Vaccine for vaccinating a canine

Abstract

The present invention relates to a vaccine for use in a method of inducing an immune response in a canine against infectious canine hepatitis and / or infectious tracheobronchitis. To combine a convenient way of administering the vaccine and a good protection against ICH and infectious tracheobronchitis, the vaccine is a first vaccine comprising a canine adenovirus type 2, and the method comprises - administration of an immunologically effective dose of the first vaccine, - subcutaneous administration of an immunologically effective dose of a second vaccine comprising a canine adenovirus type 2 7-42 days after the first vaccine, and - oral administration of an immunologically effective dose of a third vaccine comprising a canine adenovirus type 2 10-14 months after the first vaccine.

Description

[0001] VACCINE FOR VACCINATING A CANINE

[0002] Field of the invention

[0003] The present invention relates to a vaccine for use in a method of inducing an immune response in a canine against infectious canine hepatitis and / or infectious tracheobronchitis.

[0004] Background of the invention

[0005] Canine adenovirus type 1 (CAV-1) and 2 (CAV-2) are double stranded DNA viruses responsible for two major canine infectious diseases. CAV-1 causes infectious canine hepatitis (ICH), while CAV-2 is one of the viruses causing infectious tracheobronchitis.

[0006] ICH is an acute liver infection in dogs, but also in wolves, foxes, coyotes, bears and skunks. CAV-1 spreads through feces, urine, blood, saliva, and nasal discharge of infected animals. After contraction through the mouth or nose, it replicates in the tonsils and then infects the liver and kidneys. Although most animals recover spontaneously from ICH, bleeding disorders and liver disease as a result of CAV-1 infection are lethal in some.

[0007] Infectious tracheobronchitis caused by CAV-2 is a mild but contagious disease restricted to the respiratory tract of dogs. It is mainly characterized by coughing, nasal discharge, bronchitis and bronchiolitis and the virus is considered to be one of the contributors of canine infectious respiratory disease complex (kennel cough). CAV-2 spreads through aerosols produced by sneezing and coughing dogs.

[0008] In order to prevent dogs from developing ICH or infectious tracheobronchitis, dogs from the age of 6 weeks are vaccinated using a vaccine comprising CAV-2. Given the high degree of genetic similarity between CAV-1 and CAV-2, these vaccines also provide protection against CAV-1 induced ICH. Vaccines against ICH and infectious tracheobronchitis are, together with vaccines against canine distemper virus (CDV) and canine parvovirus (CPV), considered to be the core vaccines recommended for all dogs worldwide. Typically, puppies are initially vaccinated at an age of six to eight weeks, followed by a second vaccination within a period of 3-4 weeks after the first vaccination. After this, the vaccination is typically repeated once every year to three years.

[0009] In addition to the viruses contained in the core vaccines, vaccination against other viruses is recommended depending on the geographical location, local environment or lifestyle of a particular dog. These non-core vaccines include, for example, vaccines against canine parainfluenza virus (CPiV), or the bacteria Bordetella bronchiseptica, Leptospira interrogans serovars Canicola, Icterohaemorrhagiae, Grippotyphosa and Australis. CPiV and B. bronchiseptica are other contributors in the canine infectious respiratory disease complex, while bacteria of the genus Leptospira cause a blood infection that can develop into severe lung disease.

[0010] While subcutaneous administration is the traditional way of administering core and non-core vaccines, other ways are being explored in order to facilitate vaccine delivery and / or to mimic the natural route of infection of the viruses. For example, EP2762163 describes a method of vaccinating a dog against ICH caused by CAV-1 using a vaccine comprising CAV-2. This vaccine is administered subcutaneously in a first dose, orally in a second dose 7-42 days after the first dose and orally in one or more annual doses.

[0011] However, there remains a need for vaccines against ICH and infectious tracheobronchitis that combine a convenient way of administering the vaccine and a good protection against ICH and infectious tracheobronchitis.

[0012] Description of the invention

[0013] To this end, a vaccine for use in a method of inducing an immune response in a canine against infectious canine hepatitis and / or infectious tracheobronchitis is provided, wherein the vaccine is a first vaccine comprising a canine adenovirus type 2, and the method comprises: administration of an immunologically effective dose of the first vaccine, subcutaneous administration of an immunologically effective dose of a second vaccine comprising a canine adenovirus type 2 7-42 days after the first vaccine, and oral administration of an immunologically effective dose of a third vaccine comprising a canine adenovirus type 2 10-14 months after the first vaccine.

[0014] It was found that administering a first vaccine comprising CAV-2, administering a second vaccine comprising CAV-2 subcutaneously 7-42 days after the first vaccine, and administering a third vaccine comprising CAV-2 orally 10-14 months after the first vaccine resulted in a strong antibody response against both CAV-1 and CAV-2.

[0015] As shown in Fig. 1 , the concentration of CAV-2 neutralising antibodies increased gradually following subcutaneous administration of the second vaccination to dogs. Administration of the third vaccine subcutaneously boosted the CAV-2 neutralising antibody concentration. Surprisingly however, this boost was much stronger when the third vaccine was administered orally. As shown in Fig. 2, neutralising CAV-1 antibodies were also produced after administration of the first and second vaccine comprising CAV-2. As for CAV-2, the CAV-1 neutralising antibody concentration increased significantly following administration of the third vaccine comprising CAV-1 subcutaneously. However, oral administration of the third vaccine again led to a much stronger antibody response than when the third vaccine was administered subcutaneously.

