HK1190638B - Compositions for canine respiratory disease complex - Google Patents

Description

Composition for canine respiratory disease syndrome

Technical Field

The present invention relates to the field of immunology, in particular to immunogenic and vaccine compositions. It relates to compositions for the prevention and treatment of canine respiratory diseases including canine infectious respiratory disease syndrome (CIRDC). The invention also relates to methods of vaccinating, treating, or preventing respiratory diseases in canines.

Background

Canine infectious respiratory disease syndrome (CIRDC) is a very contagious disease that is common in dogs that live in crowded conditions, such as in reception centers and boarding or training kennels. Many dogs exhibit only a mild cough and then recover in a short period of time. In some cases, however, severe bronchopneumonia may develop.

The pathogenesis of CIRDC is considered to be multifactorial, involving several viruses and bacteria. Known infectious agents of CIRDC pathogens include canine respiratory coronavirus (CRCoV) (Erles et al, Virology,310(2):216-223,2003), Canine Influenza Virus (CIV) (Crawford et al, Science,310(5747): 482-.

CRCoV can cause highly contagious respiratory disease, which is transmitted by direct canine-to-canine contact, aerosols of respiratory secretions, and contact with contaminated environments and humans. Mild symptoms in dogs include coughing, sneezing, nasal discharge. Some dogs have subclinical infections and do not have any clinical symptoms, but they can transmit viruses to infect other dogs. Some dogs infected with CRCoV develop pneumonia, particularly if also infected with other respiratory pathogens.

With respect to CIV, the equine influenza virus was considered to be the major respiratory pathogen in horses since about 1956. Equine influenza virus can cause severe disease symptoms and often secondary bacterial infections. Two subtypes of Equine influenza virus have been identified, designated subtype-1 (prototype A/Equinone/Prague/1/56 (H7N7)) and subtype-2 (prototype A/Equinone/Miami/1/63 (H3N 8)). Currently, the major viral subtype is subtype-2, H3N8 strain. The H3N8 equine influenza virus can infect dogs, with a mortality rate as high as 36% in some cases. One explanation is that the whole or partial interspecies transfer of equine influenza virus to canines produces a new canine specific influenza virus associated with acute respiratory disease (Crawford et al, 2005).

CPIV causes diseases that are usually in the upper respiratory tract. The disease caused by CPIV alone is very mild or presents subclinical, and symptoms become more severe if complicated by infection with other respiratory pathogens.

CAV-2 causes respiratory diseases, which in severe cases may include pneumonia and bronchopneumonia.

Bordetella bronchiseptica (b.bronchia) is reported to be the major pathogen in the respiratory disease tracheobronchitis or "kennel cough". It leaves the dog vulnerable to other respiratory agents and often co-exist with them. Kennel cough usually occurs in the upper respiratory tract and is characterized by runny nose and cough. To date, some vaccines have been used to treat tracheobronchitis caused by bordetella bronchiseptica (Bordetella aborhiccups), including NobivacBronchi-Shield、BronchicineCAe、VanguardB、Univac2、RecombitekKC2、Naramune^TM-2 and Kennel-Jec^TM2. However, most of the existing commercial vaccines require cumbersome nasal administration and the addition of adjuvants, which may lead to harmful side effects such as burning and irritation. VieraScheibner et al, NexusDec2000(Vol8, No 1). Subunit vaccines, such as those involving the use of p68 protein from bordetella bronchiseptica (pertactin), have been studied, but have not been included to date in any commercial canine vaccine, possibly because of insufficient immunogenicity, adverse effects, and/or formulation stability.

The pathology of circc suggests that it is involved in lung injury, in some cases leading to bronchopneumonia, but it differs from kennel cough (the main pathogen: bordetella bronchiseptica b. bronchinsis), which is mainly involved in upper respiratory tract changes. Kennel cough is a much milder syndrome than CIRDC and does not have the extensive lesions indicated in CIRDC. CIRDC is also characterized by increased severity and mortality.

CIRDC is rarely fatal, but delays the dogs in rescue centers from being adopted, disrupting kennel training schedules, resulting in considerable treatment costs and welfare problems. The vaccine may be used to treat some infectious agents associated with CIRDC. However, despite the use of these vaccines, CIRDC is still prevalent worldwide, possibly due to the lack of effective vaccines against all pathogens associated with CIRDC.

Thus, there remains a need for an immunogenic prototype composition that can be safely used in canines, while providing long-term effective immune protection against CIRDC-eliciting pathogens, without deleterious side effects or interference with other antigens when used with other vaccines. The present invention addresses these and other related needs.

Disclosure of Invention

The present invention relates generally to immunogenic compositions that can provide antigens for the treatment or prevention of CIRDC. In one embodiment, the immunogenic composition comprises Canine Influenza Virus (CIV) and canine respiratory coronavirus (CRCoV). In another embodiment, the immunogenic composition further comprises bordetella bronchiseptica. In another embodiment, the immunogenic composition further comprises a pertactin antigen alone. In another embodiment, the immunogenic composition comprises p68 pertactin antigen. In another embodiment, the pertactin antigen is a recombinant protein. In yet another embodiment, the pertactin antigen is between about 1 μ g to about 30 μ g. In another embodiment, the pertactin antigen is prepared by dissolving pertactin antigen inclusion bodies in urea and optionally purifying by a chromatography column. The pertactin antigen is soluble and preferably substantially free of aggregates. In another embodiment, bordetella bronchiseptica is a vaccine or bacterial extract.

In one embodiment, the immunogenic composition comprises CIV, CRCoV, bordetella bronchiseptica, and one or two antigens selected from the group consisting of canine parainfluenza virus (CPIV) and canine adenovirus type 2 (CAV-2). In another embodiment, the immunogenic composition further comprises p68 pertactin antigen. In another embodiment, bordetella bronchiseptica is a vaccine or bacterial extract.

In another embodiment, an immunogenic composition is provided comprising CIV, CRCoV, a bordetella bronchiseptica component comprising bordetella bronchiseptica, a pertactin antigen alone, and one or both antigens selected from the group consisting of canine parainfluenza virus (CPIV) and canine adenovirus type 2 (CAV-2). In a further embodiment, the immunogenic composition comprises both CPIV and CAV-2.

In another embodiment, any of the immunogenic compositions of the above embodiments further comprises Bsp22 antigen alone.

In another embodiment, any one of the immunogenic compositions of the above embodiments is unadjuvanted. In another embodiment, any one of the immunogenic compositions of the above embodiments includes an adjuvant.

In another embodiment, any of the immunogenic compositions of the above embodiments does not contain a non-respiratory antigen.

In yet another embodiment, the immunogenic composition of any one of the above embodiments induces an immune response in a canine against a canine respiratory pathogen. In another embodiment, the canine respiratory pathogen is at least one of CIV, CRCoV, CPIV, CAV-2, Bordetella bronchiseptica, and Mycoplasma canis (Mycoplasma acnnos).

In another embodiment of the present invention is provided the use of any one of the immunogenic compositions of the above embodiments for treating or preventing infection of a canine respiratory pathogen in a canine. In another embodiment, the canine respiratory pathogen is at least one of CIV, CRCoV, CPIV, CAV-2, Bordetella bronchiseptica, and Mycoplasma canis. In another embodiment, the composition prevents the infection for a period of about 6 months or longer. In another embodiment, the composition prevents the infection for a period of about 1 year. In another embodiment, the present invention provides the use of any one of the immunogenic compositions of the above embodiments in the manufacture of a medicament for the treatment or prevention of infection by a canine respiratory pathogen in a canine.

In another embodiment of the present invention is provided the immunogenic composition of any one of the above embodiments, wherein the composition treats or prevents canine infectious respiratory disease syndrome (CIRDC) in vivo in a canine. In another embodiment of the invention, there is provided a method of treating or preventing CIRDC in a canine comprising administering to said canine an immunogenic composition of any one of the above embodiments. In another embodiment, the composition can prevent CIRCD for about 6 months or more. In another embodiment, the composition can prevent CIRCD over a period of about 1 year. Another embodiment provides the use of any one of the immunogenic compositions of the above embodiments for the manufacture of a medicament for treating or preventing CIRDC in a canine.

Brief description of the drawings

FIG. 1 serum neutralizing antibody responses against CRCoV.

When dogs are treated with saline, adjuvanted aluminum hydroxide (AlOH) or adjuvanted EmulsigenComponents for vaccination, serum neutralization antibody responses against canine respiratory coronavirus (CRCoV) were measured.

Figure 2 virus shedding from the nose after challenge. The dogs are treated with saline, adjuvanted aluminum hydroxide (AlOH) or adjuvanted EmulsigenThe fractions were vaccinated and then challenged with CRCoV and the outflow of CRCoV from the nasal passages was measured.

Figure 3 percentage of animals positive for CRCoV tissue virus on day 4 post challenge. With saline, adjuvanted aluminum hydroxide (AlOH) or adjuvanted EmulsigenThe fractions were vaccinated and then challenged with CRCoV to assess the number of dogs in respiratory tissues that were positive for CRCoV.

Detailed Description

The following definitions apply to the present disclosure. Which supersedes any conflicting definition contained in each individual reference cited herein. The undefined terms have the meanings commonly used by those skilled in the art. Further, unless specifically mentioned herein, singular nouns include plural, plural nouns include singular.

"about" and "approximately" when used in connection with a measurable numerical variable means that the indicated value of the variable and all values of the variable are within experimental error of the indicated value (e.g., within 95% confidence interval of the mean) or within 10% (or greater than 10%) of the indicated value. If "about" is used to refer to weekly intervals, "about 3 weeks" is 17 to 25 days, "about 2 weeks to about 4 weeks" is 10-40 days.

"adjuvant" refers to any substance that can act as a non-specific stimulator in an immune response. See further description below for adjuvants.

The term "animal" as used herein includes any animal that can be infected with canine respiratory disease syndrome, including domestic and wild mammals.

"antibody" refers to any polypeptide that includes an antigen binding site, regardless of its source, method of production, and other characteristics. It refers to an immunoglobulin molecule or fragment thereof that specifically binds to an antigen and thereby generates an immune response against the antigen. Immunoglobulins are serum proteins composed of "light" and "heavy" polypeptide chains with "constant" and "variable" regions, and are divided into classes based on the composition of the constant regions (e.g., IgA, IgD, IgE, IgG, and IgM). An antibody that is "specific" for a given antigen means that the variable region of the antibody can specifically recognize and bind to the particular antigen. The term includes, but is not limited to: polyclonal antibodies, monoclonal antibodies, monospecific antibodies, multispecific antibodies (multispecific antibodies), humanized antibodies, tetrameric antibodies, multispecific antibodies (multispecific antibodies), single-chain (single-chain) antibodies, specific domains (dom)ain-specific) antibodies, single domain antibodies, domain-deleted antibodies, fusion proteins, ScFc fusion proteins, single-chain (single-chain) antibodies, chimeric antibodies, synthetic antibodies, recombinant antibodies, hybrid antibodies, mutant antibodies, and CDR-grafted antibodies. The antibody may be an intact immunoglobulin from a natural source or a recombinant source, or an immunoreactive portion of an intact immunoglobulin. An "antibody" can be converted into a protein that binds to an antigen, including, but not limited to, antibody fragments, including, but not limited to, Fab, F (ab')₂Fab' fragments, Fv fragments, single chain Fv (scfv), Fd fragments, dAb fragments, diabodies, CDR3 polypeptides, constrained FR3-CDR3-FR4 polypeptides, nanobodies, diabodies, Small Modular Immunopharmaceuticals (SMIPs), low molecular weight antibodies (minibodies), and the above-mentioned fragments, chemically or genetically manipulated pairs thereof, and other antibody fragments that retain antigen binding function. Typically, such fragments comprise a domain that binds an antigen. As known to those skilled in the art, the molecules may be designed (e.g., germline) to reduce their immunogenicity, increase affinity, alter their specificity, or for other purposes.

