COMPOSITIONS FOR CANINE RESPIRATORY DISEASE COMPLEXFIELD OF THE INVENTIONThe present invention relates to the field of immunology, and in particular tothe field of immunogenic and vaccine compositions. It relates to such compositionsfor use against canine respiratory diseases, including canine infectious respiratorydisease complex (CIRDC). The present invention also relates to methods forvaccinating against, treating, or preventing canine respiratory diseases in a canine.
BACKGROUND OF THE INVENTIONCanine infectious respiratory disease complex (CIRDC) is a highly contagiousdisease that is common in dogs housed in crowded conditions, such as rehomingcenters and boarding or training kennels. Many dogs suffer only from a mild coughand recover after a short time. However in some cases, a severe bronchopneumoniacan develop.
The pathogenesis of CIRDC is considered to be multifactorial, involvingseveral viruses and bacteria. Infectious agents known to be causative agents ofCIRDC include canine respiratory coronavirus (CRCoV) (Erles et al., Virology,310(2):216223, 2003), canine influenza virus (CIV) (Crawford et al., Science,310(5747):482485, 2005), canine parainfluenzavirus (CPIV) (Binn et al., Exp. Biol.
Med., 126:1401 45, 1967), canine adenovirus serotyp e 2 (CAV2 ) (Ditchfield et al.,Can. Vet. J., 3:2382 47, 1962), Mycoplasma cynos (C halker et al., Microbiology,150:34913497, 2004), and the bacterium Bordetella bronchiseptica (Bemis et al.,Lab. Anim. Sci., 29:4852, 1977).
CRCoV causes a highly contagious respiratory infection which is is spread bydirect dogt odog contact, aerosols of respiratory secretions, and contact withcontaminated environments or people. Some dogs have a mild disease withsymptoms consisting of cough, sneezing, and nasal discharge. Some dogs have asubclinical infection with no clinical signs, yet they shed virus that can infect otherdogs. Some dogs infected with CRCoV progress to pneumonia, particularly if coinfected with other respiratory pathogens.
Regarding CIV, equine influenza virus has been recognized as a majorrespiratory pathogen in horses since about 1956. Disease symptoms caused byequine influenza virus can be severe, and are often followed by secondary bacterialinfections. Two subtypes of equine influenza virus are recognized, namely subtype1,the prototype being A/Equine/Prague/1/56 (H7N7), and subtype2 , the prototypebeing A/Equine/Miami/1/63 (H3N8). Presently, the predominant virus subtype issubtype2, the H3N8 strain. An influenza virus, H3N 8 equine influenza virus, is ableto infect canines, with fatalities in some cases as high as 36%. One explanation isthat an interspecies transfer of the complete or a portion of the equine influenza virusto the dog resulted in a new canine specific influenza virus associated with acuterespiratory disease (Crawford et al., 2005).
Disease caused by CPIV is common in the upper respiratory tract. Diseasecaused by CPIV alone can be mild or subclinical, with signs becoming more severe ifconcurrent infection with other respiratory pathogens occurs.
CAV2 causes respiratory disease which, in severe c ases, can includepneumonia and bronchopneumonia.
B. bronchiseptica has been reported as being a primary etiological agent in therespiratory disease tracheobronchitis or "kennel cough". It predisposes dogs to theinfluence of other respiratory agents, and frequently exists concurrently with them.
Kennel cough is typically a condition of the upper airways, and is characterized bynasal discharge and coughing. To date, a number of vaccines are available fortreatment of tracheobronchitis caused by Bordetella bronchiseptica, includingNobivac®, BronchiS hield®, Bronchicine® CAe, Vangua rd® B, Univac 2,TM TMRecombitek® KC2, Naramune 2 and KennelJ ec 2. However, the majority ofexisting commercial vaccines require cumbersome intranasal administration as wellas the addition of adjuvants, which can result in deleterious sideeffects, such asburning and irritation. Viera Scheibner et al., Nexus Dec 2000 (Vol 8, No1). Subunitvaccines, such as those involving the use of p68 protein of Bordetella bronchiseptica(pertactin), have been explored but to date have not been included in anycommercial canine vaccines, possibly due to insufficient immunogenicity, adversereactions, and/or formulation stability.
The pathology of CIRDC indicates that it is involved in lung damage and, insome cases, bronchopneumonia, but it is distinct from kennel cough (primaryetiological agent: B. bronchiseptica) which mainly involves upper respiratory tractchanges. Kennel cough is a milder syndrome than CIRDC, and does not have thewide range of pathology noted in CIRDC. CIRDC is also distinguished by anincreased severity and mortality.
CIRDC is rarely fatal, but it delays rehoming of d ogs at rescue centers,disrupts schedules in training kennels, and results in considerable treatment costsand welfare concerns. Vaccines are available against some of the infectious agentsassociated with CIRDC. However, despite the use of these vaccines, CIRDC is stillprevalent worldwide, possibly due to the lack of e fficacious vaccines against all theinfectious agents involved in CIRDC.
Accordingly, there remains a need for an immunogenic composition, capableof being safely administered to a canine, which provides longa ctingimmunoprotection against the agents that cause CIRDC without deleterious sideeffects or interference with other antigens in a combination vaccine. It is an object ofthe present invention to go some way to fulfilling these and other related needs;and/or to at least provide the public with a useful choice.
