Vaccines for vaccinating canines

Abstract

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

Description

[Technical Field]

[0001] The present invention relates to vaccines for use in methods of inducing an immune response against infectious canine hepatitis and / or infectious tracheobronchitis in canines. [Background technology]

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

[0003] ICH is an acute liver infection not only in dogs but also in wolves, foxes, coyotes, bears, and skunks. CAV-1 spreads through the feces, urine, blood, saliva, and nasal secretions of infected animals. After oral or nasal infection, it replicates in the tonsils and then infects the liver and kidneys. While most animals recover naturally from ICH, the bleeding disorders and liver disease resulting from CAV-1 infection can be fatal in some cases.

[0004] Infectious tracheobronchitis caused by CAV-2 is a mild but contagious disease limited to the respiratory tract of dogs. Infectious tracheobronchitis is primarily characterized by coughing, nasal discharge, bronchitis, and bronchiolitis, and the virus is thought to be one of the causes of the canine infectious respiratory disease complex (kennel cough). CAV-2 spreads through aerosols produced by sneezing and coughing dogs.

[0005] To prevent dogs from developing ICH or infectious tracheobronchitis, dogs from 6 weeks of age are vaccinated with vaccines containing CAV-2. Given the high genetic similarity between CAV-1 and CAV-2, these vaccines also provide protection against CAV-1-induced ICH. Vaccines against ICH and infectious tracheobronchitis, along with vaccines against canine distemper virus (CDV) and canine parvovirus (CPV), are considered core vaccines recommended for all dogs worldwide. Typically, puppies are first vaccinated at 6 to 8 weeks of age, followed by a second vaccination within 3 to 4 weeks after the first vaccination. After this, vaccinations are typically repeated every 1 to 3 years.

[0006] In addition to the viruses included in core vaccines, vaccination against other viruses is recommended depending on the geographic 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 and Leptospira interrogans serovars Canicola, Icterohaemorrhagiae, Grippotyphosa, and Australis. CPiV and B. bronchiseptica are other contributors to the canine infectious respiratory disease complex, while Leptospira species cause blood infections that can develop into severe pulmonary disease.

[0007] Subcutaneous administration is the traditional method for administering core and non-core vaccines, but other methods are being explored to facilitate vaccine delivery and / or mimic the virus's natural route of infection. For example, European Patent No. 2762163 describes a method for vaccinating dogs against ICH caused by CAV-1 using a vaccine containing CAV-2. The vaccine is administered subcutaneously as an initial dose, orally as a second dose 7-42 days after the initial dose, and orally as one or more annual doses. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] European Patent No. 2762163 Summary of the Invention [Problem to be solved by the invention]

[0009] However, there remains a need for a vaccine against ICH and infectious tracheobronchitis that combines a convenient method of administering the vaccine with good protection against ICH and infectious tracheobronchitis. [Means for solving the problem]

[0010] To this end, there is provided a vaccine for use in a method of inducing an immune response against infectious canine hepatitis and / or infectious tracheobronchitis in a canine, the vaccine being a first vaccine comprising canine adenovirus type 2, the method comprising: administration of an immunologically effective dose of a first vaccine; - Subcutaneous administration of a second vaccine containing an immunologically effective dose of canine adenovirus type 2 7 to 42 days after the first vaccine; and - oral administration of a third vaccine containing an immunologically effective dose of canine adenovirus type 2 10 to 14 months after the first vaccine.

[0011] It was found that administering a first vaccine containing CAV-2, followed by a subcutaneous administration of a second vaccine containing CAV-2 7 to 42 days after the first vaccine, and then an oral administration of a third vaccine containing CAV-2 10 to 14 months after the first vaccine, resulted in strong antibody responses against both CAV-1 and CAV-2.

[0012] As shown in Figure 1, after the second vaccination was administered subcutaneously to dogs, the concentration of CAV-2 neutralizing antibodies gradually increased. Subcutaneous administration of the third vaccine increased the CAV-2 neutralizing antibody concentration. Surprisingly, however, this boost was much stronger when the third vaccine was administered orally.

[0013] As shown in Figure 2, neutralizing CAV-1 antibodies were also produced after administration of the first and second vaccines containing CAV-2. For CAV-2, CAV-1 neutralizing antibody concentrations increased significantly after subcutaneous administration of the third vaccine containing CAV-1. However, oral administration of the third vaccine resulted in a much stronger antibody response than subcutaneous administration of the third vaccine.

[0014] In contrast, as shown in Figure 3, administration of the first and second vaccines containing canine distemper virus (CDV) resulted in increased CDV neutralizing antibody concentrations. However, CDV neutralizing antibody concentrations were boosted only by subcutaneous administration of the third vaccine containing CDV. Oral administration of the third vaccine containing CDV did not significantly affect CDV neutralizing antibody concentration levels.