An "antigen" or "immunogen" refers to a molecule that includes one or more epitopes that, upon exposure to a subject, will elicit an immune response specific for that antigen. An epitope is a specific site of an antigen that binds to a T-cell receptor or a specific antibody and typically contains from about 3 amino acid residues to about 20 amino acid residues. The term antigen refers to subunit antigens-isolated and discrete from antigens throughout an organism associated with antigenic properties-as well as killed, attenuated or inactivated bacteria, viruses, fungi, parasites or other microorganisms. The term antigen also refers to antibodies, such as anti-idiotypic antibodies or fragments thereof, that can mimic synthetic peptide mimotopes of an antigen or antigenic determinant (epitope). The term antigen also refers to oligonucleotides or polynucleotides that express an antigen or antigenic determinant in vivo, such as in DNA immunization applications.

"antigenic" as used herein refers to the ability of a protein or polypeptide to immunospecifically bind to antibodies that are increased for protection against the protein or polypeptide.

The term "bordetella bronchiseptica" or "bordetella bronchiseptica" refers to attenuated live cells of bordetella bronchiseptica, an inactivated whole-cell extract (vaccine) of bordetella bronchiseptica or a cellular bacterial extract of bordetella bronchiseptica.

"buffer" refers to a chemical system that prevents a change in the concentration of another chemical. The proton donor and acceptor system act as a buffer to prevent significant changes in hydrogen ion concentration (pH). An example of a buffer is a buffer containing a weak acid and its salt (conjugate base) or a mixture of a weak base and its salt (conjugate acid).

As used herein, "canine" includes what is commonly referred to as a dog, but also includes other members of the canine family.

The term "cell line" or "host cell" as used herein refers to a prokaryotic or eukaryotic cell in which a virus can replicate or be fed.

The term "culture," as used herein, refers to a population of cells or microorganisms that grow in the absence of other species or types.

"dose" refers to a vaccine or immunogenic composition administered to a subject. "first dose" or "priming dose" refers to the dose of the composition administered on day 0. The "second dose" or "third dose" or "annual dose" refers to the amount of the composition administered after the first dose is administered, which may be, but does not require, the same vaccine or immunogenic composition as the first dose.

"epitope" refers to a specific position on an antigen that can bind to a T-cell receptor or a specific antibody, and typically comprises from about 3 to about 20 amino acid residues.

As used herein, "excipient" refers to the inactive carrier component of a non-antigenic vaccine or immunogenic composition.

"fragment" refers to a portion of a protein or gene that is truncated. "functional fragment" and "biologically active fragment" refer to a fragment that retains the biological properties of a full-length protein or gene.

"homology" or "percent homology" refers to the percentage of nucleotide or amino acid residues of a candidate sequence that are identical or similar to the residues of the comparator sequence after gaps have been introduced into its sequence, and if necessary, conservative substitutions are also contemplated as part of the sequence homology in order to obtain the greatest percent sequence homology.

"homologs" or "species homologs" include genes found in 2 or more different species having a substantial amount of polynucleotide sequence homology and having the same or similar biological function and/or performance. Preferred polynucleotide sequences representing species homologues hybridize under relatively stringent conditions, as described in the examples, and have the same or similar biological activity and/or properties. In another aspect, polynucleotides representing species homologs share greater than about 60% sequence homology, greater than about 70% sequence homology, greater than about 80% sequence homology, greater than about 90% sequence homology, greater than about 95% sequence homology, greater than about 96% sequence homology, greater than about 97% sequence homology, greater than about 98% sequence homology, greater than about 99% sequence homology.

"identity" or "percent identity" refers to the percentage of nucleotides or amino acids of a candidate sequence that are identical to residues in the sequence and comparator sequence after the introduced gap ordering, without regard to any conservative substitutions as part of the sequence identity, if necessary, in order to obtain the maximum percent sequence identity.

The "immune response" as used herein, in a subject, refers to the development of a humoral immune response, a cellular immune response, or both a humoral and a cellular immune response to an antigen. By "humoral immune response" is meant an immune response mediated by antibodies. "cellular immune response" refers to an immune response mediated by T-lymphocytes or other leukocytes or both, and includes the production of cytokines, trending factors, or similar molecules produced by active T-cells, leukocytes, or both. The immune response can be determined using standard immunoassays and neutralization assays known in the art.

As used herein, "immunogenic" refers to the ability of a protein or polypeptide to elicit an immune response specifically from an antigen.

By "immunogenic composition" is meant a composition prepared to contain an immunogen, including, for example, a protein, polypeptide, whole cell, inactivated, subunit or attenuated virus, or polysaccharide, or combinations thereof, administered to stimulate the development of a humoral and cellular immune system in the recipient relative to one or more of the immunogenic compositions. "immunization" refers to the process of administering an immunogenic composition, thereby stimulating an immune or immunogenic response to an antibody in a host. Preferred hosts are mammals, such as dogs. Preferably, the immunogenic composition is a vaccine.

As used herein, an "immunoprotective amount" refers to an amount of antigen effective to elicit an immunogenic response in a recipient that prevents or ameliorates signs or symptoms of disease, including adverse health effects or complications. Either humoral immunity or cell-mediated immunity or both may be induced. The immunogenic response of an animal to a composition can be assessed indirectly by measuring antibody titers, lymphoproliferation assays, or directly by monitoring the conditions and symptoms following challenge with the wild type strain. The protective immunity conferred by the composition or vaccine can be measured, for example, by a reduction in shedding of challenge microorganisms, a reduction in mortality, morbidity, temperature, overall physical condition, and overall health and performance of the subject. Immune responses include, but are not limited to, induction of cellular and/or humoral immunity. The amount of composition or vaccine that can be effectively treated will vary and will depend upon the particular organism used, or the condition of the animal being treated or immunized, and can be determined by a veterinarian.

As used herein, "intranasal" administration refers to the introduction of a substance, such as a vaccine or composition thereof, into the subject's body through or via the nose, which includes the transport of the substance primarily through the nasal mucosa.

"isolated", as used herein, refers to removal from its naturally occurring environment, whether alone or in a heterologous host cell, or a chromosome, or a vector (e.g., plasmid, phage, etc.). "isolated bacteria," "isolated anaerobes," "isolated bacterial strains," "isolated viruses," "isolated viral strains," and the like, refer to compositions in which only bacteria or viruses, but no other microorganisms, such as when cultured after isolation from their naturally occurring environment. "isolated", when used to describe any specifically defined substance, such as a polynucleotide or polypeptide, refers to a substance that has been isolated from the original cellular environment in which the substance, such as a polypeptide or nucleic acid, is normally found. As used herein, by way of example only, recombinant cell lines constructed using polynucleotides of the present invention utilize "isolated" nucleic acids. Alternatively, if a particular protein or specific immunogenic fragment is claimed or used as a vaccine or other composition, it will be considered isolated because it has been identified, isolated and purified to some extent compared to its presence in nature. A protein or specific immunogenic fragment is considered to exist as an isolated protein or nucleic acid if it is produced in a recombinant bacterial or eukaryotic expression vector (which can produce antibodies). For example, recombinant cell lines constructed with polynucleotides utilize "isolated" nucleic acids.

"agent" refers to an agent that can be used to prevent, treat or ameliorate a medical condition, or to prevent a certain physiological condition or emergent condition.

As used herein, "monoclonal antibody" refers to antibodies produced by a single line of hybridoma cells, all directed against an epitope on a particular antigen. The antigen used to prepare the monoclonal antibody can be provided as a protein of an isolated pathogen or the entire pathogen. A "hybridoma" is a clonal cell line comprising a hybrid of myeloma cells fused to specific antibody-producing cells. Typically, monoclonal antibodies are of murine origin. However, monoclonal antibody also refers to a clonal population of antibodies to a particular epitope, which antigens are generated by phage display technology or methods equivalent to phage display or non-murine hybrid cells.

As used herein, "oral" or "peroral" administration refers to the introduction of a substance, such as a vaccine or other composition, into the body of a subject through or via the mouth, and includes swallowing or delivery via the oral mucosa (e.g., sublingual or intrabuccal absorption) or both. Intratracheal (endotracheal) is also a form of oral (oral) or peroral (peroral) administration.

As used herein, "oronasal" administration refers to the introduction of a substance, such as a composition or vaccine, into the body of a subject through or via the nose and mouth, such as by placing one or more drops intranasally. Oronasal administration includes delivery procedures associated with oral and intranasal administration.

As used herein, "parenteral administration" refers to the introduction of a substance, such as a composition or vaccine, into the body of a subject by or via a route that does not include the digestive tract. Parenteral administration includes subcutaneous, intramuscular, intraarterial, and intravenous administration. For the purposes of this disclosure, parenteral administration does not include routes of administration that primarily include delivery of substances to mucosal tissues in the mouth, nose, trachea, and lungs.

The term "pathogen" or "pathogenic microorganism" as used herein refers to a microorganism, e.g., CPIV, CAV-2, CRCoV or Bordetella bronchiseptica, that is capable of inducing or causing a disease, illness, or abnormal condition of its host animal.

As used herein, "pertactin" refers to the outer membrane protein of Bordetella bronchiseptica. The pertactin is preferably derived from bordetella bronchiseptica, most preferably "p 68", encoded by gene, pmA. Pertactin can be isolated from the native form of bordetella bronchiseptica, or it can be recombinantly produced. Sequences and examples of pertactin are provided in U.S. patent No.7736658, the contents of which are incorporated herein by reference. Antigens of pertactin include lipidated forms of the protein.

"pharmaceutically acceptable" means, within the scope of sound medical judgment, a substance that is suitable for use in contact with the tissues of the subject without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit to risk ratio, and effective for its intended use.

"polyclonal antibody" as used herein refers to a mixed population of antibodies against a particular pathogen or antigen. Generally, a population comprises multiple antibody panels, each directed to a particular epitope of a pathogen or antigen. To make polyclonal antibodies, the whole pathogen, or an isolated antigen, is introduced into the host by vaccination or infection, inducing the host to produce antibodies against the pathogen or antigen.

The term "polynucleotide" as used herein refers to an organic polymer molecule comprising nucleotide monomers covalently bound to one strand. DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) are examples of polynucleotides having different biological functions.

The term "polypeptide" as used herein refers to an organic polymeric molecule comprising two or more amino acids bonded in one chain.

As used herein, "preventing infection" refers to preventing or preventing the replication of bacteria or viruses that may cause a defined disease, preventing the transmission of bacteria or viruses, preventing the establishment of bacteria or viruses in a host, or alleviating the symptoms of a disease caused by infection. Treatment is considered effective if the bacterial or viral load is reduced.

As used herein, "protection", "protecting", "protective immunity" and the like, with respect to a vaccine or other composition, means that the vaccine or composition can prevent or reduce the symptoms of a disease caused by an organism from which the vaccine or composition is derived. The terms "protection", "protecting", and the like, mean that the vaccine or composition may be used to "treat" a disease, or one or more symptoms of a disease already present in a subject.

