus; c) inactivated bovine coronavirus; and d) E. coli fimbrial adhesins F5 and F41. In addition, the invention relates to methods for the preparation of the vaccine, methods for vaccinating ruminants against infection by Cryptosporidium parvum, bovine rotavirus, bovine coronavirus and E. coli, and to a kit-of-parts at least comprising a container comprising the at least one antigen according to the invention.

Description

[0001] VETERINARY VACCINES AGAINST ENTERIC DISEASES

[0002] TECHNICAL FIELD

[0003] The invention pertains to new combination vaccines comprising the antigens Cryptosporidium parvum gp40, inactivated bovine rotavirus, inactivated bovine coronavirus and E. coli fimbrial adhesins F5 and F41 . Methods of making and using the vaccine are also provided.

[0004] BACKGROUND

[0005] Bovine enteric disease is the result of an enteropathogenic intestinal infection that most often manifests itself in some form of diarrhea. This disease, also commonly referred to as neonatal calf diarrhea, is responsible for substantial economic loss in the farming industry. Neonatal calf diarrhea remains the most important cause of death in calves under one month of age. 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 et al. (Brunauer M. et al. Prevalence of Worldwide Neonatal Calf Diarrhoea Caused by Bovine Rotavirus in Combination with Bovine Coronavirus, Escherichia coli K99 and Cryptosporidium spp.: A Meta-Analysis. 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. Prevalence, prediction and risk factors of enteropathogens in normal and non-normal faeces of young Dutch dairy calves. 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 more than one of these pathogens.

[0006] 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 to a pregnant bovine. Immunization of bovines and newborn calves to the enteric pathogen Cryptosporidium parvum today may done by administering the commercially available Bovilis Cryptium vaccine to a pregnant bovine. The pregnant bovine is vaccinated before parturition to stimulate antibody production to specific pathogens of the neonatal calf diarrhea complex; these antibodies are then passed on to the newborn calves, for example through the feeding of colostrum of the vaccinated cows. It has been shown to induce protection against enteric disease, such as neonatal calf diarrhea, in the calves of the pregnant bovine. Whilst it is well-known in the field that multiple enteric pathogens are potentially causative for neonatal calf diarrhea, there remains a dire need to provide for vaccines protecting against these multiple enteric pathogens. However, despite this need, there is no available combination vaccine against rotavirus, coronavirus, E. coli and C. parvum. There have been efforts in the art to arrive at a combination vaccine against rotavirus, coronavirus, E. coli and C. parvum:

[0007] - WO2011056175A1 discloses methods for protecting newborn calves against neonatal calf diarrhea by vaccinating pregnant bovines. The document discloses vaccine combinations. However, no combination vaccine containing the antigen Cryptosporidium parvum gp40 is described nor suggested.

[0008] - WO0145735A2 discloses vaccines containing antigens of C. parvum, including a hypothetical example of a combination vaccine against Cryptosporidium parvum, rotavirus, coronavirus and E. coli. However, no combination vaccine containing the antigen Cryptosporidium parvum gp40 is described nor suggested.

[0009] A particular problem commonly encountered in the preparation of combination vaccines is the phenomenon of so-called “efficacy interference” in vaccines complicates development of effective combination vaccines, and should be considered during vaccine development, because the efficacy of one antigen in the combination may be diminished or reduced by another antigen. It is recommended by studies (see, for example, Venkatesan MM, Van de Verg LL. Combination vaccines against diarrheal diseases. Hum Vaccin Immunother. 2015; 11 (6): 1434-48. doi: 10.4161 / 21645515.2014.986984) to consider for human combination diarrheal vaccines to evaluate this so-called “interference” and detect the effects of the combination of antigens on any one of the antibody titers to the separate antigens during the development of a combination diarrheal vaccine for humans. In other words, any combination of antigens and a site of administration is not straightforward and requires experimentation to determine safety and efficacy. That combining vaccines is not straightforward and prone to development issues is similarly valid for veterinary vaccines as for example demonstrated by the “Note for guidance: requirements for combined veterinary products” of the European Agency for the Evaluation of Medicinal Products (EMEA CVMP / IWP / 52 / 97-FINAL), wherein is stated that “[the] development of combined vaccines is not straightforward. Each combination should be developed and studied individually in terms of quality, safety and efficacy” and “In combined vaccines, the presence of more than one component can often cause an interaction, leading to either a diminished or an increased response to individual components, compared to when the specific component(s) is administered alone."

[0010] Despite the above-mentioned efforts in the art and available commercial vaccines, there is no available combination vaccine against rotavirus, coronavirus, E. coli and C. parvum that is effective and safe. Consequently, there remains a continued need in the field to further optimize vaccines against bovine enteric disease, such as neonatal calf diarrhea. For example, it remains a need to provide vaccines comprising a combination of antigens able to induce protection against multiple enteric pathogens causative for bovine enteric disease, such as neonatal calf diarrhea, wherein the enteric pathogens are at least any one of rotavirus, coronavirus, E. coli and C. parvum. Further, it would be highly desirable to provide such combination vaccines as a ready-to-use vaccine. Hence, it is an object of the invention to at least meet one of the above-mentioned needs.

[0011] SUMMARY OF INVENTION

[0012] To meet the object of the invention there is provided, in a first aspect, a combination vaccine to aid in the prevention of enteric disease due to an infection with an enteric pathogen in a bovine comprising: a) Cryptosporidium parvum gp40; b) inactivated bovine rotavirus; c) inactivated bovine coronavirus; and d) E. co / / fimbrial adhesins F5 and F41.

[0013] The invention is also embodied in a method for protecting a bovine against an infection with Cryptosporidium parvum, bovine rotavirus, bovine coronavirus and E. coli, the method comprising administering to the bovine a vaccine comprising: a) Cryptosporidium parvum gp40; b) inactivated bovine rotavirus; c) inactivated bovine coronavirus; and d) E. coli fimbrial adhesins F5 and F41.

[0014] Preferably, the vaccine is administered subcutaneously.

[0015] DESCRIPTION OF DRAWINGS

[0016] Figure 1 : Fig. 1 A - D show an overview of average antibody responses in the serum samples for, respectively, BRV, BCV, E. coli F5 / F41 and C. parvum gp40 exhibited by the different adjuvated vaccines and determined by ELISA.

