Vaccine for vaccinating canine animals

Abstract

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

Description

Technical Field

[0001] The present invention relates to a vaccine for use in a method of inducing an immune response against canine infectious hepatitis and / or infectious tracheobronchitis in canines. Background Art

[0002] Canine adenovirus type 1 (CAV-1) and type 2 (CAV-2) are double-stranded DNA viruses that cause two major canine infectious diseases. CAV-1 causes canine infectious hepatitis (ICH), while CAV-2 is one of the viruses that cause infectious tracheobronchitis.

[0003] ICH is an acute liver infection in dogs, but it is also present in wolves, foxes, coyotes, bears, and skunks. CAV-1 is transmitted through the feces, urine, blood, saliva, and nasal secretions of infected animals. After being contracted through the mouth or nose, it replicates in the tonsils and then infects the liver and kidneys. Although most animals recover spontaneously from ICH, hemorrhagic and liver diseases caused by CAV-1 infection are fatal in some.

[0004] Infectious tracheobronchitis caused by CAV-2 is a mild but contagious disease limited to the canine respiratory tract. Its main characteristics are coughing, nasal discharge, bronchitis, and bronchiolitis, and the virus is considered one of the causative agents of the canine infectious respiratory disease complex (kennel cough). CAV-2 spreads through aerosols produced by sneezing and coughing dogs.

[0005] To prevent canines from developing ICH or infectious tracheobronchitis, canines at 6 weeks of age are vaccinated with a vaccine containing CAV-2. Considering the high genetic similarity between CAV-1 and CAV-2, these vaccines also provide protection against ICH induced by CAV-1. Vaccines against ICH and infectious tracheobronchitis, as well as vaccines against canine distemper virus (CDV) and canine parvovirus (CPV), are considered core vaccines recommended for all canines worldwide. Typically, puppies are initially vaccinated at six to eight weeks of age, followed by a second vaccination within 3 - 4 weeks after the first vaccination. Then, vaccination is usually repeated annually to three years.

[0006] In addition to the viruses included in the core vaccines, vaccination against other viruses is recommended depending on the geographical location, local environment, or lifestyle of a particular canine. These non-core vaccines include, for example, vaccines against canine parainfluenza virus (CPiV), or the bacterium Bordetella bronchiseptica, Leptospira canicola type, Leptospira icterohaemorrhagiae type, Leptospira grippotyphosa type, and Leptospira australis type. CPiV and Bordetella bronchiseptica are other causative agents in the canine infectious respiratory disease complex, while bacteria of the genus Leptospira cause blood infections that can develop into severe lung diseases.

[0007] Although subcutaneous administration is the traditional way to administer core and non-core vaccines, other ways to facilitate vaccine delivery and / or mimic the natural pathways of viral infection are being explored. For example, EP2762163 describes a method of vaccinating dogs with a vaccine comprising CAV-2 to resist ICH caused by CAV-1. The vaccine is administered subcutaneously in a first dose, a second dose is administered orally 7 - 42 days after the first dose, and annual doses are administered orally.

[0008] However, there is still a need for vaccines against ICH and infectious tracheobronchitis that combine a convenient way of administering the vaccine with good protection against ICH and infectious tracheobronchitis. SUMMARY OF THE INVENTION

[0009] To this end, there is provided a method for inducing an immune response against canine infectious hepatitis and / or infectious tracheobronchitis in canines, wherein the vaccine is a first vaccine comprising canine adenovirus type 2, and the method comprises:

[0010] - administering an immunologically effective dose of the first vaccine,

[0011] - administering an immunologically effective dose of a second vaccine comprising canine adenovirus type 2 subcutaneously 7 - 42 days after the first vaccine, and

[0012] - administering an immunologically effective dose of a third vaccine comprising canine adenovirus type 2 orally 10 - 14 months after the first vaccine.

[0013] It has been 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 results in a strong antibody response against CAV-1 and CAV-2.

[0014] As shown in Figure 1, the concentration of CAV-2 neutralizing antibodies gradually increases after immunization by the second subcutaneous administration to dogs. Subcutaneous administration of the third vaccine increases the concentration of CAV-2 neutralizing antibodies. However, surprisingly, when the third vaccine is administered orally, this enhancing effect is stronger.

[0015] As shown in Figure 2, neutralizing CAV-1 antibodies are also produced after administering the first and second vaccines comprising CAV-2. For CAV-2, after subcutaneous administration of the third vaccine comprising CAV-1, the concentration of CAV-1 neutralizing antibodies increases significantly. However, compared with subcutaneous administration of the third vaccine, oral administration of the third vaccine again results in a stronger antibody response.

[0016] In contrast, as Figure 3As shown, administration of a first vaccine and a second vaccine containing canine distemper virus (CDV) results in an increase in the concentration of CDV-neutralizing antibodies. However, the concentration of CDV-neutralizing antibodies increases only when a third vaccine containing CDV is administered subcutaneously. Oral administration of the third vaccine containing CDV has no significant effect on the concentration level of CDV-neutralizing antibodies.