[0015] As shown in Figure 4, administration of the first vaccine and the second vaccine containing canine parvovirus (CPV) also resulted in the induction of anti-CPV antibodies. However, oral administration of the third vaccine containing CPV did not increase anti-CPV antibody levels above the levels induced by subcutaneous administration of the third vaccine containing CPV.

[0016] In view of the above, it is highly surprising that oral administration of a third vaccine containing CAV-2 resulted in such a strong and unexpected increase in antibody levels against CAV-1 and CAV-2. [Brief explanation of the drawings]

[0017] [Figure 1A] 1 shows the mean CAV-2 VN50 titers in dogs in response to the first, second, and third vaccinations. [Figure 1B] 1 shows the mean CAV-2 VN50 titers in dogs in response to the first, second, and third vaccinations. [Figure 2A] 1 shows the mean CAV-1 VN50 titers in dogs in response to the first, second, and third vaccinations. [Figure 2B] 1 shows the mean CAV-1 VN50 titers in dogs in response to the first, second, and third vaccinations. [Figure 3] The mean CDV VN50 titers of dogs in response to the first, second and third vaccinations are shown. [Figure 4] The mean CPV HAI units for dogs responding to the first, second, and third vaccinations are shown. [Figure 5] The mean CPi VN50 titers of dogs in response to the first and third vaccinations are shown. [Figure 6] 1 shows the mean B. bronchiseptica antibody titers in dogs in response to the first and third vaccinations. DETAILED DESCRIPTION OF THE INVENTION

[0018] 1. A vaccine for use in a method of inducing an immune response against infectious canine hepatitis and / or infectious tracheobronchitis in a canine, wherein the vaccine is a first vaccine comprising canine adenovirus type 2, and the method comprises: administration of an immunologically effective dose of a first vaccine; - Subcutaneous administration of a second vaccine containing an immunologically effective dose of canine adenovirus type 2 7 to 42 days after the first vaccine; - oral administration of a third vaccine comprising an immunologically effective dose of canine adenovirus type 2 10 to 14 months after the first vaccine.

[0019] In one embodiment, the immune response is a protective immune response.

[0020] In one embodiment of the present invention, additional vaccines containing canine adenovirus type 2 are administered annually at intervals of 11 to 13 months after the third vaccine.

[0021] In one embodiment of the invention, the first vaccine, the second vaccine, the third vaccine and / or the further vaccine additionally comprise a NADES.

[0022] In one embodiment of the present invention, the first vaccine is administered to canines that are 2 to 12 weeks of age.

[0023] In one embodiment of the present invention, the second vaccine is administered 1 to 6 weeks after the first vaccine.

[0024] In one embodiment of the invention, the second vaccine is administered 7, 10, 15, 20, 25, 30, 35 or 40 days after the first vaccine.

[0025] In one embodiment of the present invention, the third vaccine is administered 10 to 14 months after the first vaccine.

[0026] In one embodiment of the present invention, the third vaccine is administered 42 to 60 weeks after the first vaccine.

[0027] In one embodiment of the invention, the first administration of the vaccine is subcutaneous.

[0028] In one embodiment of the present invention, the canine adenovirus type 2 included in the first vaccine, the second vaccine, the third vaccine and / or the further vaccine is an attenuated canine adenovirus type 2.

[0029] In one embodiment of the invention, the first vaccine, the second vaccine, the third and / or further vaccines additionally comprise at least one of canine distemper virus, canine parvovirus, canine parainfluenza virus, Bordetella species, and Leptospira interrogans.

[0030] In one embodiment of the invention, the first vaccine, the second vaccine, the third vaccine and / or the further vaccine additionally comprise canine distemper virus and canine parvovirus.

[0031] In one embodiment of the invention, the first vaccine, the second vaccine, the third vaccine and / or the further vaccine additionally comprise canine parainfluenza virus and a Bordetella species.

[0032] In one embodiment of the present invention, the first vaccine, the second vaccine, the third vaccine and / or the further vaccine comprises a canine adenovirus type 2 vaccine containing 10 2 ~10 6 TCID 50 Contains in an amount of

[0033] In one embodiment of the invention, the first vaccine, the second vaccine, the third vaccine and / or the further vaccine are administered in a volume of 0.1 to 5 mL.

[0034] In one embodiment of the present invention, the oral administration of the third vaccine is about one year after the administration of the first vaccine.

[0035] In one 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 one embodiment of the invention, the first vaccine, second vaccine, third and / or further vaccines additionally comprise a pharmaceutically acceptable carrier.