[0017] 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

[0018] DEFINITIONS

[0019] For purposes of the present invention, the following terms are defined below.

[0020] The term “adjuvant” is used here in its common meaning of a composition capable of stimulating an immune response in a target animal in a nonspecific manner.

[0021] An “antigen" for the invention refers to a protein that can -in the right circumstances- induce a protective immunological response in a target animal.

[0022] 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.

[0023] As is well-known, “colostrum" is the milk that is secreted by the mammary glands of a mammal in the period around delivery.

[0024] As used herein a “combination" when referring to a “combination vaccine" means that multiple immunologically active components, e.g., multiple antigens, are merged into a single vaccine product. A “combination vaccine", also sometimes referred to as “multivalent vaccine” is generally described as a vaccine containing multiple antigens to prevent different diseases or to protect or immunize against multiple strains of infectious agents causing the same disease. As meant herein, the “combination vaccine’’ is to refer to a vaccine to protect or immunize against multiple strains of infectious agents, i.e., multiple enteric pathogens, causing the same disease in a bovine, i.e., bovine enteric disease such as neonatal calf diarrhea.

[0025] 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”.

[0026] 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.

[0027] 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.

[0028] As used herein “prior to parturition" is equivalent to “prior to calving". Similarly, “after parturition’’ is equivalent to “after calving’’.

[0029] 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.

[0030] 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” and may also refer to that a simple pre-treatment may be required to make it “ready-to-administer”.

[0031] 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.

[0032] 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.

[0033] DESCRIPTION OF EMBODIMENTS

[0034] The invention is defined herein and in the accompanying claims. Subject-matter which is not encompassed by the scope of the claims does not form part of the present claimed invention.

[0035] 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.

[0036] The inventors surprisingly found that a combination vaccine comprising the antigens: Cryptosporidium parvum gp40, inactivated bovine rotavirus, inactivated bovine coronavirus and E. coli fimbrial adhesins F5 (K99) and F41 , could be formulated without suffering from interference between the antigens, such as “efficacy interference” of the antigens. The inventors found that the combination of these antigens in a combination vaccine formulation was able to stimulate the immune system of animals that have been administered said combination vaccine. In animals, for example calves and pregnant bovines, serum and / or colostrum antibody titers and / or antigen-specific T-cell responses were found that suggest a strong immune response against the antigens. For example, in an experiment testing four different doses of the antigens in four combination vaccines (see, Example 1 ), it was found that when the antigens were formulated in subcutaneously administered combination vaccines, acceptable serology levels were found (i.e., antibody levels). In other words, all doses tested of the antigens in combination were acceptable, therewith leading the inventors to conclude that the antigens do not interfere with one another in their ability to raise antibody levels in the serum. In another example, i.e., another experiment, the most preferred dose in a dose-finding study (i.e., in the dose-finding study (see Example 1 ) referred to as the “50% dose”, which is based on the dose of the commercial product Bovilis® Rotavac® corona (MSD Animal Health) and Bovilis® Cryptium® (MSD Animal Health), and comprising 2000 AU / dose of inactivated bovine rotavirus, 110 CVU / dose of inactivated bovine coronavirus, 2200 AU / dose of E. coli F5 / F41 and 5 AU / dose of C. parvum GP40) was found to result in desirable serological responses when combined with vaccines adjuvanted with adjuvants having a continuous phase that is aqueous. In one further example, the inventors found in an example that a combination of the antigens provided in the combination vaccine of the current invention, when administered twice subcutaneously to pregnant heifers before calving, resulted in desirable antibody titers for all of the antigens (inactivated bovine rotavirus, inactivated bovine coronavirus, E. coli F5 / F41 and C. parvum GP40) in the serum of heifers. In addition, it was found that this administering the combination vaccine to pregnant heifers results in heightened antibody titers in the colostrum of said heifers after parturition. This suggests that the combination vaccine provided herein may be suitable for passively -via colostrum intake- providing (neonatal) calves with colostrum containing heightened antibody titers against enteric pathogens such as rotavirus, coronavirus, E. coli and C. parvum. These results obtained in heifers lead to the conclusion that, again, the combination vaccine did not result in an interference in the efficacy of the separate antigens. Also, parasite loads in both serum and colostrum were significantly reduced in groups having received a combination vaccine comprising the antigen C. parvum gp40.

[0037] All in all, it was surprising that the antigens, when formulated in one combination vaccine, did not show any efficacy interference and were this effective (i.e., were able to result in such desirable antibody titers in both serum (calves and heifers) and colostrum (heifers) against all four antigens in bovines). Further, the inventors were surprised that none of the animals to which the combination vaccine of the current invention was administered in the performed experiments suffered from local reaction that affected the general wellbeing and behavior of the animals. Thus, overall, the safety of the herein provided combination vaccines was found to be acceptable.

[0038] In other words, the inventors found a combination vaccine of more than one different antigens, preferably antigens derived from four different (pathogenic) microbes, i.e., preferably at least four, more preferably at least five antigens, is effective and safe for administration to bovines. Therefore, in a first aspect the invention provides for a combination vaccine comprising: a) Cryptosporidium parvum gp40; b) inactivated bovine rotavirus; c) inactivated bovine coronavirus; and d) E. co / / fimbrial adhesins F5 and F41.

[0039] In some embodiments, the combination vaccine according to the invention elicits an immune response to at least one, more preferably 2, 3 or 4 of the microbes, preferably the microbes are pathogens, from which the combination vaccine comprises an antigen that is derived from said microbes, and wherein the microbes comprise Cryptosporidium parvum, bovine rotavirus, bovine coronavirus and E. coli. In some embodiments, the combination vaccine according to the invention comprises antigens that elicit an immune response to Cryptosporidium parvum, rotavirus, coronavirus and / or E. coli. In certain embodiments, the combination vaccine according to the invention comprises antigens that elicit an immune response to Cryptosporidium parvum gp40, rotavirus, coronavirus and / or E.coli fimbrial adhesins F5 and / or F41. In preferred embodiments, the combination vaccine according to the invention comprises antigens that elicit an immune response to Cryptosporidium parvum gp40, rotavirus, coronavirus and E.coli fimbrial adhesins F5 and F41 .