[0017] As Figure 4 shown, administration of a first vaccine and a second vaccine containing canine parvovirus (CPV) also results in the induction of anti-CPV antibodies. However, oral administration of the third vaccine containing CPV does not increase the anti-CPV antibody level to a level higher than that induced by subcutaneous administration of the third vaccine containing CPV.

[0018] In view of the above, it is very surprising that oral administration of the third vaccine containing CAV-2 results in such a strong and unexpected increase in the antibody levels against CAV-1 and CAV-2. Detailed Description

[0019] A vaccine for use in a method of inducing an immune response against canine infectious hepatitis and / or infectious tracheobronchitis in canines, wherein the vaccine is a first vaccine containing canine adenovirus type 2, and the method comprises:

[0020] - Administering an immunologically effective dose of the first vaccine,

[0021] - Subcutaneously administering an immunologically effective dose of a second vaccine containing canine adenovirus type 2 7 - 42 days after the first vaccine, and

[0022] - Orally administering an immunologically effective dose of a third vaccine containing canine adenovirus type 2 10 - 14 months after the first vaccine.

[0023] In one embodiment, the immune response is a protective immune response.

[0024] In one embodiment of the present invention, wherein an additional vaccine containing canine adenovirus type 2 is administered annually at an interval of 11 - 13 months after the third vaccine.

[0025] In one embodiment of the present invention, wherein the first vaccine, the second vaccine, the third vaccine, and / or the additional vaccine further comprise NADES.

[0026] In one embodiment of the present invention, wherein the first vaccine is administered to canines at 2 - 12 weeks of age.

[0027] In one embodiment of the present invention, the second vaccine is administered 1 - 6 weeks after the first vaccine.

[0028] In one embodiment of the present invention, the second vaccine is administered 7, 10, 15, 20, 25, 30, 35 or 40 days after the first vaccine.

[0029] In one embodiment of the present invention, the third vaccine is administered 10 - 14 months after the first vaccine.

[0030] In one embodiment of the present invention, the third vaccine is administered 42 - 60 weeks after the first vaccine.

[0031] In one embodiment of the present invention, the administration of the first vaccine is subcutaneous administration.

[0032] In one embodiment of the present invention, the canine adenovirus type 2 contained in the first vaccine, the second vaccine, the third vaccine and / or the additional vaccine is an attenuated canine adenovirus type 2.

[0033] In one embodiment of the present invention, the first vaccine, the second vaccine, the third vaccine and / or the additional vaccine further comprises at least one of canine distemper virus, canine parvovirus, canine parainfluenza virus, Bordetella and Leptospira interrogans.

[0034] In one embodiment of the present invention, the first vaccine, the second vaccine, the third vaccine and / or the additional vaccine further comprises canine distemper virus and canine parvovirus.

[0035] In one embodiment of the present invention, the first vaccine, the second vaccine, the third vaccine and / or the additional vaccine further comprises canine parainfluenza virus and Bordetella.

[0036] In one embodiment of the present invention, the first vaccine, the second vaccine, the third vaccine and / or the additional vaccine contains 10 2 -10 6 TCID 50 of canine adenovirus type 2.

[0037] In one embodiment of the present invention, the first vaccine, the second vaccine, the third vaccine and / or the additional vaccine is administered in a volume of 0.1 - 5 mL.

[0038] In one embodiment of the present invention, the oral administration of the third vaccine is about 1 year after the administration of the first vaccine.

[0039] In one embodiment of the present invention, the first vaccine, the second vaccine, the third vaccine and / or the additional vaccine further comprises a pharmaceutically acceptable adjuvant.

[0040] In one embodiment of the present invention, the first vaccine, the second vaccine, the third vaccine, and / or the additional vaccine further comprises a pharmaceutically acceptable carrier.

[0041] In one embodiment, a canine is a dog.

[0042] Definition

[0043] A "vaccine" is a composition comprising an antigen. Administering a vaccine to a subject results in the induction of an immune response against the antigen in the subject. It can be administered in various ways, including subcutaneous, oral, or intranasal administration. The induction of the immune response can be humoral and / or cell-mediated. The immune response can be evaluated, for example, by measuring antibody titers.

[0044] "Vaccination" is the administration of a vaccine to a subject.

[0045] "Vaccination" is the act of administering a vaccine to a subject.

[0046] A "primary vaccine" is a vaccine that is used to induce an initial protective immune response in a subject and results in at least temporary protective immunity. Multiple doses may be required to achieve protective immunity.

[0047] A "booster vaccine" is a vaccine that is used to stimulate the immune response previously induced by a primary vaccine in a subject to extend the protective immunity conferred by the primary vaccine.

[0048] An "antigen" is a molecule containing one or more epitopes that, when administered to a subject, will induce an immune response specific to that antigen. Antigens are, for example, inactivated, attenuated, or killed viruses or bacteria, or their antibodies or fragments.

[0049] An "epitope" is a specific site on an antigen to which a T cell receptor or a specific antibody binds.