In this specification where reference has been made to patent specifications,other external documents, or other sources of information, this is generally for thepurpose of providing a context for discussing the features of the invention. Unlessspecifically stated otherwise, reference to such external documents is not to beconstrued as an admission that such documents, or such sources of information, inany jurisdiction, are prior art, or form part of the common general knowledge in theart.
SUMMARY OF THE INVENTIONIn one aspect, the present invention relates to a vaccine compositioncomprising a canine influenza virus (CIV) and a canine respiratory coronavirus(CRCoV).
In another aspect, the present invention relates to a use of the vaccinecomposition of the present invention for the treatment or prevention of infection froma canine respiratory pathogen in a canine.
In another aspect, the present invention relates to a use of the vaccinecomposition of the present invention in the manufacture of a medicament for thetreatment or prevention of infection from a canine respiratory pathogen in a canine.
In another aspect, the present invention relates to a use of:a canine influenza virus (CIV), anda canine respiratory coronavirus (CRCoV)in the manufacture of a vaccine for the treatment or prevention of infectionfrom a canine respiratory pathogen in a canine.
In another aspect, the present invention relates to a method of treating orpreventing infection from a canine respiratory pathogen in a canine comprisingadministering to said canine the vaccine composition of the present invention.
In another aspect, the present invention relates to a method of treating orpreventing CIRDC in a canine comprising administering to said canine the vaccinecomposition of the present invention.
In another aspect, the present invention relates to a use of the vaccinecomposition of the present invention for the treatment or prevention of CIRDC in acanine.
In another aspect, the present invention relates to a use of the vaccinecomposition of the present invention in the manufacture of a medicament for thetreatment or prevention of CIRDC in a canine.
In another aspect, the present invention relates to a use of:a canine influenza virus (CIV), anda canine respiratory coronavirus (CRCoV)in the manufacture of a vaccine for the treatment or prevention of CIRDC in acanine.
Described herein are immunogenic compositions which provide antigens thattreat or prevent CIRDC. In one embodiment, an immunogenic composition comprisesa canine influenza virus (CIV) and a canine respiratory coronavirus (CRCoV). Inanother embodiment, the immunogenic composition further comprises a Bordetellabronchiseptica. In another embodiment, the immunogenic composition furthercomprises an isolated pertactin antigen. In another embodiment, the immunogeniccomposition comprises a p68 pertactin antigen. In another embodiment, the pertactinantigen is a recombinant protein. In yet another embodiment, the pertactin antigen ispresent at between about 1 μg and about 30 μg. In another embodiment, saidpertactin antigen is prepared by solubilizing pertactin inclusion bodies in urea andoptionally purifying by column chromatography. Said pertactin antigens are solubleand preferably substantially free of aggregates. In another embodiment, theBordetella bronchiseptica is a bacterin or a bacterial extract.
In one embodiment, the immunogenic composition comprises a CIV, aCRCoV, a Bordetella bronchiseptica and one or both antigens selected from canineparainfluenza virus (CPIV) and canine adenovirus type 2 (CAV2 ). In anotherembodiment, said immunogenic composition further comprises a p68 pertactinantigen. In another embodiment, the Bordetella bronchiseptica is a bacterin or abacterial extract.
Another embodiment described herein is an immunogenic compositioncomprising a CIV, CRCoV, a Bordetella bronchiseptica component comprisingBordetella bronchiseptica and an isolated pertactin antigen, and one or both antigensselected from canine parainfluenza virus (CPIV) and canine adenovirus type 2 (CAV2). In a further embodiment, the immunogenic composition comprises both CPIV andCAV2 .
In another embodiment, the immunogenic composition of any one of theforegoing embodiments further comprises an isolated Bsp22 antigen.
In another embodiment, the immunogenic composition of any one of theforegoing embodiments is nonadjuvanted. In another embodiment, the immunogeniccomposition of any one of the foregoing embodiments comprises an adjuvant.
In another embodiment, the immunogenic composition of any one of theforegoing embodiments does not contain a nonr espir atory antigen.
In yet another embodiment, the immunogenic composition of any one of theforegoing embodiments induces an immune response to a canine respiratorypathogen in a canine. In another embodiment, said canine respiratory pathogen is atleast one of CIV, CRCoV, CPIV, CAV2 , Bordetella bronchiseptica, and Mycoplasmacynos.
Also described herein is a use of the immunogenic composition of any one ofthe foregoing embodiments for the treatment or prevention of infection from a caninerespiratory pathogen in a canine. In another embodiment, said canine respiratorypathogen is at least one of CIV, CRCoV, CPIV, CAV2 , Bordetella bronchiseptica,and M. cynos. In another embodiment, said composition prevents said infection for aperiod of about 6 months or more. In another embodiment, said composition preventssaid infection for a period of about one year. Also described herein is a use of theimmunogenic composition of any one of the foregoing embodiments in themanufacture of a medicament for the treatment or prevention of infection from acanine respiratory pathogen in a canine.
Also described is the immunogenic composition of any one of the foregoingembodiments wherein said composition treats or prevents canine infectiousrespiratory disease complex (CIRDC) in a canine. Also described is a method oftreating or preventing CIRDC in a canine comprising administering to said canine theimmunogenic composition of any one of the foregoing embodiments. In anotherembodiment, said composition prevents CIRDC for a period of about 6 months ormore. In another embodiment, said composition prevents CIRDC for a period ofabout one year. Also described is a use of the immunogenic composition of any oneof the foregoing embodiments in the manufacture of a medicament for the treatmentor prevention of CIRDC in a canine.