[0040] The antigen Cryptosporidium parvum gp 40 is a mucin-type glycoprotein of 40 kDa from Cryptosporidia. 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, in some embodiments, the Cryptosporidium parvum gp40 is inactivated, for example as a result of alkylation of gp40 by a method of WO2021122896A1. Therefore, in some embodiments there is provided for a combination vaccine comprising: a) inactivated Cryptosporidium parvum gp40; b) inactivated bovine rotavirus; c) inactivated bovine coronavirus; and d) E. co / / fimbrial adhesins F5 and F41.

[0041] Rotaviruses are the most common cause of neonatal diarrhoea in calves. 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, Rotaviruses, 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. Review of group A rotavirus strains reported in swine and cattle, 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 inactivated bovine rotavirus as provided herein at least comprises strain UK-compton (serotype G6P5). The 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

[0011] ), 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 combination vaccine 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 P1 , P5, or P11. Accordingly, in some embodiments, the current combination vaccine 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.

[0042] In the current invention the antigen bovine rotavirus can be an attenuated, inactivated, or killed bovine rotavirus and preferably is an inactivated bovine rotavirus. In certain embodiments, the rotavirus antigen may be isolated or derived from rotavirus that have been inactivated or killed by any suitable method available to one of ordinary skill in the art. Examples of such methods include, but are not limited to, heat, formaldehyde, formalin, binary ethylenimine, radiation, and betapropiolactone treatment. In preferred embodiments, bovine rotavirus is an inactivated bovine rotavirus.

[0043] Bovine coronaviruses (BCV) are betacoronaviruses associated with neonatal calf diarrhea. Therefore, in embodiments, an antigen is a bovine coronavirus, preferably an attenuated, inactivated, or killed bovine coronavirus. In certain embodiments, the coronavirus antigen may be isolated or derived from coronavirus that have been inactivated or killed by any suitable method available to one of ordinary skill in the art. Examples of such methods include, but are not limited to, heat, formaldehyde, formalin, binary ethylenimine, radiation, and beta-propiolactone treatment. In certain embodiments, the antigen may be an attenuated, inactivated, or killed BCV. The inactivated bovine coronavirus as provided herein preferably is the inactivated bovine coronavirus, strain Mebus. The skilled person is aware of other suitable inactivated bovine coronavirus strains that may be used as alternative in the combination vaccine of the current invention. Bovine coronavirus is a singlestranded 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).

[0044] In view of the above, in some embodiments there is provided for a combination vaccine comprising: a) Cryptosporidium parvum gp40; b) inactivated bovine rotavirus; c) inactivated bovine coronavirus; and d) E. co / / fimbrial adhesins F5 and F41.

[0045] 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. Enteropathogenic E coli contains two virulence factors associated with production of diarrhea that are fimbrial antigens enabling them to attach to and colonize the villi of the small intestine of neonatal calves in the first days of life. Strains in calves most commonly possess F5 (K99) or F41 fimbrial antigens or both. These E. coli fimbrial antigens are fimbrial adhesins and may also be referred to herein as pili or fimbriae. Thus, in some embodiments, the E. coli antigen preferably is an antigen isolated or derived from E. coli that has been inactivated. E. coli may have been inactivated by methods known to one of ordinary skill in the art. Examples of such methods include, but are not limited to, heat, formaldehyde, formalin, binary ethylenimine, radiation, and beta-propiolactone treatment. For example, the E. coli may be inactivated by formalin treatment. Therefore, in some embodiments there is provided for a combination vaccine comprising: a) Cryptosporidium parvum gp40; b) inactivated bovine rotavirus; c) inactivated bovine coronavirus; and d) inactivated E. co / / fimbrial adhesins F5 and F41.

[0046] In preferred embodiments an antigen provided in the combination vaccine in accordance with the invention is inactivated E. co / / fimbrial adhesins F5 and / or F41. The E. coli strain providing said fimbrial adhesis may be, for example, Strain CN7985. Other strains may be used likewise.

[0047] Thus, in some embodiments the combination vaccine of the invention may comprise inactivated antigens. In some preferred embodiments the antigens of the combination vaccine are inactivated. As explained before, methods for the inactivation of virus particles, bacteria, or other pathogens for the use in vaccine compositions are known to a skilled person. Such methods have been broadly described in the art, such as in the handbook Vaccine Analysis: Strategies, Principles, and Control (e.g., Sanders et al. (2015). Inactivated Viral 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_2; 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) and are commonly used to reduce the pathogenicity of the virus particles, bacteria, or other pathogens that form the vaccine composition. Accordingly, in preferred embodiments there is provided for a combination vaccine comprising, as antigens: a) inactivated Cryptosporidium parvum gp40; b) inactivated bovine rotavirus; c) inactivated bovine coronavirus; and d) inactivated E. co / / fimbrial adhesins F5 and F41.

[0048] In some embodiments, the combination vaccine is suitable for aiding in the prevention of enteric disease due to an infection with an enteric pathogen in a bovine. As described herein, there are many enteric pathogens that may cause enteric disease in a bovine by infection with said enteric pathogen. Some examples of enteric disease in a bovine are bovine viral diarrhea, winter dysentery, indigestion, neonatal calf diarrhea, etc.

[0049] In some embodiments, the enteric pathogen causing the enteric disease as a result of an infection with said enteric pathogen at least comprises one selected from the group consisting of: Cryptosporidium parvum, bovine rotavirus, bovine coronavirus, E. coli and combinations thereof. It is contemplated that enteric disease as a result of an infection with one of these aforementioned enteric pathogens and as a result of infection with a further enteric pathogen, may also be in part prevented and / or may be alleviated.

[0050] Hence, in some preferred embodiments, the enteric pathogen is selected from the group consisting of: Cryptosporidium parvum, bovine rotavirus, bovine coronavirus, E. coli and combinations thereof.