[0050] An "antibody" is an immunoglobulin molecule that binds to a specific antigen. Antibodies contain light and heavy chains with constant and variable regions and can be subclassified based on the composition of the constant region, such as IgA, IgD, IgE, IgG, IgM.

[0051] A "neutralizing antibody" is an antibody that binds to a pathogen such as a virus or bacteria and blocks the infectivity of the pathogen.

[0052] An "antibody response" is an immune response that results in the production of antibodies against an antigen.

[0053] An "immune response" is the activation of the immune system in response to...

[0054] "Immune effective dose" is the amount of an antigen or vaccine that induces an immune response in a subject to which it is administered, which is sufficient to prevent signs or symptoms of a disease caused by infection with a pathogen such as a virus or bacterium.

[0055] "Protective immune response" is an immune response that is sufficient to prevent signs or symptoms of a disease caused by infection with a pathogen such as a virus or bacterium.

[0056] "Protective immunity" is immunity that prevents signs or symptoms of a disease caused by infection with a pathogen such as a virus or bacterium.

[0057] "Antibody titer" is a measure of the level of antibodies, indicating how much a sample containing antibodies can be diluted before the antibodies are no longer detectable.

[0058] "Pharmaceutically acceptable" applies to a subject and has no undue effects relative to the benefits, such as toxicity, irritation, and / or allergic reaction.

[0059] "Adjuvant" is a pharmaceutically acceptable compound or composition that increases the immune response to an antigen.

[0060] Embodiments of the present invention

[0061] The present invention relates to a method for inducing an immune response against canine infectious hepatitis and / or infectious tracheobronchitis in canines using a vaccine, wherein the vaccine is a first vaccine comprising canine adenovirus type 2, and the method comprises:

[0062] - administering an immune effective dose of the first vaccine,

[0063] - subcutaneously administering an immune effective dose of a second vaccine comprising canine adenovirus type 2 7 - 42 days after the first vaccine, and

[0064] - orally administering an immune effective dose of a third vaccine comprising canine adenovirus type 2 10 - 14 months after the first vaccine.

[0065] The first vaccine is administered subcutaneously, for example. Preferably, the first vaccine is administered orally. The compositions of the first, second, and third vaccines may be the same or different. The first and second vaccines may be the same, for example, and the composition of the third vaccine may be different from that of the first and second vaccines.

[0066] In one embodiment, the immune response is a protective immune response.

[0067] In one embodiment of the present invention, an additional vaccine comprising canine adenovirus type 2 is administered annually at an interval of 11 - 13 months after the third vaccine.

[0068] Annual administration of additional vaccines ensures that the protective effect of vaccination is maintained for a longer period. The additional vaccines can be administered, for example, subcutaneously. Preferably, the additional vaccines are administered orally. The composition of the additional vaccines can be the same as that of the first, second, and / or third vaccines. The additional vaccines can also have a composition different from that of the first, second, and third vaccines.

[0069] In one embodiment of the present invention, an additional vaccine comprising canine adenovirus type 2 is administered every two years at an interval of 22 - 26 months after the third vaccine, for example, at an interval of 23 - 25 months or 24 months.

[0070] In one embodiment of the present invention, an additional vaccine comprising canine adenovirus type 2 is administered every three years at an interval of 34 - 38 months after the third vaccine, for example, at an interval of 35 - 37 months or 36 months.

[0071] In one embodiment of the present invention, the vaccine further comprises NADES.

[0072] It has been found that when the third vaccine contains natural deep eutectic solvents (NADES), the increase in the concentration of CAV-1 neutralizing antibodies and CAV-2 neutralizing antibodies is stronger than that in the absence of NADES. Thus, NADES enhances the production of CAV-1 neutralizing antibodies and CAV-2 neutralizing antibodies in response to a vaccine containing CAV-2.

[0073] Furthermore, it has been found that subcutaneous administration of a third vaccine containing CDV after administration of the first and second vaccines containing CDV increases the level of CDV neutralizing antibodies, while oral administration of a third vaccine containing CDV after administration of the first and second vaccines containing CDV does not increase the level of CDV neutralizing antibodies. However, as Figure 3 shown, adding NADES to the orally administered third vaccine results in a moderate increase in the level of CDV neutralizing antibodies after oral administration of the third vaccine containing CDV.

[0074] WO2019122329 describes a liquid vaccine of a live enveloped virus containing NADES. It is well known in the art that a "deep eutectic solvent" (DES) is an ionic liquid containing a mixture of at least two compounds in a molar ratio to form a eutectic mixture, whereby the eutectic point of the resulting mixture is significantly lower than the melting points of the individual compounds. This decrease in the melting point of the mixture is caused by the interaction of the compounds (one acting as a proton donor and the other as a proton acceptor), which provides stable hydrogen bonds and does not crystallize, such that the mixture is in liquid form at much lower temperatures compared to its constituent components. Generally, 'eutectic' means: easily melted.

[0075] For the present invention, each of the compounds used to form the DES of the present invention has a melting point 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, and it was found that a NADES containing some water formed at a molar ratio of betaine:sucrose of 2:1 forms a clear liquid that remains liquid even at -20 °C.

