Canine influenza virus vaccine

Abstract

The present invention provides new canine Influenza H3N2 vaccines, including multivalent vaccines. The present invention provides an alphavirus RNA replicon particle that encodes a Canine Influenza (CIV) H3N2 hemagglutinin (HA) antigen. The present invention further provides methods of making and using the vaccines.

Description

CANINE INFLUENZA VIRUS VACCINE FIELD OF THE INVENTION

[0001] The present invention relates to new vaccines for canine influenza. Methods of making and using the vaccines alone or in combination with other protective agents are also provided. CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of US Application No.63 / 683,913, filed on August 16, 2024, the contents of which are incorporated herein by reference in its entirety. SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML file, created on December 3, 2024, is named 26038-WO-PCT_SL.xml and is 8.0 KB (8,192 bytes) in size on disk. BACKGROUND

[0004] Canine influenza is an easily transmissible respiratory disease that is caused by influenza A viruses, primarily avian-origin canine influenza virus (CIV) H3N2. CIV is transmitted via direct contact or airborne particles for up to ten days post-infection, while stability of the virus outside of a host is just one to two days. Although dogs are the primary hosts for CIV, interspecies transmission puts cats, ferrets, horses, mice, guinea pigs, and other species at risk for infection as well. Animals in close contact are therefore at increased risk for exposure, such as those in shelters, boarding kennels, or dog park. Since 2015, CIV H3N2 outbreaks have been an issue throughout North America and Asia. Dogs with CIV H3N2 infection can exhibit clinical signs including cough, nasal discharge, depression, and occasionally fever. In more severe cases of infection, suppurative bronchopneumonia can develop, which can be fatal. Additionally, since CIV H3N2 is associated with canine infectious respiratory disease complex (CIRDC), the probability of coinfections in these cases is even greater. For these reasons, it is highly recommended by the American Animal Hospital Association to vaccinate dogs for CIV to prevent the spread of disease and possible epidemics.

[0005] Traditional, inactivated CIV vaccines have required the use of adjuvants to assist in accentuating the immune response, but this heightened inflammatory reaction can sometimes be deleterious and may not drive a balanced immune response.

[0006] Classification of influenza A viruses starts with subtyping of hemagglutinin (HA) and neuraminidase (NA), the two major glycoproteins on the virus surface. HA protein mediates attachment and fusion of the virus to host cells. NA is an enzyme that functions in the final stage of the influenza virus replication cycle by cleaving newly formed viral particles from the host cell, thereby enabling the new progeny virus to spread and infect other cells. There are different HA subtypes and NA subtypes. Subtypes are named by combining the H and N numbers – e.g., A(H1N1), A(H3N2).

[0007] There is therefore a need for CIV H3N2 vaccine that does not have drawbacks of adjuvant assisted vaccines but is as the same time safe and efficacious.

[0008] A number of vector strategies have been employed through the years for vaccines in an effort to protect against certain pathogens. One such vector strategy includes the use of alphavirus-derived replicon RNA particles (RP) which have been developed from several different alphaviruses, including Venezuelan equine encephalitis virus (VEE). RP vaccines deliver propagation-defective alphavirus RNA replicons into host cells and result in the expression of the desired antigenic transgene(s) in vivo. The RP platform has been used to encode pathogenic antigens and is the basis for several USDA-licensed vaccines for swine and poultry.

[0009] The citation of any reference herein should not be construed as an admission that such reference is available as "prior art" to the instant application. SUMMARY OF THE INVENTION

[0010] Accordingly, the present invention provides an alphavirus RNA replicon particle that encodes the Canine Influenza H3N2 hemagglutinin (HA) protein. In particular embodiments the Canine Influenza H3N2 HA protein is the HA from a 2015 Iowa strain (A / canine / Iowa / 22619- 4 / 2015(H3N2). In a preferred embodiment, the nucleic acids that encode the Canine Influenza H3N2 hemagglutinin (HA) protein is codon-optimized.

[0011] In still more particular embodiments, the alphavirus RNA replicon particle is a Venezuelan Equine Encephalitis (VEE) alphavirus RNA replicon particle. In yet more specific embodiments the VEE alphavirus RNA replicon particle is a TC-83 VEE alphavirus RNA replicon particle. In other embodiments, the alphavirus RNA replicon particle is a Sindbis (SIN) alphavirus RNA replicon particle. In still other embodiments, the alphavirus RNA replicon particle is a Semliki Forest virus (SFV) alphavirus RNA replicon particle.

[0012] In certain embodiments, an alphavirus RNA replicon particle of the present invention encodes a Canine Influenza H3N2 hemagglutinin (HA) protein or antigenic fragments thereof. In another embodiment of the present invention and / or embodiments thereof, the RNA replicon particles is a Venezuelan Equine Encephalitis (VEE) alphavirus RNA replicon particle encoding a Canine Influenza H3N2 hemagglutinin (HA) protein or antigenic fragments thereof. In another embodiment of the present invention and / or embodiments thereof, the RNA replicon particles is a TC-83 VEE alphavirus RNA replicon particle encoding a Canine Influenza H3N2 hemagglutinin (HA) protein or antigenic fragments.

[0013] In certain embodiments, an alphavirus RNA replicon particle of the present invention encodes a Canine Influenza H3N2 hemagglutinin (HA) protein from a 2015 Iowa strain (A / canine / Iowa / 22619-4 / 2015(H3N2). In another embodiment of the present invention and / or embodiments thereof, the RNA replicon particles is a Venezuelan Equine Encephalitis (VEE) alphavirus RNA replicon particle encoding a Canine Influenza H3N2 hemagglutinin (HA) protein from a 2015 Iowa strain (A / canine / Iowa / 22619-4 / 2015(H3N2). In another embodiment of the present invention and / or embodiments thereof, the RNA replicon particles is a TC-83 VEE alphavirus RNA replicon particle encoding a Canine Influenza H3N2 hemagglutinin (HA) protein from a 2015 Iowa strain (A / canine / Iowa / 22619-4 / 2015(H3N2).

[0014] In another aspect, the present invention provides immunogenic compositions that comprise alphavirus RNA replicon particles that encode a Canine Influenza H3N2 hemagglutinin (HA) protein or antigenic fragments thereof. In certain embodiments, the immunogenic compositions comprise an alphavirus RNA replicon particle that encodes a Canine Influenza H3N2 HA protein from a 2015 Iowa strain (A / canine / Iowa / 22619-4 / 2015(H3N2).

[0015] In more particular embodiments, the immunogenic composition comprises alphavirus RNA replicon particles that are Venezuelan Equine Encephalitis (VEE) alphavirus RNA replicon particles. In yet more specific embodiments, the immunogenic compositions comprise VEE alphavirus RNA replicon particles that are TC-83 VEE alphavirus RNA replicon particles.

[0016] In particular embodiments, an alphavirus RNA replicon particle of the present invention and / or embodiments thereof encodes a Canine Influenza H3N2 HA protein comprising an amino acid sequence comprising 95% identity or more with the amino acid sequence of SEQ ID NO: 1. In more specific embodiments of this type, Canine Influenza H3N2 HA protein comprises an amino acid sequence comprising 96% identity or more with the amino acid sequence of SEQ ID NO: 1. In more specific embodiments of this type, Canine Influenza H3N2 HA protein comprises an amino acid sequence comprising 97% identity or more with the amino acid sequence of SEQ ID NO: 1. In more specific embodiments of this type, Canine Influenza H3N2 HA protein comprises an amino acid sequence comprising 98% identity or more with the amino acid sequence of SEQ ID NO: 1. In more specific embodiments of this type, Canine Influenza H3N2HA protein comprises an amino acid sequence comprising 99% identity or more with the amino acid sequence of SEQ ID NO: 1. In even more specific embodiments of this type, the Canine Influenza H3N2 HA protein comprises the amino acid sequence of SEQ ID NO: 1. In specific embodiments of this type the Canine Influenza H3N2 HA protein is encoded by the nucleotide sequence of SEQ ID NO: 2. In specific embodiments, the RP comprises a nucleotide sequence that encodes a Canine Influenza H3N2 HA protein, where the nucleotide sequence comprises the sequence of SEQ ID NO: 2 or SEQ ID NO: 3, or a nucleic acid sequence having at least 80%, or 85%, or 90% or 95% or 97%, or 98% or 99% sequence identity to SEQ ID NO: 2 or SEQ ID NO: 3.

[0017] The present invention further comprises vaccines, including multivalent vaccines, comprising the immunogenic compositions of the present invention. In particular embodiments, the vaccines are nonadjuvanted vaccine. In certain embodiments, the vaccine aids in the prevention of disease due to CIV. In related embodiments, antibodies are induced in a canine subject when the canine is immunized with the vaccine.

[0018] The present invention also provides methods of immunizing a canine against a Canine Influenza H3N2, comprising administering to the canine an immunologically effective amount of a vaccine or multivalent of the present invention and / or embodiments thereof. In particular embodiments the vaccine is administered via intramuscular injection. In alternative embodiments the vaccine is administered via subcutaneous injection. In other embodiments the vaccine is administered via intravenous injection. In still other embodiments the vaccine is administered via intradermal injection. In yet other embodiments the vaccine is administered via oral administration. In still other embodiments the vaccine is administered via intranasal administration. In specific embodiments, the canine is a domestic dog. In some embodiments, the vaccine is administered to a companion animal, e.g., a domestic cat.

[0019] It has been found that the RP platform allows for a new CIV H3N2 vaccine to be adjuvant free, while eliciting a robust humoral and cell mediated immune response by targeting dendritic cells. In addition, the small amount of RP that is required for an efficacious dose allows for a 0.5 mL administration volume, which can provide a more comfortable vaccination experience, especially for smaller breeds. Furthermore, the RP platform allows for a more closed system manufacturing process, leaving the final vaccine formulation preservative and thimerosal free.

[0020] The vaccines and multivalent vaccines of the present invention can be administered as a primer vaccine and / or as a booster vaccine. In specific embodiments, a vaccine of the present invention is administered as a one shot vaccine (one dose), without requiring subsequent administrations. In certain embodiments, in the case of the administration of both a primer vaccine and a booster vaccine, the primer vaccine and the booster vaccine can be administeredby the identical route. In certain embodiments of this type, the primer vaccine and the booster vaccine are both administered by subcutaneous injection. In alternative embodiments, in the case of the administration of both a primer vaccine and a booster vaccine, the administration of the primer vaccine can be performed by one route and the booster vaccine by another route. In certain embodiments of this type, the primer vaccine can be administered by subcutaneous injection and the booster vaccine can be administered orally.

[0021] The invention further provides for a method of immunizing a canine against CIV comprising injecting the canine with an immunologically effective amount of the above described vaccines. In particular embodiments the vaccines can include from about 1 x 104to about 1 x 1010RPs or higher, for example. In more particular embodiments the vaccines can include from about 1 x 105to about 1 x 109RPs. In even more particular embodiments the vaccines can include from about 1 x 106to about 1 x 108RPs. In particular embodiments the canine is a domestic dog.

