WO2025157994A1

WO2025157994A1 - Vaccine against ruminant respiratory disease

Info

Publication number: WO2025157994A1
Application number: PCT/EP2025/051800
Authority: WO
Inventors: Subramaniam VAIDYANATHAN
Original assignee: Intervet International BV; Intervet Inc
Current assignee: Intervet International BV; Intervet Inc
Priority date: 2024-01-25
Filing date: 2025-01-24
Publication date: 2025-07-31
Anticipated expiration: 2026-07-25

Abstract

The present invention relates to a vaccine comprising live attenuated P. multocida bacteria and a pharmaceutically acceptable carrier, for use in protecting a ruminant against an infection with P. multocida, wherein the vaccine is deposited in the oral cavity of the ruminant. In addition, the invention relates to a single-dose vaccine, to methods for the preparation of the vaccine, methods for vaccinating ruminants against infection with P. multocida, methods of improving the respiratory function of a ruminant, methods of treating, ameliorating and/or preventing a diseased state in a ruminant and to a kit-of-parts at least comprising a container comprising the vaccine of the invention.

Description

VACCINE AGAINST RUMINANT RESPIRATORY DISEASE

TECHNICAL FIELD

The present invention relates to the field of veterinary vaccinology; specifically, the invention relates to a vaccine against an infection with Pasteurella multocida. Additionally, the invention relates to methods for the preparation of such a vaccine, to methods for the vaccination of ruminants employing such a vaccine, and to medical uses of such a vaccine.

BACKGROUND

In animal husbandry of ruminants, an important veterinary problem is ruminant respiratory disease (RRD). The disease is a complex disease having a multifactorial etiology and develops as a result of complex interactions between environmental factors, host factors, and pathogens. Environmental factors (e.g., weaning, transport, commingling, crowding, inclement weather, dust, and inadequate ventilation) serve as stressors that adversely affect the immune and nonimmune defense mechanisms of the host animal. The disease is most commonly associated with the transport and assembly of large groups of recently weaned calves into feedlots. Morbidity in these types of feeder calves often peaks within the first 7 - 10 days after feedlot arrival. Morbidity can approach 35%-50%, and case fatality is 5%- 10%. Pathogens causative for RRD include one or more bacteria and/or viruses. Infected animals, especially young animals, may develop RRD. An important pathogen, among others, is: Pasteurella multocida (P. multocida), which are aerobic, non-motile, non-spore- forming, bipolar, gram-negative rods, associated with pneumonia and septicemia in all ruminants. Other bacteria causing a similar disease may be Mycoplasma bovis, Bibersteinia trehalose, Histophilus somni or Mannheimia haemolytica. Viral pathogens may also be involved, such as bovine herpesvirus 1 , bovine respiratory syncytial virus, parainfluenza-3 virus, bovine viral diarrhea virus, bovine adenovirus and bovine coronavirus.

Several veterinary treatments are available to prevent or mitigate RRD. Generally, the primary treatment is the use of broad-spectrum antimicrobials labeled for bovine respiratory disease. Macrolides and phenicols most commonly used as first-line treatment. Combinations may be used of antibiotics and of vaccines for the viral- and bacterial pathogens. Of the commercial vaccines for some bacterial pathogens involved in RRD, most are inactivated, adjuvated vaccines with antigens of M. haemolytica and/or P. multocida, in the form of bacterins (inactivated bacteria) or toxoids (de-toxified toxins). Generally, such vaccines are intended for parenteral injection by intramuscular or subcutaneous route. For example: Bovipast™ and Ovipast™ (Merck Animal Health), Rispoval™ (Zoetis), Presponse™ (Boehringer Ingelheim), and Respishield™ (Merial). Only few vaccines against bacterial RRD comprise live attenuated bacteria. Reason is that such live attenuated bacterial vaccines cannot easily be combined with the preventive use of antibiotics. Examples are: Onset™, ONCE™, Vista™, and Respavir™ (all from Merck Animal Health). These live attenuated vaccines are administered either by subcutaneous- or intranasal route. Both these routes may be quite stressful to the animal because the intranasal route typically requires the physical restraint of the animal, and especially its head, either by a human or by mechanical means.

The bacterium P. multocida is an important pathogen in cattle. Infected animals may develop RRD. Especially young ruminants, e.g., calves, are susceptible to infection by P. multocida. The bacterium P. multocida is, in addition to RRD, also associated with a variety of other diseases, including calf and yearling meningoencephalitis, lamb lymphadenitis, horse and donkey septicemia, swine pasteurellosis, porcine septicemic pasteurellosis, pneumonia, and fowl cholera. Infection by P. multocida leads to a high mortality rate amongst cattle causing serious economic losses. Therefore, improved products to treat and/or prevent infection are desirable. The serotypes A:3, A:2, A:6 are considered the serotypes most commonly associated with the development of RRD.

US 3855408 describes a vaccine for immunizing poultry against fowl Cholera comprising a small but effective amount of live stable avirulent antibacterial encapsulated P. multocida mutant strain M-2283. However, no vaccine against RRD is described or suggested.

WO 1989/009617 describes a vaccine for immunizing humans or animals against diseases caused by a toxin produced by P. multocida, specifically porcine atrophic rhinitis, wherein the vaccine comprises a recombinant, detoxified P. multocida toxin, and further comprising an immunologically acceptable carrier or vehicle. However, no vaccine against RRD is described or suggested.

Coates et al. Poult Sci. 1977 Jan;56(1 ):273-6 describes vaccinating turkeys with a live P. multocida strain by adding three doses, i.e., high, medium and low doses containing 1.2 x 10⁷, 1.5 x 10⁵ and 1.0 x 10³ bacteria respectively, per mL of drinking water were administered. The vaccine was administered by allowing the 30 turkeys to drink from 12 L of water containing the vaccine for a period of five hours. No vaccine against RRD is described or suggested.

Consequently, there remains a need in the art for effective vaccines that can be used to provide protective immunity against diseases, such as RRD, caused by P. multocida. It is an object of the present invention to at least meet the above-mentioned needs.

SUMMARY OF INVENTION

To meet the object of the invention there is provided, in a first aspect, a vaccine comprising live attenuated P. multocida bacteria and a pharmaceutically acceptable carrier, for use in protecting a ruminant against an infection with P. multocida, wherein the vaccine is deposited in the oral cavity of the ruminant.

In a further aspect there is provided for a single-dose vaccine comprising a total volume of between 2 mL - 10 mL and comprising between about 1x10⁹ - about 1x10¹¹ CFU live attenuated P. multocida, preferably P. multocida AhyaE, and a pharmaceutically acceptable carrier.

In a further aspect there is provided for a method for the preparation of the vaccine for use, or a single-dose vaccine, in accordance with the current invention wherein the method comprises the step of admixing live attenuated P. multocida bacteria and a pharmaceutically acceptable carrier.

In a further aspect there is provided for a method for the vaccination of ruminants against infection with P. multocida, comprising the step of administering the vaccine for use, or a single-dose vaccine, in accordance with the current invention to said ruminants by depositing the vaccine in the oral cavity.

In a further aspect there is provided for a method of improving the respiratory function of a ruminant comprising depositing the vaccine for use, or a single-dose vaccine in accordance with the current invention in the oral cavity of the ruminant.

In a further aspect there is provided for a method of treating, ameliorating and/or preventing a diseased state in a ruminant comprising depositing the vaccine for use, or a single-dose vaccine in accordance with the current invention in the oral cavity of the ruminant, wherein the diseased state comprises RRD.

In a final aspect there is provided for a kit of parts comprising a container comprising the vaccine for use, or the single-dose vaccine, in accordance with the current invention.

DEFINITIONS

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

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.

As used herein “deposition" (as well as variations such as "depositing", "deposited", and the like) in the context of the current invention refers to the act of actively placing of an object (herein: a vaccine comprising live attenuated P. multocida), optionally by using a suitable applicator, at a predefined site (herein: the oral cavity of a ruminant, preferably the back of the throat).

As used herein, the term "live attenuated' in the context of live attenuated bacteria, such as live attenuated P. multocida, refers to bacteria that are alive, meaning replicative, and that have a reduced capacity to induce infection or disease in a particular host, as compared to un-attenuated, pathogenic strains of the bacterium. A synonym is: modified-live. The effect of the attenuation is that the bacterium can still replicate in a target animal and so display relevant antigens to the targets' immune system, but without itself causing (serious) disease to the target. This effectively protects the target against (the consequences of) an infection with an un-attenuated version of the bacterium.