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

[0038] definition A "vaccine" is a composition containing an antigen. Administration of a vaccine to a subject results in the induction of an immune response to the antigen in the subject. Vaccines can be administered in a variety of ways, including subcutaneously, orally, or intranasally. The induction of an immune response can be humoral and / or cell-mediated. The immune response can be assessed, for example, by measuring antibody titers.

[0039] "Vaccinating" refers to administering a vaccine to a subject.

[0040] "Vaccination" is the act of administering a vaccine to a subject.

[0041] A "primary vaccine" is a vaccine used to induce an initial protective immune response in a subject, resulting in at least temporary protective immunity. Multiple doses may be required to achieve protective immunity.

[0042] A "booster vaccine" is a vaccine used to stimulate the immune response previously induced by a primary vaccine in a subject in order to prolong the protective immunity conferred by the primary vaccine.

[0043] An "antigen" is a molecule containing one or more epitopes that, when administered to a subject, induces an immune response specific to that antigen. An antigen can be, for example, a killed, attenuated, or inactivated virus or bacterium, or an antibody or fragment thereof.

[0044] An "epitope" is a specific site on an antigen that binds to a T-cell receptor or a specific antibody.

[0045] 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 subdivided into classes such as IgA, IgD, IgE, IgG, and IgM based on the composition of the constant region.

[0046] A "neutralizing antibody" is an antibody that binds to a pathogen such as a virus or bacteria and blocks the pathogen's infectivity.

[0047] An "antibody response" is an immune response that results in the production of antibodies against an antigen.

[0048] An "immune response" is the activation of the immune system in response to a disease.

[0049] An "immunologically effective dose" is an amount of an antigen or vaccine that induces an immune response in a subject to which it is administered sufficient to prevent signs or symptoms of disease caused by infection with a pathogen such as a virus or bacteria.

[0050] A "protective immune response" is an immune response sufficient to prevent the signs or symptoms of disease caused by infection with a pathogen, such as a virus or bacterium.

[0051] "Protective immunity" is immunity that prevents the signs or symptoms of disease caused by infection with a pathogen such as a virus or bacteria.

[0052] "Antibody titer" is a measure of the level of an antibody, expressed as how far a sample containing the antibody can be diluted before the antibody is no longer detectable.

[0053] "Pharmaceutically acceptable" means suitable for use in a subject without undue effects, such as toxicity, irritation and / or allergy, compared to the benefits.

[0054] An "adjuvant" is a pharmaceutically acceptable compound or composition that increases the immune response to an antigen.

[0055] Embodiments of the invention The present invention relates to a vaccine for use in a method of inducing an immune response against infectious canine hepatitis and / or infectious tracheobronchitis in a canine, wherein the vaccine is a first vaccine comprising canine adenovirus type 2, and the method comprises: administration of an immunologically effective dose of a first vaccine; - Subcutaneous administration of a second vaccine containing an immunologically effective dose of canine adenovirus type 2 7 to 42 days after the first vaccine; and - oral administration of a third vaccine containing an immunologically effective dose of canine adenovirus type 2 10 to 14 months after the first vaccine.

[0056] The first vaccine is, for example, administered subcutaneously. Preferably, the first vaccine is administered orally. The compositions of the first, second, and third vaccines may be the same or different. For example, the first and second vaccines may be the same, and the composition of the third vaccine may be different from the first and second vaccines.

[0057] In one embodiment, the immune response is a protective immune response.

[0058] In one embodiment of the present invention, additional vaccines containing canine adenovirus type 2 are administered annually at intervals of 11 to 13 months after the third vaccine.

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

[0060] In one embodiment of the invention, additional vaccines comprising canine adenovirus type 2 are administered every other year after the third vaccine at intervals of 22 to 26 months, for example at intervals of 23 to 25 months or at intervals of 24 months.

[0061] In one embodiment of the invention, additional vaccines comprising canine adenovirus type 2 are administered every three years after the third vaccine, at intervals of 34 to 38 months, such as at intervals of 35 to 37 months or at intervals of 36 months.

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

[0063] It was found that when a third vaccine contained a natural deep eutectic solvent (NADES), there was a stronger increase in the concentrations of CAV-1 and CAV-2 neutralizing antibodies than in the absence of NADES. Thus, NADES enhance the production of CAV-1 and CAV-2 neutralizing antibodies in response to a CAV-2-containing vaccine.

[0064] Furthermore, it was found that subcutaneous administration of a CDV-containing third vaccine after the first and second CDV-containing vaccines increased the level of CDV neutralizing antibodies, whereas oral administration of a CDV-containing third vaccine after the first and second CDV-containing vaccines did not. However, as shown in Figure 3, the addition of NADES to an orally administered third vaccine resulted in a moderate increase in the level of CDV neutralizing antibodies after oral administration of the CDV-containing third vaccine.