In the description in this specification reference may be made to subject matterwhich is not within the scope of the claims of the current application. That subjectmatter should be readily identifiable by a person skilled in the art and may assist inputting into practice the invention as defined in the claims of this application.
BRIEF DESCRIPTION OF THE DRAWINGSFigure 1. Serum Neutralizing Antibody Response against CRCoV.
Measurement of serum neutralizing antibody response against canine respiratorycoronavirus (CRCoV) when dogs were vaccinated with saline, Aluminum hydroxide(AlOH)a djuvanted, or Emulsigen®adjuvanted composi tions.
Figure 2. Nasal Virus Shedding Post-Challenge. Measurement of CRCoVshed from the nasal passages when dogs were vaccinated with saline, AlOHadjuvanted, or Emulsigen®adjuvanted compositions, followed by subsequentchallenge with CRCoV.
Figure 3. Percent Animals Positive for CRCoV Tissue Virus on Day 4Post-Challenge. Assessment of number of dogs positive for CRCoV in respiratorytissue when vaccinated with saline, AlOHa djuvanted , or Emulsigen®adjuvantedcompositions, followed by subsequent challenge with CRCoV.
DETAILED DESCRIPTION OF THE INVENTIONThe definitions below apply to this disclosure. They supersede anycontradictory definitions contained in each individual reference incorporated hereinby reference. Words not defined have the meaning commonly used by one skilled inthe art. Further, unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular.
The term “comprising” as used in this specification and claims means“consisting at least in part of”. When interpreting statements in this specification andclaims which include the term “comprising”, other features besides the featuresprefaced by this term in each statement can also be present. Related terms such as“comprise” and “comprised” are to be interpreted in similar manner.
“About” or “approximately,” when used in connection with a measurablenumerical variable, refers to the indicated value of the variable and to all values of thevariable that are within the experimental error of the indicated value (e.g., within the95% confidence interval for the mean), or within 10 percent of the indicated value,whichever is greater. If “about” is used in reference to time intervals in weeks, “about3 weeks” is 17 to 25 days, and “about 2 to about 4 weeks” is 10 to 40 days.
“Adjuvant”, as used herein, refers to any substance which serves as a nonspecific stimulator of the immune response. See below for a further description ofadjuvants.
The term "animal”, as used herein, includes any animal that is susceptible tocanine respiratory disease complex, including mammals, both domesticated and wild.
“Antibody”, as used herein, is any polypeptide comprising an antigenb indingsite regardless of the source, method of production, or other characteristics. It refersto an immunoglobulin molecule or a fragment thereof that specifically binds to anantigen as the result of an immune response to that antigen. Immunoglobulins areserum proteins composed of “light” and “heavy” polypeptide chains having “constant”and “variable” regions and are divided into classes (e.g., IgA, IgD, IgE, IgG, and IgM)based on the composition of the constant regions. An antibody that is “specific” for agiven antigen indicates that the variable regions of the antibody recognize and bind aspecific antigen exclusively. The term includes, but is not limited to: a polyclonalantibody, a monoclonal antibody, a monospecific antibody, polyspecific antibody,humanized antibody, a tetrameric antibody, a tetravalent antibody, a multispecificantibody, a single chain antibody, a domains pecifi c antibody, a single domainantibody, a domaindeleted antibody, a fusion prote in, an ScFc fusion protein, asinglec hain antibody, chimeric antibody, synthetic antibody, recombinant antibody,hybrid antibody, mutated antibody, and CDRgrafted antibodies. Antibodies can beintact immunoglobulins derived from natural sources or from recombinant sources, orcan be immunoreactive portions of intact immunoglobulins. An “antibody” can beconverted to an antigenbinding protein, which incl udes but is not limited to antibodyfragments which include but are not limited to: Fab, F(ab') , an Fab' fragment, an Fvfragment, a singlec hain Fv (ScFv) fragment, an Fd fragment, a dAb fragment,diabodies, a CDR3 peptide, a constrained FR3C DR3F R4 peptide, a nanobody, abivalent nanobody, a small modular immunopharmaceutical (SMIPs), and a minibodyand any of above mentioned fragments and their chemically or geneticallymanipulated counterparts, as well as other antibody fragments that retainantigenbinding function. Typically, such fragments would comprise an antigenbinding domain. As will be recognized by those of skill in the art, any of suchmolecules may be engineered (for example “germlined”) to decrease itsimmunogenicity, increase its affinity, alter its specificity, or for other purposes.
“Antigen” or “immunogen”, as used herein, refers to a molecule that containsone or more epitopes (linear, conformational or both) that upon exposure to a subjectwill induce an immune response that is specific for that antigen. An epitope is thespecific site of the antigen which binds to a Tc el l receptor or specific antibody, andtypically comprises about 3 amino acid residues to about 20 amino acid residues.