As meant herein, the term “mucoadhesive adjuvant” is a hydrophilic polymer (e.g., comprising hydroxyl, carboxyl, amine and/or sulphate groups), preferably having a molecular weight of at least 2,000 (two thousand) Da, preferably at least 5,000 (five thousand) Da, which is to be used as delivery system and carrier of agents such as immunologically active components of a vaccine, to improve the adhesion to mucosal membranes enhancing local mucosal residence time and controlled local release of said one or more agents.

As meant herein, the term “oropharyngeally” in the context of deposition of the vaccine, e.g., “the vaccine according to the present invention is deposited oropharyngeally” , refers to the deposition of the vaccine on and/or around the tissues of the oropharynx.

As meant herein the term “(peri-)tonsillah in the context of deposition of the vaccine, e.g., “the vaccine according to the present invention is deposited (peri-)tonsillah , refers to the deposition of the vaccine on and/or around the tonsil tissue.

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, 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.

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.

As meant herein, the term "respiratory disease" for the invention refers to any disease of a ruminants' respiratory tract. This may a consequence of infection with a pathogenic P. multocida bacteria, commonly in combination with infection by one or more bacteria or viruses or in combination with any other predisposing factors (physical and/or chemical). For a description see veterinary handbooks such as: "The Merck veterinary manual" (10th ed., 2010, CM. Kahn edt., ISBN: 091 191093X). Examples of such a disease are: shipping fever, or pneumonic Pasteurellosis.

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

The term “vaccine" is herein used to refer to a composition suitable for administration to a mammal, comprising one or more 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), herein at least comprising P. multocida bacteria, that is recognized by the immune system of a subject to which the vaccine is administered and that induce a protective immunological response. The response may originate from the subjects' innate- and/or from the acquired immune system and may be of the cellular- and/or of the humoral type.

DESCRIPTION OF EMBODIMENTS

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.

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.

The current invention provides for a vaccine that is safe and efficacious in protecting against infection by P. multocida in ruminants, preferably in young ruminants. The vaccine may prevent or reduce the infection of a pathogenic P. multocida in ruminants and/or one or more (clinical) signs of RRD that are associated with such an infection. It was surprisingly found by the inventors that a vaccine in accordance with the current invention reduces lung pathology after challenge with P. multocida. It was further found that there are various advantages, not limited to improved efficacy and safety, of deposition of the vaccine in the oral cavity of the ruminant compared to subcutaneous and/or (intra)nasal administration of the vaccine. For example, the deposition of the vaccine in the oral cavity of the ruminant no longer requires significant handling of the animals, nor does it result in unwanted side effects, e.g., abscesses on the skin as a result of subcutaneous injection and/or reduces unnecessary stress to the animal and in addition saves considerably on expenses for labor and veterinary services. Since stress is such an important factor in RRD the reduction of stress to the animal by vaccination using the vaccine according to the invention (further) reduces onset of RRD. Moreover, the current invention allows for the vaccine to be administered in relatively small volumes to ruminants. Further, it was found that the vaccine is efficacious in P. multocida MDA (maternally derived antibody) positive and in P. multocida MDA negative young ruminants.

In a first aspect, the current invention provides for a vaccine comprising live attenuated P. multocida bacteria and a pharmaceutically acceptable carrier, for use in protecting a ruminant against an infection with P. multocida, wherein the vaccine is deposited in the oral cavity of the ruminant.

The vaccine according to the current invention is effective and safe in protecting a ruminant, in which the vaccine is deposited in the oral cavity, against infection by P. multocida. P. multocida are bacteria from the Pasteurellaceae family and display the characterising features of their taxonomic group-members such as the morphologic, genomic, and biochemical characteristics, as well as the biological characteristics such as physiologic, immunologic, or pathologic behaviour. As is known in the field, the classification of microorganisms is based on a combination of such characterising features. The scope of the invention therefore also includes P. multocida bacteria that are sub-classified therefrom in any way, for instance as a subspecies, strain, isolate, genotype, variant, subtype, serotype, or subgroup and the like. It will be apparent to a skilled person that while a particular P. multocida bacterium for the present invention may currently be classified in a specific species and genus, such a taxonomic classification can change in time as new insights may lead to reclassification into a new or different taxonomic group. The vaccine according to the invention is capable of reducing one or more diseased states of a ruminant resulting from an infection with a pathogenic P. multocida bacterium and/or is an aid in the reduction of RRD caused by P. multocida. It is understood that a skilled person is able to determine the effectiveness of the vaccine in accordance with the invention e.g., by monitoring the immunological response following vaccination and/or after infection, e.g., by monitoring (clinical) signs of the diseased state, e.g., of RRD, or by other ways of monitoring diseased states and/or determining bacterial infection in ruminants known in the art.

The vaccine as provided by the current invention comprises live attenuated P. multocida bacteria. An attenuated bacterium can be generated in vitro in a wide variety of ways, such as through a method of induced mutation, either directed or non-specific. Any attenuated P. multocida bacterium is encompassed herein, for example, a P. multocida bacterium may be attenuated by methods such as subsequent passaging (in vivo or in vitro), use of mutagens such as chemicals or ionising radiation, recombinant DNA technology (e.g., CRISPR-CAS9) or combinations thereof. A mutant bacterium is considered attenuated if, after exposure to the mutant bacterium, an increase in the dose of pathogenic P. multocida is required to kill half the susceptible target species (i.e., the LDso increases) and/or there is a reduction in pathologic lesions after exposure of the target species to the mutant bacterium compared with exposure to a wild-type bacterium, and/or there is a reduction in commensal colonization of mucosal surfaces where P. multocida reside after exposure of the target species to the mutant bacterium when compared with exposure to a wild-type bacterium. Attenuation can be assessed by various means as known in the art., e.g., by in vitro models and/or assays.

Without being bound by theory it is contemplated by the inventors that the deposition of the vaccine is in the oral cavity of the ruminant which provides certain advantages over subcutaneous and intranasal administration. For example, it was surprisingly found by the inventors that by using deposition of the vaccine in the oral cavity the dose of the vaccine administered to an animal could be increased compared to, for example, subcutaneous administration without causing unacceptable site reactions and/or side effects. In one descriptive and non-limiting example, animals receiving the vaccine according to the invention in a dose of about 1 x 10⁹ CFU I 6ml, or even receiving 7.6 x 10⁹ CFU 1 6ml, did not exhibit unacceptable site reactions and/or side effects, whereas animals receiving a subcutaneous vaccine in a dose of 5.7x10⁸ CFU / 2 ml resulted in considerable and unacceptable local reactions in said animals receiving subcutaneous vaccine.

Also, it is contemplated that deposition of the vaccine in the oral cavity has certain advantages over traditional oral administration, e.g., administration wherein the vaccine relocates from the oral cavity to the Gl tract of the ruminant e.g., by swallowing of (a composition, e.g., a feed or drink, comprising) the vaccine. In contrast to traditional oral administration, it was found by the inventors that for the vaccine of the invention it is not required that said vaccine is directed at drug absorption/intake in the Gl tract of the ruminant. Instead, it was found that the vaccine is efficacious (and safe) when said vaccine is deposited in the oral cavity, wherein it is not required for the vaccine to be swallowed (and does not relocate from the oral cavity to the Gl tract) by the ruminant. Preferably, the vaccine according to the invention is administered in the back of the throat of a ruminant, e.g., on and/or around the tissues of the oropharynx and/or on and/or around the tonsil tissue, without inducing swallowing the vaccine by said ruminant.

Without being bound by theory it is contemplated that the deposition of the vaccine oropharyngeally and/or (peri-)tonsillar, i.e. , in the back of the oral cavity of the ruminant, allows for the immunologically active component (or immunoactive component) such as the live attenuated Pasteurella multocida bacteria of the vaccine of the current invention to enable a protective effect against an infection with P. multocida in said ruminant. Without being bound by theory, when the vaccine of the current invention and/or the live attenuated P. multocida bacteria comprised in said vaccine contact epithelial surfaces and/or lymphoid organs where antigens can be collected a protective effect may occur in the ruminant against a (future) infection with P. multocida. In other words, it is contemplated that certain lymphoid organs and/or epithelial surfaces at the back of the oral cavity, such as (but not limited to) tonsils, of the ruminant are involved in providing protective immunity for example by inducing an immune response that protects the animal against a pathogenic infection with P. multocida. Oropharyngeal and/or (peri-)tonsillar deposition of the vaccine is understood to comprise the deposition of the vaccine composition in the oral cavity, preferably in the back of the throat or back of the oral cavity.

