Canine oral vaccines and administration methods

Abstract

This disclosure provides a method for protecting dogs against canine distemper virus, the method comprising administering a recombinant oral vaccine to the dog, the recombinant oral vaccine comprising a modified live canine adenovirus type 2 (CAV-2) vector carrying canine distemper virus (CDV) antigen, wherein the vaccine is administered orally.

Description

Technical Field

[0001] This invention belongs to the field of oral vaccines, particularly oral vaccines for dogs. Background Technology

[0002] Vaccines against major canine infectious diseases have been available for 30 to 40 years and have significantly reduced the incidence of these diseases in dogs (Appel, MJ. 1999. Adv Vet Med. 41:309-324). Zoetis sells a variety of vaccines for the prevention of various viral and bacterial diseases in dogs. The VANGUARD® series of vaccines (including the core canine vaccine) is administered to healthy dogs 6 weeks of age and older to assist in the prevention of canine distemper (CD) caused by canine distemper virus (CDV), infectious canine hepatitis (ICH) caused by canine adenovirus type 1 (CAV-1), respiratory diseases caused by canine adenovirus type 2 (CAV-2), canine parainfluenza (CPI) caused by canine parainfluenza (CPI) virus, and canine parvovirus enteritis caused by canine parvovirus (CPV) (Mouzin DE et al., 2004, JAVMA, 224: 55-60).

[0003] Canine CD (CAV) is a highly morbid and fatal viral disease occurring in unvaccinated canine populations worldwide. Approximately 50% of unvaccinated, non-immune dogs infected with CDV will develop clinical symptoms, and about 90% of those dogs will die (Swango LJ. 1983. Norden News 58:4-10). Infectious canine hepatitis (ICH) caused by CAV-1 is a prevalent and sometimes fatal canine viral disease characterized by hepatic and systemic endothelial lesions. Respiratory disease caused by CAV-2 may include pneumonia and bronchopneumonia in severe cases. CAV-2 vaccines have been shown to provide cross-protection against ICH caused by CAV-1 (Bass EP et al., 1980, JAVMA, 177:234-242). Upper respiratory tract disease caused by CPI virus may be mild or subclinical, and symptoms become more severe if other respiratory pathogens are present concurrently. Enteric diseases caused by CPV are characterized by a sudden onset of vomiting and diarrhea, which is usually hemorrhagic and may be accompanied by leukopenia (Appel MJ et al., 1979, Vet Rec, 105:156-159).

[0004] Typically, puppies receive so-called core vaccines around 6 weeks of age. These vaccines contain antigens against CDV, CAV-2, and CPV. Major animal health companies sell core canine vaccines as part of their companion animal vaccine product lines. However, all of these vaccines are...

[0005] Delivery via parenteral routes (especially subcutaneous injection). Easily deliverable canine vaccines will provide greater convenience for pets, veterinarians, and pet owners, and allow individuals without training in parenteral administration techniques to deliver canine core vaccines to animals.

[0006] Oral vaccines against CDV have been publicly reported. For example, U.S. Publication No. 2010028379 discloses an oral vaccine containing modified live canine distemper virus. However, despite significant efforts, there is still no commercially available oral vaccine against CDV. Therefore, there is a need in the field for an effective oral vaccine against CDV. Summary of the Invention

[0007] In one aspect, this disclosure provides a method for protecting MDA (maternally induced antibody) negative dogs against canine distemper virus, the method comprising administering a recombinant oral vaccine to the dog, the recombinant oral vaccine comprising a modified live canine adenovirus type 2 (CAV-2) vector carrying canine distemper virus (CDV) antigens, wherein the recombinant oral vaccine is administered orally in a first dose and in a second dose.

[0008] In a second aspect, the present invention provides a method for annually revaccinating a dog, the method comprising orally administering to the dog a vaccine comprising a recombinant oral vaccine containing a modified live canine adenovirus type 2 (CAV-2) vector carrying a canine distemper virus (CDV) antigen, wherein the recombinant oral vaccine is administered approximately one year after a previous year's revaccination or approximately one year after administering a first dose of an initial vaccine to the dog, wherein the first dose of the initial vaccine is further administered parenterally to the dog, and wherein the initial vaccine contains both CDV antigen and CAV-2 antigen. In some embodiments of this second aspect, in the initial vaccine,

[0009] a) The CDV antigen is a modified live CDV, and the CAV-2 antigen is a modified live CAV-2; or

[0010] b) The CDV antigen is encoded by a nucleic acid sequence within the modified genome of a live canarypox virus.

[0011] In an additional or alternative implementation of the second aspect, the vaccine is administered to the dogs during the previous year's revaccination, wherein

[0012] a) The CDV antigen is a modified live CDV, and the CAV-2 antigen is a modified live CAV-2; or

[0013] b) The CDV antigen is encoded by a nucleic acid sequence within the modified genome of a live canarypox virus.

[0014] In certain embodiments applicable to the first and second aspects of the present invention, in the recombinant oral vaccine, the CDV antigen is the H protein, preferably comprising SEQ ID NO: 1 or a sequence that is 90% or 95% identical to SEQ ID NO: 1. In some embodiments, in the recombinant oral vaccine, SEQ ID NO: 1 is encoded by a nucleic acid sequence comprising SEQ ID NO: 2. In other embodiments, the recombinant oral vaccine further comprises the F protein of CDV, preferably comprising SEQ ID NO: 3 or a sequence that is 90% or 95% identical to SEQ ID NO: 3. In some embodiments, in the recombinant oral vaccine, SEQ ID NO: 3 is encoded by a nucleic acid sequence comprising SEQ ID NO: 3. Preferably, the nucleic acid sequence encoding the CDV antigen according to any of the embodiments of the recombinant oral vaccine described above is inserted into the E3 region of the modified live CAV-2 vector genome.

[0015] A method according to any of the above embodiments is also described, wherein each dose of the recombinant oral vaccine contains at least 10 3 TCID50 to approximately 10 8 The modified live CAV-2 carrier carrying CDV antigen, TCID50, more preferably containing about 10 mg / dose. 3.5 TCID50 to approximately 10 4.5 The modified live CAV-2 vector carrying CDV antigen of TCID50.

[0016] A method according to any of the embodiments disclosed above is also described, wherein in the recombinant oral vaccine, the modified live canine adenovirus type 2 vector carrying the CDV antigen provides protection against canine distemper virus, canine adenovirus type 1 infection, and canine adenovirus type 2 infection upon oral administration. Preferably, the dog vaccinated according to the method of the first aspect of the invention has not previously been vaccinated against canine distemper virus, and more preferably, has not been vaccinated against canine adenovirus type 2.

[0017] In certain embodiments applicable to both the first and second aspects, the recombinant oral vaccine further comprises modified live parvovirus, wherein the recombinant oral vaccine provides additional protection against canine parvovirus infection upon oral administration. Preferably, the vaccine comprises about 10 5.0 TCID50 to approximately 10 10 Modified live canine parvovirus with TCID50, more preferably 10 7.5 TCID50 to approximately 10 9 Modified live canine parvovirus with TCID50.

[0018] In another embodiment, the recombinant oral vaccine according to any embodiment of the method of the first or second aspect of the invention further comprises a modified live canine parainfluenza virus, wherein the recombinant oral vaccine provides further protection against canine parainfluenza virus infection upon oral administration. In some embodiments of the first aspect of the invention, the dog has not previously been vaccinated against canine parainfluenza virus.