[0076] The term "natural" is used to denote the compounds used to form the natural DES (NADES) of the present invention, which are organic compounds that are present in materials from biological sources such as plants or animals in amounts far above trace levels under normal conditions. Typically, such natural compounds are or are derived from primary metabolites present in specific materials of plant or animal origin. As will be understood by those skilled in the art, the term natural is used herein only to characterize the initial source of the compounds in the NADES of the present invention, and not the manner in which the compounds are actually sourced. Thus, natural compounds can also be used in the present invention when obtained by (semi)-synthetic production. Examples of natural compounds that can be used to form the NADES of the present invention are organic acids, amines, sugars, sugar alcohols, and amino acids.

[0077] The NADES can comprise a combination of natural organic compounds (salts thereof) selected from organic acids, amines, and amino acids with polyols such as sugars and sugar alcohols. Preferably, the NADES comprises an organic salt and a polyol. In this composition, the organic salt acts as an ionic substance that is a proton donor, and the polyol acts as a proton acceptor.

[0078] "Organic salt" is a salt of any organic acid or base (including zwitterions) that is within the definition of natural compounds as presented above herein and that is capable of forming a deep eutectic solvent for the present invention as described herein. Those skilled in the art are fully capable of selecting the organic salts for the present invention and applying them to form NADES.

[0079] Furthermore, the organic salt should be a pharmaceutically acceptable excipient for the vaccine composition. Such vaccine excipients are described, for example, in government regulations such as the European Pharmacopoeia and US 9CFR and are known to those skilled in the art.

[0080] Preferably, the organic salt is a salt selected from: 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 has a CAS nr. 609-36-9. Carnitine has a CAS nr. 541-15-1. Choline has a CAS nr. 62-49-7. More preferably, carnitine is L-carnitine, and / or choline is choline chloride (CAS nr. 67-48-1). The organic salts used in the present invention can be used in different salt, isomeric forms, hydrate or anhydrous forms, etc. Those skilled in the art are fully capable of selecting and testing the appropriate forms of the organic salts for use in the present invention.

[0081] The compounds for use in the NADES of the present invention can be readily obtained from various commercial suppliers in different purities and qualities. Preferably, the compounds are used in pharmaceutical grade quality.

[0082] "Polyol" is an organic compound containing two or more hydroxyl groups. However, very large polymers such as cellulose under the definition of polyol are not effective in forming NADES as defined herein, and thus they are excluded from use in the present invention. Therefore, the polyol used in the present invention has a molecular weight of less than about 10,000 grams per mole. More preferably, the polyol used in the present invention has a molecular weight of less than 5000, or even less than 1000 grams per mole, in this preferred order.

[0083] Preferred polyols are sugars or sugar alcohols, as these have proven to be versatile components that allow for the production of various effective NADES compositions for the present invention. "Sugar" for use in the present invention is any compound from the group of relatively low molecular weight water-soluble carbohydrates that are generally sweet. The term "sugar" includes reducing sugars such as fructose and maltose, and non-reducing sugars such as sucrose and trehalose. The term sugar includes monosaccharides, disaccharides or polysaccharides, up to hexasaccharides. Preferably, the sugar is selected from: fructose, maltose, sucrose, glucose, and trehalose.

[0084] The polyol used in the present invention can also be a sugar alcohol. For the present invention, "sugar alcohol" is a hydrogenated sugar containing 3 or more carbon atoms, and can be based on monosaccharides, disaccharides or polysaccharides. Preferably, the sugar alcohol is selected from: glycerol, xylitol, mannitol, and sorbitol. Preferably, the sugar is the D-isomer. Preferably, sorbitol is D-sorbitol.

[0085] Also as described above for the organic salts, the polyol can be used in different isomeric forms, hydrate or anhydrous forms, etc. Those skilled in the art are fully capable of selecting and testing the appropriate forms of the polyol for use in the present invention.

[0086] Preferably, as defined herein, the molar ratio between the organic salt and the polyol is between 1:5 and 5:1. Even more preferably, in this order of preference, as defined herein, the molar ratio between the organic salt and the polyol is between 1:4 and 4:1, between 1:3 and 3:1, or even between 1:2 and 2:1.

[0087] In one embodiment of the invention, a first vaccine is administered to canines from 2 to 12 weeks of age.

[0088] Given the severity of canine infectious hepatitis and infectious tracheobronchitis, it is important to protect canines against these diseases at the earliest possible age and thus administer the first vaccine in the vaccine at an earlier age. Thus, the first vaccine is administered at 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks of age, or at 4 - 10, 5 - 9, or 6 - 8 weeks of age.

[0089] In one embodiment of the invention, the second vaccine is administered 1 - 6 weeks after the first vaccine, for example 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.

[0090] Typically, the antibody response to the first vaccine is not sufficient to provide protection for the canine for more than a few weeks. By administering the second vaccine within the window of 1 - 6 weeks after the first vaccine, the antibody response induced by the first vaccine is enhanced and provides protection for the canine for a longer period, such as several months or even up to a year or longer.