It is not excluded that when a vaccine according to the invention is deposited in the oral cavity that it at least partially contacts the epithelial cells of the rumen wall of the rumen a young ruminant.

In some embodiments, preferred live attenuated P. multocida bacteria for the invention comprises any mutation (substitution, deletion, insertion) that attenuates the bacteria.

In some embodiments, preferred live attenuated P. multocida bacteria for the invention, are those that are acapsular, meaning that the bacterium cannot express its normal capsule of hyaluronic acid.

The acapsular phenotype may result from any mutation in the P. multocida genomic locus for capsule biosynthesis. For example, in the region encoding one of the transporters of polysaccharide to the surface; in the region encoding one of the hya genes; or in the region encoding proteins involved in phospholipid substitution; see: Chung et al. (1998, FEMS Microbiol. Letters, vol. 166, p. 289-296). Either way, the acapsular P. multocida bacteria remain viable and replicative, and can still stimulate an immune response, but cause significantly less pathology than capsular (e.g., wild type) P. multocida can.

It is preferred that the vaccine for use in accordance with the current invention comprises live attenuated P. multocida bacteria comprising a deletion of all or a part of a hyaE gene. Said deletion attenuates the bacteria. In some embodiments the deletion comprises all, or a part, of a hyaE gene. W02005/003330 describes P. multocida bacteria comprising a mutated hyaE (named: 1062 AhyaE) and is fully incorporated by reference herein. Preferred live attenuated bacteria of P. multocida used in the invention are bacteria as described in WO 2005/003330. Similarly, methods for generating deletion mutants may be as described in WO 2005/003330, but other methods known in the art can be used likewise. Methods of confirming that the method has resulted in a desirable mutation, e.g., resulting in an acapsular phenotype, are well known in the art and are encompassed herein.

In some embodiments, the live attenuated P. multocida bacteria may comprise mutations in the nanP and hyaC gene, such as those described in WO2022/072431 , which is fully incorporated by reference herein. Similarly, methods for generating deletion mutants may be as described WO2022/072431 .

In some embodiments, the the live attenuated P. multocida bacteria may comprise mutations in any one or more, preferably at least two, genes selected from the nanP and hyaC gene and/or hyaE gene.

In some embodiments, the live attenuated P. multocida may comprise other mutations, e.g., substitutions, deletions, insertions. It is preferred that such other mutations attenuate the bacteria. It is preferred that the P. multocida bacteria remain viable and replicative. In certain embodiments, the mutant bacteria do not comprise any antibiotic resistance genes or foreign DNA. Mutations that are commonly known to attenuate P. multocida are also encompassed herein.

In some embodiments, the live attenuated P. multocida comprises a deletion of all or a part of a hyaE gene and comprises another mutation which may or may not attenuate the bacteria.

In preferred embodiments of the invention the vaccine is administered to a ruminant, said vaccine having a dose (in CFU) of at least 1x10⁶ CFU of live attenuated P. multocida. In some preferred embodiments a practical vaccine comprises a dose of about 1x10⁹ CFU. It was found by the inventors that providing young ruminants with a vaccine according to the invention (for example a vaccine comprising P. multocida and having a volume of about 6 mL) having either an average dose of about 1x10⁹ CFU/6 ml (i.e., about 1.67 x 10⁸ CFU/ml), and/or an average dose of 7.6x10⁹ CFU/6 ml (i.e., about 1.26x10⁹ CFU/ml) was safe and efficacious in reducing lung pathology after challenge with a heterologous P. multocida strain. It is contemplated that at least any one dose between those tested doses (i.e., between about 1x10⁹ CFll/6 ml to about 7.6x10⁹ CFll/6 ml) likely, similarly, provides for a protective effect against an infection and/or safety profile. Furthermore, it is contemplated that a practical minimum protective dose likely can be as low as about 1x10⁷ CFU, considering the promising results the inventors found for the vaccine deposited in the oral cavity of an animal. Also, it is well known, that in vaccine development there is a workable window between minimal effective dose and maximal effective and safe dose to allow for process variability. The inventors herein tested a practical dose of 7.6x10⁹ CFU. It is thought that, given the desirable safety profile of the tested vaccine doses, such as the dose of 7.6x10⁹ CFU, it is likely that the practical maximal effective and commonly acceptable safe dose can be at most about 2x10¹¹ CFU. If needed this maximal dose may be exceeded.

In some embodiments, there is provided for a vaccine composition that comprises a dose of at most 2x10¹¹ CFU P. multocida. Preferably, the dose of a vaccine composition according to the invention is between about 1x10⁶ CFU and about 2x10¹¹ CFU. More preferably, the dose is between about 1x10⁷ CFU and about 1x10¹¹ CFU, 1x10⁸ CFU and about 1x10¹⁰ CFU, e.g., about 1.1x10⁷, 1.2x10⁷, 1.3x10⁷, 1.4x10⁷, 1.5x10⁷, 1.6x10⁷, 1.7x10⁷, 2x10⁷, 3x10⁷, 4x10⁷, 5x10⁷, 6x10⁷, 7x10⁷, 8x10⁷, 9x10⁷, 1x10⁸, 1.1x10⁸, 1.2x10⁸, 1.3x10⁸, 1.4x10⁸, 1.5x10⁸, 1.6x10⁸, 1.7x10⁸, 2x10⁸, 3x10⁸, 4x10⁸, 5x10⁸, 6x10⁸, 7x10⁸, 8x10⁸, 9x10⁸, 1x10⁹, 1 .25 x10⁹, 2x10⁹ CFU, 3x10⁹ CFU, 4x10⁹ CFU, 5x10⁹ CFU, 6x10⁹ CFU, 7x10⁹ CFU, 7.6x10⁹ CFU, 1x10¹⁰ CFU, 1x10¹¹ CFU or any other dose in between 1x10⁶ CFU and about 2x10¹¹ CFU or between about 1x10⁷ CFU and about 1x10¹¹ CFU. It is preferred that the dose of the live attenuated P. multocida bacteria is at least immunologically effective and safe and preferably can be economically produced, and preferably provides sufficient margin for losses in titer during production, formulation and storage. In optional embodiments the concentration of P. multocida (in CFU) per ml of vaccine is selected such that is has a beneficial efficacy and safety profile and can be selected from any other concentration of between 1x10⁶ CFU/ml - 1x10¹¹ CFU/ml.

In preferred embodiments, the vaccine according to the invention is provided as a one-shot vaccine (single dose vaccine). It was found that administering one dose of vaccine to one animal (ruminant) was sufficient to provide for a protective effect against infection with P. multocida. Preferably, a one-shot vaccine is both effective in providing a protective effect and is safe, i.e. , does not result in (serious and/or unexpected) adverse reactions. Hence, the vaccine according to the invention is preferably administered only once per ruminant target, i.e. , it is a single dose vaccine. Moreover, no booster vaccination is needed to arrive at a protective effect against infection with P. multocida.