[0019] In yet other embodiments, both applicable to the first and second aspects of the invention, the recombinant oral vaccine comprises 10 4.5 TCID50 to approximately 10 8 Modified live canine parainfluenza virus with TCID50, preferably 10 6.5 TCID50 to approximately 10 7.5 Modified live canine parainfluenza virus of TCID50. In some embodiments suitable for the first aspect of the invention, the dog has not previously been vaccinated against canine parainfluenza virus.

[0020] In some embodiments of the method disclosed herein, which is particularly applicable to the first aspect of the invention, the dog is about 8 to about 16 weeks old when it is first given the recombinant oral vaccine. Alternatively, in embodiments particularly applicable to the second aspect of the invention, the dog is at least 13 months old when it is given the recombinant oral vaccine.

[0021] In some embodiments of the first aspect of the invention, the second dose is administered 7-35 days after the first dose, preferably about 21 days after the first dose.

[0022] In another embodiment, the method disclosed herein further includes the step of orally administering a third dose of the recombinant oral vaccine described herein, wherein the third dose is administered approximately 21 days after the second dose.

[0023] In the third and fourth aspects, recombinant oral vaccines are provided for use in the implementation schemes of the first and second aspects as described above. Detailed Implementation

[0024] To better understand this invention, the following definitions are provided:

[0025] The term "about" when applied to reference figures means the reference figure plus or minus 10%; unless the figure is expressed in "10"... N "Given, in this case, "approximately 10" N "or 10" N "" refers to N plus or minus 10% (inclusive). For example, if N is 2, then N is approximately 1.8 to 2.2 (inclusive).

[0026] The term “conservative substitution” refers to the replacement of one amino acid with another amino acid that has similar properties. The following six groups each contain amino acids that are typical conserved substitutions for each other: [1] alanine (A), serine (S), threonine (T); [2] aspartic acid (D), glutamic acid (E); [3] asparagine (N), glutamine (Q); [4] arginine (R), lysine (K), histidine (H); [5] isoleucine (I), leucine (L), methionine (M), valine (V); and [6] phenylalanine (F), tyrosine (Y), tryptophan (W), (see, for example, U.S. Patent Publication 20100291549).

[0027] Animals mentioned as "not vaccinated" or "not actively vaccinated" do not include those that have received maternal immunity or passive antibody transfer.

[0028] The terms “oral” or “oral” refer to administration to a subject’s mouth, where the vaccine comes into contact with the subject’s oral mucosa.

[0029] The terms “protect,” “protection,” and “protective immune response” generally refer to the ability of the vaccine disclosed herein to reduce or eliminate the duration or severity of at least one clinical symptom of the pathogen targeted by the subject. Clinical symptoms vary depending on the pathogen. A more detailed description of protective immune responses against specific viruses is provided below.

[0030] The term "subject" refers to an animal that has been given the vaccine disclosed herein. This term refers to non-human animals such as dogs, cats, horses, pigs, cattle, and swine. In some embodiments, the term may also refer to humans.

[0031] In the context of this disclosure, the term "therapeutic effective amount" refers to the amount of antigen or vaccine that induces a protective immune response in a subject receiving the antigen or vaccine.

[0032] The term "vaccine" refers to a composition containing an antigen against a specific pathogen, wherein the antigen can elicit a protective immune response against said pathogen. This response is preferably generated by the adaptive immune system and may include...

[0033] Antibody immune responses generated by B cells, cell-mediated immunity mediated by T cells, or both antibody- and cell-mediated immunity.

[0034] This disclosure provides a recombinant oral vaccine comprising a modified live canine adenovirus type 2 (CAV-2) vector carrying canine distemper virus antigen. In a preferred embodiment, the antigen of the canine distemper virus in the recombinant oral vaccine is its H protein (hemagglutinin) or F protein (fusion), as these proteins are present on the surface of the virus and are known to elicit a protective immune response. In a particularly preferred embodiment, the CDV antigen in the recombinant oral vaccine is the H protein. The corresponding H proteins of different CDV strains are available in publicly available genetic information databases. In some embodiments, in the recombinant oral vaccine, the H protein comprises at least 90% of the amino acid sequence identical to SEQ ID NO: 1, and in other embodiments, may be at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% identical to SEQ ID NO: 1. In embodiments where the H protein is at least 90% but less than 100% identical to SEQ ID NO: 1, at least half of the different amino acids (or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or 100%) are conservative substitutions.

[0035] The corresponding F proteins of different CDV strains are also available in publicly available genetic information databases. In some embodiments, the F protein contains at least 90% of the same amino acid sequence as SEQ ID NO: 3, and in other embodiments, it may be at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% identical to SEQ ID NO: 3. In embodiments where the H protein is at least 90% but less than 100% identical to SEQ ID NO: 3, at least half of the different amino acids (or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or 100%) are conserved substitutions.

[0036] One way to generate a modified live canine adenovirus type 2 (CAV-2) vector carrying canine distemper virus antigen is to use a nucleic acid sequence encoding the canine distemper virus antigen according to any of the embodiments described above.

[0037] The recombinant is inserted into the genome of the modified live CAV-2 vector. In some non-limiting embodiments, in the recombinant oral vaccine described herein, the nucleic acid sequence encoding the canine distemper virus antigen comprises SEQ ID NO: 2, or, considering redundancy in the genetic code, comprises an equivalent of SEQ ID NO: 2. In other non-limiting embodiments of the recombinant oral vaccine described herein, the nucleic acid sequence encoding the canine distemper virus antigen comprises SEQ ID NO: 4, or, considering redundancy in the genetic code, comprises an equivalent of SEQ ID NO: 4.

[0038] Methods for inserting exogenous nucleic acids into viral vectors are well known in the art. Preferably, the nucleic acid sequence encoding canine distemper virus antigen is inserted into the E3 region of the genome of a modified live CAV-2 vector using any of the methods known in the art. It is also preferred that the nucleic acid sequence encoding canine distemper virus antigen according to any of the above embodiments is effectively controlled by a promoter. A suitable promoter should be able to direct the expression of the CDV antigen in the cells of a intended host (such as canines). Suitable promoters include, but are not limited to, the SV-40 promoter, the CMV promoter, the RSV promoter, and other promoters known in the art.

[0039] In certain embodiments of the recombinant oral vaccine, the modified live CAV-2 vector carrying the CDV antigen, according to any of the embodiments described herein, was not enteric-coated. Enteric coating typically protects the virus against the harsh environment of the stomach, but the applicant has previously found that enteric coating of modified live CDV suppresses the immune response upon oral administration of modified live CDV.

[0040] In other embodiments, in addition to a modified live CAV-2 vector carrying canine distemper virus antigen according to any of the embodiments described above, the recombinant oral vaccine may also contain modified live canine parvovirus and / or modified live canine parainfluenza virus. Suitable modified live CPV and CPI viruses are known in the art. For example, but not limitingly, although the VANGUARD® DAPPi vaccine is currently approved for parenteral administration, the corresponding CPV and CPI viruses present in the VANGUARD® DAPPi vaccine (containing modified live CDV, CAV-2, CPV, and CPI) may be administered orally.

[0041] In some implementations of recombinant oral vaccines, neither the modified live CPV nor the CPI is enteric-coated.