[0016] In contrast to this and as shown in Fig. 3, administering a first vaccine and second vaccine comprising canine distemper virus (CDV) resulted in an increase in CDV neutralizing antibody concentration. However, the concentration of CDV neutralizing antibodies was only boosted when a third vaccine comprising CDV was administered subcutaneously. Oral administration of the third vaccine comprising CDV had no significant effect on the concentration level of CDV neutralizing antibodies.

[0017] As shown in Fig. 4, administering a first vaccine and a second vaccine comprising canine parvovirus (CPV) also led to the induction of anti-CPV-antibodies. However, administering a third vaccine comprising CPV orally did not boost the anti-CPV-antibody levels above that of the levels induced by a subcutaneous administration of the third vaccine comprising CPV.

[0018] Given the above, it is very surprising that oral administration of the third vaccine comprising CAV-2 led to such a strong and unexpected increase of antibody levels against CAV-1 and CAV-2.

[0019] Summary of invention

[0020] A vaccine for use in a method of inducing an immune response in a canine against infectious canine hepatitis and / or infectious tracheobronchitis, wherein the vaccine is a first vaccine comprising a canine adenovirus type 2, and the method comprises: administration of an immunologically effective dose of the first vaccine, subcutaneous administration of an immunologically effective dose of a second vaccine comprising a canine adenovirus type 2 7-42 days after the first vaccine, and oral administration of an immunologically effective dose of a third vaccine comprising a canine adenovirus type 2 10-14 months after the first vaccine.

[0021] In an embodiment, the immune response is a protective immune response.

[0022] In an embodiment of the invention, a further vaccine comprising canine adenovirus type 2 is administered annually after the third vaccine with intervals of 11-13 months. In an embodiment of the invention, the first vaccine, the second vaccine, the third and / or the further vaccine additionally comprises NADES.

[0023] In an embodiment of the invention, the first vaccine is administered to a canine of 2-12 weeks of age.

[0024] In an embodiment of the invention, the second vaccine is administered 1-6 weeks after the first vaccine.

[0025] In an embodiment of the invention, the second vaccine is administered 7, 10, 15, 20, 25, 30, 35, or 40 days after the first vaccine.

[0026] In an embodiment of the invention, the third vaccine is administered 10-14 months after the first vaccine.

[0027] In an embodiment of the invention, the third vaccine is administered 42-60 weeks after the first vaccine.

[0028] In an embodiment of the invention, the administration of the first vaccine is subcutaneously.

[0029] In an embodiment of the invention, the canine adenovirus type 2 contained in the first vaccine, the second vaccine, the third vaccine and / or the further vaccine is an attenuated canine adenovirus type 2.

[0030] In an embodiment of the invention, the first vaccine, the second vaccine, the third and / or the further vaccine additionally comprises at least one of canine distemper virus, canine parvovirus, canine parainfluenza virus, Bordetella sp, Leptospira interrogans.

[0031] In an embodiment of the invention, the first vaccine, the second vaccine, the third vaccine and / or the further vaccine additionally comprises canine distemper virus and canine parvovirus.

[0032] In an embodiment of the invention, the first vaccine, the second vaccine, the third vaccine and / or the further vaccine additionally comprises canine parainfluenza virus and Bordetella sp.

[0033] In an embodiment of the invention, the first vaccine, the second vaccine, the third vaccine and / or the further vaccine contains canine adenovirus type 2 in an amount of 102-106TCID50.

[0034] In an embodiment of the invention, the first vaccine, the second vaccine, the third vaccine and / or the further vaccine is administered in a volume of 0.1-5 mL.

[0035] In an embodiment of the invention, the oral administration of the third vaccine is about 1 year after the administration of the first vaccine. In an embodiment of the invention, the first vaccine, the second vaccine, the third vaccine and / or the further vaccine additionally comprises a pharmaceutically acceptable adjuvant.

[0036] In an embodiment of the invention, the first vaccine, the second vaccine, the third and / or the further vaccine additionally comprises a pharmaceutically acceptable carrier.

[0037] In an embodiment, the canine is a dog.

[0038] Definitions

[0039] “Vaccine” is a composition that comprises an antigen. Administration of the vaccine to a subject leads to the induction of an immune response against the antigen in the subject. It may be administered in various ways, including subcutaneously, orally or intranasally. The induction of the immune response may be humoral and / or cell-mediated. The immune response can for example be assessed by measuring antibody titres.

[0040] “To vaccinate” is to administer a vaccine to a subject.

[0041] “Vaccination” is the act of administering a vaccine to a subject.

[0042] “Primary vaccine” is a vaccine used to induce an initial protective immune response in a subject and resulting in at least temporal protective immunity. Multiple doses may be required to achieve protective immunity.

[0043] “Booster vaccine” is a vaccine used to stimulate an immune response previously induced by a primary vaccine in a subject in order to extend the protective immunity conferred by the primary vaccine.

[0044] “Antigen” is a molecule containing one or more epitopes that will induce an immune response specific for that antigen when administered to a subject. An antigen is for example a killed, attenuated or inactivated virus or bacterium, or an antibody or fragment thereof.