[0065] WO 2019122329 describes a liquid vaccine for a live enveloped virus containing a NADES. "Deep eutectic solvents" (DESs) are known in the art as ionic liquids containing a mixture of at least two compounds in 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 lowering of the melting point of the mixture is caused by the interaction of the compounds, one of which acts as a proton donor and the other as a proton acceptor, providing stable hydrogen bonds without crystallization, allowing the mixture to remain in liquid form at much lower temperatures than its components. Generally, "eutectic" means readily melting.

[0066] In the present case, the individual compounds used to form the DES of the present 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, but a NADES formed with a 2:1 molar ratio of betaine:sucrose containing some water has been found to form a clear liquid that remains fluid even at −20° C.

[0067] The term "naturally occurring" is useful to indicate that the compounds used to form the natural DES (NADES) of the present invention are organic compounds present in materials from biological sources, such as plants or animals, in amounts far greater than trace under normal conditions. Typically, such natural compounds are or are derived from primary metabolites present in a particular material of plant or animal origin. As those skilled in the art will understand, the term "naturally occurring" is used herein only to characterize the original source of a compound for use in the NADES of the present invention, and not to characterize the method by which the compound used was actually obtained. Thus, natural compounds may be used in the present invention even if they are 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.

[0068] NADES can comprise a combination of natural organic compounds selected from organic acids (salts), amines, and amino acids, and polyols such as sugars and sugar alcohols. Preferably, NADES comprise an organic salt and a polyol. In this composition, the organic salt acts as an ionic species that is a proton donor, and the polyol acts as a proton acceptor.

[0069] "Organic salt" is any salt of an organic acid or base, including zwitterions, that falls within the definition of a natural compound presented hereinabove and that can form the deep eutectic solvents of the invention described herein. Those skilled in the art are fully capable of selecting and applying organic salts for the invention to form NADES.

[0070] Additionally, the organic salt should be a pharmaceutically acceptable excipient for the vaccine composition. Such vaccine excipients are described in government regulations, such as the European Pharmacopoeia and the United States Pharmacopoeia 9 CFR, and as such are known to those skilled in the art.

[0071] Preferably, the organic salt is selected from the group consisting of betaine, proline, carnitine, and choline salts. "Betaine" refers to the compound N,N,N-trimethylglycine, CAS number 107-43-7, also known as glycine-betaine. Proline has CAS number 609-36-9. Carnitine has CAS number 541-15-1. Choline has CAS number 62-49-7. More preferably, the carnitine is L-carnitine and / or the choline is choline chloride (CAS number 67-48-1). The organic salts for use in the present invention can be used in different salt forms, isomers, hydrates, anhydrous forms, etc. Those skilled in the art are fully capable of selecting and testing suitable forms of organic salts for use in the present invention.

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

[0073] A "polyol" is an organic compound containing two or more hydroxyl groups. However, very large polymers falling under the definition of polyol, such as cellulose, are not effective in forming NADES as defined herein and are therefore excluded from use in the present invention. As a result, polyols for use in the present invention have a molecular weight of less than about 10,000 grams per mole. More preferably, polyols for use in the present invention have a molecular weight of less than 5,000 grams per mole, or even less than 1,000 grams per mole, in order of preference.

[0074] The preferred polyol is a sugar or sugar alcohol, as it has proven to be a versatile component that allows for the production of a variety of effective NADES compositions for use in the present invention. A "sugar" according to the present invention is any compound from the group of relatively low molecular weight, water-soluble carbohydrates 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 encompasses monosaccharides, disaccharides, or polysaccharides up to hexasaccharides. Preferably, the sugar is selected from fructose, maltose, sucrose, glucose, and trehalose.

[0075] The polyol of the present invention may be a sugar alcohol. In the present invention, a "sugar alcohol" is a hydrogenated sugar containing three or more carbon atoms and can be based on a monosaccharide, disaccharide, or polysaccharide. Preferably, the sugar alcohol is selected from glycerol, xylitol, mannitol, and sorbitol. Preferably, the sugar is in the D-form. Preferably, the sorbitol is D-sorbitol.

[0076] As described above for the organic salts, the polyols can be used in different isomeric forms, hydrated or anhydrous forms, etc. Those skilled in the art are fully capable of selecting and testing suitable forms of polyols for use in the present invention.

[0077] Preferably, the molar ratio of the organic salt defined herein to the polyol is 1:5 to 5:1. Even more preferably, the molar ratio of the organic salt defined herein to the polyol is 1:4 to 4:1, 1:3 to 3:1, or 1:2 to 2:1, in that order of preference.

[0078] In one embodiment of the present invention, the first vaccine is administered to canines that are 2 to 12 weeks of age.

[0079] Given the severity of infectious canine hepatitis and infectious tracheobronchitis, it is important to protect canines from these diseases as early as possible, and therefore administer the first vaccine at an early age, such as 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.