[0051] Aside from the enteric pathogens E. coli, bovine rotavirus, bovine coronavirus and Cryptosporidium parvum bovine enteric diseases may be caused by further enteric pathogens. Therefore, it is encompassed by the current invention that, in addition to the antigens inactivated Cryptosporidium parvum gp40, inactivated bovine rotavirus, inactivated bovine coronavirus and inactivated E. co / / fimbrial adhesins F5 and F41 , the combination vaccine of the current invention further comprises one or more further antigens selected from an enteric pathogen of a bovine species. As provided herein, at least one, for example two, three, four, five, six, ... etc. further antigens may be selected. In preferred embodiments, the combination vaccine according to the current invention comprises one further antigen. In some embodiments, the enteric pathogen is selected from the group consisting of: Cryptosporidium parvum, bovine rotavirus, bovine coronavirus, E. coli and combinations thereof. Thus, in other words, there may be provided one or more additional antigens from any one of Cryptosporidium parvum, bovine rotavirus, bovine coronavirus, E. coli, in addition to the antigens inactivated Cryptosporidium parvum gp40, inactivated bovine rotavirus, inactivated bovine coronavirus and inactivated E. coli fimbrial adhesins F5 and F41 as provided herein.

[0052] 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 combination vaccine of the invention may comprise a further enteric pathogen that is an antigen derived from a virus, a parasite, and 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 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. In some preferred embodiments, the one or more further antigens of the combination vaccine of the current invention is one from Clostridium spp. and more preferably is a further antigen comprising C. perfringens or an antigenic fraction thereof. In particular embodiments, the antigen may be 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 combination vaccine 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). Hence, in some embodiments, there is provided for a combination vaccine comprising, as antigens: a) inactivated Cryptosporidium parvum gp40; b) inactivated bovine rotavirus; c) inactivated bovine coronavirus; and d) inactivated E. co / / fimbrial adhesins F5 and F41 ; e) an antigen from C. perfringens, preferably C. perfringens Types C and / or D toxoid.

[0053] In further embodiments, the combination vaccine in accordance with the invention is provided as a ready-to-use liquid composition. As broadly described herein, a ready-to-use liquid composition comprising the combination vaccine 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 combination vaccine in accordance with the invention, the combination vaccine comprises a pharmaceutically acceptable carrier. 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 combination vaccine is suitable for administration to an animal by injection. Therefore, in some embodiments the liquid composition comprising the combination vaccine is a suspension or emulsion. In preferred embodiments the liquid composition comprising the combination vaccine is an oil in water (O / W) emulsion.

[0054] The ready-to-use liquid composition of the combination vaccine 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 combination vaccine 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 combination vaccine in the form of a ready-to-use (and / or ready-to-administer) 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. For example, a desirable volume may be about 2 ml. Alternatively, the combination vaccine of the invention may be provided in a container suitable to extract more than a single dose from, e.g., to administer in sequence to more than one animal. For example, in some embodiments, the combination vaccine is provided in a 10mL vial, said vial comprising sufficient doses for about 4 - 5 animals and / or comprising about 4 - 5 doses. In some embodiments, the combination vaccine 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.

[0055] In some embodiments, the combination vaccine according to the invention typically comprises one or more adjuvants. Adjuvants may stimulate the immune response of a target in a non-specific manner. Many different adjuvants are known in the art. Examples of adjuvants are: complete- or incomplete Freund’s adjuvant, vitamin E or alpha-tocopherol, non-ionic block polymers and polyamines such as dextran sulphate, Carbopol™, pyran, Saponin, such as: Quil A™, or Q-vac™. Saponin and vaccine components may be combined in an ISCOM™. Furthermore, peptides such as muramyl dipeptides, dimethylglycine, tuftsin, are often used as adjuvant, and mineral oil e.g. Bayol™, Drakeol™, Klearol™, or Marcol™, Montanide™ or light mineral (paraffin) oil; non-mineral oil such as squalene, squalane; vegetable oils or derivatives thereof, e.g. ethyl- oleate. Also, combination products such as ISA™ (Seppic), or DiluvacForte™ and Xsolve™ (both MSD Animal Health) can advantageously be used. A further option is the use of SVEA adjuvant (comprising squalane and vitamin E-acetate) as disclosed in WO 2018 / 115,435. 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). Adjuvants formulated as oil-in-water (O / W) emulsions can be advantageously used. Typically, vaccine compositions may further comprise other pharmaceutically acceptable constituents, not limited to the examples of such constituents selected from preservatives and fillers. In some embodiments, the combination vaccine of the invention comprises an adjuvant which comprises a continuous phase that is aqueous. In general, phases of a matter are known to be continuous if the matter occupies a continually connected region of space (as opposed to disperse if the phase occupies disconnected regions of space). As such, the combination vaccine is aqueous, e.g., an aqueous solution. In an embodiment the aqueous phase comprises water of pharmaceutically acceptable quality. In preferred embodiments the antigens are formulated in an adjuvant having a continuous phase that is aqueous. “Formulation” refers to the combining of substances, herein e.g., antigens, adjuvants, to create a final product, herein a combination vaccine. Preferably, the antigens inactivated Cryptosporidium parvum gp40, inactivated bovine rotavirus, inactivated bovine coronavirus and inactivated E. co / / fimbrial adhesins F5 and F41 as provided herein are comprised, e.g., admixed, in an aqueous solution. In some preferred embodiments said aqueous solution is the adjuvant.

[0056] In other preferred embodiments forms the continuous phase of the adjuvant, for example when the adjuvant is an O / W emulsion.

[0057] An “emulsion” is a mixture of at least two immiscible liquids, whereby one is dispersed in another. Fundamentals of emulsion formation and stability is, 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 an oil and an aqueous phase. 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 some embodiments, the emulsion 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 water is the continuous phase. In emulsions, there commonly is a boundary between the phases, separating these phases, commonly referred to as the "interface". 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 comprises an outer aqueous phase and a dispersed internal oil phase.

[0058] Hence, in some embodiments, the adjuvant of the combination vaccine 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 is distributed throughout the continuous phase that is aqueous. In preferred embodiments, when the adjuvant comprises an O / W emulsion, all antigens are comprised in the water phase.

[0059] In an embodiment the vaccine of the present invention is prepared / produced by admixing such that the antigens Cryptosporidium parvum gp40, inactivated bovine rotavirus, inactivated bovine coronavirus and inactivated E. co / / fimbrial adhesins F5 and F41 , respectively the aqueous solution comprising the antigens, and the adjuvant, are formed in an O / W emulsion comprising the antigens, wherein said antigens are comprised in the aqueous phase, that is the continuous phase.