[0022] In particular embodiments the vaccines of the present invention are administered in 0.05 mL to 3 mL doses. In more particular embodiments the dose administered is 0.1 mL to 2 mLs. In still more particular embodiments the dose administered is 0.2 mL to 1.5 mLs. In yet other embodiments the dose administered is 0.5 mL to 2.0 mLs. In still other embodiments the dose administered is 0.3 to 1.0 mLs. In yet more particular embodiments the dose administered is 0.4 mL to 0.8 mLs.

[0023] These and other aspects of the present invention will be better appreciated by reference to the following Detailed Description. BREIF DESCRIPTION OF THE DRAWINGS

[0024] FIG.1. Whole blood for serum was collected on study days -1, 20, 28, 33, and 40 to monitor serological titers to canine influenza virus (CIV) H3N2. All vaccinated dogs sero- converted following the second vaccination, with the highest hemagglutination inhibition (HAI) antibody titers occurring on study day 33. The test vaccine significantly increased serological titers on study days 28, 33, and 40 (*p < 0.0001).

[0025] FIG 2. Dogs were observed for clinical signs of CIV H3N2 and given a score. The test vaccine significantly reduced the clinical signs of disease on days 4-10 following virulent challenge (*p ≤ 0.006).

[0026] FIG.3. Nasal swabs were collected daily post-challenge and titrated for CIV H3N2 virus. The daily mean titer for each treatment group was calculated and compared. The testvaccine significantly reduced the amount of CIV H3N2 virus that was shed from dogs on days 2- 7 following virulent challenge (*p ≤ 0.02). DETAILED DESCRIPTION OF THE INVENTION

[0027] The present invention provides efficacious, safe CIV vaccines. In particular embodiments the vaccine is nonadjuvanted.

[0028] Accordingly, the vaccine compositions of the present invention include an immunologically effective amount of a vector encoding an antigen from one or more strains of Canine Influenza H3N2 that aids in eliciting protective immunity in the recipient vaccinated animal. Furthermore, the present invention provides new immunogenic compositions to improve the reliability of the vaccination to aid in the reduction of upper respiratory disease in dogs in a canine infected by CIV and to thereby yield more transient or mild disease and / or lead to the reduction of the infection. In a particular aspect of the present invention, the vaccines comprise an alphavirus RNA replicon particle (RP) encoding a Canine Influenza H3N2 antigen.

[0029] In more specific embodiments, the vaccines comprise alphavirus RNA replicon particles (RPs) that comprise the HA protein and glycoproteins of Venezuelan Equine Encephalitis Virus (VEE) and encode the Canine Influenza H3N2 HA protein and / or an antigenic fragment thereof. In even more specific embodiments, the vaccines comprise alphavirus RNA replicon particles (RPs) that comprise the HA protein and glycoproteins of the avirulent TC-83 strain of VEE and encode one or more CIV HA proteins and / or one or more antigenic fragments thereof.

[0030] In order to more fully appreciate the invention, the following definitions are provided.

[0031] The use of singular terms for convenience in description is in no way intended to be so limiting. Thus, for example, reference to a composition comprising "a polypeptide" includes reference to one or more of such polypeptides. In addition, reference to an "alphavirus RNA replicon particle" includes reference to a plurality of such alphavirus RNA replicon particles, unless otherwise indicated.

[0032] As used herein the term "approximately" is used interchangeably with the term "about" and signifies that a value is within fifty percent of the indicated value i.e., a composition containing "approximately" 1 x 108alphavirus RNA replicon particles per milliliter contains from 0.5 x 108to 1.5 x 108alphavirus RNA replicon particles per milliliter.

[0033] As used herein, the term " canine" is used interchangeably with the term “dog” and refers to any member of the Canines including wild, zoo, and domestic canines, such as wolves, coyotes, and foxes. Canines also include dogs, particularly domestic dogs, i.e., Canis lupus familiaris or Canis familiaris, unless otherwise indicated, and includes, such as, for example,pure-bred and / or mongrel companion dogs, show dogs, working dogs, herding dogs, hunting dogs, guard dogs, police dogs, racing dogs, and / or laboratory dogs. In a specific embodiment, the canine is a domesticated dog.

[0034] As used herein, the term “replicon” refers to a modified RNA viral genome that lacks one or more elements (e.g., coding sequences for structural proteins) that if they were present, would enable the successful propagation of the parental virus in cell cultures or animal hosts. In suitable cellular contexts, the replicon will amplify itself and may produce one or more sub-genomic RNA species.

[0035] As used herein, the term “alphavirus RNA replicon particle”, abbreviated “RP”, is an alphavirus-derived RNA replicon packaged in structural proteins, e.g., the HA and glycoproteins, which also are derived from an alphavirus, e.g., as described by Pushko et al., [Virology 239(2):389-401 (1997)]. An RP cannot propagate in cell cultures or animal hosts (without a helper plasmid or analogous component), because the replicon does not encode the alphavirus structural components (e.g., HA and glycoproteins).

[0036] The terms “originate from”, “originates from” and “originating from” are used interchangeably with respect to a given protein antigen and the pathogen or strain of that pathogen that naturally encodes it, and as used herein signify that the unmodified and / or truncated amino acid sequence of that given protein antigen is encoded by that pathogen or strain of that pathogen. The coding sequence, within a nucleic acid construct of the present invention for a protein antigen originating from a pathogen may have been genetically manipulated so as to result in a modification and / or truncation of the amino acid sequence of the expressed protein antigen relative to the corresponding sequence of that protein antigen in the pathogen or strain of pathogen (including naturally attenuated strains) it originates from.

[0037] As used herein, the terms “protecting”, or “providing protection to”, or “eliciting protective immunity to”, “aids in prevention of disease”, and "aids in the protection" do not require complete protection from any indication of infection. For example, "aids in the protection" can mean that the protection is sufficient such that, after challenge, symptoms of the underlying infection are at least reduced, and / or that one or more of the underlying cellular, physiological, or biochemical causes or mechanisms causing the symptoms are reduced and / or eliminated. It is understood that “reduced,” as used in this context, means relative to the state of the infection, including the molecular state of the infection, not just the physiological state of the infection.

[0038] As used herein, a "vaccine" is a composition that is suitable for application to an animal, e.g., canine (including, in certain embodiments, humans, while in other embodiments being specifically not for humans) comprising one or more antigens typically combined with a pharmaceutically acceptable carrier such as a liquid containing water, which upon administrationto the animal induces an immune response strong enough to minimally aid in the protection from a disease arising from an infection with a wild-type micro-organism, i.e., strong enough for aiding in the prevention of the disease, and / or preventing, ameliorating or curing the disease.

[0039] As used herein, a “multivalent vaccine” is a vaccine that comprises two or more different antigens. In a particular embodiment of this type, the multivalent vaccine stimulates the immune system of the recipient against two or more different pathogens.

[0040] As used herein, the terms “live attenuated virus” and “live modified virus” are used interchangeably and are live attenuated viral immunogens that are immunogenic, but not pathogenic.

[0041] As used herein, the terms “live attenuated bacterium”, “avirulent live culture of a bacterium”, and “avirulent live bacterium” are used interchangeably and are live attenuated bacterial immunogens (e.g., an avirulent live B. bronchiseptica) that are immunogenic, but not pathogenic

[0042] The terms “adjuvant” and "immune stimulant" are used interchangeably herein, and are defined as one or more substances that cause stimulation of the immune system. In this context, an adjuvant is used to enhance an immune response to one or more vaccine antigens / isolates. Accordingly, "adjuvants" are agents that nonspecifically increase an immune response to a particular antigen, thus reducing the quantity of antigen necessary in any given vaccine, and / or the frequency of injection necessary in order to generate an adequate immune response to the antigen of interest. In this context, an adjuvant is used to enhance an immune response to one or more vaccine antigens / isolates.

[0043] As used herein, a “nonadjuvanted vaccine” is a vaccine or a multivalent vaccine that does not contain an adjuvant.

[0044] As used herein, the term "pharmaceutically acceptable" is used adjectivally to mean that the modified noun is appropriate for use in a pharmaceutical product. When it is used, for example, to describe an excipient in a pharmaceutical vaccine, it characterizes the excipient as being compatible with the other ingredients of the composition and not disadvantageously deleterious to the intended recipient animal, e.g., canine.

[0045] Parenteral administration" includes subcutaneous injections, submucosal injections, intravenous injections, intramuscular injections, intradermal injections, and infusion.

[0046] As used herein the term “antigenic fragment” in regard to a particular protein (e.g., a protein antigen) is a fragment of that protein that is antigenic, i.e., capable of specifically interacting with an antigen recognition molecule of the immune system, such as an immunoglobulin (antibody) or T cell antigen receptor. For example, an antigenic fragment of a Canine Influenza H3N2 HA protein is a fragment of the HA protein that is antigenic. Preferably,an antigenic fragment of the present invention is immunodominant for antibody and / or T cell receptor recognition. In particular embodiments, an antigenic fragment with respect to a given protein antigen is a fragment of that protein that retains at least 25% of the antigenicity of the full-length protein. In preferred embodiments an antigenic fragment retains at least 50% of the antigenicity of the full-length protein. In more preferred embodiments, an antigenic fragment retains at least 75% of the antigenicity of the full-length protein. Antigenic fragments can be as small as 20 amino acids or at the other extreme, be large fragments that are missing as little as a single amino acid from the full-length protein. In particular embodiments the antigenic fragment comprises 25 to 150 amino acid residues. In other embodiments, the antigenic fragment comprises 50 to 250 amino acid residues.

[0047] As used herein one amino acid sequence is 100% "identical" or has 100% “identity” to a second amino acid sequence when the amino acid residues of both sequences are identical. Accordingly, an amino acid sequence is 50% "identical" to a second amino acid sequence when 50% of the amino acid residues of the two amino acid sequences are identical. The sequence comparison is performed over a contiguous block of amino acid residues comprised by a given protein, e.g., a protein, or a portion of the polypeptide being compared. In a particular embodiment, selected deletions or insertions that could otherwise alter the correspondence between the two amino acid sequences are taken into account.

[0048] As used herein, nucleotide and amino acid sequence percent identity can be determined using C, MacVector (MacVector, Inc. Cary, NC 27519), Vector NTI (Informax, Inc. MD), Oxford Molecular Group PLC (1996) and the Clustal W algorithm with the alignment default parameters, and default parameters for identity. These commercially available programs can also be used to determine sequence similarity using the same or analogous default parameters. Alternatively, an Advanced Blast search under the default filter conditions can be used, e.g., using the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin) pileup program using the default parameters.

[0049] As used herein, the term "inactivated" microorganism is used interchangeably with the term “killed” microorganism. For the purposes of this invention, an "inactivated" microorganism is an organism which is capable of eliciting an immune response in an animal, but is not capable of infecting the animal. An antigen of the present invention may be inactivated by an agent selected from the group consisting of binary ethyleneimine, formalin, beta-propiolactone, thimerosal, or heat.