[0080] In one embodiment of the invention, the second vaccine is administered 1 to 6 weeks after the first vaccine, such as 2 to 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.

[0081] Typically, the antibody response to the first vaccine is not strong enough to provide protection to the canine for more than a few weeks. By administering the second vaccine within a window of 1 to 6 weeks after the first vaccine, the antibody response elicited by the first vaccine is boosted and the canine is protected for a longer period of time, for example, several months or even up to a year or more.

[0082] In one embodiment of the present invention, the third vaccine is administered 10 to 14 months after the first vaccine, for example, 11, 12, or 13 months after the first vaccine. Alternatively, the third vaccine is administered 42 to 60 weeks after the first vaccine, for example, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, or 59 days after the first vaccine, or 300 to 450 days after the first vaccine, for example, 325 to 425, 350 to 450, 375 to 425, or 400 days after the first vaccine.

[0083] Over time, the antibody levels induced by the first and second vaccines gradually decline, and a third vaccine is administered to boost the immune system and protect the canine for an additional period of time, such as one more year, two more years, or three more years.

[0084] In one embodiment of the invention, the first administration of the vaccine is subcutaneous.

[0085] The first vaccine is typically administered to the canine at 1 to 3 months of age, for example 2 months of age, and the first vaccine is typically administered while the canine is still with its littermates.

[0086] In one embodiment of the present invention, the canine adenovirus type 2 included in the first vaccine, the second vaccine, the third vaccine and / or the further vaccine is an attenuated canine adenovirus type 2.

[0087] Wild-type CAV-2 strains stimulate immune responses but also induce disease in exposed animals. However, attenuated CAV-2 vaccine strains have been shown to stimulate protective immune responses against CAV-2 and CAV-1 in animals exposed to the attenuated CAV-2, while reducing disease symptoms after exposure to wild-type strains. Attenuated CAV-2 vaccine strains are used, for example, in the approved vaccine Nobivac® DHP. Preferably, the CAV-2 is the CAV-2 strain "Manhattan."

[0088] In one embodiment of the present 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 species, Leptospira interrogans.

[0089] Typically, canines are vaccinated against infectious canine hepatitis and infectious tracheobronchitis, as well as other diseases such as those caused by CDV, CPV, CPiV, and / or Bordetella species. By including one or more of these in the first vaccine, second vaccine, third vaccine, and / or third vaccine, the number of individual vaccines administered to the canine can be reduced.

[0090] Preferably, the CDV, CPV, and / or CPiV are provided as attenuated viruses to stimulate antibody responses in vaccinated canines without causing severe disease. 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, the Bordetella species is Bordetella bronchiseptica. Preferably, the Leptospira interrogans is at least one selected from serovars Canicola, Icterohaemorrhagiae, Grippotyphosa, and Australis.

[0091] In one embodiment of the invention, the first vaccine, the second vaccine, the third vaccine and / or the further vaccine additionally comprise canine distemper virus and canine parvovirus.

[0092] In this way, only one vaccine needs to be administered to vaccinate canines against diseases for which a core vaccine is recommended. Preferably, the first and second vaccines comprise canine distemper virus and canine parvovirus.

[0093] In one embodiment of the invention, the first vaccine, the second vaccine, the third vaccine and / or the further vaccine additionally comprise canine distemper virus, canine parvovirus and canine parainfluenza virus.

[0094] Preferably, the first and second vaccines comprise canine distemper virus, canine parvovirus and canine parainfluenza virus.

[0095] In one embodiment of the invention, the first vaccine, the second vaccine, the third vaccine and / or the further vaccine additionally comprise canine parainfluenza virus and a Bordetella species.

[0096] Preferably, the third and / or further vaccines comprise canine parainfluenza and / or Bordetella species. Even more preferably, the third and further vaccines comprise canine parainfluenza virus and / or Bordetella species.

[0097] In this way, known causative agents of canine infectious respiratory disease complex (kennel cough) are combined into one vaccine, thus facilitating vaccination against kennel cough. Preferably, the Bordetella species is Bordetella bronchiseptica.

[0098] In one embodiment of the present invention, the first vaccine, the second vaccine, the third vaccine and / or the further vaccine comprises a canine adenovirus type 2 vaccine containing 10 2 ~10 6 TCID 50 Contains in an amount of

[0099] Preferably, the first vaccine, the second vaccine, the third vaccine and / or the further vaccine are administered in a dose of 10 3 ~10 5 TCID 50 , 10 3.5 ~10 4.5 TCID 50 or about 10 4 TCID 50 10 of the above 2.5 ~10 5.5 TCID 50 Contains CAV-2 in amounts of

[0100] In one embodiment of the invention, the first vaccine, the second vaccine, the third vaccine and / or the further vaccine are administered in a volume of 0.1 to 5 mL.