In embodiments of the invention the vaccine for use according to the invention further comprises at least one additional immunoactive components. Accordingly, the vaccine for use of the invention may comprise one, two, three, four, five, etc. additional immuneactive component. As long as the vaccine remains safe and efficacious, preferably for protection against infection by P. multocida, any number of additional immunoactive components may be included in the vaccine. Typical additional immunoactive components comprise adjuvants, antigens, microorganisms, or a part thereof, biologically active molecules, immune enhancing substances, and/or vaccines, either of which may comprise adjuvants. The additional immunoactive component when in the form of an antigen may consist of any antigenic component of veterinary importance. Preferably the additional immunoactive component is based upon, or derived from, a further micro-organism that is pathogenic to a ruminant animal. It may for instance comprise a biological or synthetic molecule such as a protein, a carbohydrate, a lipopolysaccharide, a nucleic acid encoding a proteinaceous antigen. Also, a host cell comprising such a nucleic acid, or a live recombinant carrier microorganism containing such a nucleic acid, may be a way to deliver or express the nucleic acid or an additional immunoactive component. Alternatively, the additional immunoactive component may comprise a fractionated or killed micro-organism such as a parasite, bacterium or virus, or a part thereof, such as an extract, fraction, or sonicate. The additional immunoactive component(s) may also be an immune-enhancing substance e.g., a chemokine, or an immunostimulatory nucleic acid. Alternatively, the vaccine according to the invention, may itself be added to a vaccine, while assuring viability and efficacy. The additional immunoactive components can be based on, or derived from, micro-organisms that are pathogenic to ruminants. Preferred micro-organisms pathogenic to ruminants are one or more selected from the group consisting of: Mannheimia haemolitica, Mycoplasma bovis, Histophilus somni, bovine coronavirus, parainfluenza-3 virus, bovine respiratory syncytial virus, bovine viral diarrhoea virus, and bovine herpes virus 1 . More preferably the additional immunoactive components are selected from any one or more live attenuated bacteria selected from the group consisting of: Mannheimia haemolytica, Mycoplasma bovis and Histophilus somni, or a combination thereof. In some embodiments the vaccine of the invention comprises live attenuated M. haemolytica bacteria which express an avirulentform of the leukotoxin A protein. In some embodiments said live attenuated M. haemolytica is the mutant strain of M. haemolytica serotype 1 , as described in WO 1999/015670 which is completely incorporated by reference herein, named: NADC D153 IktA.

It is contemplated that for deposition in the oral cavity of a ruminant the most advantageous form of the vaccine is the dosage form of a liquid, gel or paste. Therefore, in some embodiments, the vaccine according to the invention is in liquid form. The liquid may be generally aqueous, meaning: like water, and comprises dosage forms such as a solution, syrup, elixir, suspension or emulsion, but can also be a gel or a paste. Preferably, the vaccine is provided in a dosage form that is suitable for deposition of the vaccine in the oral cavity. When released in liquid form, the vaccine formulation may comprise a stabilizer to allow prolonged storage of the live attenuated bacteria. For example: when the liquid vaccine is intended to be stored frozen at a temperature below 0 °C, the stabilizer may be a cryoprotectant, for example glycerol, to allow storage at temperatures of -20 °C or less for extended periods. Alternatively, when storing the liquid vaccine at temperatures above 0 °C, a suitable liquid stabilizer may be selected, for example as described in WO 2014/140239, or US 9,393,298.

In some embodiments, the vaccine according to the invention is in freeze-dried form, also known as: in lyophilized form. This form has several advantages over a liquid vaccine or over a vaccine formulated as a gel or as a paste, e.g., it is lighter and therefore more economical to transport. Also, a freeze-dried form of a vaccine will usually not require to be kept frozen but can be stored at a more economical 2-8 °C and/or can be stored for several days at room temperature (25 °C). Prior to use in vaccination, a freeze-dried vaccine commonly is re-suspended in a physiologically acceptable diluent. This is commonly done shortly before deposition of the vaccine in the oral cavity of the target, to ascertain the best quality of the vaccine. The diluent is typically aqueous, and can e.g., be sterile water, or a physiological salt solution. Such a diluent may comprise stabilizers, adjuvants, and the like. In some embodiments, the freeze-dried vaccine can be provided together (e.g., in the form of a kit) with the diluent.

The vaccine according to the invention, in the final version of the vaccine which is ready for deposition in the in the oral cavity of a ruminant target, can conveniently be administered to a ruminant by depositing the required volume of one animal dose, directly into the back of the throat of the animal. A wide variety of tools for the convenient dosing and deposition of vaccines to ruminants are available commercially and may be used for depositing of the vaccine according to the invention into the oral cavity of a ruminant. Typically, this will be an applicator of some sort such as a syringe or injector, with a nozzle that can be placed in the animal's mouth. Applicators are also available for repeated deposition/administration, when treating large number of animals. In some aspects, the vaccine can be administered by drenching. It is herein contemplated that the deposition of the vaccine in the oral cavity, i.e. , the act of actively placing the vaccine according to the invention in the oral cavity, is performed by a skilled person, such as a veterinary doctor, veterinarian, animal husbandry worker, cattle farmer, or veterinarian technician.

Vaccines in accordance with the current invention can comprise pharmaceutically and veterinary acceptable stabilizers, salts, adjuvants, diluents, emulsions, and the like. Many of these stabilizers, salts, adjuvants, diluents, emulsions, and the like are well known to those in the art and include, but are not limited to macromolecules, such as proteins, polysaccharides, e.g., sucrose, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles. Salts may be used in the vaccine formulation (e.g., as buffering agents) and comprise, for example, mineral salts such as hydrochlorides, hydrobromides, phosphates, or sulphates, salts of organic acids such as acetates, propionates, malonates, or benzoates. In certain embodiments, vaccines according to the invention may also contain liquids, such as water, saline, glycerol, and ethanol, as well as substances such as wetting agents, emulsifying agents, or pH-buffering agents. In some embodiments, the diluent may be, without limitation, Unisol™ (MSD Animal Health). Unisol is a diluent which comprises commonly used stabilizers, commonly used salts, preferably salts with buffering properties, and water (as solvent).

In preferred embodiments the vaccine in accordance with the invention at least comprises a substance having mucoadhesive properties, e.g., a mucoadhesive adjuvant. It is preferred that such a substance enhances the adhesion of the vaccine to mucosal membranes thereby having an advantageous effect on, for example, local mucosal residence time (e.g., increase the contact time between vaccine formulation and mucosa, preferably mucosa of the oral cavity, e.g., mucosa of the oropharynx and/or mucosa-associated lymphoid tissues, such as tonsils). Possible mucoadhesive adjuvants may be selected from any one or more of polymers, such as polysaccharides (such as, without limitation, starch, chitosan, dextran or cellulose), poly-a-hydroxyesters (such as, without limitation, poly-lactic acid, poly-glycolic acid or polycaprolactone), hyperbranched polymers, polyphosphazenes, polyanhydrides or polyethyleneimines, inorganic particles (such as, without limitation, calcium phosphate, nanoparticles, nanotubes, graphene oxide, mesoporous silica nanoparticles, mesoporous carbon nanoparticles), pathogen-like particles (such as, without limitation, outer membrane vesicles, virosomes, virus-like particles), lipidic particles (such as, without limitation, micelles, vesicular systems, solid lipids, liquid lipids, cubosomes) or combinations thereof. In preferred embodiments the mucoadhesive adjuvant does not affect the efficacy and/or safety of the vaccine comprising the live attenuated P. multocida.

In certain embodiments, the vaccine may comprise liposomes. See, for example U.S. Patent 5,422,120, WO 95/13796, WO91/14445, or EP 524,968 B1.

In some embodiments, the vaccine according to the invention is administered to young ruminants. It is favourable to vaccinate healthy targets, and to vaccinate as early as possible to prevent (the consequences of) an early infection by a pathogenic P. multocida. It is however contemplated that age, weight, sex, immunological status, etc. of the target ruminant for a vaccination according to the invention, are not critical. The term "young" refers to the period in the life of a ruminant up to its weaning. This period differs for various species of ruminants; for cattle weaning is typically at about 6 - 8 weeks of age, for lambs weaning is at about 4 - 6 weeks of age. Preferably "young" refers to 0 - 8 weeks of age, more preferably to 0 - 6 weeks of age. In preferred embodiments the vaccine is administered to the ruminant within about 14 days, more preferably within about 7 days, after birth of said ruminant. For example, the vaccine is administered to the ruminant between 1 and 14 days after birth such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13 or 14 days after birth, preferably between 3 - 9 days or between 5 - 13 days, more preferably between 5 - 7 days after birth. It is also encompassed herein that the ruminants are administered the vaccine after more than 14 days after birth, e.g., after 3 weeks, 4 weeks, etc.

Further, it is contemplated herein that the young ruminants receiving the vaccine according to the invention are P. multocida MDA (maternally derived antibody) positive or P. multocida MDA negative young ruminants. A ruminant is considered MDA-negative when the ruminant has no or low passively acquired, maternally derived antibodies (MDA). Young ruminants with no or relatively low passively acquired, maternally derived antibodies (MDA) against P. multocida, are considered the most sensitive, susceptible, category of animals for infection by P. multocida. Hence, it is contemplated that such young ruminants can benefit significantly from vaccination by the vaccine in accordance with the invention.