[0050] The alphavirus RNA replicon particles of the present invention may be lyophilized and rehydrated with a sterile water diluent. On the other hand, when the alphavirus RNA replicon particles are stored separately, but intended to be mixed with other vaccine components prior toadministration, the alphavirus RNA replicon particles can be stored in the stabilizing solution of those components, e.g., a high sucrose solution.

[0051] A vaccine of the present invention can be readily administered by any standard route including intravenous, intramuscular, subcutaneous, oral, intranasal, intradermal, and / or intraperitoneal vaccination. The skilled artisan will appreciate that the vaccine composition is preferably formulated appropriately for each type of recipient animal and route of administration.

[0052] Thus, the present invention also provides methods of immunizing a canine against CIV and / or other canine pathogens. One such method comprises injecting a canine with an immunologically effective amount of a vaccine of the present invention, so that the canine produces appropriate CIV antibodies.

[0053] Accordingly, it is contemplated that a vaccine of the present invention may be administered to the animal subject, e.g., a canine, as a single dose vaccine, or as a two-dose vaccine for which the duration of immunity is at least 6 months. In certain embodiments, the duration of immunity is at least 9 months. In other embodiments, the duration of immunity (DOI) is at least 12 months. In still other embodiments, the duration of immunity is at least 18 months.

[0054] In some aspects, a second dose of a vaccine as disclosed herein (or multivalent vaccine) is administered one (1) week, multiple weeks, or months following the primary administration over a 6 to 18-month duration after the administration of the initial dose. A booster vaccine can be administered by injection (e.g., intramuscularly, subcutaneously), intranasally, or orally.

[0055] Accordingly, in some embodiments, the vaccine is administered in at least two (2) doses. In some such embodiments, for example, the vaccine is administered twice, with the second dose (e.g., a booster vaccine) being administered at least about 2 weeks after the first. In some embodiments, the vaccine is administered twice, with the second dose being administered no greater than 8 weeks after the first. In other embodiments, the second dose is administered from about 2 weeks to about 4 months after the first dose, from about 2 to about 8 weeks after the first dose, or from about 3 weeks to about 4 weeks after the first dose. In some embodiments, the second dose is administered about 4 weeks after the first dose. The first and subsequent dosages may vary, such as, for example, in amount and / or form. Often, however, the dosages are the same with respect to the amount and form. Whether the administration is performed as a single dose vaccine or in multiple doses [i.e., booster vaccine(s)], it should be understood that subsequent administrations of the vaccine are likely to be needed to be provided to a given animal subject after the 6 to 18 month duration of immunity (or even longer), such as in a yearly or every other year administration regimen.

[0056] When a single dose of a vaccine of the present invention is sufficient to aid in the protection of (and / or provide effective protection to) the animal from the pathogen for at least six (6) months, the quantity of the antigen(s) in that dose generally comprises a therapeutically effective amount of the vaccine for the 6 month or longer duration. On the other hand, when a booster vaccine dose is required to supplement that initial dose, the combined quantity of the initial vaccine and the booster vaccine may constitute the therapeutically effective amount.

[0057] In particular embodiments, an alphavirus RNA replicon particle of the present invention and / or embodiments thereof is used in the prevention of a disease due to Canine Influenza H3N2 strain for at least 6-months, comprising administering to a canine an immunogenic composition comprising a Canine Influenza H3N2 HA protein comprising an amino acid sequence comprising 95% identity or more with the amino acid sequence of SEQ ID NO: 1. In more specific embodiments of this type, Canine Influenza H3N2 HA protein comprises an amino acid sequence comprising 96% identity or more with the amino acid sequence of SEQ ID NO: 1. In more specific embodiments of this type, Canine Influenza H3N2 HA protein comprises an amino acid sequence comprising 97% identity or more with the amino acid sequence of SEQ ID NO: 1. In more specific embodiments of this type, Canine Influenza H3N2 HA protein comprises an amino acid sequence comprising 98% identity or more with the amino acid sequence of SEQ ID NO: 1. In more specific embodiments of this type, Canine Influenza H3N2 HA protein comprises an amino acid sequence comprising 99% identity or more with the amino acid sequence of SEQ ID NO: 1. In even more specific embodiments of this type, the Canine Influenza H3N2 HA protein comprises the amino acid sequence of SEQ ID NO: 1. In specific embodiments of this type the Canine Influenza H3N2 HA protein is encoded by the nucleotide sequence of SEQ ID NO: 2. In specific embodiments, the RP comprises a nucleotide sequence that encodes a Canine Influenza H3N2 HA protein, where the nucleotide sequence comprises the sequence of SEQ ID NO: 2 or SEQ ID NO: 3, or a nucleic acid sequence having at least 80%, or 85%, or 90% or 95% or 97%, or 98% or 99% sequence identity to SEQ ID NO: 2 or SEQ ID NO: 3. In some embodiments, of this type, the prevention or reduction of a disease for 6-months due to Canine Influenza H3N2 strain is a reduction in one or more of: reduced lung lesions, reduced incidence of pneumonia, reduced duration of coughing and reduced duration of CIV viral shedding of Canine Influenza H3N2. In some embodiments of this type, the prevention or reduction of a disease for 6-months due to Canine Influenza H3N2 strain is a reduction in the occurrence of lung lesions. In some embodiments of this type, the prevention or reduction of a disease for 6-months due to Canine Influenza H3N2 strain is a reduction in the incidence of pneumonia. In some embodiments of this type, the prevention or reduction of a disease for 6- months due to Canine Influenza H3N2 strain is a reduction in the duration of coughing. In some embodiments of this type, the prevention or reduction of a disease for 6-months due to Canine Influenza H3N2 strain is a reduction in the duration of CIV viral shedding of Canine InfluenzaH3N2 after Canine Influenza H3N2 exposure. In some embodiments of this type, the prevention or reduction of a disease for 6-months due to Canine Influenza H3N2 strain is a reduction in (i) occurrence of lung lesions, (ii) incidence of pneumonia, (iii) duration of coughing and (iv) duration of CIV viral shedding of Canine Influenza H3N2 after Canine Influenza H3N2 exposure.

[0058] In particular embodiments, an alphavirus RNA replicon particle of the present invention and / or embodiments thereof is used in the prevention of a disease due to Canine Influenza H3N2 strain for at least 12-months, comprising administering to a canine an immunogenic composition comprising a Canine Influenza H3N2 HA protein comprising an amino acid sequence comprising 95% identity or more with the amino acid sequence of SEQ ID NO: 1. In more specific embodiments of this type, Canine Influenza H3N2 HA protein comprises an amino acid sequence comprising 96% identity or more with the amino acid sequence of SEQ ID NO: 1. In more specific embodiments of this type, Canine Influenza H3N2 HA protein comprises an amino acid sequence comprising 97% identity or more with the amino acid sequence of SEQ ID NO: 1. In more specific embodiments of this type, Canine Influenza H3N2 HA protein comprises an amino acid sequence comprising 98% identity or more with the amino acid sequence of SEQ ID NO: 1. In more specific embodiments of this type, Canine Influenza H3N2 HA protein comprises an amino acid sequence comprising 99% identity or more with the amino acid sequence of SEQ ID NO: 1. In even more specific embodiments of this type, the Canine Influenza H3N2 HA protein comprises the amino acid sequence of SEQ ID NO: 1. In specific embodiments of this type the Canine Influenza H3N2 HA protein is encoded by the nucleotide sequence of SEQ ID NO: 2. In specific embodiments, the RP comprises a nucleotide sequence that encodes a Canine Influenza H3N2 HA protein, where the nucleotide sequence comprises the sequence of SEQ ID NO: 2 or SEQ ID NO: 3, or a nucleic acid sequence having at least 80%, or 85%, or 90% or 95% or 97%, or 98% or 99% sequence identity to SEQ ID NO: 2 or SEQ ID NO: 3. In some embodiments, of this type, the prevention or reduction of a disease for 12-months due to Canine Influenza H3N2 strain is a reduction in one or more of: reduced lung lesions, reduced incidence of pneumonia, reduced duration of coughing and reduced duration of CIV viral shedding of Canine Influenza H3N2. In some embodiments of this type, the prevention or reduction of a disease for 12-months due to Canine Influenza H3N2 strain is a reduction in the incidence of pneumonia. In some embodiments of this type, the prevention or reduction of a disease for 12-months due to Canine Influenza H3N2 strain is a reduction in the duration of coughing. In some embodiments of this type, the prevention or reduction of a disease for 12- months due to Canine Influenza H3N2 strain is a reduction in the duration of CIV viral shedding of Canine Influenza H3N2 after Canine Influenza H3N2 exposure. In some embodiments of this type, the prevention or reduction of a disease for 12-months due to Canine Influenza H3N2 strain is a reduction in (i) occurrence of lung lesions, (ii) incidence of pneumonia, (iii) duration ofcoughing and (iv) duration of CIV viral shedding of Canine Influenza H3N2 after Canine Influenza H3N2 exposure.

[0059] In particular embodiment relate to a method to prevent a disease, or reduce a disease, or reduce a symptom due to Canine Influenza H3N2 strain for at least 6-months, or at least 12- months comprising administering to a canine an immunogenic composition comprising a Canine Influenza H3N2 HA protein comprising an amino acid sequence comprising 95% identity or more with the amino acid sequence of SEQ ID NO: 1. In more specific embodiments of this type, Canine Influenza H3N2 HA protein comprises an amino acid sequence comprising 96% identity or more with the amino acid sequence of SEQ ID NO: 1. In more specific embodiments of this type, Canine Influenza H3N2 HA protein comprises an amino acid sequence comprising 97% identity or more with the amino acid sequence of SEQ ID NO: 1. In more specific embodiments of this type, Canine Influenza H3N2 HA protein comprises an amino acid sequence comprising 98% identity or more with the amino acid sequence of SEQ ID NO: 1. In more specific embodiments of this type, Canine Influenza H3N2 HA protein comprises an amino acid sequence comprising 99% identity or more with the amino acid sequence of SEQ ID NO: 1. In even more specific embodiments of this type, the Canine Influenza H3N2 HA protein comprises the amino acid sequence of SEQ ID NO: 1. In specific embodiments of this type the Canine Influenza H3N2 HA protein is encoded by the nucleotide sequence of SEQ ID NO: 2. In specific embodiments, the RP comprises a nucleotide sequence that encodes a Canine Influenza H3N2 HA protein, where the nucleotide sequence comprises the sequence of SEQ ID NO: 2 or SEQ ID NO: 3, or a nucleic acid sequence having at least 80%, or 85%, or 90% or 95% or 97%, or 98% or 99% sequence identity to SEQ ID NO: 2 or SEQ ID NO: 3.