[0045] “Epitope” is a specific site of an antigen binding to a T-cell receptor or specific antibody.

[0046] “Antibody” is an immunoglobulin molecule binding to a specific antigen. An antibody comprises light and heavy chains having constant and variable regions, and can be subdivided in classes such as IgA, IgD, IgE, IgG, IgM based on the composition of the constant regions.

[0047] “Neutralising antibody” is an antibody that binds a pathogen such as a virus or bacterium, blocking the infectivity of the pathogen.

[0048] “Antibody response” is an immune response resulting in the production of antibodies against an antigen. “Immune response” is the activation of the immune system in response to

[0049] “Immunologically effective dose” is an amount of antigen or vaccine that induces an immune response in the subject it is administered to which is adequate to prevent signs or symptoms of a disease caused by infection with a pathogen, such as a virus or bacterium.

[0050] “Protective immune response” is an immune response adequate to prevent signs of or symptoms of a disease caused by infection with a pathogen, such as a virus or bacterium.

[0051] “Protective immunity” is immunity preventing signs of or symptoms of a disease caused by infection with a pathogen, such as a virus or bacterium.

[0052] “Antibody titre” is a measure for the level of an antibody, expressed as how much a sample comprising the antibody may be diluted before the antibody is no longer detectable.

[0053] “Pharmaceutically acceptable” is suitable for use in subjects without undue effects such as toxicity, irritation, and / or allergy relative to the benefit.

[0054] “Adjuvant” is a pharmaceutically acceptable compound or composition increasing the immune response to an antigen.

[0055] Embodiments of the invention

[0056] The invention relates to a vaccine for use in a method of inducing an immune response in a canine against infectious canine hepatitis and / or infectious tracheobronchitis, wherein the vaccine is a first vaccine comprising a canine adenovirus type 2, and the method comprises: administration of an immunologically effective dose of the first vaccine, subcutaneous administration of an immunologically effective dose of a second vaccine comprising a canine adenovirus type 2 7-42 days after the first vaccine, and oral administration of an immunologically effective dose of a third vaccine comprising a canine adenovirus type 2 10-14 months after the first vaccine.

[0057] The first vaccine is for example administered subcutaneously. Preferably the first vaccine is administered orally. The composition of the first vaccine, the second vaccine and the third vaccine may be the same or different. The first and the second vaccine may for example be the same, and the composition of the third vaccine may differ from the first vaccine and the second vaccine.

[0058] In an embodiment, the immune response is a protective immune response. In an embodiment of the invention, a further vaccine comprising canine adenovirus type 2 is administered annually after the third vaccine with intervals of 11-13 months.

[0059] The annual administration of the further vaccine ensures that the protective effect of the vaccination is sustained for a longer period of time. The further vaccine may for example be administered subcutaneously. Preferably the further vaccine is administered orally. The composition of the further vaccine may be the same as the composition of the first vaccine, the second vaccine and / or third vaccine. The further vaccine may also have a different composition than the first vaccine, the second vaccine and the third vaccine.

[0060] In an embodiment of the invention, a further vaccine comprising canine adenovirus type 2 is administered every other year after the third vaccine with intervals of 22-26 months, such as with intervals of 23-25 months or intervals of 24 months.

[0061] In an embodiment of the invention, a further vaccine comprising canine adenovirus type 2 is administered every three years after the third vaccine with intervals of 34-38 months, such as with intervals of 35-37 months or intervals of 36 months.

[0062] In an embodiment of the invention, the vaccine additionally comprises NADES.

[0063] It was found that when the third vaccine comprises natural deep eutectic solvent (NADES), there was a stronger increase in the concentration of CAV-1 neutralising antibodies and CAV- 2 neutralising antibodies than in the absence of NADES. Thus, NADES enhances the production of CAV-1 neutralising antibodies and CAV-2 neutralising antibodies in response to the vaccine comprising CAV-2.

[0064] Furthermore, it was found that subcutaneous administration of a third vaccine comprising CDV after a first and second vaccine comprising CDV boosted the level of CDV neutralising antibodies, while oral administration of the third vaccine comprising CDV after the first and second vaccine comprising CDV did not do so. However, as shown in Fig. 3, the addition of NADES to the orally administered third vaccine resulted in a moderate increase in the level of CDV neutralising antibodies after the oral administration of the third vaccine comprising CDV.

[0065] WO2019122329 describes a liquid vaccine of live enveloped viruses comprising NADES. A “deep-eutectic solvent” (DES) is well-known in the art as an ionic liquid comprising a mixture of at least two compounds at a molar ratio that forms a eutectic mixture, whereby the eutectic point of the resulting mixture is significantly lower than the melting points of the individual compounds. This reduction of the melting point of the mixture is caused by the interaction of the compounds, one acting as proton donor, and one other acting as proton acceptor, which provides stable hydrogen-bonding without crystallisation, allowing the mixture to be in liquid form at much reduced temperatures, as compared to its constituents. Commonly ‘eutectic’ means: easy melting.

[0066] For the invention, the individual compounds used to form a DES for the invention have melting points above about 80 °C, and the DES has a melting point below about 40 °C. For example, the melting points of betaine and sucrose are 310 °C and 186 °C respectively, while a NADES formed at a molar ratio for betaine:sucrose of 2:1 with some water included, was found to form a clear liquid that remained fluid even at -20 °C.