[0091] In one embodiment of the invention, the third vaccine is administered 10 - 14 months after the first vaccine, for example 11, 12, or 13 months after the first vaccine. Alternatively, it is administered 42 - 60 weeks after the first vaccine, for example at 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, or 59 weeks after the first vaccine, or 300 - 450 days after the first vaccine, for example at 325 - 425, 350 - 450, 375 - 425, or 400 days after the first vaccine.

[0092] Over time, the antibody levels induced by the first and second vaccines gradually decrease. By administering the third vaccine, the immune system is strengthened and the canine is protected for an additional period, such as an additional year, an additional two years, or an additional three years.

[0093] In one embodiment of the invention, the administration of the first vaccine is subcutaneous administration.

[0094] The first vaccine is typically administered to canines between 1 and 3 months of age, such as 2 months of age, and the first vaccine is typically administered while the canines are still in the litter.

[0095] In one embodiment of the present invention, the canine adenovirus type 2 contained in the first vaccine, the second vaccine, the third vaccine and / or the additional vaccine is an attenuated canine adenovirus type 2.

[0096] CAV-2 wild strains stimulate immune responses but also induce disease in exposed animals. However, attenuated CAV-2 vaccine strains stimulate protective immune responses against CAV-2 and CAV-1 in animals exposed to attenuated CAV-2, while reducing disease symptoms following exposure to wild strains. Attenuated CAV-2 vaccine strains are used, for example, in licensed vaccines DHP. Preferably, the CAV-2 is the CAV-2 strain "Manhattan".

[0097] In one embodiment of the present invention, the first vaccine, the second vaccine, the third vaccine and / or the additional vaccine further comprises at least one of canine distemper virus, canine parvovirus, canine parainfluenza virus, Bordetella and Leptospira interrogans.

[0098] Typically, canines are not only vaccinated against canine infectious hepatitis and infectious tracheobronchitis, but also against other diseases, such as those caused by CDV, CPV, CPiV and / or Bordetella. By including one or more of the foregoing vaccines in the first vaccine, the second vaccine, the third vaccine and / or the third vaccine, the number of vaccines administered to individual canines can be reduced.

[0099] Preferably, CDV, CPV and / or CPiV are provided as attenuated viruses to stimulate an antibody response in canines receiving the vaccination without causing severe disease. Preferably, the CDV strain is CDV "Onderstepoort", the CPV strain is CPV154 or CPV 630a and / or the CPiV strain is CPiV "Cornell". Preferably, the Bordetella species is Bordetella bronchiseptica. Preferably, Leptospira interrogans is at least one selected from serotypes Canicola, Icterohaemorrhagiae, Grippotyphosa and Australis.

[0100] In one embodiment of the present invention, the first vaccine, the second vaccine, the third vaccine and / or the additional vaccine further comprises canine distemper virus and canine parvovirus.

[0101] In this way, only one vaccine needs to be administered to vaccinate canines against diseases for which core vaccines are recommended. Preferably, the first vaccine and the second vaccine comprise canine distemper virus and canine parvovirus.

[0102] In one embodiment of the present invention, the first vaccine, the second vaccine, the third vaccine and / or the additional vaccine further comprise canine distemper virus, canine parvovirus and canine parainfluenza virus.

[0103] Preferably, the first vaccine and the second vaccine comprise canine distemper virus, canine parvovirus and canine parainfluenza virus.

[0104] In one embodiment of the present invention, the first vaccine, the second vaccine, the third vaccine and / or the additional vaccine further comprise canine parainfluenza virus and Bordetella.

[0105] Preferably, the third and / or additional vaccine comprises canine parainfluenza and / or Bordetella. Even more preferably, the third and additional vaccines comprise canine parainfluenza virus and / or Bordetella.

[0106] In this way, the known causative factors of canine infectious respiratory disease syndrome (kennel cough) are combined in one vaccine, thus facilitating vaccination against kennel cough. Preferably, the Bordetella species is Bordetella bronchiseptica.

[0107] In one embodiment of the present invention, the first vaccine, the second vaccine, the third vaccine and / or the additional vaccine comprise 10 2 -10 6 TCID 50 amounts of canine adenovirus type 2.

[0108] Preferably, the first vaccine, the second vaccine, the third vaccine and / or the additional vaccine contain 10 2.5 -10 5.5 TCID 50 , for example 10 3 -10 5 TCID 50 , 10 3.5 -10 4.5 TCID 50 or about 10 4 TCID 50 amounts of CAV-2.

[0109] In one embodiment of the present invention, the first vaccine, the second vaccine, the third vaccine and / or the additional vaccine are administered in a volume of 0.1 - 5 mL.

[0110] Preferably, the first vaccine, the second vaccine, the third vaccine, and / or the additional vaccine(s) are 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.

[0111] In one embodiment of the invention, the oral administration of the third vaccine is about 1 year after the administration of the first vaccine.

[0112] Typically, the protective effect of the primary vaccination wanes 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 extended.

[0113] In one embodiment of the invention, the first vaccine, the second vaccine, the third vaccine, and / or the additional vaccine(s) further comprise a pharmaceutically acceptable adjuvant.