[0042] Methods for preparing the virus according to any embodiment described herein are known in the art. Without limitation, the virus can be propagated on NLDK (Norden Laboratory canine kidney) cells according to standard procedures. In short, canine viruses can be grown in roller flasks containing or without gentamicin in DMEM (Durbeco Minimal Essential Medium) containing 0-5% FBS. Depending on the specific virus, NLDK cells are grown at 1.2 × 10⁻⁶ cells / mL. 4 Up to 3.5×10 4 cells / cm 2 Initially seeded at a density that will be 80-100% confluent when infected with the virus after 4-7 days of growth. Infect cells with the virus at a tissue culture infection dose of 50 (TCID50) with an infection multiple (MOI) between 0.001 and 1.0, incubate at 34.0-38.0°C, rotate at 0.2-0.5 rpm, harvest after 2-5 days, and store at -40°C or below.

[0043] When administered orally, the recombinant oral vaccines described herein elicit a protective immune response against the respective pathogens. Therefore, a recombinant oral vaccine according to any of the embodiments described herein and containing a modified live CAV-2 vector carrying the CDV antigen elicits a protective immune response against CAV-1 infection, CAV-2 infection, and CDV infection. If the recombinant oral vaccine also contains a modified live CPV, it also elicits a protective immune response against canine parvovirus. If the recombinant oral vaccine also contains a modified live CPI, it also elicits a protective immune response against canine parainfluenza virus.

[0044] A “protective immune response” against CDV refers to a shortening of the duration or a reduction in the intensity of one or more clinical symptoms of CDV infection (including, but not limited to, death caused by CDV). Most preferably, a protective immune response against CDV means that, in a challenge model, at least 95% of vaccinated dogs survive (19 out of 20) without exhibiting any clinical symptoms of the disease, 80% of unvaccinated dogs (4 out of 5 die) die from the disease, and 100% of unvaccinated dogs develop clinical symptoms after challenge.

[0045] A "protective immune response" against CAV-1 refers to a shortened duration or reduced intensity of one or more clinical symptoms of CAV-1 infection (including, but not limited to, death caused by CDV). Most preferably, a protective immune response against CAV-1 means that, in a challenge model, the survival rate of vaccinated dogs is at least 95%.

[0046] (19 out of 20 survived), and none of them showed any clinical symptoms of canine hepatitis. Among them, 80% of unvaccinated dogs would show clinical symptoms.

[0047] A “protective immune response” to CAV-2 refers to a shortened duration or reduced intensity of one or more clinical symptoms of CAV-2 infection (including, but not limited to, death caused by CDV). Most preferably, a protective immune response to CAV-2 is defined as a significant difference in clinical symptoms between vaccinated and unvaccinated dogs in a challenge model, with at least 60% of unvaccinated dogs exhibiting clinical symptoms of the disease.

[0048] A “protective immune response” to CPV refers to a shortening of the duration or a reduction in the intensity of one or more clinical symptoms of CPV infection (including, but not limited to, death caused by CDV). In the most preferred embodiment, a protective immune response to CPV is defined as a survival rate of at least 95% (19 out of 20) of vaccinated dogs during the observation period in a challenge model, and the presence of no more than one of the following infection indicators: body temperature ≥103.4°C, lymphopenia ≥50% of pre-challenge normal values, presence of mucus or blood in feces, and hemagglutination level ≥1:64 in 1% fecal dilution; wherein more than 80% of unvaccinated dogs exhibit at least three of the above symptoms.

[0049] A protective immune response to CPI refers to a significant reduction in viral isolation (shedding) in vaccinated animals compared to unvaccinated animals.

[0050] As noted above, the recombinant oral vaccine can induce a protective immune response after two oral administrations according to the first aspect of the invention, or maintain (or induce) a protective immune response after annual revaccination according to the second aspect of the invention. In other words, in the first aspect of the invention, as described above, parenteral administration is not required to induce a protective immune response against the target pathogen. When the recombinant oral vaccine is administered orally in multiple doses, according to the first aspect of the invention, a second oral dose of the recombinant oral vaccine is administered 7 to about 35 days, or 14 to 35 days, or 7 to 28 days, or 14 to 28 days, or about 21 days after the first oral dose. Optionally, in some embodiments, the second oral dose of the recombinant oral vaccine may be followed by a third oral dose, wherein the third oral dose is administered 7 to about 35 days, or 14 to 35 days, or 7 to 28 days, or 14 to 28 days, or about 21 days apart. Preferably, the recombinant oral vaccine is administered according to the first aspect of the invention.

[0051] Animals receiving the vaccine (whether in two or three oral doses) had not been actively vaccinated against CDV prior to receiving the first oral dose of CDV via the recombinant oral vaccine described herein.

[0052] In other embodiments of the first aspect of the invention, regardless of whether two or three oral doses of the recombinant oral vaccine described herein are administered, the method also requires annual revaccination of the recombinant oral vaccine by oral administration of any of the vaccines disclosed herein, approximately one year after the first administration or the previous annual oral administration of the vaccine, as understood by those skilled in the art.

[0053] In a second aspect, the recombinant oral vaccine described herein can be used for revaccination, for example, annual revaccination, which occurs approximately one year after administration of a different dose of the first and second vaccines and / or approximately one year after administration of a previous annual revaccination (using the vaccine described herein or a different vaccine). The term “different vaccine” is interchangeable with the term “initial vaccine” and refers to a vaccine containing the same antigens as the vaccine according to the invention (in this case, at least CDV antigen and CAV-2 antigen) but not in the form of a recombinant CAV-2 vector containing CDV antigens (e.g., the CDV H gene).

[0054] Suitable vaccines for use as initial vaccines are known in the art and include, but are not limited to, the VANGUARD® series (e.g., VANGUARD® DAP containing modified live CAV-2 virus, modified live CDV virus, and modified live CPV virus), the RECOMBITEC® series (e.g., RECOMBITEC® C3 or RECOMBITEC® C4), or the NOBIVAC® series (e.g., NOBIVAC® canine 1-DAPPv). In this regard, puppies do not need to be MDA negative and can be vaccinated initially as early as 4 weeks of age, as directed by the manufacturer of the respective initial vaccine. Annual revaccination of the dogs is performed one year after the administration of the first dose of the different vaccine, or one year after the previous annual revaccination with the different vaccine, comprising oral administration of a recombinant oral vaccine according to any of the above embodiments.

[0055] The modified live virus in the recombinant oral vaccine may be present in the following amounts / doses (whether it is the first dose, the second dose, the third dose optional according to the first aspect, or the annual booster dose according to the first or second aspect):

[0056] Modified live CAV-2 carrying CDV antigen according to any embodiment of the present invention: 10 2.5 TCID50 to approximately 10 8 TCID50, more preferably 10 3 TCID50 to approximately 10 8 TCID50, more preferably 10 3 TCID50 to approximately 10 6TCID50, 10 3 TCID50 to approximately 10 5.5 TCID50, 10 3 TCID50 to approximately 10 5 TCID50, 10 3 TCID50 to approximately 10 4.5 TCID50, approximately

[0057] 103.5 TCID50 to approximately 10 5.5 TCID50, 10 3.5 TCID50 to approximately 10 5 TCID50, 10 3.5 TCID50 to approximately

[0058] 104.5 TCID50, 10 3.5 TCID50 to approximately 10 4 TCID50, approximately 10 3.7 TCID50 to approximately 10 5.5 TCID50, 10 3.7 TCID50 to approximately 10 5 TCID50, 10 3.7 TCID50 to approximately 10 4.5 TCID50, 10 3.7 TCID50 to approximately 10 4 TCID50, or 10 3.7 TCID50 to approximately 10 4 TCID50;

[0059] Modified live CPV (if present): 10 5.5 TCID50 to approximately 10 10 TCID50, more preferably 10 6.5 TCID50 to approximately 10 9 TCID50, or 10 6.5 TCID50 to approximately 10 8 TCID50, or 10 7.5 TCID50 to approximately 10 10 TCID50, or 10 7.5 TCID50 to approximately 10 9 TCID50;

[0060] Modified live CPI (if present): 10 4.5 TCID50 to approximately 10 8 TCID50, more preferably 10 5.5TCID50 to approximately 10 8 TCID50, or 10 6.5 TCID50 to approximately 10 8 TCID50, or 10 7.5 TCID50 to approximately 10 8 TCID50, or

[0061] 104.5 TCID50 to approximately 10 7 TCID5010 5.5 TCID50 to approximately 10 6 TCID50, more preferably 10 6.5 TCID50 to approximately 10 8 TCID50, or 10 6.5 TCID50 to approximately 10 7 TCID50.