As used herein, "inhaled" administration refers to the introduction of a substance, such as a vaccine or other composition, into the body of a subject by or via inhalation of an aerosolized (nebulized) substance. In inhalation administration, the primary transport mechanism involves absorption of nebulized material through the mucous membranes of the trachea, bronchi and lungs, unlike intranasal or oral administration.

The terms "specific binding", "specific binding" and the like refer to two or more molecules that form a complex that is quantifiable and selective under physiological or assay conditions. An antibody or other inhibitor is considered to bind specifically to a protein, and if under appropriately selected conditions, this binding is substantially non-inhibitory, while at the same time non-specific binding may be inhibited. Specific binding is characterized by high affinity and selectivity for compounds or proteins. Nonspecific binding is generally of low affinity. For example, binding to lgG antibodies, typically has at least about 10^-7M is or greater, or at least 10^-8M is or greater, or at least 10^-9M is or greater, or at least 10^-10M is or greater, or at least 10^-11M is or greater, or at least 10^-12M or higher. For example, when the antigen-binding domain is specific for a particular epitope that is not carried by many antigensThe term also applies, in which case antibodies carrying antigen binding domains do not normally bind other antigens.

As used herein, a "specific immunogenic fragment" refers to a portion of a sequence recognized by an antibody or T cell specific for that sequence.

As used herein, "subject" refers to any animal having an immune system, including mammals, such as dogs.

As used herein, "substantially identical" refers to a degree of sequence identity of at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least greater than 99%.

As used herein, a "subunit vaccine" or "subunit composition" refers to a type of vaccine or composition that includes one or more antigens, but does not necessarily have all antigens in the vaccine or composition that are derived from or homologous to an antigen of a pathogen of interest, such as a virus, bacterium, parasite, or fungus. The composition or vaccine is substantially absent from intact pathogen cells, or from particles of the disease, or from lysates of the cells or particles. A subunit vaccine or subunit composition can be prepared from an at least partially purified, or substantially purified, immunogenic polypeptide from a pathogen or analog thereof. Methods for obtaining a single antigen or multiple antigens in a subunit vaccine or subunit composition include standard purification techniques, recombinant production, or chemical synthesis. "subunit vaccine" or "subunit composition" refers to a vaccine or composition that includes a defined antigenic component of a virus, bacterium, or other immunogen.

“TCID₅₀By "is meant the" tissue culture infectious dose "which is defined as the dilution of virus required to infect 50% of the time it is scheduled to inoculate a cell culture. TCID can be determined using various methods₅₀The Spearman-Karber method is included and is used throughout the specification. See B.W.Mahy for a description of the Spearman-Karber hair&H.O.Kangro,VirologyMethodsManual25-46(1996)。

As used herein, "therapeutic agent" refers to a molecule, compound, virus, or treatment, preferably an attenuated or killed virus, or subunit or compound, that can assist in the treatment of viral, bacterial, parasitic, or fungal infections, treatments, or conditions caused thereby.

As used herein, "therapeutically effective amount" refers to an amount of antigen or vaccine that induces an immune response in a subject (e.g., a dog) receiving the antigen or vaccine or composition sufficient to ameliorate or prevent a condition or symptom of a disease including adverse health effects or other complications resulting from infection with a pathogen, such as a virus, bacterium, parasite, or fungus. Either humoral immunity or cell-mediated immunity or both humoral and cell-mediated immunity may be induced. The immunogenic response of an animal to an antigen, vaccine or composition can be assessed indirectly via measurement of antibody titration, lymphocyte proliferation assays, or directly by monitoring the condition, i.e., symptoms, following challenge with the wild type strain. The protective immunity conferred by the vaccine or composition can be measured by a reduction in shedding of challenge microorganisms, and/or a reduction in clinical conditions such as mortality, morbidity, temperature, overall physical condition, health and performance of the subject. The therapeutically effective amount of the vaccine or composition will vary, depending on the particular immunogen used or the conditions of the subject, and can be determined by one skilled in the art.

As used herein, "treating" or "treatment" refers to reversing, alleviating, inhibiting the progression of, or preventing the disorder, condition, or disease to which the term applies, or preventing one or more symptoms of the disorder, condition, or disease.

As used herein, "treatment" refers to the act of "treating" as defined above.

As used herein, "vaccine" or "vaccine composition" refers to an immunogenic composition selected from a virus, or a bacterium, either an active, attenuated, or dead, or subunit vaccine, or a combination thereof. Administration of the vaccine to a subject results in an immune response. The vaccine may be introduced directly into the subject by known routes of administration, including parenteral, oral, or the like. The term means a composition that can prevent or attenuate an infection, or prevent or attenuate one or more symptoms of an infection. The prophylactic effect of a vaccine composition against a pathogen is usually accomplished by eliciting an immune response in the subject. In general, abolishing or reducing the incidence of infection, ameliorating a disorder or condition, or accelerating the elimination of microorganisms from an infected subject is indicative of the prophylactic efficacy of the vaccine composition. The vaccine composition of the present invention provides a prophylactic effect on infections caused by canine respiratory disease syndrome.

As used herein, "veterinarily acceptable" means that the substance is, within the scope of sound medical judgment, suitable for use in contact with the tissues of veterinary subjects without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit to risk ratio, and effective for their intended use.

As used herein, "veterinarily acceptable carrier" refers to a carrier medium that does not interfere with the efficacy of the biological activity of the active ingredient and is non-toxic to the veterinary subject to which it is administered.

Antigens, immunogenic compositions, vaccines

The present disclosure provides immunogenic compositions and vaccines comprising one or more viruses and bacteria. The present disclosure provides immunogenic compositions and vaccines comprising one or more viruses and bacteria, or subunits suitable for administration to canines for the treatment of CIRDC.

The canine respiratory coronavirus (CRCoV) described herein can be characterized as a coronavirus present in the respiratory tract of a dog suffering from an infectious respiratory disease. The CRCoV phylogenetically most closely approximates bovine coronavirus (BCoV), human coronavirus (HCoV) strain OC43 and Hemagglutinating Encephalomyelitis Virus (HEV); the enterocanine coronavirus (CCoV) is only the distant relative of CRCoV. Representative examples of CRCoV strains suitable for use in the present invention include the strain identified as CRCoV strain 4182(Erles et al, VirusRes.,124:78-87,2007).

The influenza virus antigen contained in the present invention may be any established strain of influenza virus from birds or mammals, including, but not limited to, influenza virus having hemagglutinin of the H3 subtype and neuraminidase of the N8 subtype, or influenza virus of the H3N8 subtype, more commonly referred to as H3N8 virus. Influenza may be of mammalian or avian origin, including but not limited to porcine, equine or canine origin. Canine influenza antibodies were used in one example. Equine influenza antigens are used in one embodiment. Strains having the H3 subtype or the N8 subtype glycoprotein are used in one embodiment. Glycoproteins having the H3 subtype and the N8 subtype were used in one example.

Influenza antigens included in the present invention are isolated from wild and domestic dogs, horses, pigs and poultry. The animal selected for sample collection should exhibit acute and/or subacute clinical symptoms, which may include respiratory symptoms ranging from mild to severe and fever. Animals may also exhibit symptoms of anorexia and lethargy. Virus isolation methods well known in the art are used, which include: mammalian or avian cell cultures are inoculated, together with embryonated eggs from nasal or pharyngeal mucus samples (collected by swabbing the nasal passage or throat, or by collecting tissue such as spleen, lung, tonsil, liver, lung washes) from in-situ samples. Cytopathic effects of the virus can be observed in cell culture. Allantoic fluid or cell lysates can be tested for their ability to aggregate human, chicken, adult, or guinea pig erythrocytes as putative evidence of the presence of influenza virus.

A representative example of an influenza virus strain suitable for use in the present invention includes the strain identified as A/Canine/lowa/9A1/B5/08/D12, which was deposited as PAT-7694 at the American Type Culture Collection (ATCC) of university of Manassas university of Marina (university boulevard)10801, 6.29.2006, complying with the international recognition for patent applicationsThe budapest treaty on the preservation of microorganisms of the program. A representative strain of CIV antigen is CIV virus strain, Vangard, in a commercial vaccineCIV (Pfizer, Inc). The invention also includes vaccines comprising identified Equine influenza strain A/Equinone/2/Miami/1/63. The strain is deposited with the ATCC under the accession number VR317, complying with the International recognized Budapest treaty on the preservation of microorganisms for patent procedures.

Other examples of influenza viruses useful in the present invention are A/canane/lowa/13628/2005, A/Equinone/Kentucky/1998, A/Equinone/Kentucky/15/2002, A/Equinone/Ohio/1/2003, A/Equinone/Kentucky/1/1994, A/Equinone/Massachusetts/213/2003, A/Equinone/Wisconsin/2003, A/Equinone/NewYork/1999, and A/Equinone/Newmarket/A2/1993. Other preferred strains and/or isolated CIVs include those reported in U.S. patent nos.7959929 (particularly the strains and HA sequences identified as Jacksonville/2005, Miami/2005, FL/242/03 and Florida/43/04), 7384642,7572620 and 7468,187, the contents of which include all sequences, particularly the HA sequence, as well as the strain, incorporated herein by reference as if fully included in the description of the present invention. In addition, CIV strains suitable for use in the present invention include the Colorado CIV isolate (as described in Barrell et al, J.Vet.Intern.Med.,24(6), 1524-.

Canine parainfluenza virus (CPIV) included in the present invention can be characterized as one of the viruses known to be causative factors associated with kennel cough. A representative strain of CPIV antigen is an attenuated CPI strain of a commercial vaccine, VanguardPlus5 (Pfizer). Another representative strain of CPIV antigen is an attenuated CPI virus strain with a "NL-CPI-5" (national vector associated service laboratory, Ames, IA) marker.

The canine type 2 virus (CAV-2) included in the present invention can be characterized as known to be associated with kennel coughOne of the viruses with a related pathogenic factor. A representative strain of CAV-2 antibody is an attenuated CAV-2 strain, Vanguard, of a commercial vaccinePlus5 (Pfizer). A representative strain of CAV-2 antigen is the attenuated CAV-2 strain designated as the "Manhattan" strain (national Veterinaryservice laboratory, Ames, IA).

Mycoplasma canines (M.cynos) included in the present invention are as described in Chalker et al, Microbiology,150:3491-3497,2004, and are only the Mycoplasma species that are commonly associated with respiratory disease. Immunogenic compositions for m.cynos are as described in US2007/0098739, incorporated herein by reference.

The bordetella bronchiseptica component included in the present invention can be characterized as a bacterial causative agent associated with kennel cough. The immunogenic compositions and vaccines encompassed by the present invention may be one or more of live attenuated bordetella bronchiseptica, bordetella bronchiseptica vaccines or bacterial extracts. In addition, preferred compositions also include an isolated bordetella bronchiseptica subunit antigen.

In the examples bordetella bronchiseptica was prepared by passing whole-cell ultrasonic purification through a chromatographic column (as provided in patent application No. FR2571618, filed 1984, 10/12). Another representative example of Bordetella bronchiseptica is the bacterial extract Bronchicine^TMCae (pfizer), which is prepared using antigenic material extracted from bordetella bronchiseptica cells. Another example of Bordetella bronchiseptica is the live attenuated Bordetella bronchiseptica strain B-C2, present in NobivacAnd/or Living from Intra-TracBronchi-ShieldNaramune^TM、RecombitekUnivac and/or Kennel-Jec^TMOf bronchitis/bronchial septicemia (bronchinseptica).