The term antigen refers to subunit antigens—antigens separate and discrete from awhole organism with which the antigen is associated in nature—as well as killed,attenuated or inactivated bacteria, viruses, fungi, parasites or other microbes. Theterm antigen also refers to antibodies, such as antii diotype antibodies or fragmentsthereof, and to synthetic peptide mimotopes that can mimic an antigen or antigenicdeterminant (epitope). The term antigen also refers to an oligonucleotide orpolynucleotide that expresses an antigen or antigenic determinant in vivo, such as inDNA immunization applications.
“Antigenicity”, as used herein, refers to the capability of a protein orpolypeptide to be immunospecifically bound by an antibody raised against the proteinor polypeptide.
The term “Bordetella bronchiseptica” or “B. bronchiseptica” refers to: a liveattenuated bacterium of Bordetella bronchiseptica, a killed whole cell extract(bacterin) of Bordetella bronchiseptica or a cellular bacterial extract of Bordetellabronchiseptica.
“Buffer” means a chemical system that prevents change in the concentrationof another chemical substance. Proton donor and acceptor systems serve asbuffers, preventing marked changes in hydrogen ion concentration (pH). A furtherexample of a buffer is a solution containing a mixture of a weak acid and its salt(conjugate base), or a weak base and its salt (conjugate acid).
“Canine”, as used herein, includes what is commonly called the dog, butincludes other members of the family Canidae.
The term “cell line” or “host cell”, as used herein, means a prokaryotic oreukaryotic cell in which a virus can replicate or be maintained.
The term “culture”, as used herein, means a population of cells ormicroorganisms growing in the absence of other species or types.
“Dose” refers to a vaccine or immunogenic composition given to a subject. A“first dose” or “priming dose” refers to the dose of such a composition given on Day 0.
A “second dose” or a “third dose” or an “annual dose” refers to an amount of suchcomposition given subsequent to the first dose, which can be but is not required to bethe same vaccine or immunogenic composition as the first dose.
An “epitope” is the specific site of the antigen which binds to a Tc ell receptoror specific antibody, and typically comprises from about 3 amino acid residues toabout 20 amino acid residues.
“Excipient”, as used herein, refers to a nonr eacti ve carrier component of avaccine or immunogenic composition that is not an antigen.
“Fragment” refers to a truncated portion of a protein or gene. “Functionalfragment” and “biologically active fragment” refer to a fragment that retains thebiological properties of the full length protein or gene.
“Homology” or “percent homology” refers to the percentage of nucleotide oramino acid residues in the candidate sequence that are identical or similar with theresidues in the comparator sequence(s) after aligning the sequences and introducinggaps, if necessary, to achieve the maximum percent sequence homology, and alsoconsidering any conservative substitutions as part of the sequence homology.
“Homologs” or “species homologs” include genes found in two or moredifferent species which possess substantial polynucleotide sequence homology, andpossess the same, or similar, biological functions and/or properties. Preferablypolynucleotide sequences which represent species homologs will hybridize undermoderately stringent conditions, as described herein by example, and possess thesame or similar biological activities and/or properties. In another aspect,polynucleotides representing species homologs will share greater than about 60%sequence homology, greater than about 70% sequence homology, greater thanabout 80% sequence homology, greater than about 90% sequence homology,greater than about 95% sequence homology, greater than about 96% sequencehomology, greater than about 97% sequence homology, greater than about 98%sequence homology, or greater than about 99% sequence homology.
“Identity” or “percent identity” refers to the percentage of nucleotides or aminoacids in the candidate sequence that are identical with the residues in the comparatorsequence after aligning both sequences and introducing gaps, if necessary, toachieve the maximum percent sequence identity, and not considering anyconservative substitutions as part of the sequence identity.
“Immune response”, as used herein, in a subject refers to the development ofa humoral immune response, a cellular immune response, or a humoral and acellular immune response to an antigen. A “humoral immune response” refers to onethat is at least in part mediated by antibodies. A “cellular immune response” is onemediated by Tl ymphocytes or other white blood cell s or both, and includes theproduction of cytokines, chemokines and similar molecules produced by activated Tcells, white blood cells, or both. Immune responses can be determined usingstandard immunoassays and neutralization assays, which are known in the art.
“Immunogenicity”, as used herein, refers to the capability of a protein orpolypeptide to elicit an immune response directed specifically against an antigen.
An "immunogenic composition" is a preparation containing an immunogen,including, e.g., a protein, a peptide, a whole cell, inactivated, subunit or attenuatedvirus, or a polysaccharide, or combination thereof, administered to stimulate therecipient's humoral and cellular immune systems to one or more of the antigenspresent in the immunogenic composition. "Immunization" is the process ofadministering an immunogenic composition and stimulating an immune orimmunogenic response to an antigen in a host. Preferred hosts are mammals, suchas dogs. Preferably, the immunogenic composition is a vaccine.
“Immunologically protective amount”, as used herein, is an amount of anantigen effective to induce an immunogenic response in the recipient that isadequate to prevent or ameliorate signs or symptoms of disease, including adversehealth effects or complications thereof. Either humoral immunity or cellm ediatedimmunity or both can be induced. The immunogenic response of an animal to acomposition can be evaluated, e.g., indirectly through measurement of antibodytiters, lymphocyte proliferation assays, or directly through monitoring signs andsymptoms after challenge with wild type strain. The protective immunity conferred bya composition or vaccine can be evaluated by measuring, e.g., reduction of shed ofchallenge organisms, reduction in clinical signs such as mortality, morbidity,temperature, and overall physical condition, health and performance of the subject.