[0060] In some embodiments, of this type, the prevention or reduction of a disease due to Canine Influenza H3N2 strain is a reduction in one, or 2 or 3 or all four of: reduced lung lesions, reduced incidence of pneumonia, reduced duration of coughing and reduced duration of CIV viral shedding. Multivalent Vaccines:

[0061] The present invention also provides multivalent vaccines. For example, the coding sequence of a protein antigen or antigenic fragment thereof, or combination of such coding sequences of protein antigens useful in a canine vaccine can be added to an alphavirus RNA replicon particle (RP) or combined in the same RP as one that encodes a canine antigen of the CIV [e.g., the CIV HA protein] in the vaccine.

[0062] Examples of pathogens that one or more of such protein antigens can originate from include canine distemper virus, canine adenovirus type 2, canine parvovirus type, canineparainfluenza virus, canine coronavirus, and / or canine pneumovirus, Leptospira spp. and / or a Bordetella bronchiseptica. In particular embodiments, a coding sequence for a antigenic protein or analogous protein from one or more of these canine or canine pathogens can be inserted into the same RP as the CIV antigen. Alternatively, or in combination therewith, a coding sequence for an antigenic protein or analogous protein from one or more of these canine or canine pathogens can be inserted into one or more other RPs, which can be combined in a vaccine with an RP that encodes the CIV H3N2 HA protein or an antigenic fragment.

[0063] In addition, an alphavirus RNA replicon particle (RP) that encodes one or more antigens of the CIV [e.g., the H3N2 HA protein or an antigenic fragment thereof] can be added together with one or more other live, attenuated, or killed pathogen isolates, e.g., canine distemper virus, canine adenovirus type 2, canine parvovirus type, canine parainfluenza virus, canine coronavirus, and / or canine pneumovirus, Leptospira spp. and / or a Bordetella bronchiseptica.

[0064] In some embodiments, the immunogenic compositions comprise alphavirus RNA replicon particles that encode one or more antigens of CIV (e.g., the H3N2 HA protein or an antigenic fragment thereof) and a modified live canine pathogen. The immunogenic compositions and vaccines of the present invention can comprise a modified live canine parainfluenza virus, with or without an avirulent live B. bronchiseptica, and optionally with one or more additional immunogens. In certain embodiments, immunogenic compositions and vaccines can further comprise a live attenuated canine influenza virus. In other embodiments, the immunogenic compositions or vaccine as disclosed herein further comprises a live attenuated canine parvovirus. In still other embodiments, the immunogenic compositions or vaccine as disclosed herein further comprises a live attenuated canine distemper virus. In yet other embodiments, the immunogenic compositions or vaccine as disclosed herein further comprises a live attenuated canine adenovirus type 2. In still other embodiments, the immunogenic compositions or vaccine as disclosed herein further comprises a live attenuated respiratory canine coronavirus. In yet other embodiments, the immunogenic compositions or vaccine as disclosed herein further comprises a live attenuated canine pneumovirus. In still other embodiments, the immunogenic compositions or vaccine as disclosed herein further comprises a viral vector encoding one or more protein immunogens from a canine influenza virus (CIV), canine parvovirus, canine distemper virus, canine adenovirus (Type 1 or 2), a respiratory canine coronavirus, canine pneumovirus, Steptococcus equi zooepidemicus and / or Mycoplasma cynos. In yet other embodiments, the immunogenic compositions or vaccine as disclosed herein further comprises a live attenuated Steptococcus equi zooepidemicus. In still other embodiments, the vaccine further comprises a live attenuated Mycoplasma cynos. In addition, immunogenic compositions or vaccines as disclosed herein that comprise either amodified live canine parainfluenza virus or a modified live canine parainfluenza virus with an avirulent live B. bronchiseptica can further comprise two or more of these other immunogens. The immunogenic compositions or vaccine as disclosed herein of the present invention may be adjuvanted or non adjuvanted.

[0065] In other embodiments, the methods of aiding in the protection of (and / or providing effective protection to) a canine from CIV, e.g., canine influenza H3N2 strain, comprising administering a vaccine that comprises a RP encoding a canine influenza H3N2 HA protein and / or antigenic fragment thereof, as disclosed herein. In some embodiments of the methods, the vaccine can optionally comprise one or more additional immunogens. In certain embodiments of the methods, the vaccine further comprises a second RP encoding a non-CIV antigen. In certain embodiments of the methods, the vaccine further comprises a second RP encoding a CIV antigen that is not from the H3N2 strain of CIV. In other embodiments of the methods, the vaccine further comprises a live attenuated canine parvovirus. In still other embodiments of the methods, the vaccine further comprises a live attenuated canine distemper virus. In yet other embodiments of the methods, the vaccine further comprises a live attenuated canine adenovirus type 2. In still other embodiments of the methods, the vaccine further comprises a live attenuated respiratory canine coronavirus. In yet other embodiments of the methods, the vaccine further comprises a live attenuated canine pneumovirus. In still other embodiments of the methods, the vaccine further comprises one or more viral vectors encoding one or more protein immunogens from a canine influenza virus, canine parvovirus, canine distemper virus, canine adenovirus (Type 1 or 2), respiratory canine coronavirus, canine pneumovirus, Steptococcus equi zooepidemicus and / or Mycoplasma cynos. In still other embodiments of the methods, the vaccine further comprises a live attenuated Steptococcus equi zooepidemicus. In yet other embodiments of the methods, the vaccine further comprises a live attenuated Mycoplasma cynos. In addition, the methods of aiding in the protection of (and / or providing effective protection to) a canine from upper respiratory diseases and infectious tracheobronchitis can comprise orally administering a vaccine that comprises a modified live canine parainfluenza virus in combination with two or more of these immunogens.

[0066] In related embodiments, the method of aiding in the protection of (and / or providing effective protection to) a canine from CIV, e.g., canine influenza H3N2 strain, comprising administering to the canine a vaccine that comprises a RP encoding a canine influenza H3N2 HA protein and / or antigenic fragment thereof, and one or more additional live attenuated immunogens. In other embodiments of the methods, the vaccine further comprises a live attenuated canine parvovirus. In still other embodiments of the methods, the vaccine further comprises a live attenuated canine distemper virus. In yet other embodiments of the methods, the vaccine further comprises a live attenuated canine adenovirus type 2. In still other embodiments of the methods, the vaccine further comprises a live attenuated respiratory caninecoronavirus. In yet other embodiments of the methods, the vaccine further comprises a live attenuated canine pneumovirus. In still other embodiments of the methods, a vaccine that comprises a RP encoding a canine influenza H3N2 HA protein and / or antigenic fragment thereof, can comprise a vector that expresses an antigen from a canine influenza virus, canine parvovirus, canine distemper virus, canine adenovirus (Type 1 or 2), respiratory canine coronavirus, canine pneumovirus, Steptococcus equi zooepidemicus and / or Mycoplasma cynos. In still other embodiments of the methods, the vaccine further comprises a live attenuated Steptococcus equi zooepidemicus. In yet other embodiments of the methods, the vaccine further comprises a live attenuated Mycoplasma cynos. In addition, the methods of aiding in the protection of (and / or providing effective protection to) a canine from a canine from CIV, e.g., canine influenza H3N2 strain, comprising administering to the canine a vaccine that comprises a RP encoding a canine influenza H3N2 HA protein can further comprise a modified live canine parainfluenza virus and an avirulent B. bronchiseptica in combination with two or more of these immunogens.

[0067] In specific embodiments, the method of aiding in the protection of (and / or providing effective protection to) a canine from CIV, e.g., canine influenza H3N2 strain, comprising administering to the canine a vaccine that comprises a RP encoding a canine influenza H3N2 HA protein and / or an antigenic fragment thereof, can further comprise a modified live canine parainfluenza virus, a live attenuated canine parvovirus, a live attenuated canine distemper virus, a live attenuated canine adenovirus type 2, and an avirulent live B. bronchiseptica.

[0068] In certain embodiments, a method of aiding in the protection of (and / or providing effective protection to) a canine from CIV, e.g., canine influenza H3N2 strain, comprising administering to the canine a vaccine that comprises a RP encoding a canine influenza H3N2 HA protein and / or an antigenic fragment thereof of the present invention can comprise orally administering to the canine a single-dose vaccine. Accordingly, in specific embodiments, the method comprises administering to the canine a single-dose vaccine comprising a RP encoding a canine influenza H3N2 HA protein and / or an antigenic fragment thereof. In related embodiments, the method comprises administering to the canine a single-dose vaccine comprising a RP encoding a canine influenza H3N2 HA protein and / or an antigenic fragment thereof, and one or more of canine parainfluenza virus and an avirulent live B. bronchiseptica.

[0069] In alternative embodiments, a method of aiding in the protection of (and / or providing effective protection to) from CIV, e.g., canine influenza H3N2 strain, comprising administering to the canine a vaccine that comprises a RP encoding a canine influenza H3N2 HA protein and / or an antigenic fragment thereof of the present invention can comprise orally administering to the canine two or more doses of the vaccine. Accordingly, in specific embodiments, the methodcomprises orally administering to the canine two or more doses of a vaccine that comprises a RP encoding a canine influenza H3N2 HA protein and / or an antigenic fragment thereof.

[0070] In particular embodiments, the method of aiding in the protection of (and / or providing effective protection to) a canine from CIV, comprising administering to the canine a non-adjuvanted vaccine comprising a RP encoding a canine influenza H3N2 HA protein and / or an antigenic fragment thereof. Specific embodiments of these methods comprise administering to the canine a vaccine comprising a RP encoding a canine influenza H3N2 HA protein and / or an antigenic fragment thereof, in which the vaccine is a non-adjuvanted vaccine. More particular embodiments of these methods comprise administering to the canine a vaccine comprising a RP encoding a canine influenza H3N2 HA protein and / or an antigenic fragment thereof, and further comprising one or more additional antigens as disclosed herein, in which the vaccine is a non-adjuvanted vaccine. In particular embodiments of this type, the vaccines are administered as a single-dose vaccine.

[0071] Alternative embodiments of methods of aiding in the protection of (and / or providing effective protection to) from CIV, e.g., canine influenza H3N2 strain, comprising administering to the canine a vaccine that comprises a RP encoding a canine influenza H3N2 HA protein and / or an antigenic fragment thereof, where the vaccine is an adjuvanted vaccine. Particular embodiments of these methods comprise administering to the canine a vaccine comprising a RP encoding a canine influenza H3N2 HA protein and / or an antigenic fragment thereof, in which the vaccine comprises an adjuvant. Related embodiments of these methods administering to the canine a vaccine comprising both a a RP encoding a canine influenza H3N2 HA protein and / or an antigenic fragment thereof and an additional antigen, in which the vaccine comprises an adjuvant. In particular embodiments of this type, the vaccines are administered as a single-dose vaccine.

[0072] It also is to be understood that this invention is not limited to the particular configurations, process steps, and materials disclosed herein as such configurations, process steps, and materials may vary somewhat. It further is to be understood that the terminology employed herein is used for the purpose of describing particular embodiments only and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims and equivalents thereof.