In addition, subunits are also preferably present (e.g., complementary), in combination with bordetella bronchiseptica components. Representative examples of subunits are isolated pertactin antigens, preferably, bordetella bronchiseptica p68 antigen, in particular recombinant bordetella bronchiseptica p68 antigen, which can be recognized by p 68-specific monoclonal antibody Bord2-7 (as described in US7736658, which is incorporated herein by reference), and in a preferred embodiment, have the amino acid sequence described in US7736658 or have homology thereto.

The recombinant bordetella bronchiseptica p68 antigen is preferably prepared in a soluble form so that the native-like structure is preserved or restored during processing. Accordingly, it is an aspect of the present invention to provide recombinant p68 that is substantially free (less than about 80%, 90%, 95%, or even 99%) of aggregates. In another embodiment, recombinant p68 is solubilized with urea, preferably a urea solution greater than 0.1M, 0.5M, 1M, 2M, 3M, or 6M. Thereafter, the p68 antigen can be purified, such as by chromatography. Such a dissolution process is described in Surender et al, J.bioscience and Bioengineering, v.99(4), pgs303-310 (2005).

The pertactin antigen used in the present invention also includes lipidated forms. An example of the production of lipidated proteins is provided by Erdile et al, InfectiondImmnity, (1993) v.61(1), p.81-90, which is incorporated herein by reference. The disclosed method can be used to prepare post-translationally modified pertactin proteins, which comprise an attached lipid moiety.

In addition, in another embodiment, the immunogenic composition comprises bordetella bronchiseptica and an isolated Bsp22 antigen. In another embodiment, the immunogenic composition comprises bordetella bronchiseptica, an isolated pertactin antigen, and an isolated Bsp22 antigen. Bsp22 antigen can be prepared as described in Medhekar et al, molecular microbiology (2009)71(2), 492-. Preferably, the isolated Bsp22 antigen is present in cooperation (e.g. additionally) with a bordetella bronchiseptica extract and an isolated pertactin, in particular recombinant p 68.

"Bsp 22" also includes lipidated forms of the antigen. An example of the production of lipidated proteins is provided in Erdile et al, InfectiondImmnity, (1993) v.61(1), p.81-90, incorporated herein by reference. The disclosed method can be used to prepare a post-translationally modified Bsp22 protein, which contains an attached lipid moiety.

The viruses included in the present invention can be propagated in cells, cell lines, host cells. Such cells, cell lines, host cells may be, for example, but are not limited to, mammalian cells and non-mammalian cells, including insect and plant cells.

Cells, cell lines and host cells in which the viruses encompassed by the present invention can be propagated are known and available to those of skill in the art.

In another embodiment, the immunogenic compositions described herein do not include non-respiratory antigens. Accordingly, in one embodiment of the invention, a composition is provided, provided that it does not include a non-respiratory antigen. The non-respiratory antigens are unable to cause respiratory diseases in the subject. Non-limiting examples of non-respiratory antigens include rabies virus, canine parvovirus, enterocanine coronavirus, leptospirosis species (leptospira), and lyme disease spirochete (Borreliaburgdorferi).

The bacteria encompassed by the present invention can be cultured and propagated using a variety of media known to those skilled in the art, including broth (liquid) and agar (solid; semi-solid) media. Some bacteria can also be cultured and propagated in mammalian cells or non-mammalian cells.

The viruses and bacteria included in the present invention can be attenuated or inactivated prior to use in an immunogenic composition or vaccine. Methods of attenuation and inactivation are well known to those skilled in the art. Methods of attenuation include, but are not limited to, serial passage in cell culture on a suitable cell line (viral or bacterial), serial passage in broth culture (bacterial), ultraviolet radiation (viral and some bacteria), chemical mutation (viral and bacterial). Methods for inactivation of viruses or bacteria include, but are not limited to, treatment with formalin, beta-propiolactone (BPL), or diethylene imine (BEI), or other methods known to those skilled in the art.

Formalin inactivation can be achieved by mixing the suspension containing the microorganisms with 37% formaldehyde to a final concentration of 0.5% formaldehyde. The microorganism-formaldehyde mixture was mixed by constant stirring for about 24 hours at room temperature. The inactivated mixture of microorganisms is tested for residual viable microorganisms by testing for growth in a suitable cell line or broth medium.

For certain antigens, inactivation by BEI was achieved by mixing a suspension containing the microorganism of the invention with 0.1MBEI (2-bromo-ethylamine in 0.175 NNaOH) to a final concentration of 1mM BEI. For other antigens, the final concentration of BEI was 2 mM. The appropriate concentration is guided by the person skilled in the art. The virus-BEI mixed solution was mixed by continuous stirring at room temperature for about 48h, followed by addition of 1.0M sodium thiosulfate to a final concentration of 0.1 mM. The mixture was stirred for 2 h. The mixture containing the inactivated microorganisms is tested for residual acquired virus by measuring growth in an appropriate cell line or broth.

The immunogenic compositions and vaccines encompassed by the present invention can include one or more veterinarily acceptable carriers. As used herein, "veterinarily acceptable carrier" includes any and all solvents, dispersion media, coatings, adjuvants, stabilizers, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption delaying agents, and the like. Diluents include water, saline, dextrose, ethanol, glycerol, and the like. Isotonic agents include sodium chloride, glucose, mannitol, sorbitol, and lactose, and others known to those skilled in the art. Stabilizers include albumin, and others known to those skilled in the art. Preservatives include thimerosal, and others known to those skilled in the art.

Adjuvants are metabolisable, involving adjuvants comprising such components that can be metabolised by the target species, such as vegetable oil-based adjuvants. The metabolic adjuvant can be a metabolizable oil. Metabolizable oils are fats and oils that commonly occur in plants and animals, and typically comprise a mixture of large amounts of triacylglycerols, also referred to as triglycerides or neutral fats. These nonpolar, water-insoluble substances are fatty acids and triglycerides. Triacylglycerols differ according to the identity and position of their three fatty acid residues or side chains.

Adjuvants can also be non-metabolizable, involving those adjuvants that contain components that cannot be metabolized by the body of the animal subject to which the emulsion is administered. Non-metabolizable oils suitable for use in the compositions of the present invention include alkanes, alkenes, alkynes and their corresponding acids and alcohols, ethers and their esters, and mixtures thereof. Preferably, the individual oil compounds are light hydrocarbon compounds, for example, these components having 6 to 30 carbon atoms. Oils can be synthetically prepared or purified from petroleum products. Preferred non-metabolizable oils for use in the compositions described herein include, for example, mineral oil, paraffin oil, naphthenes. The term "mineral oil" refers to a non-metabolizable adjuvant which is a mixture of liquid hydrocarbons obtained from petrolatum by distillation techniques. The term is synonymous with "liquefied paraffin", "liquid paraffin", and "white mineral oil". The term is also intended to include "light mineral oils", for example, oils similar to those obtained from the distillation of petrolatum, but which have a slightly lower specific gravity relative to white mineral oil. Mineral oils are available from various commercial sources, for example, j.t. baker (phillips burg, PA), USBCorporation (Cleveland, OH). Light mineral oil from DRAKEOLThe brand is commercially available.

Adjuvants include, but are not limited to, EmulsigenAdjuvant system (MVP laboratories; Ralston, NE), RIBI adjuvant system (RibiInc.; Hamilton, MT), alum, aluminum hydroxide gel, oil-in-water emulsions, water-in-oil emulsions, e.g., Freund's complete and incomplete adjuvant, Block copolymer (CytRx; Atlanta, GA), AF-M (Chiron; Emeryville, Calif.), AMPHIGENAdjuvants, saponins, quine A, QS-21(Cambridge Biotech Inc.; Cambridge, MA), GPI-0100(galenic pharmaceuticals, Inc.; Birmingham, AL) or other saponin components, monophosphate A, Avridine lipoamine adjuvant, thermolabile enterotoxin (recombinant or otherwise) from E.coli, cholera toxin, muramyl dipeptide, block copolymers/surfactants of squalene/propylene glycol and ethylene oxide addition polymers (SP-oil), sulfurized β cyclodextrin (sulpholipobeta-cyclodextrin) (SL-CD), immunomodulator-containing lipids (such as CpG or poly: C), Muramyl Dipeptide (MDP), ISCOMATRIX (Quila A/phosphorylcholine), CpG/DEAE-glucose/mineral oil (CpG O), polyacrylic acid, triterpenoids (e.g., purified, quine A or other partially purified or partially prepared cholesterol), CpG-cholesterol (such as a dimethyl-alkyl-cholesterol), immunomodulator (such as a dimethyl-alkyl-benzophenone), and immune modulators (DDR), such as benzyl alcohol, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable carrier, such as a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable carrier, a pharmaceutically) And Th2 stimulants (e.g., glycolipids, such as BayR1005) And their compositions, including other adjuvants known to those skilled in the art.

Various combinations that can be usedNon-limiting examples of such include triterpenoids plus sterone (e.g., quinila/cholesterol, also known as QAC), triterpenoids plus sterone, immunomodulators and polymers (e.g., QuilA/cholestrol/DDA/CARBOPOL)Also known as QCDC), triterpenoid mixtures plus sterones, immunomodulators, polymers and Th2 stimulants (e.g., QuilA/cholestrol/DDA/CARBOPOL)And BayR1005Also known as QCDCR).

The amounts and concentrations of adjuvants and additives used in the context of the present invention can be readily determined by those skilled in the art. In one embodiment, the invention contemplates immunogenic compositions and vaccines comprising from about 20 μ g to about 2000 μ g adjuvant. In another embodiment, from about 100 μ g to about 2500 μ g, or from about 250 μ g to about 1000 μ g, or from about 350 μ g to about 750 μ g of adjuvant is included. In another embodiment, an adjuvant is included in an amount of about 500 μ g/2ml dose of the immunogenic composition or vaccine.

The immunogenic compositions and vaccines also include antibiotics. The antibiotic includes, but is not limited to, antibiotics selected from the group consisting of aminoglycosides, carbapenems, cephalosporins, glycopeptides, macrolides, penicillins, polypeptidoles, sulfonamides, and tetracyclines. In one embodiment, the invention contemplates immunogenic compositions and vaccines comprising from about 1 μ g/ml to about 60 μ g/ml antibiotic. In another embodiment, the immunogenic compositions and vaccines comprise from about 5 μ g/ml to about 55 μ g/ml, or from about 10 μ g/ml to about 50 μ g/ml, or from about 15 μ g/ml to about 45 μ g/ml, or from about 20 μ g/ml to about 40 μ g/ml, or from about 25 μ g/ml to about 35 μ g/ml of antibiotic. In yet another embodiment, less than about 30 μ g/ml of antibiotic is included in the immunogenic compositions and vaccines.

Immunogenic compositions and vaccines encompassed by the present invention can include one or more polynucleotide molecules that encode a virus, or a bacterium, or a viral or bacterial protein. DNA or RNA molecules can be used in immunogenic compositions or vaccines. The DNA or RNA molecule may be administered in the absence of other drugs, or it may be administered with agents that promote cellular uptake (e.g., liposomes or cationic liposomes). The total polynucleotides in the immunogenic composition or vaccine is between about 0.1 μ g/ml to about 5.0 mg/ml. In another embodiment, the total polynucleotides in the immunogenic composition or vaccine can be between about 1 μ g/ml to about 4.0mg/ml, or between about 10 μ g/ml to about 3.0mg/ml, or between about 100 μ g/ml to about 2.0 mg/ml. Vaccines and vaccination procedures using nucleic acids (DNA or RNA) have been described in the prior art, for example, u.s.pat. No.5703055, u.s.pat. No.5580859, and u.s.pat. No.5589466, all of which are incorporated herein by reference.