[0060] 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 combination vaccine 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 combination vaccine 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, the two of the antigens, e.g., inactivated Cryptosporidium parvum gp40 and inactivated bovine rotavirus may be comprised in the internal water phase, and the other antigens, e.g., inactivated bovine coronavirus and inactivated E. coli fimbrial adhesins F5 and F41 may be comprised in the outer water phase. In preferred 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, all antigens are comprised in the water phase.

[0061] In some embodiments, emulsions can be formed and stably maintained by selecting an appropriate kind and concentration of emulsifier(s). An emulsifier 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 preferred emulsifier for the adjuvant of the invention is polysorbate 80, also known as polyoxyethylene sorbitan monooleate, and commercially available as Tween® 80.

[0062] In some embodiments, when the combination vaccine comprises an adjuvant having a continuous phase that is aqueous (e.g., water) and a discontinuous phase that is an oil, said adjuvant is selected from:

[0063] - an emulsion of a mineral oil and a tocopherol in water;

[0064] - an emulsion of a non-mineral oil and a tocopherol in water.

[0065] The inventors found desirable antibody responses (as exemplified in, but not limited to, Example 2 wherein as shown by determining average antibody titer levels in bovines subsequent to a first and / or second (booster) vaccination), when administering to bovines combination vaccines 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 comprises the mineral oil or non-mineral oil and the 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.

[0066] 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).

[0067] 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. Preferably, the non- mineral 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. 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 combination vaccine of the invention may be:

[0068] • 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);

[0069] • An O / W emulsion comprising light liquid paraffin oil and Vitamin E-acetate;

[0070] • An O / W emulsion comprising squalane and Vitamin E-acetate.

[0071] In some other embodiments the aforementioned aqueous solution comprising the antigens inactivated Cryptosporidium parvum gp40, inactivated bovine rotavirus, inactivated bovine coronavirus and inactivated E. coli fimbrial adhesins F5 and F41 may further comprise matrix-like particles. Such matrix-like particles are comprised in the continuous aqueous phase. Preferably, the matrix-like particles comprise saponins. 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 combination vaccine 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, the inventors found that by using a dispersion of saponin matrix particles in water as adjuvant for a combination vaccine 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 combination vaccine comprising the antigens of C. parvum gp40 antigen and / or bovine coronavirus. Further, the combination vaccine 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 combination vaccine comprising: a) Cryptosporidium parvum gp40; b) inactivated bovine rotavirus; c) inactivated bovine coronavirus; and d) E. co / / fimbrial adhesins F5 and F41 , and comprising an adjuvant, wherein the adjuvant is a dispersion of saponin matrix particles in water. In some embodiments, the adjuvant comprises the commercially available Matrix-M™. In some embodiments, the combination vaccine 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, about 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 combination vaccine. In one illustrative and non-limiting example, the combination vaccine 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 combination vaccine 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 combination vaccine comprises per dose an amount of:

[0072] - between 0.25 - 25 AU of Cryptosporidium parvum gp40;

[0073] - between 500 - 10.000 AU of inactivated bovine rotavirus;

[0074] - between 25 - 500 CVU of inactivated bovine coronavirus; and / or

[0075] - between 500 - 10.000 AU of E. coli fimbrial adhesins F5 and F41 .

[0076] Thus, a combination vaccine having a volume of about 2 mL may comprise, for example, 2000 AU / 2 mL of the antigen inactivated bovine rotavirus, 110 CVU / 2 mL of inactivated bovine coronavirus, the E. coli F5 / F41 , 2200 AU / 2 mL of E. co / / fimbrial adhesins F5 and F41 , and about 0,75 AU / 2 mL or about 5 AU / 2 mL of C. parvum GP40. Preferably the combination vaccine comprises effective amounts of the antigens Cryptosporidium parvum gp40, inactivated bovine rotavirus, inactivated bovine coronavirus and / or E. coli fimbrial adhesins F5 and F41. In some embodiments, the effective amounts may vary depending upon the strain or strains of E. coli, bovine rotavirus, bovine coronavirus and / or 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.

[0077] As is known to a skilled person in the art, “AU” refers to arbitrary units and “CVU“ refers to collective viral units, which are both commonly used to indicate the amount of viral, parasitic and / or bacterial antigens present in a composition such as an immunological composition, e.g., a vaccine.

[0078] The amounts of the antigens comprised in the combination vaccine of the invention may be determined by using methods known in the art, such as a BRV potency ELISA, BCV potency ELISA, E. coli F5 (K99) and F41 potency ELISA and / or Cryptosporidium parvum GP40 potency ELISA.

[0079] Further provided herein are methods for the preparation of the combination vaccine according to the current invention. The method may comprise the steps of i) providing the antigens, ii) admixing the antigens with a pharmaceutically acceptable carrier to obtain the combination vaccine.

[0080] It may be that the method for preparing the combination vaccine comprises the inactivation of any one or more of the antigens Cryptosporidium parvum gp40, bovine rotavirus, bovine coronavirus and E. coli fimbrial adhesins F5 and F41 by using methods known in the art for inactivating any one of these antigens, e.g., heatbased treatment, solvent / detergent inactivation etc.

[0081] In some preferred embodiments, the method for the preparation of the combination vaccine comprises the step of admixing the antigens and an adjuvant. 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 emulsion of a mineral oil and a tocopherol in water, an 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 combination vaccine provided herein preferably is “ ready-to-use" . Therefore, it is contemplated that the methods for the preparation of the combination vaccine 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 combination vaccine for use in the immunization comprising the composition of a combination vaccine as provided herein 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.

[0082] In some embodiments, the immunization comprises administering to the bovine said combination vaccine according to the invention. The administering may comprise the parental e.g., intramuscular, subcutaneous, intraperitoneal, or intradermal, preferably subcutaneous, administering of the combination vaccine to a bovine. Preferably, the combination vaccine 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.

[0083] In some illustrative examples provided herein, to which the invention is not limited, after administration of the combination vaccine to bovine serum-antibody titers were achieved that are illustrative for immune protection by the combination vaccine against at least one, two, three, four or all the enteric pathogens for which antigens are comprised in the combination vaccine, in the vaccinated cow.