[0073] All patents and other publications identified are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based onthe information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

[0074] Some embodiments of the technology described herein can be defined according to any of the following numbered paragraphs: 1. An alphavirus RNA replicon particle that encodes a Canine Influenza (CIV) H3N2 hemagglutinin (HA) antigen. 2. The alphavirus RNA replicon of paragraph 1, wherein the alphavirus RNA replicon particle is a Venezuelan Equine Encephalitis (VEE) alphavirus RNA replicon particle. 3. The alphavirus RNA replicon of paragraph 1 or 2, wherein the HA protein is the HA protein from a CIV 2015 Iowa strain (A / canine / Iowa / 22619-4 / 2015(H3N2). 4. The alphavirus RNA replicon of any of paragraphs 1-3, wherein the CIV HA protein comprises an amino acid sequence comprising at least 85%, or at least 90% or at least 95% identity with the amino acid sequence of SEQ ID NO: 1. 5. The alphavirus RNA replicon of any of paragraphs 1-4, wherein the CIV HA protein is encoded by a nucleic acid sequence comprising at least 85%, or at least 90% or at least 95% identity with the sequence of SEQ ID NO: 2 or SEQ ID NO: 3. 6. An immunogenic composition comprising an alphavirus RNA replicon of any of paragraphs 1-6. 7. The immunogenic composition of claim 6, wherein the composition does not include an adjuvant. 8. A vaccine to aid in the prevention of disease due to CIV in a canine comprising the immunogenic composition of any of paragraphs 6-7, and a pharmaceutically acceptable carrier. 9. The vaccine composition of paragraph 8, that further comprises at least one non-CIV antigen for eliciting protective immunity to a non-CIV canine pathogen. 10. The vaccine of paragraph 9, wherein the non-CIV canine pathogen is selected from the group consisting of canine distemper virus, canine adenovirus type 2, canine parvovirus type, canine parainfluenza virus, canine coronavirus, and / or canine pneumovirus, Leptospira spp. and / or a Bordetella bronchiseptica, and any combination thereof. 11. The vaccine of any of paragraphs 8-10, wherein the vaccine is a nonadjuvanted vaccine. 12. A method of immunizing a canine against a pathogenic CIV comprising administering to the canine an immunologically effective amount of the vaccine of any of paragraphs 8-11, or the alphavirus RNA replicon of any of paragraphs 1-5, or immunogenic composition of claims 6-7. 13. A method to prevent a disease due to Canine Influenza (CIV) H3N2 strain, or to reduce the severity of a disease due to Canine Influenza (CIV) H3N2 strain, comprising administering to a canine the immunogenic composition of paragraph 6 or 7, or the vaccine of any of claims 8-11, or the alphavirus RNA replicon of any of paragraphs 1-5.14. A method to induce an immune response to Canine Influenza (CIV) H3N2 strain comprising administering to a canine the immunogenic composition of paragraph 6 or 7, or the vaccine of any of claims 8-11, or the alphavirus RNA replicon of any of paragraphs 1-5. 15. The method of any of paragraphs 12-14, wherein the vaccine or immunogenic composition further comprises at least one non-CIV antigen for eliciting protective immunity to a non-CIV canine pathogen. 16. The method of paragraph 15, wherein the non-CIV canine pathogen is selected from the group consisting of canine distemper virus, canine adenovirus type 2, canine parvovirus type, canine parainfluenza virus, canine coronavirus, and / or canine pneumovirus, Leptospira spp. and / or a Bordetella bronchiseptica, and any combination thereof. 17. The method of any of paragraphs 12-16, wherein the vaccine is a nonadjuvanted vaccine. 18. The method of any of paragraphs 12-17, wherein the method prevents or reduces the severity of a disease due to Canine Influenza (CIV) H3N2 strain for at least 6-months after administration of the vaccine. 19. The method of any of paragraphs 12-18, wherein the method prevents, or reduces the severity of a disease due to Canine Influenza (CIV) H3N2 strain for at least 12-months after the administration of the vaccine. 20. The method of any of paragraphs 12-19, wherein the method prevents or reduces, any one or more of: (i) occurrence of lung lesions (ii) incidence of pneumonia, (iii) duration of coughing and (iv) duration of CIV viral shedding of Canine Influenza H3N2 virus after Canine Influenza H3N2 exposure. 21. The vaccine of any of paragraphs 8-11, wherein the vaccine aid in the prevention of disease due to CIV in a canine for at least 6-months. 22. The vaccine of any of paragraphs 8-11, wherein the vaccine aid in the prevention of disease due to CIV in a canine for at least 12-months. 23. The vaccine composition of paragraph 8-11, 21 or 22, wherein the vaccine prevents or reduces, any one or more of: (i) occurrence of lung lesions (ii) incidence of pneumonia, (iii) duration of coughing and (iv) duration of CIV viral shedding of Canine Influenza H3N2 virus after Canine Influenza H3N2 exposure. 24. The vaccine composition of paragraph 8-11, 21 or 22, wherein the vaccine prevents or reduces (i) the occurrence of lung lesions (ii) the incidence of pneumonia, (iii) the duration of coughing and (iv) the duration of CIV viral shedding of Canine Influenza H3N2 virus after Canine Influenza H3N2 exposure.

[0075] Table 1: SEQUENCE TABLESEQ ID NO: Description Type 1 Canine Influenza H3N2 HA amino acid 2 Canine Influenza H3N2 HA codon optimised nucleic acid DNA 3 Canine Influenza H3N2 HA nucleic acid A / canine / Iowa / 22619-4 / 2015(H3N2)_HA_wild- type DNA SEQUENCES Canine Influenza H3N2 HA SEQ ID NO: 1 MKTVIALSYIFCLAFGQNLLGNENNAATLCLGHHAVPNGTMVKTITDDQIEVTNATELVQNSSTGKICNNP HKILDGRDCTLIDALLGDPHCDVFQNETWDLFVERSNAFSNCYPYDVPDYASLRSIVASSGTLEFITEGFT WAGVTQNGGSGACKRGPANSFFSRLNWLTKSGNTYPVLNVTMPNNNNFDKLYIWGVHHPSTNQEQTSLYIQ ASGRVTVSTRRSQQTIIPNIGSRPLVRGQSGRISVYWTIVKPGDILVINSNGNLIAPRGYFKMHIGKSSIM RSDAPIDTCISECITPNGSIPNEKPFQNVNKITYGACPKYVKQNTLKLATGMRNVPERQTRGLFGAIAGFI ENGWEGMVDGWYGFRHQNSEGTGQAADLKSTQAAIDQINGKLNRVIEKTNEKFHQIEKEFSEVEGRIQDLE RYVEDTKVDLWSYNAELLVALENQNTIDLTDSEMNKLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESI RNGTYDHNIYRDEAVNNRFQIKGVELKSGYKDWILWISFAISCFLLCVVLLGFIMWACQRGNIRCNICI Canine Influenza H3N2 HA SEQ ID NO: 2 atgaaaactgtcattgctctttcctatatcttctgcctcgcatttggtcagaatttgcttggaaacgaaaa caatgctgcaacactttgcctgggacatcatgcagtgcctaacggtactatggtgaaaacaatcactgacg atcaaattgaggtgaccaacgccaccgaacttgtccaaaactcctcaacagggaaaatctgcaacaatccc cacaagattcttgatggaagggactgtactcttattgatgcccttctcggggaccctcactgtgacgtgtt ccaaaatgagacttgggacctttttgtggagagatccaatgcttttagcaattgttacccttatgatgtcc cggactatgcttctctccggtctattgtcgcttcatcaggaacacttgagttcatcacagaaggtttcact tgggcaggagtcacacaaaatggaggaagcggagcttgtaaaagaggtcctgctaactcattcttctctcg gttgaattggttgacaaaatctggtaatacttatccagtgttgaatgtgactatgcctaacaacaacaatt tcgacaaactttacatttggggagtccatcacccgagtacaaatcaagaacaaaccagcctgtatatccag gcctcgggtcgggtcactgtgtcgaccagaaggagccaacagaccattatcccgaacatcggttcaagacc ccttgtgcggggccaatctggcagaatttctgtctattggactattgtcaaaccgggagacattctggtca ttaactcaaatggtaatcttatcgcaccacggggatacttcaaaatgcacattgggaaaagctcgattatg cggtcagatgctccaattgacacttgcatttctgaatgtatcaccccaaacgggagcatccccaatgagaa gcccttccaaaatgtgaataagatcacatacggtgcatgtcccaagtatgtcaagcaaaacacccttaaactggcaactggaatgcggaatgtgccggaacggcaaaccagagggctgttcggagcaattgcagggttcatc gaaaatggatgggaaggaatggtggacggttggtatggcttccggcatcaaaactcagagggaactggtca agctgctgacctcaagtctacacaggctgccattgaccagattaacggaaaactcaatagggtgattgaaa agactaacgaaaagttccatcaaattgagaaggagttttcagaggtcgaggggaggattcaagaccttgag aggtacgtcgaggacactaaagtggatttgtggtcatacaatgccgaacttttggtcgccttggagaacca gaacactattgatcttactgattcagagatgaacaagctttttgagaaaactagaaggcaacttagggaaa atgcagaggacatggggaatggttgcttcaagatctaccacaagtgtgacaatgcttgcattgaatcaatt cggaacggtacttatgaccataacatctaccgggatgaggcagtgaacaataggttccagatcaaaggtgt cgaattgaagtcaggatacaaagactggatcctttggatttcctttgccatttcatgctttttgttgtgtg tcgtccttctgggattcatcatgtgggcctgccagagggggaatatccggtgcaacatttgcatt Canine Influenza H3N2 HA SEQ ID NO: 3 atgaaaactgttattgctttaagctatattttctgcctggcttttggtcagaatcttctaggaaatgaaaa taatgctgcaacactatgcctgggacatcatgcagtgccgaacgggacaatggtgaaaactatcacagacg atcaaattgaggtgaccaacgccaccgagctagtccaaaactcctcaacagggaaaatatgcaacaatccc cacaagattcttgatgggagggactgcacactaatagatgccctactaggggacccacactgtgacgtctt ccaaaatgagacatgggacctttttgtggaacgaagcaatgcttttagcaattgttacccttatgatgtac cagactatgcatccctccgatccatagttgcatcatcaggcacattggagttcatcactgaaggtttcact tgggcaggagtaactcaaaatggaggaagcggtgcttgtaaaaggggacctgctaatagtttcttcagtag attaaattggttaactaaatcaggaaatacatatccagtgttgaatgtgactatgccaaacaacaacaatt tcgacaaattatacatttggggagttcatcacccaagcactaatcaagaacaaaccagcctgtatattcag gcctcaggaagagtcacagtctctaccaggagaagccaacagaccataatcccaaacattggatctagacc cttggtaaggggccaatctggcagaataagcgtatattggacaatagtcaaacctggagacatactggtaa taaacagtaatggaaacctaatcgctcctcgaggatacttcaaaatgcacattgggaaaagctcaataatg agatcagatgcacctattgacacctgcatttccgaatgtatcaccccgaacgggagcatccccaatgaaaa gcccttccaaaatgtaaacaagatcacatacggagcatgtcccaaatatgttaagcaaaacaccttgaaac tggcaacaggaatgcggaatgtccctgagaggcaaaccagaggcctgttcggcgcaatagcaggcttcata gaaaatggatgggaagggatggtagacggttggtatggcttcaggcaccaaaattccgaaggtacaggaca agcagcagaccttaaaagcactcaggcagccattgaccagattaatgggaaattgaacagagtgattgaaa aaacgaatgagaagttccatcaaattgaaaaggagttttccgaagtagaagggaggattcaagaccttgag agatacgttgaagacacaaaagtagatctttggtcttacaatgccgagcttcttgttgccttagaaaacca gaacacaattgatttaactgattcagaaatgaacaaattgtttgaaaagactaggaggcaattgagggaaa atgctgaagacatgggcaatggctgcttcaagatataccacaagtgtgacaatgcttgcatagaatcgatt agaaacggaacttatgaccataacatatatagagatgaggcagtgaacaatcggttccagatcaaaggtgt tgagctaaagtctggatacaaagactggatcttgtggatttcctttgccatatcatgctttttgctttgtg ttgtcttgctgggtttcattatgtgggcctgccagagaggcaacattaggtgcaacatttgcatt