The immune response can comprise, without limitation, induction of cellular and/orhumoral immunity. The amount of a composition or vaccine that is therapeuticallyeffective can vary, depending on the particular organism used, or the condition of theanimal being treated or vaccinated, and can be determined by a veterinarian.
“Intranasal” administration, as used herein, refers to the introduction of asubstance, such as a vaccine or other composition, into a subject’s body through orby way of the nose, and involves transport of the substance primarily through thenasal mucosa.
“Isolated”, as used herein, means removed from its naturally occurringenvironment, either alone or in a heterologous host cell, or chromosome or vector(e.g., plasmid, phage, etc.). “Isolated bacteria,” “isolated anaerobic bacteria,”“isolated bacterial strain,” “isolated virus” “isolated viral strain” and the like refer to acomposition in which the bacteria or virus are substantial free of othermicroorganisms, e.g., in a culture, such as when separated from it naturally occurringenvironment. “Isolated,” when used to describe any particularly defined substance,such as a polynucleotide or a polypeptide, refers to the substance that is separatefrom the original cellular environment in which the substance such as a polypeptideor nucleic acid is normally found. As used herein therefore, by way of example only,a recombinant cell line constructed with a polynucleotide described herein makes useof the “isolated” nucleic acid. Alternatively, if a particular protein or a specificimmunogenic fragment is claimed or used as a vaccine or other composition, it wouldbe considered to be isolated because it had been identified, separated and to someextent purified as compared to how it may exist in nature. If the protein or a specificimmunogenic fragment thereof is produced in a recombinant bacterium or eukaryoteexpression vector that produces the antigen, it is considered to exist as an isolatedprotein or nucleic acid. For example, a recombinant cell line constructed with apolynucleotide makes use of an “isolated” nucleic acid.
“Medicinal agent” refers to any agent which is useful in the prevention, cure, orimprovement of a medical condition, or the prevention of some physiologicalcondition or occurrence.
"Monoclonal antibody", as used herein, refers to antibodies produced by asingle line of hybridoma cells, all directed towards one epitope on a particularantigen. The antigen used to make the monoclonal antibody can be provided as anisolated protein of the pathogen or the whole pathogen. A “hybridoma” is a clonalcell line that consists of hybrid cells formed by the fusion of a myeloma cell and aspecific antibodyproducing cell. In general, mono clonal antibodies are of mouseorigin. However, monoclonal antibody also refers to a clonal population of anantibody made against a particular epitope of an antigen produced by phage displaytechnology, or method that is equivalent to phage display, or hybrid cells of nonmouse origin.
“Oral” or “peroral” administration, as used herein, refers to the introduction of asubstance, such as a vaccine or other composition, into a subject’s body through orby way of the mouth and involves swallowing or transport through the oral mucosa(e.g., sublingual or buccal absorption) or both. Intratracheal is also a means of oralor peroral administration.
“Oronasal” administration, as used herein, refers to the introduction of asubstance, such as a composition or vaccine, into a subject’s body through or by wayof the nose and the mouth, as would occur, for example, by placing one or moredroplets in the nose. Oronasal administration involves transport processesassociated with oral and intranasal administration.
“Parenteral administration”, as used herein, refers to the introduction of asubstance, such as a composition or vaccine, into a subject’s body through or by wayof a route that does not include the digestive tract. Parenteral administration includessubcutaneous, intramuscular, intraarterial, and intravenous administration. For thepurposes of this disclosure, parenteral administration excludes administration routesthat primarily involve transport of the substance through mucosal tissue in the mouth,nose, trachea, and lungs.
The term “pathogen” or “pathogenic microorganism”, as used herein, means amicroorganism for example, CPIV, CAV2 , CRCoV, CI V, or Bordetellabronchiseptica which is capable of inducing or causing a disease , illness, orabnormal state in its host animal.
“Pertactin”, as used herein, refers to an outer membrane protein of Bordetella.
Preferably the pertactin is from B. bronchiseptica and most preferably, “p68”, and isencoded by the gene, prnA. Pertactin can be isolated in its native form fromBordetella bronchiseptica, or it can be produced recombinantly. Sequences andexamples of pertactin are provided in U.S. Patent No. 7,736,658, the content ofwhich is hereby incorporated by reference. The pertactin antigen used hereinincludes lipidated forms of the protein.
“Pharmaceutically acceptable” refers to substances which, within the scope ofsound medical judgment, are suitable for use in contact with the tissues of subjectswithout undue toxicity, irritation, allergic response, and the like, commensurate with areasonable benefitt or isk ratio, and effective for their intended use.
"Polyclonal antibody", as used herein, refers to a mixed population ofantibodies made against a particular pathogen or antigen. In general, the populationcontains a variety of antibody groups, each group directed towards a particularepitope of the pathogen or antigen. To make polyclonal antibodies, the wholepathogen, or an isolated antigen, is introduced by inoculation or infection into a host,which induces the host to make antibodies against the pathogen or antigen.
The term “polynucleotide”, as used herein, means an organic polymermolecule composed of nucleotide monomers covalently bonded in a chain. DNA(deoxyribonucleic acid) and RNA (ribonucleic acid) are examples of polynucleotideswith distinct biological function.
The term “polypeptide”, as used herein, means an organic polymer moleculecomposed of two or more amino acids bonded in a chain.