[0084] In some illustrative examples, serum-antibody titers were obtained for at least one, preferably all, of the antigens comprised in the combination vaccine 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 combination vaccine provided herein prior to parturition. In some illustrative examples, to which the invention is not limited, the booster (secondary) vaccination with the combination vaccine improved antibody titers, against at least one, two, three, four or all the enteric pathogens for which antigens are comprised in the combination vaccine, to a level illustrative for improved immune protection by the combination vaccine in the vaccinated cow. In one further illustrative example, non-limiting for the invention, it was shown that the combination vaccine according to the invention had a desirable effect on the average parasite load (C. parvum) of serum and colostrum. In other words, in said non-limiting examples, the vaccination reduced parasite load compared to a control.

[0085] In some other embodiments, the immunization comprises administering to the bovine, preferably to a calf, colostrum comprising maternally derived antigens (MDA) comprising antigens to an enteric pathogen, preferably comprising at least one, two, three four, more preferably all, of the antigens of the combination vaccine according to the invention, wherein the colostrum is derived from a bovine to which the combination vaccine according to the invention has been administered. Preferably, the combination vaccine and / or colostrum is administered in effective amounts. Preferably, the colostrum is administered in effective amounts. Thus, a variant of a combination vaccine according to the invention is the colostrum, comprising Cryptosporidium parvum gp40, inactivated bovine rotavirus, inactivated bovine coronavirus and inactivated E. coli fimbrial adhesins F5 and F41 , that is generated by a pregnant heifer or pregnant cow that was immunized with the combination vaccine according to the invention. The colostrum can be fed to or drunk by the calf. Alternatively, antibodies derived from such colostrum can be administered.

[0086] 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.

[0087] Thus, methods provided by the present invention also can be used to provide passive immunity against enteric disease, preferably neonatal calf diarrhea, in calves.

[0088] In related embodiments, a pregnant cow or pregnant heifer may be administered a primary and a booster (secondary) vaccine of the combination vaccine provided herein prior to parturition.

[0089] 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 the combination vaccine 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, bovine coronavirus and / or Cryptosporidium infection (and optionally C. perfringens infection) in a bovine that comprises administering parentally, preferably subcutaneously, to a bovine a combination vaccine 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 a colostrum of a vaccinated bovine, wherein said bovine has been vaccinated with the combination vaccine 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, bovine coronavirus and / or Cryptosporidium infection (and optionally C. perfringens infection) in a bovine calf that comprises administering to a heifer and / or cow pregnant with the calf a combination vaccine of the present invention.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] EXAMPLES

[0094] - Example 1 is a dose-response study of a vaccine with the antigens provided herein.

[0095] - Example 2 is a safety & efficacy study of several combination vaccines comprising the antigens provided herein.

[0096] - Example 3 is a serology study in serum and colostrum of ISCOM-adjuvanted vaccines with the antigens provided herein.

[0097] Example 1 : Dose-response study of a vaccine of combination vaccines containing bovine rotavirus, bovine coronavirus, Escherichia Coli and Cryptosporidium parvum antigens

[0098] Study Design

[0099] In this experiment thirty-two calves (Friesian-Holstein or cross breed) at 3 - 8 months of age were divided in four groups of eight animals each. All animals will be vaccinated once subcutaneous (SC) in the right side of the neck with the RCC antigens formulated in oil (ISA 70) + aluminum (Alhydrogel) at 20%, 50%, 100% and 200% of the doses used in the commercial vaccines Bovilis Rotavec corona and Bovilis Cryptium (see, Table 1 ).

[0100] Table 1 : Study design

[0101] Treatment

[0102] Animals received a 2m L SC injection of the described vaccine preparations according to Table 2, at approximately 3 - 8 month of age in the right side of the neck. Vaccines were adjuvanted with Alu-oil (oil (ISA 70) + aluminum (Alhydrogel)). Table 2: Vaccine preparations

[0103] Procedures

[0104] Daily observations

[0105] Animals were inspected on a daily basis for general wellbeing and signs of disease or abortions.

[0106] Blood sampling

[0107] Plain blood samples for serum preparation were taken according to standard procedures. Samples were drawn prior to vaccination (DO) and 2, 4 and 6 weeks thereafter.

[0108] Local reactions

[0109] On D37 all the animals were checked for local reactions by visual inspection and palpation to determine the sizes L x B x H [cm] (estimate), boundary definition and consistency.

[0110] Morbidity, veterinary treatment

[0111] No animals required treatment by the Responsible Veterinarian for any clinical sign attributable to vaccination.

[0112] Processing of plain blood samples

[0113] Blood was stored in the lab for clotting at 2-8°C overnight or at ambient temperature for 1 -8 hours. The samples were centrifuged for 10-20 minutes at 3,000 x g, serum was collected. The serum samples were heat-inactivated for 30 minutes at 56°C and stored at <-15°C until use.

[0114] Testing of sera for Rotavirus, coronavirus and E. coli K99 antibody ELISA

[0115] Serum samples were analysed with commercial tests from Bio-X Diagnostics for rotavirus (BIO K126, Monoscreen Abel ISA Bovine rotavirus I competition), coronavirus (BIO K 392, Monoscreen AbELISA Bovine coronavirus I competition) and E. coli F5 (K99) (BIO K 295, Monoscreen AbELISA E. coli F5 (K99) / blocking) antibodies according to the manufacturer’s instructions.

[0116] Testing of sera for C. parvum specific antibodies

[0117] Briefly, the wells of a polystyrene microtiter plate were coated with a fixed amount of Gp40. Then serial dilutions of the serum samples were added and the goat-anti- bovine lgG(H+L) peroxidase labeled conjugate was used to detect the presence of antibodies against Gp40. The amount of peroxidase conjugate bound correlated with the amount of bovine IgG captured to Gp40 and was determined by the enzymatic conversion of a colorless substrate into a blue reaction product. The reaction was stopped by adding H2SO4 and the color changed from blue to yellow. The plates were read at an absorbance of 450 nm by using an ELISA reader and the antibody titer was calculated with CBA (Calculations of Biological Assays) according to the Abendvertical v1.22 (Inside Interpolation) method.

[0118] Results

[0119] Overall, all doses resulted in acceptable serology results. The highest serology results were found at one SC administration of the combination vaccine with the antigens at 50% dose of the commercial vaccines Bovilis Rotavec and Bovilis Cryptium.