In addition to the viruses or bacteria described above, the immunogenic compositions and vaccines encompassed by the present invention include other added antigens. The antigen can be in a completely or partially inactivated form of the prepared microorganism, or in the form of an antigenic molecule obtained by genetic engineering techniques or chemical synthesis. Other antigens suitable for use in the present invention include, but are not limited to, pathogenic viruses from such sources as canine distemper virus, canine herpes virus, canine influenza virus, rabies virus, pathogenic viruses such as Bordetella bronchiseptica, Leptospira bratislava, Leptospira canicola, Leptospira influenzae, Leptospira icterohaeae, Leptospira pomona, Leptospira boviensis, Leptospira bovis, Porphyromonas, Bacteroides, Borrelia, Streptococcus, including Streptococcus equisimilis, Ehrlichia, Mycoplasma, including M. Antigens may also be derived from pathogenic fungi, such as Candida, protozoa, such as Cryptosporidium, Neosporon Canitis, Toxoplasma, Eimeria, Babylonia, Giardia, Leishmania, or parasites, such as Taenia, Flaviviridae, Echinococcus and Parazobium.

Form, dosage and route of administration

The immunogenic compositions and vaccines contained in the present invention can be administered to animals to elicit an effective immune response to CIRDC. Accordingly, the present invention provides methods for stimulating an effective immune response by administering to an animal a therapeutically effective amount of an immunogenic composition or vaccine described herein.

The immunogenic compositions and vaccines described herein can be administered to an animal to vaccinate the animal subject against CIRDC. The immunogenic compositions and vaccines can be administered to an animal to prevent or treat CIRDC in the animal. Thus, described herein are methods of vaccinating an animal against CIRCD, and preventing or treating CIRCD, comprising administering to the animal a therapeutically effective amount of an immunogenic composition or vaccine described herein.

The immunogenic compositions and vaccines encompassed by the present invention can be prepared in a variety of forms depending on the needs of the route of administration. For example, immunogenic compositions and vaccines can be prepared in the form of sterile aqueous solutions or dispersions suitable for injection or in lyophilized form using freeze-drying techniques. Lyophilized immunogenic compositions and vaccines are typically maintained at about 4 ℃ and can be reconstituted in a stable solution, such as saline or HEPES, with or without adjuvant. Immunogenic compositions and vaccines can also be prepared in the form of suspensions or emulsions.

The invention encompasses immunogenic compositions and vaccines comprising a therapeutically effective amount of one or more of the microorganisms described above²To about 1 × 10¹²Viral or bacterial particles in betweenOr at about 1 × 10³To about 1 × 10¹¹In the middle, or at about 1 × 10⁴To about 1 × 10¹⁰In the middle, or at about 1 × 10⁵To about 1 × 10⁹In the middle, or at about 1 × 10⁶To about 1 × 10⁸In between. The exact amount of microorganism in an immunogenic composition or vaccine that can be effective to provide protective efficacy can be determined by one skilled in the art.

The pertactin antigen is between about 1 μ g to about 30 μ g. More specifically, the pertactin is between about 5 μ g and about 20 μ g, more specifically between about 7 μ g and about 15 μ g, and even more specifically about 5 μ g, 10 μ g, 15 μ g, or 20 μ g.

Immunogenic compositions and vaccines typically contain a veterinarily acceptable carrier in a volume of between about 0.5ml to about 5 ml. In another embodiment, the volume of the carrier is between about 1ml to about 4ml, or between about 2ml to about 3 ml. In another embodiment, the volume of the carrier is about 1ml, or about 2ml, or about 5 ml. The veterinarily acceptable carrier suitable for use in the immunogenic compositions and vaccines can be any of those described above.

One skilled in the art can readily determine whether a virus or bacterium needs to be attenuated or inactivated prior to administration. In another embodiment of the invention, the virus or bacteria can be administered directly to the animal without additional attenuation. The amount of a therapeutically effective amount of a microorganism will vary and can be determined by one skilled in the art depending on the particular microorganism used, the condition of the animal, and/or the extent of infection.

According to the methods of the invention, the animals may be administered a single dose, or, alternatively, 2 or more vaccinations may occur at intervals of about 2 to about 10 weeks. A stimulation regimen may be required and the dosing regimen can be adjusted to provide optimal immunity. The optimal dosing regimen can be readily determined by one skilled in the art.

The immunogenic compositions and vaccines can be administered directly into the blood, muscle, internal organs, or subcutaneously. Suitable methods for parenteral administration include intravenous, intraarterial, intramuscular, and subcutaneous administration. Suitable devices for parenteral administration include needle (including microneedle) type injectors and needleless injectors.

Parenteral formulations are generally aqueous solutions containing excipients such as salts, carbohydrates, proteins and buffer solutions (preferably having a pH of from about 3 to about 9, or from about 4 to about 8, or from about 5 to about 7.5, or from about 6 to about 7.5, or from about 7 to about 7.5), but in some applications can more suitably be presented as sterile non-aqueous solutions or in dry form for formulation with suitable excipients such as sterile, pyrogen-free water or saline.

Parenteral formulations can be prepared under sterile conditions, for example, by freeze-drying, and can be readily prepared by standard pharmaceutical techniques known to those skilled in the art.

The solubility of materials for preparation without enteric solutions can be enhanced by using suitable formulation techniques that have been known to those skilled in the art, such as incorporating solubility enhancing agents, including buffers, salts, surfactants, liposomes, cyclodextrins, and the like.

Compositions for parenteral administration can be formulated for direct or modified release. Modified release formulations include delayed, sustained, pulsed, controlled, targeted, and programmed release. Thus, the immunogenic compositions and vaccines can be formulated as solid, semi-solid, or thixotropic liquids for use as injection sites to provide modified release of the immunogenic compositions and vaccines.

Other methods of administration of the immunogenic composition or vaccine include via microneedles or needle-free (e.g., Powderject)^TM,Bioject^TMEtc.) are released by injection.

In the case of subcutaneous or intramuscular injection, an isotonic formulation is preferred. In general, additives for isotonicity can include sodium chloride, dextrose, mannitol, sorbitol, and lactose. In certain cases, isotonic solutions, such as phosphate buffered saline, are used. The formulation can further include stabilizers such as gelatin and albumin. In some embodiments, a vasoconstrictor is added to the formulation. The pharmaceutical formulations according to the present invention are generally provided under sterile and pyrogen-free conditions. However, formulations for pharmaceutically acceptable carriers known to those skilled in the art are those approved by regulations promulgated by the U.S. department of agriculture, or by foreign equivalent governmental agencies such as canada, mexico, or any of the european countries, for use in canine vaccines, polypeptide (antigen) subunit immunogenic compositions and vaccines, recombinant viral vector vaccines, and DNA vaccines. Thus, a pharmaceutically acceptable carrier for the commercial production of an immunogenic composition or vaccine is one that has been or will be approved by the appropriate governmental agency in the united states or foreign countries. The immunogenic composition and vaccine can be further mixed with pharmaceutically acceptable adjuvants. In certain immunogenic composition and vaccine formulations, the immunogenic composition or vaccine is combined with other canine immunogenic compositions or vaccines to produce a multivalent product that can protect canines from a variety of diseases caused by other canine pathogens.

The immunogenic compositions described herein are capable of preventing infection by a canine respiratory pathogen or are capable of preventing infection of a canine with CIRCD over a period of about 3 months or more. The composition is capable of preventing infection by said canine respiratory pathogen, or preventing infection of the canine with CIRCD for a period of about 6 months or more. The composition is capable of preventing infection by said canine respiratory pathogen, or preventing infection of the canine with CIRCD for a period of about 1 year or more.

Method of detection and diagnosis

The extent and nature of the immune response elicited in an animal can be assessed using a variety of techniques. For example, by collecting serum from the vaccinated animal and then detecting the presence or absence of antibodies to the immunogen. Detection of responsive T-lymphocytes (CTLs), which indicate the induction of a cellular immune response, in lymphoid tissues can be accomplished by detection of, for example, T cell proliferation. Related techniques have been reported in the art.

Reagent kit

Because it is desirable to administer an immunogenic composition or vaccine in combination with an additional composition or compound-e.g., for the purpose of treating a particular disease or condition-hereinafter included within the scope of the present invention, the immunogenic composition or vaccine can be conveniently contained or incorporated in the form of a kit suitable for administration of the compositions or for simultaneous administration.

Thus, the kits encompassed by the present invention can comprise one or more separate pharmaceutical compositions, at least one of which is an immunogenic composition or vaccine according to the invention, and means for separately holding the compositions, such as containers, vials, or divided aluminum foil packets. One example of such a kit is a syringe and needle, and the like. The kits of the invention are particularly suitable for administration of different dosage forms, e.g. oral or parenteral, for administration of separate compositions at different dosage intervals, or for titration of the components independently of one another. To facilitate use of the compositions contained in the present invention, the kit will generally include instructions for administration.

Another kit encompassed by the present invention can include one or more reagents for detecting an infected animal. The kit can include reagents for assaying for the presence of all microorganisms, polypeptides, epitopes or polynucleotide sequences in a sample. The presence of viral, bacterial, polypeptide, or polynucleotide sequences can be determined using antibody, PCR, hybridization, and other detection methods known in the art.

Another kit encompassed by the present invention can provide reagents for detecting antibodies to a particular epitope. The reagent is used to analyze a sample for the presence of antibodies, which are well known and available to those skilled in the art. The presence of antibodies can be determined by standard detection methods known to those skilled in the art.

In certain embodiments, the kit can include a set of printed instructions, or a label indicating that the kit is for use in detecting an infected animal.

Antibodies

The antibody can be monoclonal, polyclonal, or recombinant. Antibodies can be prepared to the immunogen or a portion thereof. For example, polypeptides synthesized based on the amino acid sequence of the immunogen, or recombinants prepared by cloning techniques, or natural gene products and/or portions thereof, can be isolated and used as immunogens. The immunogen can be used to produce antibodies by standard antibody production techniques well known to those skilled in the art. Antibody fragments can also be prepared from antibodies by methods well known to those skilled in the art, including F (ab')₂And Fv fragments.

In the production of antibodies, screening for the desired antibody can be accomplished by standard methods of immunology known in the art. in general, ELISAs and Western blotting are the preferred types of immunoassays, both assays are well known to those skilled in the art. polyclonal and monoclonal antibodies can be used for detection.¹⁴C, and iodination.

The invention is further illustrated, but is not limited, by the following examples.

Examples

Example 1 evaluation of vaccines containing CRCoV

60 beagle dogs with approximately good health status at 8-9 weeks of age were used in this disclosure. All animals received a physical examination upon arrival and were again examined on study day 2 or day 1. Animals were observed once daily for their general health, from study day 8 to study day 39. Tympanic membrane temperatures were collected from study day 1 and prior to inoculation. Blood samples (approximately 5mL) were collected on SST tubes for serology on study day 0 and day 21 prior to each vaccination.