[0076] The following examples serve to provide further appreciation of the invention but are not meant in any way to restrict the effective scope of the invention. EXAMPLES EXAMPLE 1 INCORPORATION OF THE CODING SEQUENCES FOR CIV HA PROTEINS INTO THE ALPHAVIRUS RNA REPLICON PARTICLES

[0077] RNA viruses have been used as vector-vehicles for introducing vaccine antigens, which have been genetically engineered into their genomes. However, their use to date has been limited primarily to incorporating viral antigens into the RNA virus and then introducing the virus into a recipient host. The result is the induction of protective antibodies against the incorporated viral antigens. Alphavirus RNA replicon particles have been used to encode pathogenic antigens. Such alphavirus replicon platforms have been developed from several different alphaviruses, including Venezuelan equine encephalitis virus (VEE) [Pushko et al., Virology 239:389-401 (1997)], Sindbis (SIN) [Bredenbeek et al., Journal of Virology 67:6439-6446 (1993) the contents of which are hereby incorporated herein in their entireties], and Semliki Forest virus (SFV) [Liljestrom and Garoff, Biotechnology (NY) 9:1356- 1361 (1991), the contents of which are hereby incorporated herein in their entireties]. Moreover, alphavirus RNA replicon particles are the basis for several USDA-licensed vaccines for swine and poultry. These include: Porcine Epidemic Diarrhea Vaccine, RNA Particle (Product Code 19U5.P1 ), Swine Influenza Vaccine, RNA (Product Code 19A5.D0), Avian Influenza Vaccine, RNA (Product Code 19O5.D0), and Prescription Product, RNA Particle (Product Code 9PP0.00). ALPHAVIRUS RNA REPLICON PARTICLE CONSTRUCTION

[0078] Amino acid sequences for CIV HA proteins were used to generate codon-optimized (canine codon usage) nucleotide sequences in silico. Optimized sequences were prepared as synthetic DNA by a commercial vendor (ATUM, Newark, CA). Accordingly, synthetic genes were designed based on the amino acid sequences of a 2015 Iowa strain (A / canine / Iowa / 22619- 4 / 2015(H3N2) HA protein, The propagation-defective replicon RNA particles contain alphavirus replicon RNA expressing the Canine Influenza H3N2 hemagglutinin (HA) gene, packaged with the capsid protein and glycoproteins of the avirulent TC-83 strain of VEEV. The wild-type CanineInfluenza H3N2 HA sequence was obtained from a 2015 Iowa strain (A / canine / Iowa / 22619- 4 / 2015(H3N2), and a synthetic, codon-optimized gene for the HA open reading frame and flanking restriction sites was prepared by a supplier (ATUM, Newark, CA). The synthetic gene was cloned into the DNA plasmid vector, pVHV, which is derived from the avirulent human vaccine strain of VEEV (strain TC-83). Batches of experimental RPs were prepared as previously described [Langereis, M.A.; et al. Vaccines 2021, 6]. Briefly, Vero cells were electroporated with individual replicon RNA and promoterless helper RNAs encoding the VEEV strain TC-83 glycoproteins and capsid. The RPs were combined with stabilizer [NZ Amine, sucrose, and Dulbecco’s Modified Eagles Medium (DMEM)], lyophilized, and reconstituted with 0.5 mL vaccine diluent prior to use. The placebo vaccine used in the efficacy study consisted of all components of the test vaccine except the RP-CIV H3N2 antigen.

[0079] The VEE replicon vectors designed to express CIV HA proteins were constructed as previously described [see, U.S.9,441,247 B2; the contents of which are hereby incorporated herein by reference], with the following modifications. The TC-83-derived replicon vector “pVEK” [disclosed and described in U.S.9,441,247 B2] was digested with restriction enzymes AscI and PacI.

[0080] Production of TC-83 RNA replicon particles (RP) was conducted according to methods previously described [U.S.9,441,247 B2 and U.S.8,460,913 B2; the contents of which are hereby incorporated herein by reference in their entireties]. Briefly, pVHV replicon vector DNA and helper DNA plasmids were linearized with NotI restriction enzyme prior to in vitro transcription using MegaScript T7 RNA polymerase and cap analog (Promega, Madison, WI). Importantly, the helper RNAs used in the production lack the VEE subgenomic promoter sequence, as previously described [Kamrud et al., J Gen Virol.91(Pt 7):1723-1727 (2010)]. Purified RNA for the replicon and helper components were combined and mixed with a suspension of Vero cells, electroporated in 4 mm cuvettes, and returned to OptiPro®SFM cell culture media (Thermo Fisher, Waltham MA). Following overnight incubation, alphavirus RNA replicon particles were purified from the cells and media by passing the suspension through a ZetaPlus BioCap depth filter (3M, Maplewood, MN), washing with phosphate buffered saline containing 5% sucrose (w / v), and finally eluting the retained RP with 400 mM NaCl buffer. Eluted RP were formulated to a final 5% sucrose (w / v), passed through a 0.22 micron membrane filter, and dispensed into aliquots for storage. Titer of functional RP was determined by immunofluorescence assay on infected Vero cell monolayers. EXAMPLE 2 EVALUATION OF EFFICACY AND SAFETY OF ACIV H3N2 VACCINE IN DOGS Challenge Material

[0081] The heterologous canine influenza H3N2 challenge strain was originally isolated in eggs from a field sample collected in 2017 from a dog in Louisville, Kentucky suffering from canine respiratory disease. On the day of challenge, frozen challenge virus was thawed and diluted in sterile, cold DMEM. Efficacy Study Design

[0082] Conventional beagles, 7-8 weeks old, were housed in an isolation facility in barrier rooms and randomized into two treatment groups using litter size and housing units as randomization factors. All dogs were vaccinated subcutaneously twice, 21 days apart (study days 0 and 21), with a 0.5 mL dose of RP-CIV H3N2 vaccine (n = 20) at minimum protective dose or a 0.5 mL dose of a placebo vaccine that consisted of all components of the test vaccine except the RP-CIV H3N2 antigen (n = 20). Rectal temperatures were recorded on the day prior to each vaccination, on the day of each vaccination prior to administration, and for 2 days following each vaccination. In addition, all dogs were monitored for injection site reactions starting at 2-4 hours post-vaccination and for 2 days post-vaccination. Injection sites were assessed for visibility, size, and description including thickening, soft (edema), hard, tender, visible, or scratching at the injection site. Whole blood for serum was collected on the day prior to each vaccination (study days -1 and 20) and then on study days 28, 33, and 40 (day before challenge). Nasal swabs were also collected on study day 40.

[0083] Three weeks following the second vaccination, 19 vaccinates and 20 placebo- vaccinated control dogs were challenged by the intranasal route with virulent CIV H3N2. One vaccinate had to be humanely euthanized prior to challenge due to welfare concerns following a blood collection-associated adverse event. During the 10-day post-challenge observation phase of the study, nasal swabs were collected daily, and clinical signs including spontaneous coughing, retching, sneezing, nasal discharge, dyspnea, and fever (rectal temperature ≥ 103.5°F) were monitored daily. Dogs were humanely euthanized on day 10 post-challenge, and the lungs were immediately evaluated for consolidation. Each lobe of the right (cranial, middle, caudal, and accessory lobes) and the left (cranial-cranial, cranial-caudal, and caudal lobes) lungs were scored individually for percent consolidation by a veterinarian blinded to the treatment group designation of each dog. The percent consolidation of each lung lobe was then converted into a weighted score, and a total score for each dog was calculated

[0023] . The weighted lung lesion score was based on the size of each lobe; right cranial (x 0.152), right middle (x 0.1), right caudal (x 0.248), accessory lobes (x 0.09), left cranial-cranial (x 0.091), leftcranial-caudal (x 0.06), and left caudal (x 0.259). Lung swabs and lung tissue were also collected from each dog. Immunofluorescence Assay for Vaccine Titer

[0084] The titer of the RP-CIV H3N2 vaccine was determined by serially diluting the vaccine which was added to a Vero cell monolayer culture in 48-well plates (Greiner; Monroe, NC) and incubated at 37±1°C in 4-6% CO2for 18-22 hours. After incubation, the cells were fixed and stained with the primary antibody specific for CIV H3N2 HA followed by an Alexa Fluor® 488 conjugated secondary antibody (Invitrogen; Carlsbad, CA). RPs were quantified by counting positive, fluorescent stained cells. The titer was calculated from the known dilution and inoculation volume. Hemagglutination Inhibition Assay for Serology

[0085] Whole blood collected into serum separation tubes (BD; Franklin Lakes, NJ) was allowed to clot for a minimum of 2 hours at 15-30°C, then was centrifuged at 1000 x g to separate the serum. Serum samples were tested in a hemagglutination inhibition assay (HAI) to evaluate the serological response post-vaccination. Briefly, CIV antigen was added to 1:2 dilutions of the test serum. Following incubation, a 0.5% rooster red blood cell suspension was added to the serum / virus mixtures. After another incubation, the results were read, and the titer was recorded as the reciprocal of the highest dilution of serum showing hemagglutination inhibition. CIV Titration for Viral Shedding