[0067] The term “natural” serves to indicate that the compounds used to form the natural DES (NADES) for the invention, are organic compounds that under normal conditions are present in material from biological sources such as plants or animals, in amounts well above trace amounts. Typically such a natural compound is, or is derived from, a primary metabolite that is present in a specific material of vegetable or animal origin. As the skilled person will appreciate, the term natural is only used herein to characterise the initial origin of a compound for use in a NADES for the invention, and not to characterise the way the compound being used was actually sourced. Thus the natural compound may also be employed for the invention when obtained via (semi-) synthetic production. Examples of natural compounds that can be used to form a NADES for the invention, are organic acids, amines, sugars, sugar alcohols, and amino acids.

[0068] The NADES can comprise a combination of a natural organic compound selected from (salts of) organic acids, amines, and amino acids, combined with a polyol such as a sugars and sugar alcohol. Preferably, the NADES comprises an organic salt and a polyol. In this composition, the organic salt acts as the ionic species which is the proton donor, and the polyol acts as the proton acceptor.

[0069] The “organic salt” is a salt of any organic acid or base, including zwitterions, that is within the definition of being a natural compound as presented herein above, and that is capable of forming a deep-eutectic solvent for the invention as described herein. A skilled person is perfectly capable of selecting an organic salt for the present invention, and applying that to form a NADES.

[0070] Further the organic salt should be a pharmaceutically acceptable excipient of a vaccine composition. Such vaccine excipients are for example described in governmental regulations such as the European Pharmacopoeia and the American 9 CFR, and are as such known to the skilled person.

[0071] Preferably, the organic salt is selected from salts of: betaine, proline, carnitine, and choline. ‘Betaine’ refers to the compound N,N,N-trimethylglycine, CAS nr. 107-43-7, which is also known as glycine-betaine. Proline is CAS nr. 609-36-9. Carnitine is CAS nr. 541-15-1. Choline is CAS nr 62-49-7. More preferably, the carnitine is L-carnitine, and / or the choline is cholinechloride (CAS nr. 67-48-1). The organic salt for use in the invention can be used as different salts, isomeric forms, hydrate- or anhydrous forms, etcetera. The skilled person is perfectly capable of selecting and testing a suitable form of the organic salt for use in the invention.

[0072] The compounds for use in the NADES for the invention are readily available in different purities and qualities from a variety of commercial suppliers. Preferably the compound is used in a pharmaceutical grade quality.

[0073] A “polyol” is an organic compound containing two or more hydroxyl groups. However, very large polymers that are under the definition of a polyol, such as cellulose, are not effective at forming a NADES as defined herein, thereby they are excluded for use in the invention. Consequently, polyols for use in the invention have a molecular weight of less than about 10.000 grams per Mole. More preferably polyols for use in the invention have a molecular weight of less than 5000, or even less than 1000 grams per Mole, in this order of preference.

[0074] Preferred polyols are sugars or sugar-alcohols, as these have demonstrated to be versatile components that allow the generation of a variety of effective NADES compositions for use in the invention. A “sugar” for the invention is any compound from the group of water-soluble carbohydrates of relatively low molecular weight that typically have a sweet taste. The term “sugar” includes reducing sugars such as fructose and maltose, as well as non-reducing sugars such as sucrose and trehalose. The term sugar covers mono-, di-, or poly-saccharides up to and including hexa-saccharides. Preferably, the sugar is selected from: fructose, maltose, sucrose, glucose and trehalose.

[0075] The polyol for the invention can also be a sugar alcohol. For the invention “sugar alcohols” are hydrogenated sugars, that comprise 3 or more carbon atoms, and can be based on mono-, di- or polysaccharides. Preferably, the sugar-alcohol is selected from: glycerol, xylitol, mannitol, and sorbitol. Preferably, the sugar is in the D-isoform. Preferably, the sorbitol is D-sorbitol.

[0076] As also described for the organic salts above, the polyol can be used in different isomeric forms, hydrate-or anhydrous forms, etcetera. The skilled person is perfectly capable of selecting and testing a suitable form of a polyol for use in the invention.

[0077] Preferably, the molar ratio between the organic salt and the polyol, as defined herein, is between 1 :5 and 5:1. Even more preferably, the molar ratio between the organic salt and the polyol, as defined herein, is between 1 :4 and 4:1 , between 1 :3 and 3:1 , or even is between 1 :2 and 2:1 , in this order of preference. In an embodiment of the invention, the first vaccine is administered to a canine of 2-12 weeks of age.

[0078] Given the seriousness of infectious canine hepatitis and infectious tracheobronchitis, it is important to protect canines at the earliest age possible against these diseases, and therefore to administer the first vaccine of the vaccine at an early age. The first vaccine is therefore administered at the age of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12 weeks, or at the age of 4-10, 5-9, or 6-8 weeks.

[0079] In an embodiment of the invention, the second vaccine is administered 1-6 weeks after the first vaccine, such as 2-5 or 2, 3, 4 or 5 weeks after the first vaccine. Alternatively, the second vaccine is administered 7, 10, 15, 20, 25, 30, 35, or 40 days after the first vaccine.