[0062] In addition to the antigen, the recombinant oral vaccines described herein may contain other components, including but not limited to pharmaceutically acceptable excipients, such as carriers, solvents and diluents, isotonic agents, buffers, stabilizers, preservatives, immunomodulators (e.g., interleukins, interferons, and other cytokines), vasoconstrictors, antibacterial agents, and antifungal agents. Typical carriers, solvents, and diluents include water, saline, dextran, ethanol, and glycerol. Representative isotonic agents include sodium chloride, dextran, mannitol, sorbitol, and lactose. Available stabilizers include gelatin and albumin.

[0063] As used herein, "pharmaceutically acceptable carriers" include any and all solvents, dispersion media, coatings, adjuvants, stabilizers, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption delay agents, etc. Carriers

[0064] It must be "acceptable" in the sense of being compatible with the components of the invention and harmless to the subjects to be immunized. Typically, the carrier will be sterile and pyrogen-free, and selected based on the administration method to be used. It is well known to those skilled in the art that preferred formulations of pharmaceutically acceptable carriers constituting vaccines are those approved by applicable regulations issued by the U.S. Department of Agriculture or equivalent government agencies in non-U.S. countries. Therefore, a pharmaceutically acceptable carrier for the commercial production of a vaccine is one that has been or will be approved by the appropriate government agency in the U.S. or a foreign country.

[0065] The recombinant oral vaccine composition may optionally include a pharmaceutically acceptable (i.e., sterile and non-toxic) liquid, semi-solid, or solid diluent that is vaccine-compatible, serving as a drug carrier, excipient, or medium. Diluents may include water, saline, dextran, ethanol, glycerol, etc. Isotonic agents may include sodium chloride, dextran, mannitol, sorbitol, and lactose, etc. Stabilizers include albumin, etc.

[0066] The recombinant oral vaccine according to either the first or second aspect may be adjuvant-free. In other embodiments, the recombinant oral vaccine may also contain an adjuvant. Suitable adjuvants include, but are not limited to, salts of N-(2-deoxy-2-L-leucine-amino-bD-glucopyranosyl)-N-octadecyldodecylamide, such as acetate, used alone or in combination with other adjuvant compounds, such as lecithin and DDA (dimethylbis(octadecyl)ammonium bromide) or PAM3CSK4.

[0067] In other embodiments, the recombinant oral vaccines disclosed herein may also comprise a mucosal adhesive. Suitable mucosal adhesives include, but are not limited to, natural or synthetic hydrophilic substances having organic functional groups (carboxyl, hydroxyl, and amino groups) or hydrogen bonds. Some known mucosal adhesive polymers are carbomers, cellulose derivatives, alginates, lectins, and thiolized polymers. Specific mucosal adhesives include CARBOPOPL® (mildly cross-linked polyacrylic acid (PAA)) and chitosan.

[0068] Recombinant oral vaccines can be administered in doses of the following volumes: between 0.25 ml and about 5 ml, for example between about 0.5 ml and about 3 ml, or between about 0.5 ml and 2 ml, or about 1 ml.

[0069] Preferably, in the first aspect, the vaccine is administered to MDA-negative dogs that are approximately eight weeks old or older (e.g., approximately 10 weeks, approximately 12 weeks, approximately 14 weeks, etc.). Thus, the first dose is administered to the dogs at approximately four weeks of age (or approximately six weeks, approximately eight weeks, approximately 10 weeks, approximately 12 weeks, approximately 14 weeks, etc.), followed by subsequent doses according to the schedule given herein. If the recombinant oral vaccine is administered in annual booster doses, the dogs may be as young as approximately 14 months old at the time of annual booster (if puppies must wait until they are MDA-negative before oral vaccination according to the method of the first aspect of the invention).

[0070] In contrast, if puppies have received an initial vaccination with a different recombinant oral vaccine than described in this article, and if the recombinant oral vaccine is administered at an annual booster dose, the dogs may be as young as 13 months at the time of the annual booster.

[0071] All publications cited in this specification, including patent publications and non-patent publications, reflect the skill of a person skilled in the art to which this invention pertains. All such publications are incorporated herein by reference in their entirety, as if each individual publication were specifically and individually indicated as incorporated by reference.

[0072] The invention will now be described with reference to the following non-limiting embodiments.

[0073] Example

[0074] Example 1. Preparation of CAV-2 vector carrying CDV H gene

[0075] Recombinant canine adenovirus type 2 (rCAV-2::CDV-H) expressing the canine distemper virus hemagglutinin (H) protein was generated by initially transfecting a virulent Manhattan strain of CAV-2 with a plasmid containing the mCherry expression cassette and flanking sequences of the E3 gene in the CAV-2 genome, followed by culturing in Norden's laboratory canine kidney (NLDK) cells to produce rCAV-mC. Subsequently, rCAV-mC was transfected with a plasmid containing the CDV-H expression cassette and the same E3 gene flanking sequences to replace the mCherry gene with the H gene from the Onderstepoort CDV strain.

[0076] The expression plasmid pSV40 used to construct pCAV-SV40-H was obtained from GenScript by Zoetis VMRD, Kalamazoo, MI.

[0077] The expression plasmid pCAV-SV40-H, used to replace mCherry and generate CAV-SV40-H, was constructed by digesting and ligating the SV40 promoter in pSV40 to the NotI-AscI site of pCAV-CMV-H.

[0078] According to the 9CFR exogenous factor test, trypsin was confirmed to be negative for the following specific exogenous factors:

[0079] Fluorescent antibodies (FA) on MDBK cells: Bovine adenovirus I, Bovine adenovirus III, Bovine adenovirus V, Bovine parvovirus, Bovine viral diarrhea virus I, Bovine viral diarrhea virus II, Rabies, Infectious bovine rhinotracheitis, Parainfluenza-3;

[0080] FAs on Vero cells: Bovine respiratory syncytial virus, bluetongue virus, bovine reovirus, bovine rotavirus;

[0081] FAs on ST cells: porcine adenovirus, porcine hemagglutinating encephalomyelitis, porcine parvovirus, swine influenza virus H1N1 and N3N2, transmissible gastroenteritis;

[0082] FA on MA104 cells: porcine reproductive and respiratory syndrome, porcine rotavirus;

[0083] The absence of exogenous factors was confirmed by CPE and HA in MDBK, Vero, MA 104 and ST cells;

[0084] Negative for porcine circovirus strains I and II;

[0085] According to the 9CFR exogenous factor test, the pre-irradiated serum was confirmed to be negative for the following specific exogenous factors:

[0086] FAs on MDBK cells: Bovine adenovirus I, Bovine adenovirus III, Bovine adenovirus V, Bovine parvovirus, Bovine viral diarrhea virus I, Bovine viral diarrhea virus II, Rabies;

[0087] FAs on Vero cells: Bovine respiratory syncytial virus, bluetongue virus, bovine reovirus, bovine rotavirus;

[0088] FA on CRFK cells: Bovine reovirus;

[0089] The absence of exogenous factors was confirmed by CPE and HA on MDBK, Vero, and CRFK cells.