"Preventing infection", as used herein, means to prevent or inhibit thereplication of the bacteria or virus which causes the identified disease, to inhibittransmission of the bacteria or virus, to prevent the bacteria or virus from establishingitself in its host, or to alleviate the symptoms of the disease caused by infection. Thetreatment is considered therapeutic if there is a reduction in bacterial or viral load.
“Protection”, “protecting”, “protective immunity”, and the like, as used hereinwith respect to a vaccine or other composition, means that the vaccine orcomposition prevents or reduces the symptoms of the disease caused by theorganism from which the antigen(s) used in the vaccine or composition is derived.
The terms “protection”, “protecting”, and the like, also mean that the vaccine orcomposition can be used to “treat” the disease, or one or more symptoms of thedisease that already exists in a subject.
“Respiratory” administration, as used herein, refers to the introduction of asubstance, such as a vaccine or other composition, into a subject’s body through orby way of inhalation of a nebulized (atomized) substance. In respiratoryadministration, the primary transport mechanism involves absorption of the atomizedsubstance through the mucosa in the trachea, bronchi, and lungs and is thereforedifferent than intranasal or peroral administration.
The terms “specific binding,” “specifically binds,” and the like, are defined astwo or more molecules that form a complex that is measurable under physiologic orassay conditions and is selective. An antibody or other inhibitor is said to “specificallybind” to a protein if, under appropriately selected conditions, such binding is notsubstantially inhibited, while at the same time nons pecific binding is inhibited.
Specific binding is characterized by high affinity and is selective for the compound orprotein. Nonspecific binding usually has low affinity. Binding in IgG antibodies, forexample, is generally characterized by an affinity of at least about 10 M or higher,8 9such as at least about 10 M or higher, or at least about 10 M or higher, or at least11 12about 10 or higher, or at least about 10 M or higher, or at least about 10 M orhigher. The term is also applicable where, e.g., an antigenbinding domain is specificfor a particular epitope that is not carried by numerous antigens, in which case theantibody carrying the antigenb inding domain will g enerally not bind other antigens.
“Specific immunogenic fragment”, as used herein, refers to a portion of asequence that is recognizable by an antibody or T cell specific for that sequence.
“Subject”, as used herein, refers to any animal having an immune system,which includes mammals, such as dogs.
“Substantially identical”, as used herein, refers to a degree of sequenceidentity of at least about 90%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99%.
“Subunit vaccine”, and “subunit composition”, as used herein, refers to a typeof vaccine or composition that includes one or more antigens but not necessarily allantigens in the vaccine or composition which are derived from or homologous to,antigens from a pathogen of interest, such as a virus, bacterium, parasite or fungus.
Such a composition or vaccine is substantially free of intact pathogen cells orpathogenic particles, or the lysate of such cells or particles. Thus, a subunit vaccineor subunit composition can be prepared from at least partially purified, orsubstantially purified, immunogenic polypeptides from the pathogen or their analogs.
Methods of obtaining an antigen or antigens in the subunit vaccine or subunitcomposition include standard purification techniques, recombinant production, orchemical synthesis. A “subunit vaccine” or “subunit composition” thus refers to avaccine or composition consisting of a defined antigenic component or componentsof a virus, bacterium, or other immunogen.
“TCID ” refers to “tissue culture infective dose” and is defined as that dilutionof a virus required to infect 50% of a given batch of inoculated cell cultures. Variousmethods can be used to calculate TCID , including the SpearmanK arber method,which is utilized throughout this specification. For a description of the SpearmanKarber method, see B. W. Mahy & H. O. Kangro, Virology Methods Manual 2546(1996).
"Therapeutic agent", as used herein, refers to any molecule, compound, virusor treatment, preferably a virus attenuated or killed, or subunit or compound, thatassists in the treatment of a viral, bacterial, parasitic or fungal infection, disease orcondition caused thereby.
“Therapeutically effective amount”, as used herein, refers to an amount of anantigen or vaccine or composition that would induce an immune response in asubject (e.g., dog) receiving the antigen or vaccine or composition which is adequateto prevent or ameliorate signs or symptoms of disease, including adverse healtheffects or complications thereof, caused by infection with a pathogen, such as a virus,bacterium, parasite or fungus. Humoral immunity or cellm ediated immunity, or bothhumoral and cellm ediated immunity, can be induced. The immunogenic response ofan animal to an antigen, vaccine, or composition can be evaluated indirectly throughmeasurement of antibody titers, lymphocyte proliferation assays, or directly throughmonitoring signs and symptoms after challenge with the wild type strain. Theprotective immunity conferred by a vaccine or composition can be evaluated bymeasuring reduction of challenge organism shed, and/or reduction in clinical signs,such as mortality, morbidity, temperature, and overall physical condition, health, andperformance of the subject. The amount of a vaccine or composition that istherapeutically effective can vary, depending on the particular immunogen used, orthe condition of the subject, and can be determined by one skilled in the art.
“Treat” or “treating”, as used herein, refers to reversing, alleviating, inhibitingthe progress of, or preventing a disorder, condition or disease to which such termapplies, or to preventing one or more symptoms of such disorder, condition ordisease.
“Treatment”, as used herein, refers to the act of “treating”, as definedimmediately above.