[0101] Preferably, the first vaccine, second vaccine, third vaccine and / or further vaccine is administered in a volume of 0.5 to 4 mL, for example in a volume of 1, 1.5, 2, 2.5, 3 or 3.5 mL.

[0102] In one embodiment of the present invention, the oral administration of the third vaccine is about one year after the administration of the first vaccine.

[0103] Generally, the protective effect of a primary vaccination decreases over time. By administering a third vaccine approximately one year after the first, the protective effect conferred by the primary vaccination is extended.

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

[0105] The adjuvant is, for example, aluminum hydroxide or saponin, or an oil-based adjuvant such as Freund's complete or incomplete adjuvant.

[0106] In one embodiment of the invention, the first vaccine, the second vaccine, the third vaccine and / or the further vaccine additionally comprises a pharmaceutically acceptable carrier.

[0107] Carriers are, for example, solvents, dispersion media, or diluents, which are commonly known in the art. Typically, carriers are sterile, pyrogen-free, and selected based on the mode of administration to be used.

[0108] In one embodiment, the canine is a dog.

[0109] [Example] [Example 1] This study will evaluate the use of a third vaccine containing CAV-2, with a first vaccine containing CAV-2 administered subcutaneously, a second vaccine containing CAV-2 administered subcutaneously, and a third vaccine containing CAV-2 administered orally.

[0110] 1.1.Vaccines The first and second vaccines used as primary vaccines in this study are listed in Table 1. [Table 1] The third vaccine used as a booster vaccine in this study is listed in Table 2. [Table 2] 1.2. Vaccine Schedule and Preparation Sixteen beagle pups from four litters were used in this study. Table 1 shows the vaccination schedule. [Table 3] The primary DHP vaccine was supplied in lyophilized form. Each vaccine vial was reconstituted with 1 ml of Nobivac® L4 diluent.

[0111] The primary Nobivac® KC vaccine was supplied in lyophilized form. Each vial of lyophilized Nobivac® KC vaccine was resuspended in 1.0 ml of Nobivac® KC diluent (supplied with the vaccine).

[0112] The booster DHP vaccine was supplied in lyophilized form. Each vial of lyophilized booster DHP vaccine was resuspended in 1.0 ml of Nobivac® Solvent. The resuspended vaccine was then pooled.

[0113] The booster Nobivac® KC vaccine was supplied in lyophilized form. Each vial of lyophilized Nobivac® KC vaccine was resuspended in 1.0 ml of Nobivac® KC diluent (supplied with the vaccine). The resuspended vaccine was then pooled.

[0114] The NADES DHPPi vaccine was supplied as a ready-to-use liquid in multi-dose vials containing proline, sorbitol and methionine.

[0115] Nobivac® Respira Bb is supplied as a ready-to-use liquid in multi-dose vials.

[0116] At 5-6 weeks of age, all pups received an initial dose of 1 mL of primary DHP vaccine resuspended in Nobivac® L4 and 0.4 mL of primary Nobivac® vaccine administered intranasally. 28 days after the first vaccination, dogs received a second dose of 1 mL of primary DHP vaccine resuspended in Nobivac® L4 subcutaneously.

[0117] 1.3. Processing and sample collection Before the third dose, the dogs were divided into three groups based on their serological status against CDV, CAV, and CPi, so as to achieve similar serological responses (after one year) among all groups.

[0118] On day 417 after administration of the initial dose and day 389 after completion of the primary vaccination course, dogs in Group 1 were vaccinated subcutaneously with a 1 mL booster DHP vaccine and orally with a 1 mL booster Nobivac® KC vaccine intrabuccally. Dogs in Group 2 were vaccinated orally with a 1 mL booster DHP vaccine intrabuccally and subcutaneously with a 1 mL Nobivac® Respira® Bb vaccine. Dogs in Group 3 were vaccinated orally with a 1 mL NADES DHPPi vaccine intrabuccally and subcutaneously with a 1 mL Nobivac® Respira® Bb vaccine. Blood was collected from a superficial vein at designated 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.

[0119] 1.4. Serological analysis CAV-1 and CAV-2 Antibodies to CAV-1 and CAV-2 were determined by virus neutralization assays. CAV-1 and CAV-2 neutralizing antibody titers were detected by preparing serial serum dilutions and incubating them with equal volumes of suspensions of known titers of CAV-1 or CAV-2, respectively. After incubation, samples were inoculated into wells pre-seeded with MDCK cells in a 96-well plate and incubated at 37°C + 5% CO2 for 6 days. The presence of virus in the tissue culture medium was measured by hemagglutination assay (HA) of human type O erythrocytes. Wells were scored as positive or negative for agglutination. VN of sera based on HA 50 The titers were calculated by Reed & Muench. VN of neutralizing antibodies against CAV-1 or CAV-2 in serum 50 The titer is the reciprocal of the dilution calculated to be the 50% endpoint.