[0120] Bovine rotavirus antibody levels

[0121] At the start of the study the average rotavirus antibody levels were comparable for all four groups and were respectively 23.9, 23.9, 18.9 and 18.3% inhibition. Fourteen days (D14) post vaccination the average antibody responses in all groups increased to 41.4 till 64.7% inhibition. On D28 / D42 the average antibody levels in all groups decreased till respectively titers of 35.6, 50.3, 50.8 and 55.8% inhibition at day 42 (D42). The highest average response was found in group 2 (50% vaccine dose) at D14.

[0122] Table 3: Average bovine rotavirus antibody levels (% inhibition)

[0123] Bovine coronavirus antibody levels

[0124] At the start of the study the average coronavirus antibody levels were respectively 29.8, 57.0, 39.4 and 43.3% inhibition for the four groups. After vaccination, the average antibody levels of all groups increased to maximal 36.4 (D14), 93.2 (D28), 83.6 (D28) and 68.4% (D28) inhibition for respectively group 1 , 2, 3 and 4. For all groups a decrease in average antibody level was observed at D42. The highest average response was found in group 2 (50% vaccine dose) throughout the study and highest at D28.

[0125] Table 4: Average bovine coronavirus antibody levels (% inhibition)

[0126] E. coli F5 antibody levels

[0127] At the start of the study the average E. coli F5 (K99) antibody levels in groups 1 , 3 and 4 were comparable and were respectively 19.1 , 19.3 and 18.1 % inhibition, whilst in group 2 a lower average inhibition level (0.6%) was found. During the following timepoints the average E. coli F5 (K99) antibody levels increased to maximum responses of 77.7, 91.4, 85.4 and 80.3% inhibition for respectively group 1 , 2, 3 and 4 at D42. The highest average response was found in group 2 (50% vaccine dose).

[0128] Table 5: Average E. coli F5 (K99) antibody levels (% inhibition)

[0129] C. parvum Gp40 antibody levels

[0130] At the start of the study the average IgG antibody levels against Gp40 were comparable for all four groups: approximately 10.5 Iog2. Fourteen days post vaccination the antibody responses in all groups increased to 12.3 till 16.2 Iog2. 28 / 42 post vaccination the average antibody levels in all groups decreased roughly 1 Iog2. The highest response was found in group 4 (200% vaccine dose) at D14.

[0131] Table 6: Average C. parvum Gp40 antibody levels (in Iog2)

[0132] Safety & adverse reactions

[0133] Daily observations

[0134] No signs of ill health were observed during this experiment. Local reactions were found in five, four, six and seven animals per group for respectively group 1 till group 4. On average, the surface size and the height of local reactions were largest in group 4 followed by group 3, group 2, and group 1 . The local reaction did not affect the general wellbeing and behaviour of the animals. Overall, the safety of the tested vaccines was found to be acceptable. Conclusion

[0135] Overall, all doses resulted in acceptable serology results. There appeared to be a positive correlation between the seventy of the local reaction and the vaccine dose. SC administration of the combination vaccine at 50% dose resulted, for BRV, BCV and E. coli F5 / F41 in the highest and thus most desirable serology results.

[0136] Example 2: Serology study of combination vaccines containing bovine rotavirus, bovine coronavirus, Escherichia Coli and Cryptosporidium parvum antigens

[0137] Study Design

[0138] 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 7) in such way that each group consisted of six calves and two cows.

[0139] Adjuvants tested herein are:

[0140] - GNE: a non-bacterial oil / water adjuvant;

[0141] - Alhydrogel: an aluminum-based wet gel suspension;

[0142] - SVEA-E: an emulsion of a non-mineral oil and a tocopherol in water;

[0143] - X-Solve: an emulsion of a mineral oil and a tocopherol in water;

[0144] - Emunade: emulsion of a mineral oil in water, and containing alhydrogel;

[0145] - ISCOM: a dispersion of saponin matrix particles in water;

[0146] - Alu-oil: oil (ISA 70) + aluminum (Alhydrogel).

[0147] Table 7: Study design

[0148] Treatment

[0149] All animals received a 2 mL SC injection of the described vaccine preparations according to Table 8, 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 the animals of group 3, 5 and 7, respectively 14 and 17 weeks after the first administration.

[0150] Table 8: Vaccine preparation

[0151] Procedures

[0152] Daily observations

[0153] Animals were inspected on a daily basis for general wellbeing and signs of disease. Local reactions

[0154] 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.

[0155] Serum samples

[0156] 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.

[0157] Blood sampling

[0158] 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.

[0159] 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.

[0160] Morbidity, veterinary treatment

[0161] No animals required treatment by the Responsible Veterinarian for any clinical sign attributable to vaccination.

[0162] Neutralization assay

[0163] 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

[0164] 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.

[0165] Results

[0166] Overall, the serological responses upon subcutaneous administrations of vaccines adjuvanted with SVEA-E (Group 3), X-Solve (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.

[0167] Bovine rotavirus antibody levels

[0168] At the start of the study the average BRV antibody level was 58.3% inhibition. After the first vaccination (see, Table 9), the average antibody levels of all groups increased slowly to maximal 55.4 (4w post V1 ), 65.5 (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 ).

[0169] Table 9: Average inactivated bovine rotavirus antibody levels (% inhibition)

[0170] Two weeks after the booster vaccination (V2) (see, Table 10) of the animals in group 3 (SVEA-E), 5 (X-Solve) 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.

[0171] Table 10: Average inactivated bovine rotavirus antibody levels (% inhibition) after after V2

[0172] Bovine coronavirus antibody levels

[0173] 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.

[0174] Table 11 : Average inactivated bovine coronavirus antibody levels (% inhibition)

[0175] Table 12: Average inactivated bovine coronavirus antibody levels (% inhibition) after

[0176] V2 E. coli F5 antibody levels

[0177] 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) 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.

[0178] Table 13: Average E. coli F5 (K99) antibody levels (% inhibition)

[0179] Table 14: Average E. coli F5 (K99) antibody levels (% inhibition) after booster

[0180] 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) 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 15: Average C. parvum gp40 antibody antibody levels (Iog2)

[0181] Table 16: Average C. parvum gp40 antibody antibody levels (Iog2) after booster

[0182] 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) the level remains around 8 Iog2 during the study, but 2 weeks after the second vaccination it increases to 9.3 Iog2.