[0114] The adjuvant is, for example, aluminum hydroxide or saponin, or an oil-based adjuvant such as Freund's complete adjuvant or Freund's incomplete adjuvant.

[0115] In one embodiment of the invention, the first vaccine, the second vaccine, the third vaccine, and / or the additional vaccine(s) further comprise a pharmaceutically acceptable carrier.

[0116] The carrier is, for example, a solvent, a dispersion medium, or a diluent. These are generally known in the art. Typically, the carrier will be sterile and pyrogen-free and will be selected based on the mode of administration to be used.

[0117] In one embodiment, the canine is a dog.

[0118] Example

[0119] Example 1

[0120] This study evaluated the use of a third vaccine comprising CAV-2, wherein the first vaccine comprising CAV-2 was administered subcutaneously, the second vaccine comprising CAV-2 was administered subcutaneously, and the third vaccine comprising CAV-2 was administered orally.

[0121] 1.1. Vaccines

[0122] The first and second vaccines used as primary vaccines in this study are described in Table 1.

[0123]

[0124] *Table 1. Components of the primary vaccines.

[0125] The third vaccine used as a booster vaccine in this study is described in Table 2.

[0126]

[0127] *Table 2. Components of the booster vaccine.

[0128] 1.2. Vaccine regimens and formulations

[0129] Sixteen beagle puppies from four litters were used in this study. Table 1 shows the vaccination regimens.

[0130]

[0131] *Table 3. Vaccination regimens used in the study of Example 1.

[0132] The primary DHP vaccine was provided in lyophilized form. Each vaccine vial was reconstituted with 1 ml of L4 as a diluent.

[0133] Primary The KC vaccine was provided in lyophilized form. Each vial of lyophilized KC vaccine was resuspended in 1.0 ml of KC diluent (provided with the vaccine).

[0134] The booster DHP vaccine was provided in lyophilized form. Each vial of lyophilized booster DHP vaccine was resuspended in 1.0 ml of solvent. The resuspended vaccines were then combined.

[0135] Booster The KC vaccine was provided in lyophilized form. Each vial of lyophilized KC vaccine was resuspended in 1.0 ml of KC diluent (provided with the vaccine). The resuspended vaccines were then combined.

[0136] The NADESDHPPi vaccine was provided as a ready-to-use liquid in multi-dose vials containing proline, sorbitol, and methionine.

[0137] Respira Bb was provided as a stock solution in multi-dose vials.

[0138] At 5 - 6 weeks of age, all puppies received subcutaneously the first dose of 1 mL of the primary DHP vaccine reconstituted in L4, and intranasally 0.4 mL of the primary vaccine. Twenty-eight days after the first vaccination, the dogs received subcutaneously the second dose of 1 mL of the primary DHP vaccination reconstituted in L4.

[0139] 1.3. Treatments and sample collection

[0140] Before administering the third dose, the dogs were divided into three groups according to their serological status against CDV, CAV, and CPi. The individual groups were formed in such a way as to obtain similarity in the serological responses (after 1 year) of the dogs in all groups.

[0141] On the 417th day after the first dose was administered, 389 days after the primary vaccination process was completed, the dogs in Group 1 were subcutaneously inoculated with 1 mL of booster DHP vaccine and orally administered 1 mL of booster KC vaccine in the buccal sac. The dogs in Group 2 were orally inoculated with 1 mL of Booster DHP vaccine in the buccal sac and subcutaneously inoculated with 1 mL Respira Bb vaccine. The dogs in Group 3 were orally inoculated with 1 mL of NADESDHPPi vaccine in the buccal sac and subcutaneously inoculated with 1 mL RespiraBb vaccine. Blood was sampled from the superficial vein at the scheduled time points and allowed to clot at ambient temperature for several hours or overnight at 2 - 8°C. After clotting, the serum was collected by centrifugation.

[0142] 1.4. Serological analysis

[0143] 1.4.1. CAV - 1 and CAV - 2

[0144] Antibodies against CAV - 1 and CAV - 2 were determined in a virus neutralization test. The CAV - 1 neutralizing antibody titer and CAV - 2 neutralizing antibody titer were detected by preparing serial serum dilutions and incubating them separately with an equal volume of CAV - 1 or CAV - 2 suspension of known titer. After incubation, in a 96 - well plate, the samples were inoculated into the wells pre - inoculated with MDCK cells and incubated at 37°C + 5% CO2 for 6 days. The presence of virus in the tissue culture medium was determined by hemagglutination assay (HA) of human type O red blood cells. The agglutination of the wells was scored as positive or negative. Based on HA, the serum VN 50 titer was calculated by Reed & Muench. The VN 50 titer of the neutralizing antibody against CAV - 1 or CAV - 2 in the serum was calculated as the reciprocal of the dilution at the 50% end - point.

[0145] Positive reference serum, negative reference serum, virus retitration, and cell - only negative wells were used as test controls.