A ruminant is considered MDA-positive when the ruminant, preferably the calf, has acquired maternally derived antibodies against P. multocida. Commonly, such MDAs are provided to the young ruminant through the feeding of colostrum. In general, young ruminants reared for domestical or agricultural purposes are seropositive (i.e., are MDA-positive) for P. multocida antibodies. Therefore, in preferred embodiments, the vaccine in accordance with the invention is administered to a ruminant that is MDA-positive against P. multocida.

In some embodiments, the volume of the vaccine in accordance with the current invention is between 0.1 mL - 50 mL. Preferably, the volume of one animal dose is between 1 mL - 50 mL. The volume may be any volume between 1 mL or 50mL, but preferably is between 1 mL - 10 mL or, even more preferably, is between about 2 mL and about 7 mL, such as 3 mL, 4 mL, 5 mL or 6 mL. Although it is possible to provide commercial vaccines for ruminants in packaging for a single animal, this is not very cost efficient, nor is it practicable for use on large number of animals. Therefore, in some embodiments, there is provided for commercial forms of packaging of ruminant vaccines wherein the commercial forms are containers that comprise the animal doses for 2, 5, 10, 20, 25, 50, 100, 200, 250, 500, or even 1000 animals. Hence, herein encompassed is also a vaccine having a volume suitable for any amount of doses, such as any one dose selected from 2, 5, 10, 20, 25, 50, 100, 200, 250, 500, or even 1000 animal doses or any amount of doses in between 2 - 1000 doses. Further encompassed herein is a container comprising a volume suitable for any amount of doses, such as at least 2, 5, 10, 20, 25, 50, 100, 200, 250, 500, or even 1000 animal doses or any amount of doses in between 2 - 1000 doses.

In further embodiments of the invention there is provided for a vaccine for use as described herein, wherein before and/or after the deposition of the vaccine in the oral cavity of the ruminant, said ruminant is prevented from eating and/or drinking for a period of time, wherein said period of time is sufficient for absorption of the vaccine by the ruminant in the oral cavity and/or through the mucosa of the oral cavity.

The inventors found that in some embodiments of the invention it was advantageous to prevent the ruminants from eating and/or drinking before and/or after the after the deposition of the vaccine according to the invention in the oral cavity of the ruminant. Without being bound by theory, the inventors found that preventing the ruminants from eating and/or drinking accordingly prevented for the vaccine deposited in the oral cavity to be at least partially swallowed by the ruminant to which the vaccine was administered, such that the vaccine would not end up in the Gl tract but remains in the oral cavity, e.g., in the back of the throat (e.g., on and/or around the tissues of the oropharynx, and/or on and/or around the tonsil tissue). It was found that preventing the ruminants from eating and/or drinking before and/or after the deposition of the vaccine was advantageous for the efficacy. Further, it is contemplated that preventing the ruminant from eating and/or drinking has a beneficial effect on the adhesion of a vaccine according to the invention and comprising a mucoadhesive adjuvant to mucosal tissues.

In some preferred embodiments the eating and/or drinking is prevented before the deposition of the vaccine according to the invention to the ruminant. In some preferred embodiments the eating and/or drinking is prevented after the deposition of the vaccine according to the invention to the ruminant. In more preferred embodiments, the eating and/or drinking is prevented before and after the deposition of the vaccine according to the invention to the ruminant. In such preferred embodiments it is understood that due to the prevention of drinking and/or eating the amount of saliva produced in the oral cavity of the ruminant, which may affect the intake and swallowing of the vaccine composition according to the invention, is limited. In some optional embodiments, for example to limit saliva production in the oral cavity of a ruminant, the vaccine composition does not comprise an substance having a pleasant taste, such as sucrose. Both active feeding of the ruminant by providing a drink and/or food to the ruminant, or allowing the ruminant to drink freely e.g., from a trough of fresh water and/or eat freely e.g., from hay or other types of feeds are contemplated to be avoided for a certain period of time before and/or after the vaccination, thereby allowing sufficient absorption of the vaccine by the ruminant. Preferably, sufficient absorption of the vaccine composition in the oral cavity and/or through the mucosa of the oral cavity of the ruminant comprises that a sufficient amount (for example in CFU) of antigen(s), i.e., live attenuated P. multocida, is absorbed such that an advantageous effect, e.g., a protective effect against an infection of P. multocida can be observed and/or measured in the ruminant by using methods known in the art.

According to the invention the vaccine is not administered as a drink, thus it is not provided to a ruminant as a mixture of the vaccine with a liquid such as a milk or water, e.g., having a volume of between 500 mL and 2L, and feeding said mixture to the ruminants. Instead, a drink, such as a milk replacer, is only provided to the ruminant before and/or after the vaccination and is provided a period of time before and/or after said vaccination, thus allowing the vaccine to be absorbed by (mucosal) tissues in the oral cavity of the ruminant.

It is contemplated that the ruminants, to which the vaccine according to the invention has been deposited, are prevented from eating and/or drinking prior and/or before vaccination for a period of time of at least 15 minutes, preferably at least 30 minutes. The period of time provided herein should be sufficient for enabling the absorption of the vaccine by the ruminant in the oral cavity and/or through the mucosa of the oral cavity. It is contemplated that a period of time of at least about 15 minutes or at least about 30 minutes is sufficient for the vaccine to have a protective effect in the vaccinated ruminant. In some preferred embodiments, the ruminants are fed with a liquid nutritional product such as a milk replacer about 30 minutes after deposition of the vaccine.

In view of the above, in particular preferred embodiments, there is provided for a vaccine for use according to the invention, wherein before and/or after the deposition of the vaccine in the oral cavity of the ruminant, said ruminant is prevented from eating and/or drinking for at least 15 minutes, preferably at least 30 minutes, thereby providing for a period of time that is sufficient for absorption of the vaccine by the ruminant in the oral cavity and/or through the mucosa of the oral cavity. It will be apparent to a skilled person that feed and/or water should be available to the ruminants within a reasonable amount of time before and/or after the deposition of the vaccine in the oral cavity, such as up to about at least 30 minutes, or at least 15 minutes before and/or after deposition of the vaccine in the oral cavity. As a non- limiting descriptive example: to maintain a healthy homeostasis in the ruminants there is provided continuous access to feed and water until 30 minutes before deposition of the vaccine according to the invention and after 30 minutes after deposition of the vaccine.

In a preferred embodiment, a "ruminant" for the invention relates to any ruminant of relevance to veterinary science or to commercial farming operations. Preferably this refers to bovine, caprine, ovine or cervine animals. The economic relevance of bovine-, and in particular of cattle farming has the largest global relevance. Therefore, in an embodiment of the vaccine for use according to the invention, the ruminant is a bovine animal. In preferred embodiments, the ruminant is a bovine animal, preferably selected from the group consisting of: taurine cattle (Bos taurus), zebu cattle (Bos indicus), buffalo, bison, yak and wisent. The ruminant, preferably bovine, can be of any type: dairy or beef, or parental stock for dairy- or beef type. Since in preferred embodiments the vaccine for use is administered in young ruminants, it is preferred that said ruminants are calves.

In an aspect there is provided for a single-dose vaccine for deposition in the oral cavity of a ruminant, said single-dose vaccine comprising a total volume of between 2 mL - 10 mL and comprising between about 1x10⁷ - about 1x10¹¹ CFU live attenuated P. multocida, preferably P. multocida AhyaE, and a pharmaceutically acceptable carrier. Preferably, said single dose for deposition in the oral cavity comprises any concentration of between 1x10⁶ CFU/ml - 1x10¹⁰ CFU/ml. As previously described herein, the vaccine according to the invention preferably is a one-shot vaccine. The vaccine may be provided as a ready-to- administer product suitable for the deposition of the vaccine to a single ruminant. Such vaccines may be provided in several single-dose vaccines suitable to administer to multiple ruminants, e.g., a herd of several cows. For example, a single-dose vaccine according to the current invention can be provided in a container containing one or multiple, e.g., 10, 100, 1000, etc., separate single-dose vaccines. Therefore, commercial forms of packaging of herein provided ruminant vaccines can be in containers that comprise the animal doses for one or multiple animals, e.g., 1 , 2, 5, 10, 20, 25, 50, 100, 200, 250, 500, or even 1000 animals. In some instances, the vaccine is provided as a single-use vaccine, i.e. , the vaccine may only be used for a single animal and therefore is provided in a volume that is suitable only for a single animal, e.g., a total volume of between about 2 mL - about 10 mL, such as 6 mL or 8 mL, and/or having a dose of live attenuated P. multocida of between about 1x10⁷ - about 1x10¹¹ CFU as provided herein.