“Vaccine” or “vaccine composition,” as used herein, refers to an immunogeniccomposition selected from a virus or bacteria, either modified live, attenuated, orkilled, or a subunit vaccine, or any combination of the aforementioned.
Administration of the vaccine to a subject results in an immune response. Thevaccine can be introduced directly into the subject by any known route ofadministration, including parenterally, perorally, and the like. The terms mean acomposition which prevents or reduces an infection, or which prevents or reducesone or more signs or symptoms of infection. The protective effects of a vaccinecomposition against a pathogen are normally achieved by inducing in the subject animmune response. Generally speaking, abolished or reduced incidences of infection,amelioration of the signs or symptoms, or accelerated elimination of themicroorganism from the infected subjects are indicative of the protective effects of avaccine composition. The vaccine compositions of the present invention provideprotective effects against infections caused by canine respiratory disease pathogens.
“Veterinarily acceptable”, as used herein, refers to substances which are,within the scope of sound medical judgment, suitable for use in contact with thetissues of veterinary subjects without undue toxicity, irritation, allergic response, andthe like, commensurate with a reasonable benefitt o r isk ratio, and effective for theirintended use.
“Veterinarily acceptable carrier", as used herein, refers to a carrier mediumthat does not interfere with the effectiveness of the biological activity of the activeingredient, and is not toxic to the veterinary subject to whom it is administered.
Antigens, Immunogenic Compositions, and VaccinesThe present disclosure provides immunogenic compositions and vaccinescomprising one or more viruses and bacteria. The present disclosure providesimmunogenic compositions and vaccines comprising one or more viruses andbacteria or subunits that are suitable for administration to a canine for treatmentagainst CIRDC.
The canine respiratory coronavirus (CRCoV) described herein can becharacterised as a coronavirus present in the respiratory tracts of dogs with infectiousrespiratory disease. CRCoV is phylogenetically most closely related to bovinecoronavirus (BCoV), human coronavirus (HCoV) strain OC43 and hemagglutinatingencephalomyelitis virus (HEV); enteric canine coronavirus (CCoV) is only distantlyrelated to CRCoV. A representative example of a CRCoV suitable for use in thepresent invention includes a strain identified as CRCoV strain 4182 (Erles et al., VirusRes., 124:7887, 2007).
The influenza virus antigens encompassed by this invention can be anyidentified influenza virus strain, from any bird or mammal, including but not limited to,influenza virus having the subtype H3 hemagglutinin and subtype N8 neuraminidase,or the H3N8 subtype, more commonly referred to as an H3N8 virus. The influenzacan be of mammalian or avian origin, including but not limited to swine, equine orcanine origin. In one embodiment a canine influenza antigen is used. In oneembodiment an equine influenza antigen is used. In one embodiment, a strain havingthe subtype glycoproteins designated H3 or N8 is used. In one embodiment, a strainhaving both subtype H3 and N8 glycoproteins is used.
The influenza antigens encompassed by this invention can be isolated fromdogs, horses, pigs, and fowl, both domestic and wild. The animals chosen for samplecollection should display acute and/or subacute cl inical syndromes, which caninclude mild to severe respiratory symptoms and fever. Animals can also exhibitsigns of anorexia and lethargy. Methods of virus isolation are well known to thoseskilled in the art including: inoculating mammalian or avian cell cultures, inoculatingembryonated eggs with nasal or pharyngeal mucus samples from clinical specimens,collection by swabbing of the nasal passage or throat, or by collecting tissues suchas spleen, lung, tonsil and liver and lung lavage. The cytopathic effect of the viruscan be observed in cell culture. Allantoic fluid or cell lysates can be tested for theirability to agglutinate human, chicken, turkey or guinea pig red blood cells,presumptive evidence for the presence of an influenza virus.
A representative example of an influenza strain suitable for use in the presentinvention includes a strain identified as A/canine/Iowa/9A1/B5/08/D12, which wasdeposited as PTA7 694 on 29 June 2006 at the Americ an Type Culture Collection(ATCC), 10801 University Boulevard, Manassas, VA 201102 209, in compliance withBudapest Treaty on the International Recognition of the Deposit of Microorganismsfor the Purposes of Patent Procedure. A representative strain of the CIV antigen isthe CIV virus strain in the commercial vaccine, Vanguard® CIV (Pfizer, Inc). Thisinvention also encompasses vaccines comprising a strain identified as EquineInfluenza Strain A/Equine/2/Miami/1/63. This strain was deposited at the ATCC, withaccession number VR 317, in compliance with Budapest Treaty on the InternationalRecognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.
Additional examples of influenza viruses for use in the present invention areA/canine/Iowa/13628/2005, A/Equine/Kentucky/1998, A/Equine/Kentucky/15/2002,A/Equine/Ohio/1/2003, A/Equine/Kentucky/1/1994,A/Equine/Massachusetts/213/2003, A/Equine/Wisconsin/2003,A/Equine/NewYork/1999, and A/Equine/Newmarket/A2/1993. Other preferred strainsand/or isolates of CIV include those disclosed in U.S. Patent Nos. 7,959,929(particularly strains and HA sequences identified therein as Jacksonville/2005,Miami/2005, FL/242/03 and Florida/43/04), 7,384,642, 7,572,620 and 7,468,187, thecontents of which, including all sequences, particularly HA sequences, and strains,are hereby incorporated by reference as if set forth fully herein. Additonally, a CIVstrain suitable for use herein includes the Colorado CIV isolate described in Barrell etal., J. Vet. Intern. Med., 24 (6), 15241527 (2010) , having accession numberADW41784.