[0086] Polyester fiber tipped swabs (Fisherbrand; Pittsburgh, PA) were wetted in transport media [DMEM (Corning; Corning, NY), gentamicin (Gibco; Grand Island, NY), and amphotericin- B (Corning; Corning, NY)] and inserted into each nostril of the dog. In the lab, swabs were removed from the transport media, and the liquid contents centrifuged at 500 x g. The supernatant was used to inoculate a confluent monolayer of canine kidney cells that were planted on 96-well tissue culture plates (Falcon; Corning, NY). Plates were incubated at 36±2°C in 4-6% CO2for 7 days, and monolayers were then observed for cytopathic effect. The virus titer was calculated by the Spearman-Karber method. Lung Swab Testing

[0087] Polyester fiber tipped swabs (Fisherbrand; Pittsburgh, PA) were used to swab the interior of the lung tissue and placed into collection media. One swab was placed into supplemented media containing tryptose phosphate broth (BD; Franklin Lakes, NJ) and glycerol (Fisher; Hampton, NH), which was tested for Bordetella bronchiseptica (B. bronchiseptica) and Streptococcus equi subspecies zooepidemicus (S. equi subsp. zooepidemicus). For B.bronchiseptica, the lung swab material was streaked onto MacConkey agar plates (Thermo Scientific; Waltham, MA), and the plates incubated at 36±2°C for 48-96 hours. For S. equi subsp. zooepidemicus, the lung swab material was streaked onto anaerobic reducible blood agar with colistin and nalidixic acid agar plates (Thermo Scientific; Waltham, MA), and the plates incubated at 36±2°C for 24-48 hours. The second swab was placed into supplemented media containing trypticase soy broth (BD; Franklin Lakes, NJ), glycerol (Fisher; Hampton, NH), and carbenicillin (Teknova; Hollister, CA), which was tested for Mycoplasma by streaking the lung swab material onto PPLO agar plates (Thermo Scientific; Waltham, MA) and incubating at 36±2°C in 4-6% CO2for 21 days. The third swab was placed into supplemented media containing Dulbecco’s Modified Eagles Medium (Corning; Corning, NY), gentamicin (Gibco; Grand Island, NY), and amphotericin-B (Corning; Corning, NY), which was tested for canine parainfluenza (CPI) virus. Dilutions of lung swab material were inoculated onto dog kidney cells, and after 4-6 days, monolayers were fixed and stained with fluorescein-conjugated CPI antiserum (VMRD; Pullman, WA). Titers were calculated by the Spearman-Karber method. Histopathology of Lung Tissue Fresh lung tissue was collected after lung scoring and placed into 10% buffered formalin (Fisherbrand; Pittsburgh, PA) for histopathology testing performed by the University of Nebraska-Lincoln Veterinary Diagnostic Center. The slides were read by a board- certified pathologist (American College of Veterinary Pathologists) who was blinded to the treatment group information. The lesion types were assigned a score, and the nature of the lesion was described as suppurative or not. Statistical Analysis

[0088] Statistical analysis was performed in R-3.5.3 and Microsoft® Excel® version Microsoft 365® to evaluate efficacy. The duration of cough, duration of shedding, and lung scores were compared between treatment groups by Wilcoxon Rank Sum Test. The proportion of affected dogs with pneumonia in the two groups was calculated and compared using Fisher’s Exact Test. Statistical significance for serologic geometric means, clinical sign scores, and viral load were calculated and compared using a two-tailed Welch’s T-test. Significance was declared for two-sided p-values < 0.05. Field Safety Study Design

[0089] A total of 654 dogs (330 males; 324 females) were enrolled from 5 different geographic locations, and 644 dogs completed the study. The age of the dogs ranged from 8- weeks to 15-years, with 210 being purpose-bred beagles and 434 being client-owned dogs of various breeds. Two hundred seventeen dogs (217; 33%) were 8 weeks of age at the time ofvaccination. Owners were informed of the details of the study and signed a consent form prior to enrollment.

[0090] Each dog was vaccinated by the subcutaneous route with a 0.5 mL typical field dose vaccine on study day 0 and then another 0.5 mL dose was administered 3-4 weeks later for a total of 1301 doses administered. Owners were instructed to closely observe their dog(s) daily for 14 days following the first and second vaccinations and document any adverse local or systemic events in a diary. An adverse event was defined as any observation in the dogs, whether considered to be product related or not, that was unfavorable and unintended and occurred after the use of the test vaccine. A serious adverse event was defined as an adverse event which was fatal or life-threatening, resulted in significant disability, incapacity, or a congenital anomaly / birth defect, or resulted in permanent or prolonged signs or required professional intervention beyond routine prevention measures and common first aid. The relationship to the vaccine was evaluated as “related” or “not related” by a study veterinarian. Serology

[0091] Prior to vaccination, all dogs were sero-negative (HAI antibody titer < 10) to CIV H3N2, indicating they were susceptible to vaccination. Following the second vaccination, all vaccinated dogs sero-converted, with the highest HAI antibody titers occurring on study day 33, ranging from 10 to 320 with a geometric mean of 106. Meanwhile, the placebo- vaccinated control dogs remained sero-negative until challenge. On study days 28, 33, and 40, the geometric mean titer was significantly higher (p-value < 0.0001) in the vaccinate group compared to the placebo-vaccinated control group. (FIG.1). Post-Challenge Monitoring

[0092] Following challenge, fevers were sparse with only one placebo-vaccinated control dog having a fever (104.2°F and 103.6°F) on 2 separate days, and one vaccinated dog having a fever (103.5°F) on one day. Spontaneous cough, with or without retching, was the primary clinical sign for analysis, but other clinical signs of respiratory disease such as sneezing and serous nasal discharge were also observed. Of the 20 placebo-vaccinated control dogs, 18 (90%) coughed on multiple days and 1 (5%) coughed on 1 day. In contrast, only 1 (5%) of the 19 vaccinates coughed on multiple days and another 5 (26%) coughed on 1 day. In addition, the overall duration and severity of clinical signs were more apparent in the placebo-vaccinated control dogs compared to the vaccinated dogs. Duration of coughing (in days), from first to last occurrence, was determined for each dog. The median duration of coughing for the placebo- vaccinated control group was 6 days, which was significantly more than the 0 days for the vaccinated group (p-value <0.0001). Furthermore, a scoring system was implemented to evaluate clinical signs. Sneezing, serous nasal discharge, and spontaneous coughing were allassigned a score of 1, while spontaneous cough with retching was assigned a score of 2. For dogs with multiple clinical signs on a given day, the scores were added together. The mean clinical sign scores were significantly higher, and thus more severe, in the placebo-vaccinated control group on study days 4 (p-value = 0.001), 5, 6, 7 (p-value <0.0001), 8 (p-value = 0.006), 9 (p-value = 0.0005), and 10 (p-value = 0.0008) post-challenge compared to the vaccinated group (FIG.2). CIV Viral Shedding

[0093] Prior to challenge, dogs were not shedding any CIV, indicating they were susceptible to the challenge virus. After the CIV H3N2 challenge, the duration of viral shedding (in days), from first to last occurrence, was determined for each dog. Virus shedding peaked 1 day after challenge, and the median number of days during which virus was shed in the placebo- vaccinated control group was 6 days, which was significantly more than the 1 day in the vaccinated group (p-value <0.0001). By day 6 post-challenge, none (0%) of the 19 vaccinated dogs were shedding virus, compared to 18 (90%) of 20 placebo-vaccinated control dogs who were still shedding a mean viral load of 1.18 log10TCID50 / mL. The viral load was significantly higher in the placebo-vaccinated control dogs on days 2, 3, 4, 5, 6 (p-value <0.0001), and 7 (p- value = 0.02) post-challenge compared to the vaccinated dogs (Figure 3). Lung Consolidation and Histopathology

[0094] All dogs were necropsied 10 days post-challenge to examine the lungs and score for consolidation. The range of weighted lung scores for the placebo-vaccinated control dogs was 0 to 22.6, with a median of 7.2. In contrast, the range of weighted lung scores for the vaccinated dogs was 0 to 1.0, with a median of 0 (p-value <0.0001). Lung tissue was collected at the time of necropsy, preserved in formalin, and sent to the University of Nebraska-Lincoln for histopathology. Twelve (60%) of 20 dogs in the placebo-vaccinated control group exhibited varying degrees of suppurative pneumonia, which was significantly more than none of the dogs in the vaccinated group (p-value <0.0001). The degree of suppurative pneumonia was divided as either less than 50% (2 of 12 affected dogs) or greater than 50% (10 of 12 affected dogs) of the lung displaying severe consolidation. The lung consolidation and suppurative pneumonia were a direct result of CIV H3N2 infection, as the lung swabs collected during necropsy were negative for many of the CIRDC associated pathogens such as B. bronchiseptica, Mycoplasma, S. equi subsp. zooepidemicus, and CPI. Vaccine Safety

[0095] In the efficacy study, there were no injection site reactions or systemic adverse events following either vaccination. In addition, none of the dogs ran a fever following vaccination. In the 654-dog field safety trial (1,301 doses), there were 117 adverse events documented duringthe study, with 85 [occurring in 64 (9.8%) dogs] of them considered “not related” to the test vaccine and 32 [occurring in 24 (3.7%) dogs] considered to be “related” to the test vaccine. Lethargy was the most common adverse event attributed to the test vaccine at a rate of 1.6%. Other adverse events included diarrhea (0.3% of doses) and polydipsia (0.2% of doses). There were only two adverse events associated with the injection site including swelling (0.1% of doses) and pain (0.1% of doses), which resolved by the next day. Finally, there was one adverse event characterized as anaphylaxis that presented as lethargy and excessive salivation, which resolved promptly following treatment with diphenhydramine and dexamethasone. EXAMPLE 3

[0096] To determine the Duration of Immunity (DOI), a study was done to determine efficacy after 1 year. On study days 0 and 21, 7-to-8-week-old dogs were vaccinated with a 0.5 mL dose of Canine Influenza (H3) RNA-Particle vaccine (referred to herein interchangeably as RP-CIV H3N2 vaccine or RNA-P CIV H3) at minimum potency or placebo vaccine, according to the same procedure as in Example 2. One year after the second vaccination, dogs were challenged with a heterologous strain of Canine Influenza Virus (CIV) H3N2. Clinical observations and temperatures were recorded for 10 days post-challenge, and nasal swabs were collected for 10 consecutive days to determine viral shedding. At 10 days post-challenge, all dogs were euthanized, and the lungs were scored for consolidation.

[0097] The RP-CIV H3N2 vaccine was efficacious at 1-year post-vaccination since there was a significant reduction in the lung lesions of the dogs that received the test vaccine compared to the dogs that received the placebo vaccine. The Stratified Mitigated Fraction (stratified on challenge room and litter) was 0.64 with a 95% confidence interval of [0.29, 0.96] (Table 2).

[0098] Table 2. Total Lung Lesion Scores Treatment Group Median Total Lung Score Vaccinates (n = 20) 0* Placebo-vaccinated Controls (n = 20) 3.0 * p-value = 0.0005

[0099] This indicates that all dogs vaccinated with the test vaccine had significantly reduced lung lesions as compared to the placebo vaccinated control group.