The CRCoV vaccine strain is derived from the strain crcov.669, which was deposited at the ATCC under the budapest treaty on the international recognition of the microbial preservation for patent procedures, with the deposit number PTA-11444. CIV vaccine strains from VanguardThe vaccine line (Pfizer) prepares the viral seeds for the vaccine. The antigen was prepared in the highest channel of the virus (MasterSeeVirus + 5). The vaccine composition comprises QuilA (20 μ g), cholesterol (20 μ g), dimethyl dioctadecyl ammonium bromide (DDA; 10 μ g) and Carbopol(polyacrylic acid, 0.05% v/v) adjuvant. The CRCoV antigen is formulated at 1.3 Relative Antigen Units (RAUs) per dose targeted. The experimental vaccine was sterilized.

Heterologous CRCoV isolates ("Max" strain; channel 1) were used as challenge material. Virus storage material was propagated and titrated on HRT18G cells and was determined to have a 10^7.1TCID₅₀Titer in/mL. Challenge materials were tested and confirmed to be satisfactorily sterile, with no mycoplasma or canine/feline in vitro agents.

One animal was vaccinated on day 21; the remaining animals were vaccinated on study day 22. Animals were subcutaneously vaccinated with the appropriate vaccine or placebo according to the experimental design shown in table 1. The first vaccination was administered in the right shoulder area (study day 0) and the second vaccination was administered in the left shoulder area (study day 22).

TABLE 1 Experimental design

¹The study was administered Subcutaneously (SC) with veterinary products (IVP).

²Challenge dose of intracoronary 1 channel CRCoVMax isolate.

RTU: instant liquid vaccine

After the first vaccination, animals were observed daily (from study day 1 to day 8) for injection swelling after vaccination. After the second inoculation, animals were observed daily for post inoculation injection swelling to day 29. Animals with injection site swelling/pain beyond the days listed above were observed 2 times a week continuously until swelling/pain disappeared. Tympanic membrane temperature was collected daily one week after each injection.

Blood samples (approximately 8mL) were collected from SST tubes for serology on study day 42 prior to challenge. Tympanic temperatures were collected on study days 40, 41, and 42 prior to challenge. 2 types of pharyngeal swabs (VTM for virus isolation (viral transport medium), and Amies for bacterial isolation) were collected from each dog on study day 42 prior to challenge. Animals were observed daily for clinical symptoms of respiratory disease on study days 40, 41, and 42 prior to challenge to establish baseline values.

On study day 42, all animals were challenged with CRCoV at target challenge dose 10⁶A intranasal challenge was performed per mL/dog. All animals were injected intravenously with Domitor prior to challenge administrationAnd (6) carrying out sedation. After sedation, each animal received 10mL of challenge virus and was slowly injected with a syringe without a needleApproximately 0.5mL per nostril was administered. Following challenge application, Antisedan was injected intramuscularlySedation was reversed.

Tympanic membrane temperature, clinical observations and pharyngeal swabs were collected daily from study days 42-56 post challenge. Blood samples (approximately 5mL) were collected daily for serology on study day 46 and study day 56 (prior to necropsy).

In necropsy, intact lungs and trachea were removed under sterile conditions and placed on a sterile drape to roughly assess lung lesions (consolidation) of the lung lobes. Each lobe was scored according to the percent of curing of the lung. One lung group was under-exsanguinated and not evaluated. The trachea was transected and the lumen assessed for gross pathology and any findings were recorded.

After the lungs were scored, the right tail lung lobes were rinsed with approximately 30.0mL of media for bacterial analysis and virus isolation. A pair of tissue samples were taken from the trachea, and nasal cavity, one for virus isolation and the second for histopathology. The right cranial lobe was divided into 3 samples, including one for bacteriological sampling. Blood samples for serology were collected on a predetermined study day.

And (6) obtaining the result. All animals were confirmed to be negative for antibodies against CRCoV by IFA assay (IFA titer <40) by study day 0. Pharyngeal swabs isolated for evaluation of the CRCoV virus confirmed that all animals did not have CRCoV on study day 0 prior to inoculation. Controls vaccinated with placebo still had CRCoV until study day 42. Experimental groups were confirmed to be free of CRCoV by virus isolation on study day 42, indicating a lack of foreign CRCoV in the device. All dogs were confirmed to be free of bordetella bronchiseptica on study day 42.

The concentration of the CRCoV antigen in the vaccine was measured using an ELISA assay to assign a batch of reference antigens CRCoV specific to a pair of Relative Antigen Units (RAUs). The CRCoV antigen was identified as 0.5 RAU/dose.

After vaccination, the majority of vaccinated dogs, including the placebo-vaccinated groups, developed injection swelling at the injection site. For a monovalent vaccinated group, the size of the swelling is usually small in most vaccinated groups (2 cm or less on the long scale). Swelling in most dogs was eliminated within 2 weeks. There was no pain or systemic reaction to the vaccine on any of the vaccinated dogs. No clinical fever (. gtoreq.39.5 ℃) was observed except for one dog, and the temperature rise in dogs was independent of vaccination (temperature was taken before the second vaccination). These findings indicate that the monovalent vaccine elicits only injection swelling, within the range that can be expected with an adjuvant vaccine.

Serum neutralization titers were tabulated and compared between groups. All monovalent vaccinated dogs (100%) developed SN titers (GMT371) at 3 weeks after the second vaccination, indicating active immunity. Significant post challenge (recalled) serum neutralization responses were detected on day 56 in combination vaccinated dogs (GMT6915) relative to placebo vaccinated dogs (GMT 471). The results of these studies were statistically significantly different, indicating that the CRCoV vaccine antigen effectively stimulated and elicited an immune response in dogs against CRCoV infection.

After challenge, all placebo-vaccinated animals (100%) shed virus in their respiratory secretions on at least one of days 1-6 post challenge, indicating initiation of a CRCoV infection. When compared to placebo (3.3 days), the monovalent vaccine significantly decreased (p =0.0237) the average number of days with airway secretions (2.1 days), indicating that the vaccine was effective in attenuating CRCoV infection.

All placebo-vaccinated dogs (100%) were positive for tracheal, nasal, pulmonary tests relative to virus isolation on day 4 post challenge, indicating respiratory organ infection with CRCoV. No virus isolated from any organ at day 14 post challenge, indicated a typical respiratory viral infection similar to canine influenza. In contrast, a monovalent vaccine can prevent infection in 90% and 50% of the lungs (p-value <0.0001) and trachea (p-value <0.0237) of vaccinated dogs, respectively. It indicates that a monovalent vaccine is able to induce sufficient immunity to prevent viral infection of these critical organs. There was no significant difference in nasal infection rates between the vaccinated and control groups.

CRCoV challenge caused only mild clinical symptoms under laboratory conditions. Eye and nasal discharge and conjunctivitis were reported in each treatment group of dogs. During the post-challenge period, 5 animals were reported to have a clinical fever (. gtoreq.39.5 ℃ C.), 2 in the placebo group, 1 in the monovalent vaccine group, and 2 in the combination vaccine group.

General assessments of the lungs, trachea and nasal turbinates were performed on days 4 and 14 post challenge. No significant severe lesions were reported, except 2 dogs-one at T05 and one at T01-with a lower level of lung consolidation; 2 dogs-one at T01 and one at T03 had necrosis in the focal region of the turbinate.

For histopathology, lung, trachea, and nasal tissues were examined and scored. The score (0-4) is set according to the degree of change observed. The changes due to feeding are most pronounced in the nasal concha, then in the trachea, and finally in the lungs. It is consistent with the aggressive viruses of the respiratory tract primarily affecting the upper respiratory tract (nasal concha and trachea), with a subsequent and weaker effect on the lower respiratory tract (lungs). It indicates that CRCoV infection triggers histopathology of respiratory organs.

Previous studies have shown that tracheal cilia damage is a characteristic pathological consequence of CRCoV infection on day 4 post challenge. The data indicate that the monovalent vaccine prevented tracheal cilia damage in 60% of the animals compared to placebo (30%) vaccination, but the reduction was not significant (P = 0.1538). Bacteriological diagnosis on lung and lung lavage confirmed that all animals were negative for bordetella bronchiseptica, pasteurella, staphylococcus standerdius and streptococcus canis. The lungs, lung lavage, or both were positive for mycoplasma in only 4 animals. The results of this study indicate that the lesions are specific for and caused by viral infection.

In summary, all CRCoV vaccinated dogs (100%) at T03-T06 developed serum neutralizing titers at 3 weeks after the second vaccination, indicating active immunity. Monovalent vaccines induce an immune response in vaccinees that reduces viral shedding in oropharyngeal secretions and respiratory organs. It also reduced tracheal cilia damage in the vaccinees relative to the placebo-vaccinated control group. Histopathological examination showed that the monovalent vaccine prevented tracheal cilia damage in 60% of the animals relative to placebo-vaccinated animals (30%).

Example 2 efficient detection of bivalent CRCoV/CIV vaccines in dogs

60 beagle dogs with approximately good health status at 8-9 weeks of age were used in this disclosure. All animals received a physical examination on study day 9 after arrival. All animals, except one dog removed on study day 7, received a second physical examination on study day 2 and were considered suitable for the study.

From study day 7 to study day 39, animals were observed once daily for general health. Blood samples for serology were taken on study day 0 and study day 21 Serum Separation Tubes (SST) prior to each vaccination. 2 sets of nasal swabs were collected from each dog on day 0 prior to vaccination-one set for CRCoV and one set for CIV virus isolation-for confirmation of absence of CRCoV and CIV. Tympanic membrane temperatures were collected and recorded on day 1 and 0 prior to vaccination and used to establish a pre-vaccination baseline. Tympanic membrane temperature was collected on day 21 prior to the second inoculation. Palpation was performed on the shoulder area of the animals on days 0 and 21 prior to inoculation to confirm that there were no preexisting lesions in the area of the injection site.

One dog at T04 was removed from the study because of respiratory distress on study day 7. One dog in T05 was removed from the study because of respiratory distress on study day 0 prior to vaccination. In addition, 2 animals were removed from the study because of conditions unrelated to the study performed. One dog in T06 was removed from the study on study day 21 before receiving the second vaccination because of respiratory distress. One dog in T02 was removed from the study on study day 21 before receiving the second vaccination because there was no healed keratoconjunctivitis and drooping tail.

Two bivalent vaccines, adjuvant 5% v/v EmulsigenInactivated CRCoV/inactivated CIV vaccine, adjuvant 5% v/v (Table 2) REHYDRAGEL^TMThe inactivated CRCoV/inactivated CIV vaccine of (1) is prepared. The CRCoV vaccine strain is from the strain deposited with ATCC as accession number PTA-11444. The CIV vaccine strain is from the strain deposited with ATCC as accession number PTA-7694. The antigens used to prepare vaccines are mostly produced in the largest pathways of the virus or cell to meet the immunogenicity requirements. The CRCoV antigen was formulated according to the target 1.55 RAU/dose. CIV antigen was formulated as 640HA units/dose of interest.

TABLE 3 Experimental design

¹The study was administered Subcutaneously (SC) with veterinary products (IVP).

²Target challenge dose for CRCoVMax isolate (channel 1), administered intranasally.

AlOH: aluminum hydroxide gel

Heterologous CRCoV isolates ("Max" strain; channel 1) were used as challenge material. Virus storage material was propagated and titrated on HRT18G cells and was determined to have a 10^7.1TCID₅₀Titer in/mL. Challenge materials were tested and confirmed to be satisfactorily sterile, with no mycoplasma or canine/feline in vitro agents.