The vaccine according to the invention can be prepared from live attenuated P. multocida, by methods well known in the art, and within the routine capabilities of a person skilled in the art. For example: P. multocida is cultured in fermenters using standard culture medium with monitoring of temperature and use of variable stirrer speed and oxygen level. The culture is harvested at an appropriate time, such as upon reaching a specified culture density, measurable e.g., by optical density. The bacteria are then harvested for example by centrifugation or (d ia)fi Itration and are taken up into a pharmaceutically acceptable carrier such as water for injection combined with the necessary stabilizers. In some embodiments, the method for the preparation can involve the admixing with further pharmaceutically acceptable stabilizers, adjuvants, diluents, emulsions, and the like. Next, the vaccine product is apportioned into appropriately sized containers, and can be further formulated such as by freeze-drying, or the product can be released on the market in liquid or in the form of a gel or paste. Hence, in a further aspect there is provided for a method for the preparation of the vaccine for use according to the invention, or a single-dose vaccine according to the invention comprising the step of admixing live attenuated P. multocida bacteria and a pharmaceutically acceptable carrier. At different points in this method, additional steps may be added, for example for additional treatments such as for purification or storage.

Various steps of the method for the preparation of the vaccine can be monitored by adequate tests, e.g., by microbiological and/or immunological tests for the quality and quantity of the bacteria or any further antigens; by tests for absence of extraneous agents; and/or by in vitro or in vivo experiments to determine vaccine efficacy and/or -safety. All these tests are well known to a skilled person and are prescribed in governmental regulations such as the Pharmacopoeia, and 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 ).

The vaccine according to the invention, ready for deposition in the oral cavity of a ruminant target, can conveniently be administered to a ruminant by depositing the required volume of one animal dose, directly into the animal's oral cavity. As described above this route of providing the vaccine to a ruminant has various advantages over other methods of administration, such as, without limitation, improved efficacy of the vaccine, improved safety of the vaccine (e.g., reduced side-effects or reduced site reactions, for instance compared to subcutaneous administration), improved dose of the vaccine, less stressful conditions of administration compared to other methods of administration, allows mass administration of the vaccine etc. Hence in an embodiment there is provided for a method comprising the step of administering the vaccine by depositing the vaccine in the oral cavity of the ruminant.

Conveniently, the vaccine is administered to a ruminant by depositing the required volume of one animal dose of the vaccine directly into the animal's oral cavity. For the deposition various applications known in the art may be used in the depositing of the vaccine composition in the oral cavity. For example, such deposition of the vaccine in the oral cavity comprises providing the vaccine as a fluid to an animal by means of using an apparatus commonly called a drench. Alternatively, a wide variety of tools for the convenient dosing and oral deposition of fluids are available commercially. Typically, this will be an applicator of some sort such as a syringe or injector, with a nozzle that can be placed in the animal's mouth. Such applicators are also available for repeated administration, when treating large number of animals. It is preferred that the vaccine, as a one-shot vaccine, is actively deposited, e.g., by using an applicator available in the art, in the oral cavity of the animal, thus meaning that the vaccine preferably is not administered as a drink (e.g., through the drinking of water or through the admixed with milk), or with the feed (e.g., as a top-dressing of feed, or as a bait, treat, chew, or lick). In some embodiments, the vaccine according to the invention is administered in the back of the throat of a ruminant, e.g., on and/or around the tissues of the oropharynx, and/or on and/or around the tonsil tissue, without inducing swallowing the vaccine by said ruminant.

As described above and broadly exemplified herein, the vaccine according to the invention provides for protective effect against RDD. As such, it is contemplated that the administration of the vaccine to the ruminant by depositing the vaccine in the oral cavity of the ruminant prevents reduction of the respiratory function of a ruminant. For example, in case the animal is infected with P. multocida, the vaccine according to the invention reduces lung pathology in vaccinated animals. Therefore, in one aspect, there is provided for a method for the vaccination of ruminants against infection with P. multocida, the method comprising the step of administering the vaccine for use to said ruminants by depositing the vaccine in the oral cavity. Further, in another aspect, there is provided herein for a method for improving the respiratory function of a ruminant comprising the step of depositing in the oral cavity of said ruminant the vaccine or a single-dose vaccine in accordance with the invention. As described herein it is contemplated that through the vaccine according to the invention, in case of infection by P. multocida and/or RRD, the respiratory function of the ruminant is improved compared to a non-vaccinated ruminant.

In a further aspect there is provided for methods of treating, ameliorating and/or preventing a diseased state in a ruminant comprising the depositing in the oral cavity of the ruminant the vaccine or a single-dose vaccine in accordance with the invention, wherein the diseased state comprises RRD. In preferred embodiments of the method for the treating, ameliorating and/or preventing of a diseased state in a ruminant, the vaccine is deposited in the oral cavity of the ruminant. In further embodiments the diseased state treated, ameliorated and/or prevented by deposition of the vaccine in the oral cavity comprises any one or more diseased state selected from: calf and yearling meningoencephalitis, lamb lymphadenitis and pneumonia.

The vaccine can be provided to the market as a commercial product, such as a kit of parts. Therefore, in a further aspect of the invention there is provided for a kit of parts comprising a container comprising the vaccine or the single-dose vaccine according to the current invention. Optionally said kit of parts comprises at least one further container (e.g. , a second container) comprising at least one pharmaceutically acceptable carrier.

The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art (including the contents of the references cited herein), readily modify and/or adapt for various applications, such as specific embodiments, without undue experimentation, without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. All references cited herein, including journal articles or abstract, published, or corresponding patent applications, patents, or any other references, are incorporated by reference herein in its entirety, including all data, tables, figures, and text presented in the cited references. Additionally, the entire contents of the references cited within the references cited herein are also entirely incorporated by reference.

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

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

EXAMPLES

EXAMPLE 1 : EFFICACY OF THE PASTEURELLA MULTOCIDA AHYAE MONOVALENT LIVE STRAIN GIVEN BY THE ORAL OR INTRANASAL ROUTE

1.1. Objective

The objective of this study was to investigate the efficacy of the live, freeze-dried vaccine strain P. multocida AhyaE after deposition in the oral cavity or after intranasal administration against experimental challenge with virulent P. multocida (P. multocida 17336).

1.2. Study design

1.2.1. General

Thirty 1 week old, healthy MDA- calves were used in this study. Three groups were made: Group 1 was Control, Group 2 received the vaccine deposited in the oral cavity, Group 3 received the intranasal vaccine. Twenty calves were vaccinated, in which 10 calves were vaccinated through deposition of the vaccine in the oral cavity of the calves and 10 calves were vaccinated intranasally with the same dose. The other 10 calves were non-vaccinated controls. Rectal temperatures were recorded in the morning. The study was divided in 2 cohorts. Average age of the calves at day of vaccination was 5-13 days in each group. General health observations were done after vaccination. No unacceptable site reactions signs were observed due to vaccination.

Table 1 : Cohorts, age of vaccination and vaccination dose (CFU)

Twenty days after vaccination all 30 calves were challenged with P. multocida 17336, via intratracheal administration. After challenge calves were monitored for clinical symptoms (general and respiratory) and rectal body temperatures were recorded. Blood samples were taken to measure antibody titers against P. multocida. Seven days after challenge the animals were euthanized, and necropsy was performed to determine lung pathology (lung lesion scores, LLS). Lung samples were collected to confirm the presence of the challenge strain. At pre-set time points throughout the experiment plain blood samples were taken to measure P. multocida specific antibodies. Also, nasal swabs were collected to determine the presence of P. multocida in the nose.

1.2.2. Experimental animals

The animals received approximately 2.7 L colostrum (Pm ELISA Ab titer 8.8) immediately after arrival at the animal facilities. Then calves received at least 2 L milk replacer (Sprayfo Royal milkpowder) twice or three times daily. From an age of approximately 7 days onwards, they were provided with hay and feed and had free access to fresh water in accordance with standard procedures. 1.2.3. Vaccination

On day 0 the calves in group 2 and 3 received a vaccine dose of 1 x10⁹ CFU or 7.6x10⁹ CFU in 6 ml. The deposition of the vaccine in the oral cavity was done by giving 6 ml at the back of the throat (e.g., oropharyngeally and/or (peri-)tonsillar) by deposition of the vaccine using a suitable applicator. The intranasal (IN) vaccination was done by giving 3 ml in each nostril (total for both nostrils: 6 ml) directly from the tip of the syringe without a spraying device. Vaccination was done about a half hour before feeding of the calves with milk replacer.