[0080] Typically, the antibody response to the first vaccine is not sufficiently strong to provide protection to the canine for a period of longer than several weeks. By administering the second vaccine within a window of 1-6 weeks after the first vaccine, the antibody response elicited by the first vaccine is reinforced and the canine is protected for a longer period of time, such as for a period of months or even up to a year or longer.

[0081] In an embodiment of the invention, the third vaccine is administered 10-14 months after the first vaccine, such as 11 , 12 or 13 months after the first vaccine. Alternatively, the third vaccine is administered 42-60 weeks after the first vaccine, such as 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, or 59 after the first vaccine, or 300-450 days after the first vaccine such as 325-425, 350-450, 375-425 or 400 days after the first vaccine.

[0082] Over time, the antibody level induced by the first and second vaccine gradually reduces. By administering the third vaccine, the immune system is boosted and the canine is protected for an additional period of time, such as for an additional year, an additional two years or an additional three years.

[0083] In an embodiment of the invention, the administration of the first vaccine is subcutaneously.

[0084] The first vaccine is typically administered to canines at the age of 1-3 months, such as at the age of 2 months old, and the first vaccine is typically administered while the canines are still together in a litter.

[0085] In an embodiment of the invention, the canine adenovirus type 2 comprised in the first vaccine, the second vaccine, the third vaccine and / or the further vaccine is an attenuated canine adenovirus type 2.

[0086] CAV-2 field strains stimulate the immune response, but also induce sickness in exposed animals. Attenuated CAV-2 vaccine strains, however, have been shown to stimulate a protective immune response against CAV-2 and CAV-1 in animals exposed to the attenuated CAV-2 whilst reducing the symptoms of illness after exposure to field strains. An attenuated CAV-2 vaccine strain is for example used in the approved vaccine Nobivac® DHP. Preferably, the CAV-2 is CAV-2 strain “Manhattan”.

[0087] In an embodiment of the invention, the first vaccine, the second vaccine, the third vaccine and / or the further vaccine additionally comprises at least one of canine distemper virus, canine parvovirus, canine parainfluenza virus, Bordetella sp, Leptospira interrogans.

[0088] Typically, a canine is not only vaccinated against infectious canine hepatitis and infectious tracheobronchitis, but also against other diseases, such as those caused by CDV, CPV, CPiV and / or Bordetella sp. By including one or more of these in the first vaccine, the second vaccine, the third vaccine and / or the third vaccine, the number of individual vaccines to be administered to a canine can be reduced.

[0089] Preferably, the CDV, CPV and / or CPiV are provided as an attenuated virus in order to stimulate an antibody response in the canine receiving the vaccination without causing serious illness. Preferably, the CDV strain is CDV “Onderstepoort”, the CPV strain is CPV 154 or CPV 630a and / or the CPiV strain is CPiV “Cornell”. Preferably Bordetella species is Bordetella bronchiseptica. Preferably Leptospira interrogans is at least one chosen from serovar Canicola, Icterohaemorrhagiae, Grippotyphosa and Australis.

[0090] In an embodiment of the invention, the first vaccine, the second vaccine, the third vaccine and / or the further vaccine additionally comprises canine distemper virus and canine parvovirus.

[0091] In this way, only one vaccine needs to be administered in order to vaccinate the canine against the diseases against which core vaccines are recommended. Preferably, the first vaccine and the second vaccine comprise canine distemper virus and canine parvovirus.

[0092] In an embodiment of the invention, the first vaccine, the second vaccine, the third vaccine and / or the further vaccine additionally comprises canine distemper virus, canine parvovirus and canine parainfluenza virus.

[0093] Preferably, the first vaccine and the second vaccine comprise canine distemper virus, canine parvovirus and canine parainfluenza virus.

[0094] In an embodiment of the invention, the first vaccine, the second vaccine, the third vaccine and / or the further vaccine additionally comprises canine parainfluenza virus and Bordetella sp. Preferably, the third and / or further vaccine comprise canine parainfluenza and / or Bordetella sp. Even more preferably, the third and further vaccine comprise canine parainfluenza virus and / or Bordetella sp.

[0095] In this way, the known contributors of the canine infectious respiratory disease complex (kennel cough) are combined in one vaccine, thus facilitating vaccination against kennel cough. Preferably, the Bordetella species is Bordetella bronchiseptica.

[0096] In an embodiment of the invention, the first vaccine, the second vaccine, the third vaccine and / or the further vaccine contains canine adenovirus type 2 in an amount of 102-106TCIDso.

[0097] Preferably, the first vaccine, the second vaccine, the third vaccine and / or the further vaccine contains CAV-2 in an amount of 102 5-1055TCID50, such as 103-105TCID50, 1035-1045TCIDso or about 104TCIDso.

[0098] In an embodiment of the invention, the first vaccine, the second vaccine, the third vaccine and / or the further vaccine is administered in a volume of 0.1-5 mL.

[0099] Preferably, the first vaccine, the second vaccine, the third vaccine and / or the further vaccine is administered in a volume of 0.5-4 mL, such as in a volume of 1 , 1.5, 2, 2.5, 3 or 3.5 mL.

[0100] In an embodiment of the invention, the oral administration of the third vaccine is about 1 year after the administration of the first vaccine.