On days 0 and 21 (table 2) animals were vaccinated with the respective vaccine or placebo. The first vaccination was administered from the right shoulder region on day 0 and the second vaccination was administered from the left shoulder region on day 21.

Animals were observed for injection swelling/pain from study day 1 to study day 8 after the first vaccination, then on study days 12, 15, 19, 21, 22 and 26. On study day 8, observations of swelling in 18 animals were inadvertently not recorded. Additional observations were recorded for the right shoulder (first vaccination) observations of some animals on study day 21.

Animals were observed daily for injection swelling/pain on study day 21 post-inoculation, on study days 21-29 post-inoculation, and then on study days 33, 36, and 40. All swelling due to the second inoculation was eliminated by study day 40. Tympanic membrane temperature was collected on days 0-7 and on days 21-28, approximately 3 hours after each inoculation.

Daily SST tubes were collected for serology (approximately 6mL) prior to challenge at 42. Also prior to challenge, tympanic membrane temperatures were collected on study days 40, 41 and 42 to establish baseline values. 2 types of nasal swabs (VTM for CRCoV virus isolation, Amies for bacterial isolation) were collected from each dog on study day 42 prior to challenge. Animals were observed daily for clinical symptoms of respiratory disease on study days 40, 41 and 42 prior to challenge for establishing baseline values.

All treatment groups had 6 dogs in each group and challenge virus was administered by aerosolizing 19mL challenge substance in the plexiglass chamber for approximately 30 minutes. When less than six dogs are challenged at one time, the volume of the challenge virus nebulized in the chamber is adjusted accordingly. Virus titration on CRCoV challenge samples taken after challenge dosing confirmed that the amount of live challenge virus nebulized in the chamber contained 10^5.1TCID₅₀and/mL. Following challenge, tympanic temperature, clinical observations and nasal swabs (steriledacron swabs, Puritan25-806-1PD) were collected daily from dogs for virus isolation (VTM tubes) from study days 42 to 56. Blood samples (approximately 6mL) were collected for serology on study days 46 and 56 prior to necropsy.

In necropsy, intact lungs and trachea were removed under sterile conditions and placed on a sterile drape. Lung lobes were used to roughly assess lung lesions (consolidation). Each lobe was scored according to the percent of curing of the lung. Lung groups from 2 animals with inadequate exsanguination were not evaluated and scored. The trachea was transected and the lumen assessed for gross pathology and any findings were recorded. After the lungs were scored, each right tail lung lobe was washed with approximately 30.0mL of VTM (no antibiotics) for diagnostic bacterial analysis and virus isolation.

After the lungs were scored, tissue samples taken from the trachea, nasal cavity, and entire left middle lung lobe were used for histopathology. Tissue samples were collected from trachea, nasal cavity, right cranial lung lobes for virus isolation and bacteriology.

Blood for serology was collected on a predetermined study day.

Nasal swabs (charcoal-free Amies transmission medium) were collected from each dog only on study day 42 (prior to challenge) for diagnosis of bacteriology. These swabs were tested for the presence of bordetella, pasteurella, staphylococci, mycoplasma and streptococcus canis.

And (6) obtaining the result.

On study day 0 before inoculation, 59 beagle dogs were negative for antibodies to CRCoV (IFA titer <40) as confirmed by IFA assay. Serum samples were also tested by serum neutralization and confirmed negative for antibodies to CRCoV (SN titer < 20). Nasal swabs used to evaluate CRCoV virus isolation confirmed the absence of CRCoV virus in all animals on study day 0 prior to vaccination. The detection of CIV virus and antibody on study day 0 confirmed the absence of CIV virus and CIVHAI antibody in the animals (HAI titer < 8). According to these two criteria, animals proved to be susceptible and therefore suitable for the evaluation of the effectiveness and safety of the CRCoV and CIV vaccines. Saline-inoculated control groups remained seronegative for CRCoV until study day 42. All animals were confirmed to be absent of CRCoV by the study day 42 virus isolation study, indicating the lack of exposure of the external CRCoV in the device. All dogs on study day 42 (before challenge) were confirmed to be free of bordetella bronchiseptica.

Dogs were vaccinated with a vaccine comprising inactivated CRCoV and inactivated CIV antigen formulated in 2, supplemented with EmulsigenOr Rehydragel^TM. The potency of the CRCoV antigen in the vaccine was determined by a double antibody sandwich ELISA using CRCoV-specific serum neutralizing monoclonal antibody 41.1.1. The potency was determined to be 1.14 RAU/dose relative to the indicated reference antigen. The CIVHAI titer of guinea pigs was 955. (the grid criteria are HAI Titers>161)

Emulsigen was received after the first vaccination10 of the 19 animals of the formulation (T05 and T06) developed measurable injection swelling. There were scratches in most dogs reported immediately after vaccination. Only 2 dogs were reported to be painful to the touch. Except for one dog, the swelling of the group was eliminated the next day. There was a slight numerical increase in the magnitude and frequency of swelling after the second vaccination, but it was within the expected range as a typical adjuvant vaccine response. None of the vaccinated dogs were reported to have systemic adverse effects, confirming the lack of clinical fever (39.5 ℃). The results of these studies indicate that the vaccine formulation is safe for administration to dogs of this age group, with safety within the expected range of adjuvant vaccines.

Rehydragel was received after each inoculation^TMMost dogs of the formula (T03 and T04) developed injection swelling. Swelling appeared 3 days after the first inoculation and most of the swelling was eliminated on study day 19. A similar response was seen after the second vaccination, with most swelling eliminated on study day 36. Injection swelling was small in scale and is typical of alum adjuvant reactions. No pain and fever were reported, confirming that no systemic response to the vaccine was produced. The results of these studies indicate that the vaccine formulation is safe for administration to dogs of this age group, with safety within the expected range of adjuvant vaccines.

Seroneutralization titration has been tabulated and performedThe components compare (see figure 1). Both vaccine formulations elicited a serum neutralizing antibody (SN) response in all vaccinated dogs after the first vaccination, indicating active immunity (fig. 1). SN reaction (Rehydragel)^TMGMT =552, EmulsigenGMT =2030) increased after the second vaccination, indicating a growth promoting effect after the second vaccination. Both vaccine formulations resulted in a strong recall SN response after challenge (Rehydragel in the dogs remaining on study day 56 experiment^TMGMT =10725, EmulsigenGMT =11584), indicating a potent immunological memory response. It is important to note that the antibody response to CRCoV is completed in the presence of CIV antigen, indicating a lack of interference in the antigen of bivalent vaccines.

The remaining 56 dogs in the experiment were challenged by nebulization on experimental day 42. After challenge, rhinovirus isolation confirmed that saline vaccinated dogs (100%) shed challenge virus on at least 3 of days 1 to 6 post challenge, indicating that dogs were infected with CRCoV, with an average number of shed days of 4.5 (fig. 2). Use of Rehydragel^TMAnd EmulsigenThe two vaccine formulations each significantly reduced viral shedding to 2.6 days (p)<0.0001) and 3.4 days (p)<0.0042). These findings indicate that vaccine induction is effective, which results in attenuation of viral infection.

Tissue virus isolation data showed 90-100% in dogs vaccinated with saline groups, positive for virus in nasal, tracheal and lung tissues on study day 4 post challenge, indicating infection of respiratory organs (figure 3). In contrast, the vaccine significantly reduced lung (P)<0.002) neutralizing nasal cavity (P)<0.002) percentage of animals positive for virus isolation. Although the vaccine reduced trachea (Rehydragel)^TMFrom 70% of the group virus isolates, EmulsigenGroup 44%) virus isolates, but only Emulsigen compared to saline control group (P =0.0089)The virus caused by the formula is obviously reduced. No virus isolated from any animal at day 14 post challenge, indicating that CRCoV infection rapidly entered and exited respiratory tissues, similar to the canine influenza protocol. Virus isolation data indicate that both vaccine formulations significantly reduced viral infection in dogs.

CRCoV challenge caused only mild clinical symptoms of the respiratory tract under the experimental conditions. All treatment groups of dogs were reported to have ocular and nasal discharge.

One animal on study day 41 (day before challenge) in the saline control group was removed, and all animals were warm before challenge. The saline control group had 2 animals reported to have clinical fever after challenge. The temperature of 2 dogs was 39.6 ℃ after challenge (on study day 44). One of the dogs exhibited a fever (40 ℃) again on day 4 post challenge. The dog was treated for concurrent gastroenteritis. This might explain the febrile response following a canine CRCoV challenge, since this virus was not previously shown to trigger fever under experimental conditions. No clinical fever was reported in vaccinated dogs.

Gross autopsies of lungs, trachea and turbinates were performed on days 4 and 14 post challenge. No significant severe lesions were reported, except that lung consolidation occurred in 2 dogs in T05, 2 dogs in T01, and two dogs in T02. The cause of the lesion is not clear, but is unlikely to be due to CRCoV, since the lesion is not uniform and CRCoV does not appear to initiate pulmonary solidification. Diagnostic bacteriological examination of tissues showed no involvement of other pathogens.

Lung, tracheal and nasal tissue sections were examined and scored. A score (0-4 points) was designed based on the degree of change observed. Previous studies have shown that attack is occurringCiliary damage of the airway epithelium on day 4 post-stroke is a characteristic pathological effect associated with CRCoV infection. (Priestnall et al, 2009) pathological tissue data showed that 70% of saline-inoculated dogs experienced some degree of tracheal epithelial cilia damage on day 4 post challenge. In contrast, both vaccines reduced the number of infected dogs to Rehydragel^TM(P =0.1184)40% and Emulsigen(p =0.0003) 0%. This indicates the effectiveness of the vaccine induction in preventing or reducing tracheal mucociliary damage, an important innate defense mechanism in infected dogs.

To assess potential involvement of other respiratory pathogens in the experiment, animals were tested for diagnostic cytology before challenge (nasal swab) and after challenge (lung tissue/lavage). The results obtained indicate that the animals are largely free of other respiratory pathogens, indicating that the clinical outcome measured after challenge is specific for CRCoV infection.

In summary, all CRCoV-CIV vaccinated dogs (100%) developed CRCoV serum neutralizing antibody titers 3 weeks after the second vaccination, indicating active immunity after recall response after a strong challenge, indicating a good immune system. Both vaccine formulations significantly reduced viral shedding. Both vaccine formulations significantly reduced pulmonary (P)<0.0001) and in the nasal cavity (P)<0.002) percentage of animals positive for virus isolate. Both vaccines reduced virus isolates in the trachea, although only Emulsigen when compared to the saline control group (p0.0089)The formulation of (a) results in a significant reduction of virus isolates. Both vaccines also reduced the number of dogs infected with tracheal epithelial cilia. The effectiveness of the CRCoV antigen in these vaccines was achieved in the presence of the CRCoV antigen, indicating the lack of interference of the CIV fraction with the CRCoV.

Example 3 safety and efficacy of Bordetella bronchiseptica-containing vaccines against dogs

Fifty (50) dogs, divided into 5 groups, were selected as subjects. Animals were determined whether they were used in the study based on a physical examination on day 4.

Blood samples (approximately 8mL) for serology were collected from all animals with SST tubes on study days 2, 21 and 28 prior to each vaccination. Serum samples collected on day 2 were used to confirm the presence of b.bronchi septica in the animals. Nasal swabs were collected on day 0 prior to vaccination and tested for the presence of b. Tympanic membrane temperature was collected from day 4 to establish a pre-vaccination baseline.

TABLE 4 Experimental design

¹The study was administered Subcutaneously (SC) with veterinary products (IVP).