[0120] Positive reference serum, negative reference serum, virus back titration and negative cell only wells were included as assay controls.

[0121] CDV Antibodies to CDV were determined by virus neutralization assay. CDV neutralizing antibody titers were detected by making serial serum dilutions and incubating them with an equal volume of a CDV suspension of known titer. After incubation, samples were inoculated into wells pre-seeded with Vero cells in a 96-well plate and incubated at 37°C + 5% CO2 for 5 days. Infection of Vero cells by CDV virus results in a cytopathic effect (CPE) recognizable by microscopy. Wells were scored as positive or negative for CPE, and the VN of the sera was determined by Reed & Muench. 50 The titer was calculated as follows: VN of neutralizing antibodies against CDV in the test samples 50 The titer is the reciprocal of the dilution calculated to be the 50% endpoint.

[0122] Positive reference serum, negative reference serum, virus back titration and negative cell only wells were included as assay controls.

[0123] CPV Antibodies to CPV were determined by hemagglutination inhibition assay (HAI) according to standard procedures. The HAI assay measures the ability of serum CPV antibodies to inhibit the agglutination of porcine red blood cells. Briefly, prediluted serum samples were adsorbed to porcine red blood cells to block nonspecific hemagglutination. Test serum samples were serially diluted in a 96-well plate and incubated first with a fixed amount of CPV antigen (8 HA units) and then with porcine red blood cells. Inhibition of hemagglutination was characterized by tear-dropping of porcine red blood cells. The HAI titer of a sample was calculated as the reciprocal of the last dilution at which agglutination inhibition occurred (<50% agglutination).

[0124] Positive and negative reference sera were included as assay controls. In addition, positive and negative HA controls were present on each plate.

[0125] 1.4.4.CPi Antibodies to CPiV were determined by virus neutralization assay. 50Antibody titers were detected by making serial serum dilutions and incubating them with an equal volume of a suspension of known titer of CPi. After incubation, samples were inoculated into wells pre-seeded with Vero cells in a 96-well plate and incubated at 37°C + 5% CO2 for 6 days. Infection of Vero cells with CPiV virus results in a cytopathic effect (CPE) recognizable by microscopy. Wells were scored as positive or negative for CPE, and the VN of the serum was determined by Reed & Muench. 50 The titer was calculated as VN of neutralizing antibodies against CPi in the test samples. 50 The titer is the reciprocal of the dilution calculated to be the 50% endpoint.

[0126] Positive reference serum, negative reference serum, virus back titration and negative cell only wells were included as assay controls.

[0127] 1.4.5. Bordetella bronchiseptica Bordetella bronchiseptica antibody titers were 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, ELISA plates were coated with purified Bordetella antigen and incubated overnight at 37°C. The plates were blocked, washed, and prediluted test serum was added. Positive and negative serum controls were included. Test serum samples were diluted across the plate and incubated for 1 hour at 37°C. Anti-canine peroxidase conjugate was added, and the plate was incubated for 30 minutes at 37°C. 3,3',5,5'-tetramethylbenzidine substrate was then added, and the plate was incubated for 15 minutes at room temperature in the dark. The color reaction was stopped by the addition of 4N sulfuric acid, and absorbance was read at an optical density of 450 nm. Antibody titers were calculated as the log2 dilution value (absorbance 450 nm) with the intercept at the cutoff, relative to the negative reference serum value.

[0128] Positive and negative reference sera were included as assay controls.

[0129] 1.4.6. Leptospira interrogans Sera were tested for agglutinating antibodies against Leptospira interrogans (sensu lato) serogroups using the Microscopic Agglutination Test (MAT). Titers of agglutinating antibodies against four serogroups were determined: Canicola, Icterohaemorrhagiae, Grippotyphosa, and Australis.

[0130] Briefly, MAT was performed as follows: MAT is used to detect agglutinating serum antibodies specifically directed against Leptospira serogroups and to determine their titers. Serial dilutions of dog serum were incubated with live antigens of the relevant serogroup of Leptospira. The titer was then determined as the reciprocal log2 value of the highest dilution at which the serum-antigen mixture showed 50% agglutination (non-motile) leptospira. A serogroup-specific positive rabbit antiserum and one negative rabbit antiserum were used as control sera.

[0131] A positive reference serum, a negative reference serum and reference wells were included as assay controls.