[0090] To generate CAV-mC, the transfer plasmid pCAV-CMV-mC was linearized by digestion with PacI restriction enzyme. The linearized plasmid was transfected into NLDK cells using Lipofectamine 3000 (Life Technologies) and then infected in 6-well plates with parental CAV-2 virus. Four days post-transfection / infection, transfected / infected cells were harvested and purified four times using agarose coating in 6-well plates to obtain red fluorescent plaques. One purified fluorescent plaque was further tested by PCR to confirm the absence of wild-type CAV virus contamination. This plaque was then scaled up in T-75 culture flasks. Cells and supernatant from the T-75 culture were harvested, aliquoted, and stored at -80°C. This recombinant virus was named CAV-mC.

[0091] To generate pCAV-SV40-H, the SV40 promoter from pSV40 was digested and ligated into the NotI-AscI site of pCAV-CMV-H. The transfer plasmid pCAV-SV40-H was linearized by digestion with PacI restriction enzyme. The linearized plasmid was transfected into NLDK cells using Lipofectamine 3000 and infected with CAV-mC virus in 6-well plates. Four days post-transfection / infection, transfected / infected cells were harvested and purified four times using agarose coating in 6-well plates to obtain non-fluorescent plaques. Seven purified non-fluorescent plaques were further confirmed by PCR to contain the SV40-H insert.

[0092] Three purified plaques were further passaged / amplified, and all cultures were harvested and stored at -80°C. These three plaques were named CAV-SV40-H clones 1, 2, and 3. The third generation of the three clones was further sequenced to confirm the inserts. The PCR-amplified insert region (SV40-H) of each clone was confirmed to have the correct sequence. IFA staining of the cell cultures infected with the recombinant virus using available canine anti-CDV serum failed to definitively confirm CDV-H expression due to high background.

[0093] A CAV-SV40-H clone was selected as a candidate to advance to the construction of the master seed pre-batch viral library. This clone was further amplified by two passages (p4 and p5) and cultured in DMEM medium containing 2 mM L-glutamine and 10 µg / mL gentamicin to infect NLDK cells for amplification of pre-MSV culture. The fifth-generation material was then seeded into NLDK cells cultured in DMEM medium containing L-glutamine and 0.05% gentamicin. This pre-MSV was named...

[0094] “CAV-SV40-H #1 p6 batch number 221202-011”. P6 indicates 6 passages (after clonal purification) used for scale-up culture. This pre-MSV was frozen by storage at -80°C.

[0095] Purity, potency, and identification of CAV-SV40-H pre-MSV candidate batch number 221202-011 were tested. Purity was tested using a modified sterility / purity assay, and mycoplasma was tested using the Sigma Aldrich Lookout Mycoplasma qPCR Detection Kit (catalog number: MP0400A-1KT). Potency and identification were tested using IFA on NLDK cells infected with CAV-SV40H.

[0096] In summary, potency and identification were determined by IFA on CAV-SV40-H-infected NLDK cells. CAV-SV40-H pre-MSV batch number 221202-011 was titrated on NLDK cells to determine potency. CAV-2 in wells containing CPE was tested using a CAV-2-specific goat polyclonal antibody as the primary antibody and detected with bovine anti-goat IgG Alex Fluor 488 probe (green fluorescence). Identification of CDV-H was determined using a canine polyclonal antibody as the primary antibody and detected with Cy3 probe-labeled rabbit anti-canine (red fluorescence). The dual-labeling assay allowed for simultaneous assessment of CAV-2 and CDV-H expression in CAV-SV40-H-infected cultures and showed both fluorescence in each well.

[0097] Example 2: Generation of CAV-2 virus expressing CDV F protein.

[0098] The materials and methods used in this embodiment are similar to those disclosed in Example 1. The nucleic acid sequence of the CDV F protein (SEQ ID NO: 4). pCAV-CMV2-F, containing the CDV F gene under the control of a truncated CMV promoter, was synthesized by GenScript. The transfer plasmid pCAV-CMV2-F was linearized by digestion with PacI restriction enzyme. The linearized plasmid was transfected into NLDK cells using Lipofectamine 3000 (Life Technologies) and infected with CAV-mC virus in 6-well plates. Four days after transfection / infection, the transfected / infected cells were harvested and purified four times using agarose capping in 6-well plates to obtain non-fluorescent plaques. Six non-fluorescent plaques were picked from the fourth plaque purification. PCR analysis of the N-terminus and C-terminus of the CMV2-F insert showed positive results for all plaques. Three of these plaques were passaged three times in T-25 culture flasks and stored at -80°C. o Refrigerator C. These three recombinant viruses were named CAV-CMV2-F clones 1, 2, and 3. DNA was extracted from the third-generation virus culture and PCR was performed to confirm stability.

[0099] Example 3. Canine adenovirus type 2 carrying CDV antigen confers a protective response to CDV challenge.

[0100] The aim of this study was to evaluate the efficacy of two modified live vaccines administered orally to dogs approximately three weeks after the third vaccination against canine distemper virus challenged intravenously (IV) at a dilution of 1:50. The vaccines utilized recombinant canine adenovirus-2 (rCAV-2) expressing either the H or F protein of canine distemper virus (CDV).

[0101] Thirty (30) beagle dogs aged approximately 7–10 weeks on day 0 were randomly assigned to three treatment groups of 10 dogs each using a randomized complete block design, with blocks assigned based on maternal age first, followed by date of birth. Animals in treatment group T01 served as controls and received saline. Animals in treatment group T02 received rCAV-2::CDV H. Animals in treatment group T03 received rCAV-2::CDVH and rCAV-2::CDVF. Vaccinations were administered orally to the cheek pouches on days 0, 21, and 42. All animals were challenged on day 63 by intravenous injection of a 1:50 dilution of CDV Snyder Hill strain.

[0102] At the time of the first vaccination, the puppy's age ranges from 7 weeks and 4 days to 8 weeks and 6 days. The dog is healthy and seronegative for CDV (SN titer <2).

[0103] The feed conformed to the standard operating procedures of the testing facility. The diet was a dry ration appropriate for the animals' age and nutritional needs, moistened as necessary, and administered at least daily throughout the study. free Feeding. Canned food or non-drug nutritional supplements may also be provided as needed. Batch numbers of dry food, canned food, and supplements must be recorded. Water should always be provided. free Supply. For the first few weeks after dogs enter the vaccination facility, water bowls are provided in addition to the automatic watering system.

[0104] At the supplier’s facility, antiparasitic and / or antibiotic treatment is administered in accordance with their operating procedures. Upon arrival, the animals receive additional pretreatment as needed, as determined by the ARS veterinarian, for a maximum of three (3) days.

[0105] Serological samples (approximately 6 mL) from all animals were collected in SST tubes on days 0, 21, and 42 (before each vaccination), day 63 (before challenge), and day 84. Nasal swabs were collected using polyester swabs containing approximately 3.0 g of [unspecified substance].