²Biological target challenge dose of Bordetella bronchiseptica strain 10^9

The response of the injection sites of all animals was observed on the 0 th, 21 th and 28 th vaccination days after vaccination. The injection response was observed daily from days 1 to 7 and days 22-35 after vaccination. Tympanic membrane temperatures were collected on days 0-7 and days 21-35.

Blood samples (approximately 6mL) were collected for serology the day before challenge, day 55. Tympanic membrane temperatures were collected on days 54, 55 and 56 prior to challenge. Nasal swabs were collected on day 55 prior to challenge and tested for the presence of bordetella bronchiseptica. Animals were observed 2 times daily for clinical symptoms of their respiratory illness, each for approximately 30 minutes in the morning on days 54 and 55, and 56, for establishing baseline values.

Application of Bordetella bronchiseptica challenge strain in preparation of 10⁹CFU/4 mL/target challenge dose for dog. In the first placeOn day 56, all treatment groups of dogs were challenged intranasally with Bordetella bronchiseptica by nebulization in the plexiglas chamber for a total banning period of 30 minutes per challenge. 5 dogs in the same bans (one from each treatment group) at a time were challenged.

Tympanic membrane temperatures were recorded each time from day 56-77 after challenge. Each time 2 clinical observations (am and pm) were made, approximately 30 minutes each time in each room, from day 56 until day 76, once on day 77 (am). Briefly, coughing, runny nose, sneezing, eye secretions, retching, and depression were observed using the following scoring system: none (0), mild (1), moderate (2), severe (3). Nasal swabs were collected on days 59, 62, 66, 69, 74, 76 and 77 for determination of efflux of challenge microorganisms.

Blood samples (approximately 6mL) were collected for serology on day 77. Nasal swabs for isolation of bordetella bronchiseptica were collected with swabs and transmission media.

The aggregated antibody of bordetella bronchiseptica was determined by the Microgel Aggregation Test (MAT). Serum samples collected from treatment groups T04 and T05 on days 0, 28, 55, and 77 were titrated for CRCoV, as well as CIV and HAI by serum neutralization IFA. The bordetella bronchiseptica isolates obtained from nasal swabs were performed according to standard procedures. Each set of samples was qualitatively tested for the presence or absence of bacteria.

ResultsFifty (50) healthy beagle puppies of approximately 8 weeks of age were confirmed to be free of bordetella bronchiseptica on day 0 by nasal swab culture isolation. Serum samples used to evaluate agglutinating antibodies to bordetella bronchiseptica by MAT demonstrated MAT titers of ≦ 8 on day 2 for all puppies.

All experimental vaccines used in the present disclosure for evaluation produced slight to no injection swelling after the first vaccination. For most vaccinations, injection swelling was limited to day 0. Slight to no injection swelling was also reported after the second vaccination. When swelling of the injection site occurred, it was eliminated within 1 to 3 days after the second vaccination. Flex marks were mainly reported in the 5-combination group (T04). No clinical fever was reported after vaccination. No injection swelling was reported in the saline injection group. This data demonstrates the safety of the vaccine.

Colony counts performed before and after challenge inoculation confirmed an average of 1.45 × 10 per dog⁸The bacteria bordetella CFU were atomized in an atomization chamber. Challenge vaccination induced cough in the saline control dogs (T02), with an average observed percentage of cough of 43.5% and cough for 12.2 days. Treatment group T05, vaccinated with 4-virus only (CRCoV/CIV/CPIV/CAV2) developed a cough similar to the saline control group without bordetella antigen, with an average observed percentage of cough of 43.4% and 12.2 days of cough. These findings indicate that challenge is appropriate and consistent with evaluating test vaccines.

Dogs in treatment group T01 vaccinated with bordetella vaccine were significantly protected against challenge (cough 3.6 days, p <0.0001) compared to control (cough 12.2 days). The same vaccine also clearly protected dogs in the T03 group when given a 3 week interval treatment regimen (cough 5.8 days, P = 0.0004). There was no significant difference in the reduction of cough scores in both groups (T01vsT03) (p value =0.1883), indicating that the level of protection given to the vaccine at 3 or 4 weeks apart was similar.

Dogs in the T04 group receiving the 5-combination vaccine without adjuvant were significantly (p value =0.0016) protected against bordetella challenge (cough 6.6 days) compared to the saline control group (cough 12.2 days) and to the T05 group receiving the 4-virus (CRCoV/CIV/CPIV/CAV2) combination (cough 12.1 dean, p =0.0019), indicating the efficacy of the bordetella fraction in the combination vaccine lacking the adjuvant.

Serological evaluation of virus fractions in a 5-combination vaccine may only be demonstrated seronegative on study day 2 for 2 fractions, CIV and CRCoV, dogs. On study day 56, the CIVHAI response for the 4-vaccine group (T04) was similar to its value in the 5-vaccine group (T05), indicating interference with CIV antigen in the absence of the Bordetella fraction. The value of the 4-vaccine group (T04) was higher than the 5-vaccine group (T05) in the CRCoVSN response on study day 56, indicating that bordetella may interfere with the CRCoV fraction. However, the results of these studies are not conclusive, as these vaccines are not adjuvanted and the formulation is not optimized and the efficacy of the CRCoV challenge is not tested.

Monovalent bordetella vaccines have proven to be safe and effective. The efficacy of a monovalent vaccine was demonstrated when the vaccine was given at intervals of 21-or 28 days. The bordetella fraction also proved to be effective when given as 5-combination vaccines without adjuvant.

Example 4 multivalent serological Studies

40 dogs, approximately 8 weeks old and approximately good health, were pre-screened for Bordetella bronchiseptica using the Microgel Assay (MAT) and canine respiratory coronavirus (CRCoV) by the indirect fluorescent antibody assay (IFA). Serum Neutralization (SN) was also used to assess antibody levels. On day 0, all dogs were negative for bordetella bronchiseptica antibodies by MAT (<16) and CRCoV antibodies by IFA (IFA < 40). All dogs were determined to be free of bordetella bronchiseptica and CRCoV by nasal swab isolation detection prior to the first vaccination (day 0).

Dogs were divided into 5 treatment groups of 8 dogs each, and vaccinated according to the experimental design of table 1. Each dog was vaccinated in the right shoulder area for the first vaccination and in the left shoulder area for the second vaccination.

TABLE 2 Experimental design

¹EMA = ethylene-maleic anhydride

After the second vaccination, the T04 and T05 groups were removed from the study due to complications. Dogs in the remaining group (T01, T02, T03) were observed daily after the vaccination response and the body (tympanic membrane) temperature was measured 7 days after each vaccination. Blood samples were collected from dogs on days 0, 21, 42 and 56 for measurement of antibody responses.

Blood samples on days 0, 21, 42 and 56 were tested for agglutinating antibodies to bordetella bronchiseptica using MAT detection. Serum samples on the same date were also titrated for CRCoV antibodies by serum neutralization, as well as for CIV by HAI and CAV-2 and CPI antibodies by serum neutralization. Geometric mean antibody titers were obtained from each treatment group.

After the second dose, the test vaccines of T02 and T03 elicited antibody responses in all (100%) vaccinated dogs, indicating active immunity to viral antigens. After the second vaccination, antibody responses increased in the absolute majority of vaccinated dogs, indicating the growth promoting effect of the second vaccination. It is important to note that the antibody response in the viral fraction was completed in the presence of multiple viral and bacterial (bordetella bronchiseptica) antigens, indicating a lack of immune interference. MAT serology is not associated with protection against bordetella, but is a valuable screening tool for recruiting appropriate research animals. Finally, the efficacy of viral antigens was predicted in 5-multivalent vaccines based on the immune response in vaccinated dogs.

Example 5 duration of immunization study

The purpose of this study was to demonstrate the duration of immunization of multivalent respiratory combination vaccines in dogs. The vaccine comprises the following antigenic components: modified-live CAV-2, modified-live CPIV, inactivated CIV, inactivated CRCoV, and Bordetella bronchiseptica extracts supplemented with recombinant antigens (pertactin, or Bsp22, or both).

All animals were well-conditioned and did not receive any vaccination for any pathogen against which the vaccine was designed. Dogs were divided into multiple treatment groups. Each set consisted of 2 treatment groups, one control group receiving placebo vaccine, and one vaccination group receiving test vaccine. Animals were vaccinated 2 times, approximately 2-4 weeks apart. Their injection site response was observed after each vaccination.

Approximately 3-12 months after vaccination, 2 treatment groups (immune and control) per set were challenged with one of the pathogens against which the vaccine was designed. Clinical observations were made before and after challenge. Nasal swabs for isolation of challenge pathogens were collected during the post challenge period. Blood samples were collected from each animal for serum, which was used for subsequent analytical analysis. Clinical symptoms of respiratory disease, pathogens shed after challenge, and serological responses are used as criteria for judging vaccine efficacy.

All of the above references are incorporated by reference as if fully set forth herein.

Although the invention has been described in detail in various embodiments thereof, it is to be understood that the invention defined by the appended claims is not to be limited by particular details set forth in the above description as many apparent variations thereof are possible without departing from the spirit or scope thereof.

Claims (20)

1. A vaccine composition comprising Canine Influenza Virus (CIV) and canine respiratory coronavirus (CRCoV), wherein said CIV is deposited with the ATCC as PTA-7694 and said CRCoV is deposited with the ATCC as PTA-11444.

2. The vaccine composition of claim 1, further comprising bordetella bronchiseptica.

3. The vaccine composition of claim 2, further comprising p68 pertactin antigen.

4. The vaccine composition of claim 2, wherein said bordetella bronchiseptica is a bacterium or a bacterial extract.

5. The vaccine composition of claim 2, further comprising one or two antigens selected from the group consisting of canine parainfluenza virus (CPIV) and canine adenovirus serotype 2 (CAV-2).

6. The vaccine composition of claim 5 wherein the one or both antigens are CPIV and CAV-2.

7. The vaccine composition of claim 2, further comprising an isolated Bsp22 antigen.

8. The vaccine composition of claim 2, wherein said composition is devoid of an adjuvant.

9. The vaccine composition of claim 2, further comprising an adjuvant.

10. The vaccine composition of claim 1, which does not contain a non-respiratory antigen.

11. The vaccine composition of claim 1, which induces an immune response in canines against a canine respiratory pathogen.

12. The vaccine composition of claim 11, wherein the canine respiratory pathogen is at least one of CIV, CRCoV, CPIV, CAV-2, bordetella bronchiseptica, and mycoplasma canis (m.

13. Use of the vaccine composition of any one of claims 1 to 10 in the manufacture of a medicament for preventing infection of a canine respiratory pathogen in a canine.

14. The use according to claim 13, wherein the canine respiratory pathogen is at least one of CIV, CRCoV, CPIV, CAV-2, bordetella bronchiseptica and mycoplasma canis (m.

15. The use of claim 13 or 14, said composition preventing said infection for a period of 6 months or more.

16. The use of claim 13 or 14, said composition preventing said infection over a period of 1 year.

17. The vaccine composition of any one of claims 1 to 10, which prevents canine infectious respiratory disease syndrome (CIRDC) in a canine.

18. Use of the vaccine composition of any one of claims 1 to 10 in the manufacture of a medicament for preventing CIRDC in a canine.

19. The use of claim 18, the composition prevents CIRDC for 6 months or more.

20. The use of claim 18, the composition prevents CIRDC over a period of 1 year.