[0101] Generally, the protective effect of the primary vaccination diminishes over time. By administering the third vaccine about 1 year after the administration of the first vaccine, the protective effect conferred by the primary vaccination is prolonged.

[0102] In an embodiment of the invention, the first vaccine, the second vaccine, the third vaccine and / or the further vaccine additionally comprises a pharmaceutically acceptable adjuvant.

[0103] The adjuvant is for example aluminum hydroxide or saponin, or an oil-based adjuvants, such as Freund’s complete adjuvant or Freund’s incomplete adjuvant.

[0104] In an embodiment of the invention, the first vaccine, the second vaccine, the third vaccine and / or the further vaccine additionally comprises a pharmaceutically acceptable carrier.

[0105] The carrier is for example a solvent, dispersion medium or diluent. These are generally known in the art. Typically, the carrier will be sterile and pyrogen-free, and selected based on the mode of administration to be used.

[0106] In an embodiment, the canine is a dog. Examples

[0107] Example 1

[0108] This study evaluated the use of a third vaccine comprising CAV-2, where a first vaccine comprising CAV-2 is administered subcutaneously, a second vaccine comprising CAV-2 is administered subcutaneously and a third vaccine comprising CAV-2 is administered orally.

[0109] 1.1. Vaccines

[0110] The first vaccine and second vaccine used as primary vaccines in this study are described in table 1.

[0111] The third vaccines used as booster vaccine in this study are described in table 2.

[0112] Table 2. Composition of booster vaccines. 7.2. Vaccine schedule and preparation

[0113] 16 beagle pups from four litters were used for this study. Table 1 shows the vaccination schedule.

[0114] The Primary DHP vaccine was supplied in a freeze-dried form. The vaccine vials were each reconstituted in 1 ml of Nobivac® L4 as diluent.

[0115] The Primary Nobivac® KC vaccine was supplied in freeze-dried form. Each vial of freeze-dried Nobivac® KC vaccine was resuspended in 1.0 ml Nobivac® KC Diluent (supplied with the vaccine).

[0116] The Booster DHP vaccine was supplied in freeze-dried form. Each vial of freeze-dried booster DHP vaccine was resuspended in 1.0 ml Nobivac® Solvent. The resuspended vaccines were then pooled.

[0117] The Booster Nobivac® KC vaccine was supplied in freeze-dried form. Each vial of freeze-dried Nobivac® KC vaccine was resuspended in 1.0 ml Nobivac® KC Diluent (supplied with the vaccine). The resuspended vaccines were then pooled.

[0118] The NADES DHPPi vaccine was supplied as a ready to use liquid in a multidose vial comprising proline, sorbitol and methionine.

[0119] The Nobivac® Respira Bb was supplied as a ready to use liquid in a multidose vial. At the age of 5-6 weeks, all pups received a first dose of 1 mL Primary DHP vaccine resuspended in Nobivac®L4 subcutaneously, and 0.4 mL Primary Nobivac® vaccine intranasally. 28 days after the first vaccination, the dogs received a second dose of 1 mL Primary DHP vaccine resuspended in Nobivac®L4 subcutaneously.

[0120] 1.3. Treatment and sample collection

[0121] Before administration of the third dose, the dogs were divided in three groups, based on their serological status against CDV, CAV and CPi. Groups were composed in such a way that a similarity of serological responses (after 1 year) of the dogs in all groups was achieved.

[0122] On day 417 after administration of the first dose, 389 days after completion of the primary vaccination course, the dogs in group 1 were vaccinated subcutaneously with 1 mL Booster DHP vaccine and orally in the buccal pouch with 1 mL Booster Nobivac® KC vaccine. The dogs in group 2 were vaccinated orally in the buccal pouch with 1 mL Booster DHP vaccine and subcutaneously with 1 mL Nobivac® Respira Bb vaccine. The dogs ingroup 3 were vaccinated orally in the buccal pouch with 1 mL NADES DHPPi vaccine and subcutaneously with 1 mL Nobivac® Respira Bb vaccine. Blood was sampled from a superficial vein at predetermined time points and allowed to clot for several hours at ambient temperature or overnight at 2-8 °C. After clotting, serum was collected by centrifugation.

[0123] 1.4. Serological analysis

[0124] 1.4.1. CAV-1 and CAV-2

[0125] Antibodies to CAV-1 and CAV-2 were determined in a virus neutralisation assay. CAV-1 neutralising antibody titres and CAV-2 neutralising antibody titres were detected by making serial serum dilutions and incubating them with an equal volume of a known titre suspension of CAV-1 or CAV-2, respectively. After incubation, the samples were inoculated into wells preseeded with MDCK cells in 96-well plates and incubated at 37 °C + 5% CO2 for 6 days. The presence of virus within the tissue culture media was measured by a haemagglutination assay (HA) of Human type O red blood cells. The wells were scored positive or negative for agglutination. The VN50 titre of the serum, based on HA, was calculated by Reed & Muench. The VN50 titre of neutralising antibodies against CAV-1 or CAV-2 in serum is the reciprocal of the dilution calculated to be the 50% end point.