[0146] 1.4.2. CDV

[0147] Antibodies against CDV were determined in a virus neutralization test. CDV neutralizing antibody titers were detected by preparing serial serum dilutions and incubating them with an equal volume of CDV suspension of known titer. After incubation, in a 96-well plate, the samples were inoculated into wells pre-inoculated with Vero cells and incubated at 37 °C + 5% CO2 for 5 days. CDV virus infection of Vero cells results in a cytopathic effect (CPE) recognizable by microscopy. The CPE of the wells was scored as positive or negative, and the VN of the serum was calculated by Reed & Muench 50 titer. The VN 50 titer of the neutralizing antibodies against CDV in the test samples was calculated as the reciprocal of the dilution at the 50% end point.

[0148] Positive reference serum, negative reference serum, virus retitration, and cell-only negative wells were used as test controls.

[0149] 1.4.3.CPV

[0150] Antibodies against CPV were determined by hemagglutination inhibition test (HAI) according to standard methods. The HAI test measures the ability of serum CPV antibodies to inhibit the agglutination of porcine red blood cells. Briefly, pre-diluted serum samples were adsorbed onto porcine red blood cells to block any non-specific hemagglutination. Test serum samples were serially diluted on a 96-well plate, first incubated with a constant amount of CPV antigen (8 HA units), and then with porcine red blood cells. Inhibition of hemagglutination was characterized by the tearing-off of porcine blood cells. The HAI titer of the sample would be the reciprocal of the last dilution at which agglutination inhibition occurred (<50% agglutination).

[0151] Positive reference serum and negative reference serum were used as test controls. In addition, positive HA and negative HA controls were present on each plate.

[0152] 1.4.4.CPi

[0153] Antibodies against CPiV were determined in a virus neutralization test. CPiVVN 50 antibody titers were detected by preparing serial serum dilutions and incubating them with an equal volume of CPi suspension of known titer. After incubation, in a 96-well plate, the samples were inoculated into wells pre-inoculated with Vero cells and incubated at 37 °C + 5% CO2 for 6 days. CPiV infection of Vero cells results in a cytopathic effect (CPE) recognizable by microscopy. The CPE of the wells was scored as positive or negative, and the VN of the serum was calculated by Reed & Muench 50 titer. The VN 50 titer of the neutralizing antibodies against CPi in the test samples was calculated as the reciprocal of the dilution at the 50% end point.

[0154] Positive reference serum, negative reference serum, virus retitration, and cell-only negative wells were used as test controls.

[0155] 1.4.5. Bordetella bronchiseptica

[0156] The antibody titer of Bordetella bronchiseptica was determined by enzyme-linked immunosorbent assay (ELISA). ELISA is a colorimetric assay that measures the ability of serum antibodies to bind to a known amount of antigen. Briefly, an ELISA plate was coated with purified Bordetella antigen and incubated overnight at 37 °C. The plate was blocked, washed, and pre-diluted test serum was added. Positive and negative serum controls were included. The test serum samples were diluted on the plate and incubated at 37 °C for 1 hour. Anti-canine peroxidase conjugate was added, and the plate was incubated at 37 °C for 30 minutes. Subsequently, 3,3’,5,5’-tetramethylbenzidine substrate was added, and the plate was incubated in the dark at room temperature for 15 minutes. The color reaction was terminated by adding 4N sulfuric acid, and the absorbance was read at 450 nm optical density. The antibody titer was calculated against the negative reference serum value and the cut-off intercept as the log2 dilution value (absorbance at 450 nm).

[0157] Positive reference serum and negative reference serum were used as test controls.

[0158] 1.4.6. Leptospira interrogans

[0159] Microscopic agglutination test (MAT) was used to examine agglutinating antibodies against the serogroups of Leptospira interrogans (sensu lato) in serum. The titers of agglutinating antibodies against the following four serogroups were determined: Canicola type, Icterohaemorrhagiae type, Grippotyphosa type, and Australis type.

[0160] Briefly, MAT was performed as follows: MAT is used to detect agglutinating serum antibodies specific for Leptospira serogroups and to determine their titers. Serial dilutions of canine serum were incubated with live antigen of the relevant Leptospira serogroups. Then, the titer was determined as the log2 value of the reciprocal of the highest dilution at which the serum-antigen mixture showed 50% agglutination of (non-motile) Leptospira. Serum-group specific positive rabbit anti-serum and a negative rabbit anti-serum were used as control sera.

[0161] Positive reference serum, negative reference serum, and reference wells were used as test controls.

[0162] 1.5. Results

[0163] 1.5.1. CAV-1 and CAV-2

[0164] Figure 1A and 1B showed the mean CAV-2 VN responses to the first, second, and third vaccinations in dogs50 Titer. As Figure 1A shown, the CAV-2 VN 50 titer increased after administration of the second vaccine, but decreased at approximately 270 days after administration of the first vaccine. As Figure 1B shown, subcutaneous administration of a booster at 417 days after administration of the first vaccine resulted in a slight increase in the CAV-2 VN 50 titer (Group 1). However, when the booster was administered orally, the increase in the CAV-2 VN 50 titer was much stronger than the increase in titer with subcutaneous administration of the booster (Group 2), and this was even more the case when an NADES-containing booster was administered orally (Group 3).