[0106] Sterile collection tubes containing 3.0 mL of virus transport medium (VTM, supplemented with antibiotics) were collected before Day 0. Stool swabs (one swab per 3.0 mL VTM tube) were collected before Day 0. During the vaccination phase, clinical observations were recorded at the following time points: Day-1, twice on Day 0 (before vaccination and 3–6 hours post-vaccination), once daily on Days 1–7 and 20, twice on Day 21 (before vaccination and 3–6 hours post-vaccination), once daily on Days 22–28 and 41, twice daily on Day 42 (before vaccination and 3–6 hours post-vaccination), and once daily on Days 43–49. During the challenge phase, clinical observations were recorded at the following time points: Day 62, twice on Days 63–68, three times on Days 69–75, twice on Days 76–83, and once on Day 84. During the vaccination phase, tympanic membrane temperature was recorded at the following time points: once on day -1, twice on day 0 (before vaccination and 3-6 hours after vaccination), once daily on days 1-7 and 20, twice on day 21 (before vaccination and 3-6 hours after vaccination), once daily on days 22-28 and 41, twice daily on day 42 (before vaccination and 3-6 hours after vaccination), and once daily on days 43-49. During the challenge phase, tympanic membrane temperature was recorded at the following time points: once on day 62, twice on day 63 (before challenge and approximately 3-6 hours after challenge), and at least once daily from days 64 to 84.

[0107] After the challenge, the veterinarian considered the humanitarian endpoint of the study.

[0108] efficacy determination

[0109] At the time of this study, no monoclonal antibody targeting the CDV H protein was available. Zoetis Study B6563 used beacon to generate this antibody. Therefore, results related to the CDV H protein utilized in T02 and T03 were not yet available. The amount of CAV-2 carrying the CDV H protein was determined by measuring CAV-

[0110] Quantification is performed using a dose of 2. The amount of antigen per dose is as follows: In T02: Total CAV-2 is 10. 3.7 TCID50, in T03: Total CAV-2 is 10 3.7 TCID50, CAV-2 carrying F protein is 10 3.42 TCID50. The challenge material is administered at 10 mg / dose. 3.3 TCID50 dosage.

[0111] result

[0112] Results were determined based on 9 CFR 113.306: Immunogenicity of Canine Distemper Vaccines. All animals in the study must be seronegative to CDV on day 0 (before vaccination). Control animals must remain seronegative until challenge.

[0113] Disease / mortality was the primary variable. Nine out of ten vaccinated animals had to survive the observation period without exhibiting disease. Vaccine efficacy was defined as a significant difference between the vaccinated groups (T02, T03, and / or T04) and the control group (T01) at α=0.10 (two-sided).

[0114] When fever is not included as a clinical symptom of post-challenge disease, group T02 met the outcome criteria of this study. In 9 CFR section 113.306, "clinical disease" is defined as...

[0115] 1) Fever. 2) Clinical signs of canine distemper virus. 3) Any neurological symptoms or moribund behavior. 4) Death. Fever was observed in all animals at at least one time point after challenge. Fever was also recorded after vaccination in all treatment groups (including control animals receiving saline).

[0116] Differences in fever severity were observed between control and vaccinated animals. Since 80% of the control animals died 8 days after challenge (day 72 of the study), the duration of fever between animal groups could not be assessed. In addition to higher fever values ​​than the vaccinated group, control animals also exhibited the following clinical symptoms: agitation, anorexia, dehydration, depression, diarrhea, eye discharge, muscle tremors, pain, bleeding, photophobia, vomiting, and death (euthanasia due to reaching a humanitarian endpoint). Therefore, the definition of disease was revised to exclude fever from both treatment groups.

[0117] When fever was excluded from other clinical symptoms in the disease definition, all vaccination groups (T02-T04) showed significant differences in disease frequency compared to the control group (T01). All animals in the T01 control group developed the disease, while no animals in T02 developed the disease (P < 0.0001), and two animals in T03 developed the disease (P = 0.0003). The data are summarized in Table 1.

[0118] Table 1. Frequency distribution of clinical observations of diseases including and without fever

[0119]

[0120] Excluding fever, the stratified disease prevention score was 100% in animals vaccinated with rCAV-2::CDV-H, and 80% in animals vaccinated with rCAV-2::CDV-H / rCAV-2::CDV-F. The lower limit of 90% was higher than zero in all vaccination groups (Table 2).

[0121] Table 2. CMH estimates and confidence limits for treatment group comparisons and disease stratification prevention scores excluding fever.

[0122]

[0123] mortality rate

[0124] No dogs (0%) in treatment groups T02 (rCAV-2 CDV H) or T03 (rCAV-2 CDV H and rCAV-2 CDV F) withdrew from the study and / or died after challenge (Table 3). Eight dogs (80%) in the control group T01 withdrew from the study after challenge (Table 3).

[0125] Table 3. Frequency distribution of mortality rates in the treatment group

[0126]

[0127] Vaccination phase

[0128] Clinical observation

[0129] Several dogs in all three treatment groups were observed to have fever (≥39.5°C) after the first, second, and third vaccinations; however, fever was observed at the following time points: day -1 in treatment group T02 and before the first vaccination on day 0 in all four treatment groups; day 20 in T01, T03, and T04 and before the second vaccination on day 21 in T02-T04; and day 41 in T02-T03 and before the vaccination on day 42 in all four treatment groups. No additional clinical signs (restlessness, anorexia, ataxia, cough, dehydration, depression, diarrhea, near death, mucopurulent nasal discharge, muscle tremors, pain, petechiae, photophobia, respiratory distress, seizures, swimming-like movements, vomiting, or other clinical signs) were observed in any treatment group after the first vaccination.

[0130] Infectious Disease Stage

[0131] All animals in all treatment groups (100%) showed elevated body temperature or fever (≥39.5°C) during some observation periods after challenge; however, fever was also frequently observed in most animals before challenge.

[0132] The frequency distribution of clinical symptoms after challenge is shown in Table 4. The T02 group, which received the CAV-2::CDV-H construct orally, never showed clinical symptoms of canine distemper after challenge.

[0133] Table 4. Frequency distribution of clinical symptoms of disease after viral challenge in the treatment group

[0134]

[0135]

[0136] No animals in any treatment group (0%) exhibited the following clinical signs after challenge: ataxia, near death, mucopurulent nasal discharge, respiratory distress, seizures, or swimming-like movements. Other clinical signs included head tremors, arched back posture, decreased body condition, reluctance to stand, stiff gait, vocalizations, and bloody stools.

[0137] Serology

[0138] As expected, all animals in T02 (rCAV-2::CDV-H) and T03 (rCAV-2::CDV-H / rCAV-2::CDV-F) underwent seroconversion to CDV (Table 5), CAV-1 (Table 6), and CAV-2 (Table 7) prior to challenge, as measured by SN antibody titers. The levels of CAV-1 and CAV-2 titers were expected to provide disease prevention.

[0139] Table 5. Summary of geometric mean of CDV valence in treatment groups

[0140]

[0141] Table 6. Summary of geometric mean of CAV-1 titers in treatment groups

[0142]

[0143] Table 7. Summary of geometric mean of CAV-2 titers in treatment groups

[0144]

[0145] All dogs (100%) were negative for CDV, CAV-1, and CAV-2 on day -1. The control animals in T01 remained negative for all sub-items until challenge, at which point the remaining two animals became seropositive for CDV on day 84. All dogs (100%) in T02 and T03 became SN-positive for CDV, CAV-1, and CAV-2 during the vaccination phase of the study (Table 8).