1.2.4. Challenge material

P. multocida 17336 was inoculated on blood agar plates and incubated for 24 hours at 37°C. Subsequently, a bacterial suspension in physiological salt solution was made. The challenge culture suspension was made as following: 2 ml bacterial suspension with an ODeeo of 0.250 was added to 200 ml TPB (at 20°C) and incubated for 8 hours at 37°C and 90 rpm. After incubation the culture was diluted 1 :1 in TPB, aiming at 5x10⁸ CFU/ml for a total target dose of 5x10¹⁰ CFU in 100 ml. The actual viable count of the challenge culture was verified by plate counting before and after the challenge procedure.

1.2.5. Administration of the Challenge material

On day 21 the challenge suspension of P. multocida 17336 was administered to all 30 calves intratracheal with a dose of 5x10¹° CFU in 100 ml using a flexible tube. The tube was entered in the calf via a nostril and inserted until approximately 5-10 cm before the bifurcation (at a length of 50 cm tube or catheter). It was confirmed that the tube was in the trachea and not in the esophagus by pulling up air and notice respiratory reaction of the calf. The 100 ml suspension was at ambient temperature and injected slowly via the tube or catheter using a 50 ml syringe.

1.3. Experimental procedures

1.3.1. Determination of the viable counts (CFU) in samples and suspensions

Viable bacterial counts of samples and suspensions containing the vaccine or challenge strain were determined. Briefly, serial ten-fold dilutions were made in saline solution and then 0.1 ml was plated on blood agar plates. Inoculated plates were incubated for 16-24 hours at 37°C. The number of colony forming units (CFU) of P. multocida were recorded. 1.3.2. Rectal temperature

On the timepoints indicated in the time schedule, the rectal temperatures of the animals were measured and recorded. The physiological range of body temperatures for calves is 38.0 - 39.5°C. Rectal temperature measurements were performed daily at fixed time points before any handling other than feeding and several hours (±4 hours) after vaccination and challenge.

1.3.3. Necropsy

Seven days after the challenge the calves were euthanized, followed immediately by a postmortem examination. The calves were euthanized by an intravenous injection followed by complete bleeding. During this procedure blood samples of all calves were taken. Tissue samples were excised from two sites of the lungs. In case of consolidation, two samples of two different consolidation sites were taken. In case of a negative score for consolidation two random samples were taken.

1.3.4. Macroscopic pathology

The lungs were removed from the calves and inspected macroscopically. For each lung lobe the % consolidation (lung lesions) was estimated and recorded. In addition, consolidation scores were adjusted for the relative size of the lobe (weighted lung lesion score, w_LLS) in relation to the lung (cranial left: 11 %, cranial right: 11 %, middle: 7%, accessory: 4%, caudal left: 32%, caudal right: 35%, Jericho and Langford [1982] Can. J. com. Med. 46:287-292), resulting in the affected lung surface area.

1.3.5. Statistical methods

Instead of comparing all 3 groups to each other, each vaccinated group was compared separately with the control group and no adjustment for multiple comparison was made in a post-hoc test.

1.4. Results

1.4.1. Confirmation of the vaccine dose

The dose of the vaccine preparation (P.multocida AhyaE) is presented in the table below for both cohorts. The calves of cohort 1 received a dose of at least 1x10⁹ CFU in 6 ml. The vaccine solution for cohort 2 resulted in a higher dose of 7.6 x 10⁹ CFU in 6 ml (per calf).

See Table 2.

Table 2: Average dose

1.4.3. Rectal temperatures after vaccination and challenge

No statistically significant differences were found for both maximum rectal temperature and for the duration of fever when comparing groups 2 and 3 with group 1 after vaccination and challenge. The mean rectal temperature profiles of both the oral and intranasal vaccinated animals are similar to the control group.

1.4.3. Lung lesion scores

The average total lung lesion score (%) for the control group is for cohort 1 : 191 .6, for cohort 2 : 151 .0 and total average is: 171 .3. The oral cavity vaccinated group shows an average of 127.5 for cohort 1 , 102.5 for cohort 2 and overall average of 117.5, showing a reduction of 31 %. The intranasal vaccinated group shows an average of: 245.8 (cohort 1 ), 111.5 (cohort 2) and 192.1 (overall average). When the weighted lung lesion scores are compared, 21.16 (control), 14.20 (oral) and 23.46 (intranasal), a reduction of 33 % is seen for the oral cavity vaccinated animals. Lung lesions were significantly reduced for the oral cavity vaccinated animals (total LLS reduction 31 %, p=0.0068, w_LLS (weighted) reduction 33%, p=0.0467). The results after vaccination and challenge are summarized in Table 3 below. Table 3: Results after infection.

¹ Avg. total LLS is the total percentage lung lesion scores after necropsy.

² Avg. total w_LLS is the relative (weighted) consolidation of lung lesion scores after necropsy.

³ Avg. Peak Temperature (°C) is the average rectal temperature after challenge of the group.

During necropsy the lungs were also scored for the presence of pleuritis. No difference was observed between control and oral cavity (2.3 vs. 2.0). However, IN vaccinated animals show much higher pleuritis scores with an average of 5.1 . 1.5. Conclusion

In both cohort 1 and cohort 2 the animals that received vaccine deposited in the oral cavity showed a reduction in LLS in contrast to the IN vaccination. Thus, it was concluded that a single deposition of a vaccine with a dose of about 1x10⁹ CFU and of about 7.6 x 10⁹ CFU of P. multocida AhyaE to the oral cavity is effective in reducing lung pathology after challenge with P. multocida. No unacceptable safety issues for the vaccine deposited in the oral cavity were observed. A single intranasal vaccination does not seem to reduce lung pathology. EXAMPLE 2: EFFICACY OF THE PASTEURELLA MULTOCIDA AHYAE MONOVALENT LIVE STRAIN GIVEN BY THE SUBCUTANEOUS ROUTE (1-SHOT VACCINATION SCHEME)

2.1. Objective

The goal was to develop a live GMO vaccine based on P. multocida P1062 AhyaE given at a minimum age of 2 weeks by the subcutaneous (SC) route.

2.2. Study Design

Twenty animals were included in the study. The animals were MDA- calves. Ten animals were SC vaccinated once with 2 ml P. multocida monovalent vaccine at a dose of 5.7x10⁸ CFU, using Unisol as diluent. Ten animals were used as control and received no vaccination. Three weeks after the vaccination the control animals and vaccinated animals were challenged with a heterologous strain P. multiocida 671/90. An overview of the study design is shown in Table 4.

Table 4: Study design (subcutaneaous administration of vaccine comprising live attenuated P. multocida P1062 AhyaE)

2.2.1. Infection material

The P. multocida 671/90 was inoculated on blood agar plates and incubated 16 hours at 37°C. Subsequently, a bacterial suspension in physiological salt was made. Challenge culture suspension was prepared as follows: 1 ml bacteria suspension with an ODeeonm of 0.250 was added to 200 ml Tryptose Phosphate Broth and incubated for 4-5 hours at 37°C and 90 rpm. After incubation, the number of bacterial cells was counted using a Petroff Hausser counting chamber and a light microscope. For this study (MDA-negative calves, no DEX pre-treatment) the culture was diluted, aiming at 0.7-1 .3x10⁹ cells/ml, for a total target dose of 2-4x10¹° colony forming units (CFU) in 30 ml.

2.3. Experimental procedures

2.3.1. CFU determination for vaccine strain and challenge material

CFU were determined by making 10-fold serial dilutions and subsequently inoculating 0.1 ml on blood agar plates following incubation for 24-48 hours. CFU determination was done in duplicate. The number of colonies were counted and expressed as CFU per ml.

2.3.2. Observation of clinical signs, including rectal temperatures

The clinical signs of the animals were monitored and recorded on relevant time points, for example after vaccination and/or after challenge until day of necropsy. After challenge, specific symptoms of respiratory disease were monitored like coughing, type and rate of respiration, nasal and/or ocular discharge. Rectal body temperatures were measured on relevant time points in accordance with standard procedures. Temperatures were considered elevated if they were above 39.5°C.