[0183] Table 17: Average BRV neutralizing antibodies (Iog2)

[0184] BCV neutralization assay

[0185] 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) 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.

[0186] Table 18: Average BCV neutralizing antibodies (Iog2)

[0187] C. parvum I VI I

[0188] The average parasite load was tested for the groups 3 (SVEA-E), 5 (X-Solve), 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 IVII 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 IVII positive control (consisting of polyclonal serum pool): 922, 1149 and 1600 for respectively group 3, 5 and 7.

[0189] Table 19: Average of C. parvum IVII results (SQ)

[0190] Lymphocyte activation assay

[0191] 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. Thus, 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).

[0192] Safety & Adverse reactions

[0193] Mild or no clinical signs were observed after vaccination. All tested vaccines caused raised rectal temperatures for 1-2 days after each administration.

[0194] After the first vaccination, local reactions (see, Table 20). were found in eight, seven, three, five, three, seven and eight 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 .

[0195] Table 20: Average local reaction sizes in cm3 (LxWxH) after first vaccination

[0196] After the second vaccination (V2) (see, Table 21 ), 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.

[0197] Table 21 : Average local reaction sizes in cm3 (LxWxH) after booster

[0198] 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, except for Groups 1 and 2, the adjuvanted vaccines were found to be acceptable in terms of safety.

[0199] Conclusion The serological responses upon subcutaneous administrations of combination vaccines according to the invention resulted in desirable antibody responses. Most adjuvanted vaccines were found to be acceptable in terms of safety. Vaccines adjuvanted with SVEA-E (Group 3), X-Solve (Group 5) and ISCOM (Group 7) after primary vaccination, and after an additional booster vaccination, resulted in desirable safety and efficacy results.

[0200] Example 3: Serology study in serum and colostrum upon subcutaneous (SC) administration of ISCOM-adjuvanted combination vaccines

[0201] 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 22 and 23). 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).

[0202] Table 22: Study design

[0203] 20

[0204] Table 23: Vaccine preparation Treatment

[0205] All animals received a 2 mL SC injection of the described vaccine preparations according to Table 23, 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.

[0206] Procedures

[0207] Daily observations

[0208] Animals were inspected on a daily basis for general wellbeing and signs of disease.

[0209] Blood- and serum sampling

[0210] 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.

[0211] Colostrum sampling

[0212] 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.

[0213] Morbidity, veterinary treatment

[0214] No animals required treatment by the Responsible Veterinarian for any clinical sign attributable to vaccination.

[0215] ELISA

[0216] 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).

[0217] C. parvum in-vitro invasion inhibition (I VI I) test

[0218] Pools of serum samples and pools of colostrum samples were tested for neutralizing antibodies against C. parvum in an IVI I test.

[0219] Results

[0220] 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.

[0221] Bovine rotavirus antibody levels

[0222] 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.

[0223] 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.

[0224] Table 24: Summary of BRV antibody ELISA (% inhibition)

[0225] Bovine coronavirus antibody levels

[0226] 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 and were comparable for all three groups.

[0227] 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).

[0228] Table 25: Summary of BCV antibody ELISA (% inhibition)

[0229] E. coli F5 antibody levels

[0230] 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.

[0231] 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.

[0232] Table 26: Summary of E. coli F5 antibody ELISA (% inhibition)

[0233] C. parvum Gp40 antibody levels

[0234] 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 .

[0235] 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.

[0236] Table 27: Summary of C. parvum gp40 antibody ELISA (Iog2)

[0237] C. parvum I VI I

[0238] 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.

[0239] Table 28: 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

[0240] 878.9 SQ.

[0241] Table 29: Summary of C. parvum IVI I results of colostrum pools (SQ)

[0242] Conclusion

[0243] The serological responses in serum and colostrum of two ISCOM adjuvanted vaccines, containing C. parvum gp40, and containing regular and high 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 after two milkings for all antigens. For C. parvum there was a clear effect of average parasite load in the serum and colostrum after vaccination. The combination vaccines according to the invention (vaccines comprising antigens of C. parvum gp40, BRV, BCV and E. coli F5 / F41 ) and tested were found to result in a desirable antibody response and were found to be acceptable in terms of safety.

Claims

CLAIMS1. A combination vaccine comprising: a) Cryptosporidium parvum gp40; b) inactivated bovine rotavirus; c) inactivated bovine coronavirus; and d) E. co / / fimbrial adhesins F5 and F41.

2. The combination vaccine according to Claim 1 , further comprising one or more further antigens selected from an enteric pathogen of a bovine species.

3. The combination vaccine of any one of the previous claims, further comprising a further antigen from Clostridium spp.

4. The combination vaccine of any one of the previous claims, wherein the vaccine is in the form of a ready-to-use liquid composition.

5. The combination vaccine of any one of the previous claims, wherein the antigens are formulated in an adjuvant having a continuous phase that is aqueous.

6. The combination vaccine according to Claim 5, wherein the adjuvant comprises an oil as a discontinuous phase.

7. The combination vaccine according to Claim 5 or 6, wherein the adjuvant is formulated as an oil in water (O / W) emulsion.

8. The combination vaccine of any one of Claims 6 - 7, wherein the vaccine comprises an adjuvant selected from:- an emulsion of a mineral oil and a tocopherol in water;- an emulsion of a non-mineral oil and a tocopherol in water.

9. The combination vaccine of any one of Claim 8, wherein the tocopherol is Vitamin-E-acetate.

10. The combination vaccine according to Claim 5, wherein the adjuvant is a dispersion of saponin matrix particles in water.11 . A combination vaccine comprising: a) Cryptosporidium parvum gp40; b) inactivated bovine rotavirus; c) inactivated bovine coronavirus; and d) E. coli fimbrial adhesins F5 and F41 for use in protecting a bovine against an infection with Cryptosporidium parvum, bovine rotavirus, bovine coronavirus and E. coli.

12. A method for protecting a bovine against an infection with Cryptosporidium parvum, bovine rotavirus, bovine coronavirus and E. coli, the method comprising administering to the bovine a vaccine comprising: a) Cryptosporidium parvum gp40; b) inactivated bovine rotavirus; c) inactivated bovine coronavirus; and d) E. coli fimbrial adhesins F5 and F41.

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

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