[0126] A positive reference serum, negative reference serum, a virus back titration and negative cell- only wells were included as assay controls. 1.4.2. CDV

[0127] Antibodies to CDV were determined in a virus neutralisation assay. CDV neutralising antibody titres were detected by making serial serum dilutions and incubating them with an equal volume of a known titre suspension of CDV. After incubation, the samples were inoculated into wells pre-seeded with Vero cells in 96-well plates and incubated at 37 °C + 5% CO2 for 5 days. Infection of Vero cells by CDV virus results in a cytopathic effect (CPE) that is recognizable by microscopy. The wells were scored positive or negative for CPE and the VN50 titre of the serum was calculated by Reed & Muench. The VN50 titre of neutralizing antibodies against CDV in the test sample is the reciprocal of the dilution calculated to be the 50% end point.

[0128] A positive reference serum, negative reference serum, a virus back titration and negative cell- only wells were included as assay controls.

[0129] 1.4.3. CPV

[0130] Antibodies to CPV were determined by haemagglutination inhibition assay (HAI) in accordance with standard procedure. The HAI assay measures the ability of serum CPV antibodies to inhibit the agglutination of pig red blood cells. Briefly, pre-diluted serum samples were adsorbed against porcine red blood cells to block any non-specific haemagglutination. The test serum samples were serially diluted across a 96-well plate and incubated firstly with a constant amount of CPV antigen (8 HA units), followed by an incubation with porcine red blood cells. The inhibition of haemagglutination is characterised by tear-dropping of the pig blood cells. The HAI titre of the sample will be the reciprocal of the last dilution at which inhibition of agglutination occurs (<50% agglutination).

[0131] A positive reference serum and negative reference serum were included as assay controls. Furthermore, a positive HA and negative HA control were present on each plate.

[0132] 1.4.4. CPi

[0133] Antibodies to CPiV were determined in a virus neutralisation assay. CPiV VN50 antibody titre were detected by making serial serum dilutions and incubating them with an equal volume of a known titre suspension of CPi. After incubation, the samples were inoculated into wells preseeded with Vero cells in 96-well plates and incubated at 37 °C + 5% CO2 for 6 days. Infection of Vero cells by CPiV results in a cytopathic effect (CPE) that is recognizable by microscopy. The wells were scored positive or negative for CPE and the VN50 titre of the serum was calculated by Reed & Muench. The VN50 titre of neutralising antibodies against CPi in the test sample is the reciprocal of the dilution calculated to be the 50% end point. A positive reference serum, negative reference serum, a virus back titration and negative cell- only wells were included as assay controls.

[0134] 1.4.5. Bordetella bronchiseptica

[0135] Bordetella bronchiseptica antibody titres were determined by enzyme-linked immunosorbent assay (ELISA). An ELISA is a chromogenic assay that measures the ability of serum antibodies to bind to a known amount of antigen. Briefly, the ELISA plates were coated with purified Bordetella antigen and incubated at 37 °C overnight. The plates were blocked, washed and the pre-diluted test sera was added. Positive and negative sera controls were included. The test sera samples were diluted across the plate and incubated for one hour at 37 °C. An antidog peroxidase conjugate was added, and the plates were incubated for 30 minutes at 37 °C. Subsequently, 3,3',5,5'-tetramethylbenzidine substrate was added, and the plates were incubated for 15 minutes at room temperature in the dark. The colour reaction was stopped by the addition of 4 N sulphuric acid and the absorbances read at a 450 nm optical density. The antibody titres were calculated against the negative reference serum values and at the intercept of the cut-off, as a Iog2 dilution value (absorbance 450 nm).

[0136] A positive reference serum and negative reference serum were included as assay controls.

[0137] 1.4.6. Leptospira interrogans

[0138] The sera were examined for agglutinating antibodies against Leptospira interrogans (sensu lato) serogroups using the Microscopic Agglutination Test (MAT). Titres of agglutinating antibodies against the following four serogroups were determined: Canicola, Icterohaemorrhagiae, Grippotyphosa and Australis.

[0139] In short, the MAT was performed as follows: The MAT is used to detect agglutinating serum antibodies specifically directed against Leptospira serogroups and determine their titres. Serial dilutions of the dog serum were incubated with live antigen of relevant serogroups of Leptospira. After this, the titre was determined, being the Iog2 value of the reciprocal of the highest dilution in which the serum-antigen mixture shows 50% agglutinated (non-motile) leptospires. Serogroup-specific positive rabbit antisera and one negative rabbit antiserum were used as control sera.

[0140] Positive reference sera, a negative reference serum and reference wells were included as assay controls.

[0141] 1.5. Results

[0142] 1.5.1. CAV-1 and CAV-2 Figs. 1A and 1 B show the average CAV-2 VN5o titre in dogs in response to the first, second and third vaccination. As shown in Fig. 1A, the CAV-2 VN5o titre rises after administration of the second vaccine, but decreases from about 270 days after administration of the first vaccine. As shown in Fig. 1 B, subcutaneous administration of the booster 417 days after administration of the first vaccine resulted in a slight increase of the CAV-2 VN5o titre (group 1). When the booster was administered orally however, the increase in the CAV-2 VN5o titre was much stronger when compared to that of the subcutaneously administered booster (group 2), and even more so when the oral booster comprised NADES (group 3).