[0165] Figure 2A and 2B show the mean CAV-1 VN 50 titers for the responses to the first, second, and third vaccinations in dogs. As Figure 2A shown, similar to the CAV-2 VN 50 titer curve, the CAV-1 VN 50 titer increased after administration of the second vaccine, but decreased at approximately 270 days after administration of the first vaccine. As Figure 2B shown, subcutaneous administration of a booster at 417 days after administration of the first vaccine resulted in a slight increase in the CAV-1 VN 50 titer (Group 1). However, when the booster was administered orally, the increase in the CAV-1 VN 50 titer was much stronger than the increase in titer with subcutaneous administration of the booster (Group 2), and this was even more the case when an NADES-containing booster was administered orally (Group 3).

[0166] 1.5.2. CDV

[0167] Figure 3 show the mean CDV VN 50 titers for the responses to the first, second, and third vaccinations in dogs. The CDV VN 50 titer increased after administration of the first vaccine and remained almost constant until administration of the third vaccine. Subcutaneous administration of the third vaccine significantly increased the CDV VN 50 titer (Group 1), while oral administration of the third vaccine without NADES did not increase the CDV VN 50 titer (Group 2). Oral administration of the third vaccine containing NADES slightly increased the CDV VN 50 titer, but to a lesser extent than subcutaneous administration (Group 3).

[0168] 1.5.3. CPV

[0169] Figure 4Shows the mean CPV HAI units of the responses to the first, second, and third vaccinations in dogs. The HAI units increased after administration of the first and second vaccines and decreased approximately 239 days after administration of the first vaccine. After administration of the third vaccine, the HAI units increased to the same extent, regardless of whether the NADES was present in the subcutaneous or oral administration or oral formulation.

[0170] 1.5.4.CPi

[0171] Figure 5 Shows the mean CPi VN of the responses to the first and third vaccinations in dogs 50 titers. The CPi VN 50 titers increased after intranasal administration of the first vaccine. Oral administration of the third vaccine did not significantly increase the CPi VN 50 titers (Groups 1 and 3) compared to the group that did not receive a CPi booster (Group 2).

[0172] 1.5.5. Bordetella bronchiseptica

[0173] Figure 6 Shows the mean Bordetella bronchiseptica antibody titers of the responses to the first and third vaccinations in dogs. The antibody titers increased after intranasal administration of the first vaccine, while administration of the second vaccine did not result in a stronger increase. Subcutaneous administration of the third vaccine resulted in a strong increase in antibody titers (Groups 2 and 3), while oral administration only slightly increased the antibody titers (Group 1).

Claims

1. A vaccine for use in a method of inducing an immune response against canine infectious hepatitis and / or infectious tracheobronchitis in canines, wherein the vaccine is a first vaccine comprising canine adenovirus type 2, and the method comprises: - administering an immunologically effective dose of the first vaccine, - subcutaneously administering an immunologically effective dose of a second vaccine comprising canine adenovirus type 2 7 - 42 days after the first vaccine, and - orally administering an immunologically effective dose of a third vaccine comprising 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 an additional vaccine comprising canine adenovirus type 2 is administered annually at an interval of 11 - 13 months after the third vaccine.

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

5. The vaccine for use according to any one of the preceding claims, wherein the first vaccine is administered to canines aged 2 - 12 weeks.

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

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

8. The vaccine for use according to any one of the preceding claims, wherein the oral administration of the third vaccine is about 1 year after the administration of the first vaccine.

9. The vaccine for use according to any one of the preceding claims, wherein the first vaccine, the second vaccine, the third vaccine and / or the additional vaccine further comprises at least one of canine distemper virus, canine parvovirus, canine parainfluenza virus, Bordetella and Leptospira interrogans.

10. The vaccine for use according to any one of the preceding claims, wherein the first vaccine, the second vaccine, the third vaccine and / or the additional vaccine further comprises canine parainfluenza virus and / or Bordetella.

11. The vaccine for use according to any one of the preceding claims, wherein the first vaccine, the second vaccine, the third vaccine and / or the additional vaccine further comprises canine distemper virus, canine parvovirus and canine parainfluenza virus.

12. The vaccine for use according to any one of the preceding claims, wherein the first vaccine, the second vaccine, the third vaccine and / or the additional vaccine further comprises a pharmaceutically acceptable adjuvant.

13. The vaccine for use according to any one of the preceding claims, wherein the first vaccine, the second vaccine, the third vaccine and / or the additional vaccine further comprises a pharmaceutically acceptable carrier.

14. The vaccine for use according to any one of the preceding claims, wherein the first vaccine, the second vaccine, the third vaccine and / or the additional vaccine comprise an amount of canine adenovirus type 2 of 10 2 -10 6 TCID 50 .

15. The vaccine for use according to any one of the preceding claims, wherein the first vaccine, the second vaccine, the third vaccine, and / or the additional vaccine is administered in a volume of 0.1 - 5 mL.

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