[0146] Table 8. Frequency distribution of positive serum neutralizing (SN) efficacy value (>32) in the treatment group during the vaccination phase.

[0147]

[0148] in conclusion

[0149] This study was valid. All animals were healthy and serologically negative for canine distemper virus (<2) prior to day 0. Eighty percent (80%) of the control animals reached the mortality endpoint and were humanely euthanized. The remaining 20% ​​of the animals developed typical clinical symptoms of canine distemper virus disease over multiple observation periods. These criteria met the challenge outcome according to 9 CFR 113.306.

[0150] The stratified prevention score for diseases excluding fever was 100% in the T02 (rCAV-2::CDV-H) vaccination group (P<0.0001), while it was 80% in the T03 (rCAV-2::CDV-H and rCAV-2::CDV-F) group (P=0.0003). The 90% confidence limit was higher than 0 for all vaccination groups.

[0151] Because fever was observed in all treatment groups before and after challenge, analyses were performed with and without fever in the disease definition. Without fever, 100% of the control animals became ill after challenge, none of the T02 animals (rCAV-2::CDV-H) experienced disease after challenge, while two (20%) of the T03 animals (rCAV-2::CDV-H and rCAV-2CDV F) became ill after challenge.

[0152] Control animals remained seronegative for CDV before challenge; after challenge, the remaining two animals became seropositive for CDV on day 84, indicating exposure to the challenge virus. All vaccination groups (T02, T03) showed a recall response to challenge by geometric mean titer.

[0153] As expected, due to the rCAV-2 vaccine vector, animals in T02 and T03 also underwent seroconversion of CAV-1 and CAV-2.

[0154] Based on the results of this study, the rCAV-2::CDV-H construct (T02) appears to be more effective than both rCAV-2::CDV-H and rCAV-2CDV F (T03) because no animals in the former group exhibited disease when fever was excluded, and the vaccine...

[0155] It provides complete protection against intravenous CDV Snyder Hill challenge. The combination of the two rCAV-2 constructs (T03) did not outperform the monovalent construct (T02).

Claims

1. A method for protecting MDA-negative dogs against canine distemper virus, the method comprising administering to the dog a modified live canine adenovirus type 2 (CAV-2) vector carrying a recombinant oral vaccine containing canine distemper virus (CDV) antigen, wherein the recombinant oral vaccine is administered orally in a first dose and in a second dose.

2. A method for annually revaccinating a dog, the method comprising orally administering to the dog a vaccine comprising a recombinant oral vaccine comprising a modified live canine adenovirus type 2 (CAV-2) vector carrying a canine distemper virus (CDV) antigen, wherein the recombinant oral vaccine is administered approximately one year after a previous year's revaccination or approximately one year after administering a first dose of an initial vaccine to the dog, wherein the first dose of the initial vaccine is administered parenterally to the dog, and wherein the initial vaccine comprises both CDV antigen and CAV-2 antigen.

3. The method of claim 2, wherein in the initial vaccine, c) The CDV antigen is a modified live CDV, and the CAV-2 antigen is a modified live CAV-2; or d) The CDV antigen is encoded by a nucleic acid sequence within the genome of a modified live canarypox virus.

4. The method of claim 2 or claim 3, wherein the vaccine is administered to the dog during the previous year's revaccination, wherein... c) The CDV antigen is a modified live CDV, and the CAV-2 antigen is a modified live CAV-2; or d) The CDV antigen is encoded by a nucleic acid sequence within the genome of a modified live canarypox virus.

5. The method according to any one of claims 1 to 4, wherein in the recombinant oral vaccine, the CDV antigen is the H protein.

6. The method of any one of claims 1 to 5, wherein in the recombinant oral vaccine, the CDV antigen comprises SEQ ID NO: 1 or a sequence that is 90% or 95% identical to SEQ ID NO:

1.

7. The method of claim 6, wherein in the recombinant oral vaccine, the CDV antigen is a conserved substitution variant of SEQ ID NO:

1.

8. The method of any one of claims 1 to 7, wherein in the recombinant oral vaccine, the genome of the modified live CAV-2 vector contains a nucleic acid sequence encoding the CDV antigen.

9. The method of claim 8, wherein in the recombinant oral vaccine, the nucleic acid sequence encoding the CDV antigen is effectively regulated by the SV-40 promoter.

10. The method of any one of claims 8 or 9, wherein in the recombinant oral vaccine, the nucleic acid sequence comprises SEQ ID NO:

2.

11. The method of any one of claims 8 to 10, wherein in the recombinant oral vaccine, the nucleic acid sequence encoding the CDV antigen is inserted into the E3 region of the modified live CAV-2 vector genome.

12. The method of any one of claims 1 to 11, wherein each dose of the recombinant oral vaccine contains at least 10 3 TCID 50 To about 10 8 TCID 50 The modified live CAV-2 vector carrying the CDV antigen.

13. The method of claim 12, wherein each dose of the recombinant oral vaccine contains about 10 3.5 TCID 50 To about 10 4.5 TCID 50 The modified live CAV-2 vector carrying the CDV antigen.

14. The method of any one of claims 1 and 5 to 13, wherein the dog has not previously been vaccinated against canine distemper virus.

15. The method of any one of claims 1 to 14, wherein the modified live canine adenovirus type 2 vector carrying the CDV antigen provides protection against canine distemper virus, canine adenovirus type 1 infection and canine adenovirus type 2 infection upon oral administration.

16. The method of claim 15, wherein the dog has not previously been vaccinated against canine adenovirus type 2.

17. The method of any one of claims 1 to 16, wherein the recombinant oral vaccine further comprises modified live parvovirus, and wherein the recombinant oral vaccine provides further protection against canine parvovirus infection when administered orally.

18. The method of claim 17, wherein the recombinant oral vaccine comprises about 10 5.5 TCID 50 To about 10 10 TCID 50 The modified live canine parvovirus described above.

19. The method of claim 17, wherein the recombinant oral vaccine comprises 10 7.5 TCID 50 To about 10 9 TCID 50 The modified live canine parvovirus described above.

20. The method of any one of claims 17 to 19, wherein the dog has not previously been vaccinated against canine parvovirus.

21. The method of any one of claims 1 to 20, wherein the recombinant oral vaccine further comprises a modified live canine parainfluenza virus, and wherein the recombinant oral vaccine provides further protection against canine parainfluenza virus infection upon oral administration.

22. The method of claim 19, wherein the recombinant oral vaccine comprises 10 4.5 TCID 50 To about 10 8 TCID 50 The modified live canine parainfluenza virus described above.

23. The method of claim 19, wherein the recombinant oral vaccine comprises 10 6.5 TCID 50 To about 10 7.5 TCID 50 The modified live canine parainfluenza virus described above.

24. The method of any one of claims 21 to 23, wherein the dog has not previously been vaccinated against canine parainfluenza virus.

25. The method of any one of claims 1, 5 to 24, wherein the dog is about 8 to about 16 weeks old when the recombinant oral vaccine is administered; or the method of any one of claims 2 to 13, 15, 17 to 19 or 21 to 23, wherein the dog is at least 13 months old when the recombinant oral vaccine is administered.

26. The method of any one of claims 1, 5 to 25, wherein the second dose of the recombinant oral vaccine is administered 7 to 35 days after the first dose of the recombinant oral vaccine.

27. The method of any one of claims 1, 5 to 26, wherein the second dose of the recombinant oral vaccine is administered about 21 days after the first dose of the recombinant oral vaccine.