[0143] Figs. 2A and 2B show the average CAV-1 VN50 titre in dogs in response to the first, second and third vaccination. As shown in Fig. 2A and similar to the CAV-2 VN5o titre profile, the CAV- 1 VN5o titre rises after administration of the second vaccine, but decreases from about 270 days after administration of the first vaccine. As shown in Fig. 2B, subcutaneous administration of the booster 417 days after administration of the first vaccine resulted in a slight increase of the CAV-1 VN50 titre (group 1). When the booster was administered orally however, the increase in the CAV-1 VN50 titre was much stronger when compared to that of the subcutaneously administered booster (group 2), and even more so when the oral booster comprised NADES (group 3).

[0144] 1.5.2. CDV

[0145] Fig. 3 shows the average CDV VN50 titre in dogs in response to the first, second and third vaccination. The CDV VN50 titre rises after administration of the first vaccine, and remains virtually constant up to the administration of the third vaccine. Subcutaneous administration of the third vaccine strongly increases the CDV VN50 titre (group 1), while oral administration of the third vaccine not comprising NADES did not increase the CDV VN50 titre (group 2). Oral administration of the third vaccine comprising NADES slightly increased the CDV VN50 titre, but to a lesser extent than subcutaneous administration (group 3).

[0146] 1.5.3. CPV

[0147] Fig. 4 shows the average CPV HAI units in dogs in response to the first, second and third vaccination. The HAI units increase after administration of the first and second vaccine, and decreases after about 239 days after administration of the first vaccine. After administration of the third vaccine, the HAI units increased to the same degree, irrespective of subcutaneous or oral administration or the presence or absence of NADES in the oral formulation.

[0148] 1.5.4. CPi

[0149] Fig. 5 shows the average CPi VN50 titre in dogs in response to the first and third vaccination.

[0150] The CPi VN50 titre increases after intranasal administration of the first vaccine. Compared to the group which did not receive a CPi booster (group 2), oral administration of the third vaccine did not significantly increase the CPi VN50 titre (group 1 and group 3).

[0151] 1.5.5. B. Bronchiseptica

[0152] Fig. 6 shows the average B. bronchiseptica antibody titre in dogs in response to the first and third vaccination. The antibody titre increases after intranasal administration of the first vaccine, while administration of the second vaccine does not result in a stronger increase. Subcutaneous administration of the third vaccine resulted in a strong increase in the antibody titre (groups 2 and 3), while oral administration only marginally increased the antibody titre (group 1).

Claims

Claims1. A vaccine for use in a method of inducing an immune response in a canine against infectious canine hepatitis and / or infectious tracheobronchitis, wherein the vaccine is a first vaccine comprising a canine adenovirus type 2, and the method comprises: administration of an immunologically effective dose of the first vaccine, subcutaneous administration of an immunologically effective dose of a second vaccine comprising a canine adenovirus type 2 7-42 days after the first vaccine, and oral administration of an immunologically effective dose of a third vaccine comprising a canine adenovirus type 2 10-14 months after the first vaccine.

2. The vaccine for use according to claim 1 , wherein the immune response is a protective immune response.

3. The vaccine for use according to claim 1 or 2, wherein a further vaccine comprising canine adenovirus type 2 is administered annually after the third vaccine with intervals of 11-13 months.

4. The vaccine for use according to any of the preceding claims, wherein the first vaccine, the second vaccine, the third vaccine and / or the further vaccine additionally comprises NADES.

5. The vaccine for use according to any of the preceding claims, wherein the first vaccine is administered to a canine of 2-12 weeks of age.

6. The vaccine for use according to any of the preceding claims, wherein the administration of the first vaccine is subcutaneously.

7. The vaccine for use according to any of the preceding claims, wherein the canine adenovirus type 2 contained in the first vaccine, the second vaccine, the third vaccine and / or the further vaccine is an attenuated canine adenovirus type 2.

8. The vaccine for use according to any of the preceding claims, wherein the oral administration of the third vaccine is about 1 year after the administration of the first vaccine.The vaccine for use according to any of the preceding claims, wherein the first vaccine, the second vaccine, the third vaccine and / or the further vaccine additionally comprises at least one of canine distemper virus, canine parvovirus, canine parainfluenza virus, Bordetella sp, and Leptospira interrogans. The vaccine for use according to any of the preceding claims, wherein the first vaccine, the second vaccine, the third vaccine and / or the further vaccine additionally comprises canine parainfluenza virus and / or Bordetella sp. The vaccine for use according to any of the preceding claims, wherein the first vaccine, the second vaccine, the third vaccine and / or the further vaccine additionally comprises canine distemper virus, canine parvovirus and canine parainfluenza virus. The vaccine for use according to any of the preceding claims, wherein the first vaccine, the second vaccine, the third vaccine and / or the further vaccine additionally comprises a pharmaceutically acceptable adjuvant. The vaccine for use according to any of the preceding claims, wherein the first vaccine, the second vaccine, the third vaccine and / or the further vaccine additionally comprises a pharmaceutically acceptable carrier. The vaccine for use according to any of the preceding claims, wherein the first vaccine, the second vaccine, the third vaccine and / or the further vaccine contains canine adenovirus type 2 in an amount of 102-106TCID50. The vaccine for use according to any of the preceding claims, wherein the first vaccine, the second vaccine, the third vaccine and / or the further vaccine is administered in a volume of 0.1-5 mL.

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