2.3.3. Necropsy

At the end of the studies, one week after challenge, the calves were sacrificed, immediately followed by a post-mortem examination with special attention to the lungs. For each lung lobe the % consolidation was estimated by the representative of the pathologist and recorded (called: lung lesion score (LLS)). Also, the relative (weighted) lung lesion score of each animal was obtained by addition of the % lung consolidations of the individual lung lobes with a correction factor for the relative size of the lobe.

2.3.4. Re-isolation of P. multocida challenge strain from post-mortem lung samples

Tissue samples were excised from two sites of the lungs. In case of consolidation, two samples of two different consolidation sites were taken. In case of absence of consolidation two random samples were taken. Each sample was submerged in boiling water for 3 seconds, homogenized, plated on blood agar plates and then incubated for 16-24 hours at 37°C. The number of Pasteurella-\\ke colonies (after confirmation by standard microbiological techniques, like MALDI-TOF or PCR) were counted or estimated and expressed as CFU/sample, or as absent or present.

2.4 Results

2.4.1. Efficacy results

Lung pathology scores are shown in Table 5. Lung pathology was lower in the vaccinated group compared to control.

Table 5: Lung pathology scores

2.4.2. Safety results

Vaccination with a 5.7x10⁸ CFU/2 ml subcutaneous dose induced considerable local reactions with an average size of 45 cm² (measured 14 and 20 days after vaccination). At necropsy, four weeks after vaccination, abscesses were found in 7 of 10 animals with an average size varying between 10 and 210 cm³. Further, the vaccine strain could be reisolated from local reactions in 7 of 10 animals P. multocida, which is unacceptable for safety reasons.

2.5. Conclusion

It was not feasible to find a safe and efficacious, i.e., acceptable, protective dose of P. multocida AhyaE for SC vaccination using the heterologous challenge model with strain 671/90. EXAMPLE 3: DURATION OF IMMUNITY AFTER A 2-SHOT ORAL VACCINATION WITH THE PASTEURELLA MULTOCIDA AHYAE MONOVALENT LIVE STRAIN GIVEN BY THE ORAL ROUTE

3.1 Objective

The objective of this study was to investigate the duration of immunity of the live, freeze- dried vaccine strain P. multocida AhyaE after deposition in the oral cavity twice, at one week of age and at 4 weeks of age, against experimental challenge with virulent P. multocida (P. multocida 17336) at nine weeks after the second vaccination.

3.2 Study design

Thirty-six 1 week old, healthy calves with low antibody titers against P. multocida were used in this study. Two groups were made: Group 1 (N=18) was Control, Group 2 (N=18) received the vaccine deposited in the oral cavity at a dose of approx. 1x10⁹ CFU in 2 ml. The study was divided in 2 cohorts. Average age of the calves at day of first vaccination was 2-9 days in each group. No clinical signs were observed after both vaccinations.

Table 1 : Cohorts, age of vaccination and vaccination dose (CFU)

Nine weeks after the second vaccination all calves were challenged with P. multocida 17336, via intratracheal administration (1 -3 x 10¹¹ CFU in 100ml). After challenge calves were monitored for clinical symptoms (general and respiratory) and rectal body temperatures were recorded. Blood samples were taken to measure antibody titers against P. multocida. Three weeks after challenge the animals were euthanized, and necropsy was performed to determine lung pathology (lung lesion scores, LLS [consolidation] and pleuritis). 3.3 Results

Lung pathology was significantly reduced for the vaccinated animals: total LLS reduction of 46% (p=0.0077) and pleuritis score reduction of 54% (p=0.0026). Vaccinated animals had fewer days of fever (>39.5°C): 39 versus 61. Total clinical and respiratory scores were consistently lower in the vaccinated group throughout the period after challenge (data not shown).

All animals had moderate antibody titers for P. multocida at the beginning of this experiment (approx. 8.0 Iog2), which increased after challenge more profoundly in vaccinated than in control animals (immunological booster effect indicating immunological memory of the vaccinations)

3.4 Conclusion

It was concluded that nine weeks after two depositions of a vaccine with a minimal dose of about 1x10⁹ CFU of P. multocida AhyaE to the oral cavity is effective in reducing lung pathology after challenge with P. multocida and has a notable reduction of clinical symptoms. The results after infection are summarized in the table (Table 2) below. No unacceptable safety issues for the vaccine deposited in the oral cavity were observed.

Table 2: Results after infection.

¹ Avg. total LLS is the total percentage lung lesion scores during necropsy.

² Avg. pleuritis score per animal during necropsy

³ Total number of days of fever (>39.5°C) in a treatment group

⁴ Number of days of clear difference between vaccinated and control groups.

Claims

1 . A vaccine comprising live attenuated Pasteurella multocida (P. multocida) bacteria and a pharmaceutically acceptable carrier, for use in protecting a ruminant against an infection with P. multocida, wherein the vaccine is deposited in the oral cavity of the ruminant.

2. The vaccine for use according to claim 1 , wherein said vaccine is deposited oropharyngeal ly and/or (peri-)tonsillar.

3. The vaccine for use according to any one of the previous claims, wherein the live attenuated P. multocida bacteria comprise a deletion of all or a part of a hyaE gene.

4. The vaccine for use according to any one of the previous claims, wherein the vaccine deposited in the oral cavity comprises a dose of at least 1x10⁶ CFU, preferably at least 1x10⁷ CFU, more preferably between about 1x10⁷ - about 1x10¹¹ CFU.

5. The vaccine for use according to any one of the previous claims, wherein the vaccine further comprises at least one additional immunoactive component, preferably selected from any one or more live attenuated bacteria selected from the group consisting of: Mannheimia haemolitica, Mycoplasma bovis and Histophilus somni.

6. The vaccine for use according to any one of the previous claims, wherein the vaccine is a liquid vaccine or is in the form of a gel or paste.

7. The vaccine for use according to any one of the previous claims, wherein the vaccine comprises a mucoadhesive adjuvant.

8. The vaccine for use according to any one of the previous claims, wherein the vaccine is administered to the ruminant within about 14 days, more preferably within about 7 days, after birth of said ruminant.

9. The vaccine for use according to any one of the previous claims, wherein the vaccine is administered in a volume of between 1 mL - 50 mL, preferably between 1 mL - 10 mL, more preferably between 2 mL and 7m L, such as 5 mL or 6 mL.

10. The vaccine for use according to any one of the previous claims, wherein before and/or after the deposition of the vaccine to oral cavity of the ruminant, said ruminant is prevented from eating and/or drinking for a period of time, wherein said period of time is sufficient for absorption of the vaccine by the ruminant in the oral cavity and/or through the mucosa of the oral cavity, and preferably wherein said period of time is at least 15 minutes, more preferably at least 30 minutes.

11. The vaccine for use according to any one of the previous claims, wherein the ruminant is a bovine animal, preferably selected from the group consisting of: taurine cattle (Bos taurus), zebu cattle (Bos indicus), buffalo, bison, yak and wisent.

12. A single-dose vaccine for deposition in the oral cavity of a ruminant comprising a total volume of between 2 mL - 10 mL and comprising between about 1x10⁷ - about 1x10¹¹ CFU live attenuated P. multocida, preferably P. multocida AhyaE, and a pharmaceutically acceptable carrier.

13. Method for the preparation of the vaccine for use according to any one of claims 1 - 11 or a single-dose vaccine of claim 12 comprising the step of admixing live attenuated P. multocida bacteria and a pharmaceutically acceptable carrier.

14. Method for the vaccination of ruminants against infection with P. multocida, the method comprising the step of administering the vaccine for use according to any one of claims 1 - 11 or a single-dose vaccine of claim 12 to said ruminants by depositing the vaccine in the oral cavity.

15. Method of improving the respiratory function of a ruminant comprising depositing the vaccine for use according to any one of claims 1 - 11 or a single-dose vaccine of claim 12 in the oral cavity of the ruminant.

16. Method of treating, ameliorating and/or preventing a diseased state in a ruminant comprising the depositing the vaccine for use according to any one of claims 1 - 11 or a single-dose vaccine of claim 12 in the oral cavity of the ruminant, wherein the diseased state comprises ruminant respiratory disease.

17. A kit of parts comprising a container comprising the vaccine for use according to any one of claims 1 - 11 or the single-dose vaccine according to claim 12, and optionally comprising a second container comprising a pharmaceutically acceptable carrier.