28. The method of any one of claims 1, 5 to 27, comprising the step of orally administering a third dose of the recombinant oral vaccine, wherein the third dose is administered approximately 21 days after the second dose of the recombinant oral vaccine.

29. The method of any one of claims 1 to 28, wherein in the recombinant oral vaccine, the modified live CAV-2 vector is not enteric-coated, and wherein, if present in the recombinant oral vaccine, the modified live canine parvovirus is not enteric-coated, and wherein, if present, the modified live canine parainfluenza virus is not enteric-coated.

30. A recombinant oral vaccine comprising a modified live canine adenovirus type 2 (CAV-2) vector carrying canine distemper virus (CDV) antigen, in a method for protecting MDA-negative dogs against CDV infection, wherein the vaccine is administered to the dog, characterized in that... The recombinant oral vaccine is administered orally in a first dose and then orally in a second dose.

31. A recombinant oral vaccine comprising a modified live canine adenovirus type 2 (CAV-2) vector carrying canine distemper virus (CDV) antigen, for annual revaccination of dogs, the method comprising orally administering the recombinant oral vaccine to the dog about one year after a previous year's revaccination or about one year after administering a first dose of an initial vaccine to the dog, wherein the first dose of the initial vaccine is administered parenterally to the dog, and wherein the initial vaccine comprises CDV antigen and CAV-2 antigen.

32. The recombinant oral vaccine of claim 31, wherein in the initial vaccine, a) The CDV antigen is a modified live CDV, and the CAV-2 antigen is a modified live CAV-2; or b) The CDV antigen is encoded by a nucleic acid sequence within the modified live canarypox virus genome.

33. The recombinant oral vaccine of claim 31 or 32, wherein the vaccine is administered to the dog during the prior year revaccination, wherein... a) The CDV antigen is a modified live CDV, and the CAV-2 antigen is a modified live CAV-2; or b) The CDV antigen is encoded by a nucleic acid sequence within the modified live canarypox virus genome.

34. The recombinant oral vaccine for the purpose as described in any one of claims 30 to 33, wherein the CDV antigen is the H protein.

35. The recombinant oral vaccine for the purpose as claimed in any one of claims 30 to 34, wherein in the recombinant oral vaccine, the CDV antigen comprises SEQ ID NO: 1 or a sequence that is 90% or 95% identical to SEQ ID NO:

1.

36. The recombinant oral vaccine for the stated purpose as claimed in claim 35, wherein in the recombination, the CDV antigen is a conserved substitution variant of SEQ ID NO:

1.

37. The recombinant oral vaccine for the purpose as described in any one of claims 30 to 36, wherein in the recombinant oral vaccine, the genome of the modified live CAV-2 vector contains a nucleic acid sequence encoding the CDV antigen.

38. The recombinant oral vaccine for the stated purpose as claimed in claim 37, wherein in the recombinant oral vaccine, the nucleic acid sequence encoding the CDV antigen is effectively regulated by the SV-40 promoter.

39. The recombinant oral vaccine for the purpose as described in any one of claims 37 or 38, wherein in the recombinant oral vaccine, the nucleic acid sequence encoding the CDV antigen comprises SEQ ID NO:

2.

40. The recombinant oral vaccine for the purpose as described in any one of claims 37 to 39, wherein in the recombinant oral vaccine, the nucleic acid sequence encoding the CDV antigen is inserted into the E3 region of the modified live CAV-2 vector.

41. The recombinant oral vaccine for the purpose as described in any one of claims 30 to 40, wherein each dose of the recombinant oral vaccine contains at least 10 3 TCID 50 To about 10 8 TCID 50 The modified live CAV-2 vector carrying the CDV antigen.

42. The recombinant oral vaccine for the said use as claimed in claim 41, wherein each dose of the recombinant oral vaccine contains about 10 3.5 TCID 50 To about 10 4.5 TCID 50 The modified live CAV-2 vector carrying the CDV antigen.

43. The recombinant oral vaccine for the purpose as claimed in any one of claims 30, 34 to 42, wherein the dog has not previously been vaccinated against canine distemper virus.

44. The recombinant oral vaccine for the stated purpose as described in any one of claims 30 to 43, wherein the modified live canine adenovirus type 2 vector carrying the CDV antigen provides protection against canine distemper virus, canine adenovirus type 1 infection and canine adenovirus type 2 infection upon oral administration.

45. The recombinant oral vaccine for the purpose of claim 44, wherein the dog has not previously been vaccinated against canine adenovirus type 2.

46. ​​The recombinant oral vaccine for the purpose as claimed in any one of claims 30 to 45, wherein the recombinant oral vaccine further comprises modified live parvovirus, and wherein the vaccine provides further protection against canine parvovirus infection when administered orally.

47. The recombinant oral vaccine for said use as claimed in claim 46, wherein the recombinant oral vaccine comprises about 10 6.5 TCID 50 To about 10 10 TCID 50 The modified live canine parvovirus described above.

48. The recombinant oral vaccine for said use as claimed in claim 46, wherein the recombinant oral vaccine comprises about 10 5.5 TCID 50 To about 10 9 TCID 50 The modified live canine parvovirus described above.

49. The recombinant oral vaccine for the purpose as claimed in any one of claims 46 to 48, wherein the dog has not previously been vaccinated against canine parvovirus.

50. The recombinant oral vaccine for the purpose as claimed in any one of claims 30 to 49, wherein the recombinant oral vaccine further comprises a modified live canine parainfluenza virus, and wherein the recombinant oral vaccine provides further protection against canine parainfluenza virus infection upon oral administration.

51. The recombinant oral vaccine for the said use as claimed in claim 50, wherein the recombinant oral vaccine comprises about 10 4.5 TCID 50 To about 10 8 TCID 50 The modified live canine parainfluenza virus described above.

52. The recombinant oral vaccine for the said use as claimed in claim 50, wherein the vaccine comprises 10 6.5 TCID 50 To about 10 7.5 TCID 50 The modified live canine parainfluenza virus described above.

53. The recombinant oral vaccine for the purpose as claimed in any one of claims 50 to 52, wherein the dog has not previously been vaccinated against canine parainfluenza virus.

54. A recombinant oral vaccine for the purpose as claimed in any one of claims 30, 34 to 53, wherein the dog is about 8 to about 16 weeks old when the recombinant oral vaccine is administered; or a recombinant oral vaccine for the purpose as claimed in any one of claims 31 to 42, 44, 46 to 48, 50 to 52, wherein the dog is at least 13 months old when the recombinant oral vaccine is administered.

55. The recombinant oral vaccine for the purpose as described in any one of claims 30, 34 to 54, wherein the second dose is administered 7-35 days after the first dose.

56. The recombinant oral vaccine for the purpose as described in any one of claims 30, 34 to 55, wherein the second dose is administered about 21 days after the first dose.

57. The recombinant oral vaccine for the purpose as described in any one of claims 30, 34 to 56, wherein the method is further characterized by oral administration of a third dose of the vaccine, wherein the third dose is administered approximately 21 days after the second dose.

58. The recombinant oral vaccine for the purpose as claimed in any one of claims 30 to 57, wherein the modified live CAV-2 vector in the recombinant oral vaccine is not enteric-coated, and wherein, if present in the recombinant oral vaccine, the modified live canine parvovirus is not enteric-coated, and wherein, if present in the recombinant oral vaccine, the modified live canine parainfluenza virus is not enteric-coated.

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