[0100] In addition, there was a significantly higher number of dogs in the placebo-vaccinated control group that exhibited varying degrees of suppurative pneumonia than in the vaccinated group; 10 placebo-vaccinated control dogs compared to 3 vaccinates. The CoalescedPrevented Fraction Estimate was 0.76 with 95% confidence interval of [0.16, 0.93] (Table 3). Furthermore, the mean duration of coughing and mean duration of CIV viral shedding for the placebo-vaccinated control dogs was significantly more than the vaccinated dogs (Tables 4 and 5).

[0101] Table 3. Incidence of Pneumonia by Histopathology Treatment Group Median Total Lung Score Vaccinates (n = 20) 3* Placebo-vaccinated Controls (n = 20) 10 * p-value = 0.04

[0102] Table 4. Summary of Duration of Cough Treatment Group Median Number of Days Vaccinates (n = 20) 5* Placebo-vaccinated Controls (n = 20) 8 * p-value = 0.013

[0103] Table 5. CIV Viral Shedding Treatment Group Median Number Days of Viral Shedding Vaccinates (n = 20) 4* Placebo-vaccinated Controls (n = 20) 6 * p-value < 0.0001

[0104] The result in Example 3 demonstrate that one year after dogs were administered the RP-CIV H3N2 vaccine, when the dogs were challenged with a heterologous strain of Canine Influenza Virus (CIV) H3N2, there was a significant reduction in each one of: (i) the number of lung lesions, (ii) incidence of pneumonia, (iii) the duration of coughing and (iv) the duration of CIV viral shedding, in the RP-CIV H3N2 vaccine treated dogs as compared to the placebo controlled vaccinated dogs, demonstrating that the RP-CIV H3N2 vaccine was efficacious at 1- year post-vaccination. Discussion

[0105] A novel RNA Particle platform has been used to develop a new CIV H3N2 vaccine for dogs. The vaccine has been shown to be very safe and highly efficacious. The efficacy studywas valid, as all dogs were sero-negative prior to vaccination, the placebo-vaccinated control dogs remained sero-negative until challenge, all dogs were negative for CIV H3N2 shedding prior to challenge, and the virulent CIV H3N2 challenge material induced severe respiratory disease in the placebo-vaccinated control group. The complete data package demonstrates strong protection against CIV H3N2 challenge in dogs vaccinated with the non-adjuvanted RP- CIV H3N2 vaccine at minimum protective dose. The minimum protective dose establishes the lowest amount of RNA particles needed in the vaccine to provide protection against virulent challenge and serves as the baseline to ensure all commercially available vaccine is above this minimum threshold.

[0106] The RP-CIV H3N2 vaccine protected dogs by significantly reducing (i) the duration of virus shedding, (ii) the amount of virus shed, (iii) duration of coughing, (iv) severity of clinical signs, (iv) incidence of suppurative pneumonia, and (v) the development of lung consolidation. CIV H3N2 shedding and viral load were determined by quantifying the amount of virus in nasal swabs collected from dogs daily for 10 days post-challenge. The median duration of shedding (in days) was significantly lower in vaccinated dogs compared to the placebo-vaccinated control dogs. In addition, viral shedding from the vaccinates had completely subsided 2 days sooner than for the placebo-vaccinated controls. Furthermore, the amount of virus that was shed in the placebo-vaccinated control dogs was significantly higher over 6 days post-challenge than the vaccinated dogs. Therefore, vaccination with the RP-CIV H3N2 vaccine not only reduces the number of days that virus is shed, but it also reduces the amount of virus shed. This is important in reducing the risk of transmission from an infected dog to a naïve animal such as other pets or animals in close contact at a shelter or dog park.

[0107] The primary clinical sign of CIV H3N2 infection is coughing. In Example 2, 6 (32%) of the 19 vaccinated dogs coughed on just one or two occasions, while 19 (95%) of the 20 placebo-vaccinated control dogs persistently coughed anywhere from 1 to 8 days. The median duration of coughing was significantly lower in the vaccinate group (p-value <0.0001). Additionally, the severity of clinical signs in the placebo-vaccinated control group was significantly more than that of the vaccinate group. For example, only 1 (5%) of the vaccinated dogs retched once, while 18 (90%) placebo-vaccinated control dogs retched persistently. Furthermore, nasal discharge was only present in the placebo-vaccinated control group. Taken together, clinical signs of a CIV infection are significantly reduced in both duration and severity due to vaccination with the RP-CIV H3N2 vaccine even after a highly virulent CIV H3N2 challenge. Consequently, morbidity can be diminished or possibly even prevented using this vaccine.

[0108] Because CIV H3N2 causes a lower respiratory infection in dogs, lung consolidation is an important disease outcome and is the primary variable that the USDA uses when evaluatingCIV studies. Consolidation occurs when exudate from the infection fills the air spaces within the lung lobes, resulting in the clinical signs of coughing and retching. In addition, these infections can often result in pneumonia, which can be fatal. Pneumonia can put the welfare of an infected dog at great risk by increasing the severity of clinical signs as well as increasing the risk of coinfections from other viral and bacterial CIRDC pathogens. In the current study, the median weighted lung score of the vaccinate group was significantly lower than the placebo-vaccinated control (p-value <0.0001). Fourteen (74%) dogs that had been vaccinated with the RP-CIV H3N2 vaccine did not have any consolidation on any lung lobe, and the highest total lung score for the group was 1.0. In contrast, only 2 (10%) of the placebo-vaccinated control dogs had a lung score of 0, and the highest score for the group was 22.6. The higher lung scores in the placebo-vaccinated control group correlated to 60% of the dogs affected with suppurative pneumonia. Additional testing confirmed that the lung consolidation and clinical disease observed in this study were due to CIV H3N2 and no other CIRDC associated pathogens.

[0109] In summary, vaccination with the RP-CIV H3N2 vaccine at minimum protective dose is efficacious in dogs as young as 8 weeks of age, as demonstrated by the significant reduction in lung consolidation and the incidence of suppurative pneumonia. Moreover, as demonstrated in Example 3, vaccination with the RP-CIV H3N2 vaccine at minimum protective dose is efficacious in dogs for at least 1 year, as demonstrated by the significant reduction in the lung lesions, reduced incidence of pneumonia, reduced duration of coughing and reduced duration of CIV viral shedding in the RP-CIV H3N2 vaccine treated dogs as compared to the placebo vaccine treated dogs.

[0110] Demonstrating vaccine safety in the target animal is another aspect of vaccine development. In the current efficacy study, there were no local or systemic adverse events recorded. No swelling or tenderness at the injection site was observed upon palpation of each dog on the days of and after each vaccination. In addition, a field safety study was conducted in purpose bred dogs, as well as client-owned dogs, to further evaluate the safety profile of this vaccine. Of the 1,301 administrations of 2 different serials of the RP-CIV H3N2 vaccine containing an amount of antigen representative of vaccine to be used by customers, only 2 caused a local reaction that resolved within 24 hours without treatment. Overall, only 2.5% of the doses administered in the field safety trial caused adverse reactions that were primarily mild and of short duration. Lethargy was the most common event (1.6% of total administrations). In addition, the safety profile of the RP-CIV H3N2 vaccine appears to be no different in younger dogs than older dogs, as well as males versus females. The combined data demonstrates that the RP-CIV H3N2 vaccine is safe for use in dogs as young as 8 weeks of age.

[0111] In summary, a new 0.5 mL dose CIV H3N2 vaccine has been developed using a novel RNA Particle platform and confirmed to be both highly efficacious and safe in dogs as young as8 weeks of age. Efficacy and duration of immunity (DOI) lasted for at least one year, An adjuvant is not needed in this vaccine formulation to induce a highly effective and robust immune response, as demonstrated by the significant reduction of lung consolidation and incidence of suppurative pneumonia in the vaccinated dogs compared to the placebo-vaccinated control dogs. In addition, a significant reduction in the duration and severity of clinical signs, as well as the duration and amount of virus shed post-challenge was also demonstrated. The effectiveness of this new vaccine provides opportunity to use this novel platform for other vaccine development programs to address unmet needs, improve the safety and efficacy profile of current vaccines, evaluate cross protection to other strains, and quickly respond to future outbreaks of disease. REFERENCES

[0112] The references cited in the specification are incorporated herein in their entirety by reference.

Claims

CLAIMS:

1. An alphavirus RNA replicon particle that encodes a Canine Influenza (CIV) H3N2 hemagglutinin (HA) antigen.

2. The alphavirus RNA replicon of Claim 1, wherein the alphavirus RNA replicon particle is a Venezuelan Equine Encephalitis (VEE) alphavirus RNA replicon particle.

3. The alphavirus RNA replicon of Claim 1, wherein the HA protein is the HA protein from a CIV 2015 Iowa strain (A / canine / Iowa / 22619-4 / 2015(H3N2).

4. The alphavirus RNA replicon immunogenic composition of Claim 1, wherein the CIV HA protein comprises an amino acid sequence comprising at least 95% identity with the amino acid sequence of SEQ ID NO:

1.

5. An immunogenic composition comprising an alphavirus RNA replicon of Claim 1.

6. A method to prevent a disease due to Canine Influenza (CIV) H3N2 strain, comprising administering to a canine the immunogenic composition of claim 5.

7. A vaccine to aid in the prevention of disease due to CIV comprising the immunogenic composition of Claim 5 and a pharmaceutically acceptable carrier.

8. The vaccine composition of Claim 7, that further comprises at least one non-CIV antigen for eliciting protective immunity to a non-CIV canine pathogen.

9. The vaccine of Claim 8, wherein the non-CIV canine pathogen is selected from the group consisting of canine distemper virus, canine adenovirus type 2, canine parvovirus type, canine parainfluenza virus, canine coronavirus, and / or canine pneumovirus, Leptospira spp. and / or a Bordetella bronchiseptica, and any combination thereof.

10. The vaccine composition of Claim 7, that is a nonadjuvanted vaccine.

11. A method of immunizing a canine against a pathogenic CIV comprising administering to the canine an immunologically effective amount of the vaccine of Claim 7.

12. The vaccine composition of claim 7, wherein the vaccine aid in the prevention of disease due to CIV in a canine for at least 6-months.

13. The vaccine composition of claim 7, wherein the vaccine aid in the prevention of disease due to CIV in a canine for at least 12-months.

14. The vaccine composition of claim 12 or 13, wherein the vaccine prevents or reduces, any one or more of: (i) occurrence of lung lesions (ii) incidence of pneumonia, (iii) duration of coughing and (iv) duration of CIV viral shedding of Canine Influenza H3N2 virus after Canine Influenza H3N2 exposure.

15. The vaccine composition of claim 12 or 13, wherein the vaccine prevents or reduces (i) the occurrence of lung lesions (ii) the incidence of pneumonia, (iii) the duration of coughing and (iv) the duration of CIV viral shedding of Canine Influenza H3N2 virus after Canine Influenza H3N2 exposure.

Images