[0132] 1.5.Results CAV-1 and CAV-2 Figures 1A and 1B show the mean CAV-2 VN of dogs in response to the first, second, and third vaccinations. 50 As shown in Figure 1A, the CAV-2 VN 50 The titer increased after the second vaccine administration but decreased approximately 270 days after the first vaccine administration. As shown in Figure 1B, a booster given subcutaneously 417 days after the first vaccine administration significantly increased the CAV-2 VN titer. 50However, when the booster was administered orally, the CAV-2 VN 50 The increase in titer was much more potent when compared with the increase in the subcutaneously administered booster (Group 2) and even more potent when the oral booster contained NADES (Group 3).

[0133] Figures 2A and 2B show the mean CAV-1 VN of dogs in response to the first, second, and third vaccinations. 50 As shown in Figure 2A, the CAV-2 VN 50 Similar to the titer profile, CAV-1 VN 50 The titer increased after the second vaccine administration but decreased approximately 270 days after the first vaccine administration. As shown in Figure 2B, a booster given subcutaneously 417 days after the first vaccine administration significantly increased the CAV-1 VN titer. 50 However, when the booster was administered orally, the CAV-1 VN 50 The increase in titer was much more potent when compared with the increase in the subcutaneously administered booster (Group 2) and even more potent when the oral booster contained NADES (Group 3).

[0134] CDV Figure 3 shows the mean CDV VN of dogs in response to the first, second and third vaccinations. 50 The titer is shown. CDV VN 50 Titers rise after the first vaccine dose and remain essentially constant until the third vaccine dose. 50 Oral administration of a third vaccine containing no NADES strongly increased the CDV VN titer (Group 1). 50 Oral administration of a third vaccine containing NADES did not increase the CDV VN titer (Group 2). 50 It increased titers slightly, but to a lesser extent than subcutaneous administration (Group 3).

[0135] CPV Figure 4 shows the mean CPV HAI units in dogs in response to the first, second, and third vaccinations. HAI units increase after administration of the first and second vaccines and decrease approximately 239 days after administration of the first vaccine. After administration of the third vaccine, HAI units increased to a similar extent regardless of whether the vaccine was administered subcutaneously or orally or whether NADES was present in the oral formulation.

[0136] 1.5.4.CPi Figure 5 shows the mean CPi VN of dogs in response to the first and third vaccinations. 50 Titer shown. CPi VN 50 Titers increased after intranasal administration of the first vaccine. Oral administration of the third vaccine increased CPi VN compared to the group that did not receive a CPi booster (Group 2). 50 did not significantly increase the titer (Groups 1 and 3).

[0137] 1.5.5.B. bronchiseptica Figure 6 shows the mean B. bronchiseptica antibody titers in dogs in response to the first and third vaccinations. Antibody titers increase after intranasal administration of the first vaccine, but administration of the second vaccine does 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 antibody titers (Group 1).

Claims

1. 1. A vaccine for use in a method of inducing an immune response against infectious canine hepatitis and / or infectious tracheobronchitis in a canine, wherein the vaccine is a first vaccine comprising canine adenovirus type 2, and the method comprises: - administering an immunologically effective dose of a first vaccine; - subcutaneous administration of a second vaccine comprising an immunologically effective dose of canine adenovirus type 2 7 to 42 days after the first vaccine; - oral administration of a third vaccine comprising an immunologically effective dose of canine adenovirus type 2 10 to 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. 3. The vaccine for use according to claim 1 or 2, wherein a further vaccine comprising canine adenovirus type 2 is administered annually at intervals of 11 to 13 months after said third vaccine.

4. 10. A vaccine for use according to any preceding claim, wherein the first vaccine, the second vaccine, the third vaccine and / or the further vaccine additionally comprise NADES.

5. 10. The vaccine for use according to any preceding claim, wherein said first vaccine is administered to a canine animal between 2 and 12 weeks of age.

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

7. 10. A vaccine for use according to any preceding claim, 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. 10. The vaccine for use according to any of the preceding claims, wherein the oral administration of said third vaccine is about one year after the administration of said first vaccine.

9. 10. 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 species and Leptospira interrogans.

10. 10. A vaccine for use according to any preceding claim, wherein the first vaccine, the second vaccine, the third vaccine and / or the further vaccine additionally comprise canine parainfluenza virus and / or Bordetella species.

11. 10. 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 comprise canine distemper virus, canine parvovirus and canine parainfluenza virus.

12. 10. 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 comprise a pharmaceutically acceptable adjuvant.

13. 10. 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 comprise a pharmaceutically acceptable carrier.

14. The first vaccine, the second vaccine, the third vaccine and / or the further vaccine contain canine adenovirus type 2. ２ ~10 ６ T.C.I.D. ５０ 10. A vaccine for use according to any of the preceding claims, comprising:

15. 10. 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 are administered in a volume of 0.1 to 5 mL.

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