The canine parainfluenza virus (CPIV) encompassed by this invention can becharacterized as one of the viruses known to be a causative agent associated withkennel cough. A representative strain of the CPIV antigen is the attenuated CPI virusstrain in the commercial vaccine, Vanguard® Plus 5 (Pfizer). Another representativestrain of the CPIV antigen is the attenuated CPI virus strain having the designation of“NLCPI5 ” (National Veterinary Service Laboratory, Ames, IA).
The canine adenovirus, type 2 (CAV2) encompassed b y this invention can becharacterized as one of the viruses also known to be a causative agent associatedwith kennel cough. A representative strain of the CAV2 antigen is the attenuatedCAV2 virus strain in the commercial vaccine, Vangu ard® Plus 5 (Pfizer). Arepresentative strain of the CAV2 antigen is the a ttenuated CAV2 strain designatedas the “Manhattan” strain (National Veterinary Service Laboratory, Ames, IA).
The Mycoplasma cynos (M. cynos) encompassed by this invention isdescribed in Chalker et al., Microbiology, 150:34913 497, 2004 and is the onlyspecies of mycoplasma commonly associated with respiratory disease. Immunogeniccompositions against M. cynos are described in US 2007/0098739, incorporatedherein by reference.
The Bordetella bronchiseptica component encompassed by this invention canbe characterized as the bacterial causative agent associated with kennel cough. Theimmunogenic compositions and vaccines encompassed by the present invention canbe one or more of: a live attenuated Bordetella bronchiseptica, a Bordetellabronchiseptica bacterin or a bacterial extract. Additionally, the composition preferablyalso includes an isolated subunit antigen of Bordetella bronchiseptica.
In one embodiment the Bordetella bronchiseptica is prepared as a whole cellsonicate purified through column chromatography as provided in Patent ApplicationNo. FR2571618, filed October 12, 1984. Another representative example of aBordetella bronchiseptica is the bacterial extract Bronchicine™ CAe (Pfizer), which isprepared from antigenic material extracted from Bordetella bronchiseptica cells.
Another example of Bordetella bronchiseptica is the live attenuated Bordetellabronchiseptica strain BC 2 present in Nobivac® and/or the live bronchiseptica strainfrom IntraTrac®, BronchiS hield®, Naramune , Recombitek®, Univac, and/orKennelJ ec .
Additionally, a subunit is preferably also present (i.e., supplemented), incombination with the Bordetella bronchiseptica component. A representative exampleof the subunit is an isolated pertactin antigen, preferably, a Bordetella bronchisepticap68 antigen, particularly the recombinant Bordetella bronchiseptica p68 antigenwhich is recognized by the p68s pecific monoclonal antibody Bord 27 (described inUS 7,736,658, which is incorporated herein by reference) and in one preferredembodiment, has an amino acid sequence as set forth in US 7,736,658 or havinghomology thereto.
The recombinant p68 pertactin antigen is preferably prepared in a solubleform, such that nativel ike structure is preserved or restored during processing.
Accordingly, one aspect of the invention provides a recombinant p68 that issubstantially free (less than about 80%, 90%, 95% or even 99%) of aggregates. Inanother embodiment the recombinant p68 is solubilised with urea, preferably about0.1 M, 0.5 M, 1 M, 2 M, 3 M, or 6 M solution of urea. Thereafter, the p68 antigen canbe purified, such as through column chromatography. One such solubilisationprocess is described in Surinder et al., J. Bioscience and Bioengineering, v. 99(4),pgs 303310 (2005).
Pertactin antigens used herein also include lipidated forms. Examples ofproduction of lipidated proteins is provided in Erdile et al., Infection and Immunity,(1993) v.61(1), p. 8190, incorporate by reference. The methods disclosed thereincan be used to prepare posttranslationally modified pertactin proteins that contain anattached lipid moiety.
Furthermore, in another embodiment, an immunogenic composition comprisesBordetella bronchiseptica and an isolated Bsp22 antigen. In another embodiment, theimmunogenic composition comprises Bordetella bronchiseptica, an isolated pertactinantigen, and an isolated Bsp22 antigen. The Bsp22 antigen can be prepared asprovided in Medhekar et al., Molecular Microbiology (2009) 71(2), 492–504.
Preferably, the isolated Bsp22 antigen is present in conjunction with (i.e., in additionto) a Bordetella bronchiseptica extract and an isolated pertactin antigen, specificallyrecombinant p68.
“Bsp22” also includes lipidated forms of the antigen. Examples of production oflipidated proteins is provided in Erdile et al., Infection and Immunity, (1993) v.61(1),p. 819 0, incorporated by reference. The methods di sclosed therein can be used toprepare posttranslationally modified Bsp22 proteins that contain an attached lipidmoiety.
Viruses encompassed by the present invention can be propagated in cells, celllines and host cells. Said cells, cell lines or host cells can be for example, but notlimited to, mammalian cells and nonmammalian cells , including insect and plantcells. Cells, cell lines, and host cells in which viruses encompassed by the presentinvention can be propagated are readily known, and accessible to those of ordinaryskill in the art.
In another embodiment, the immunogenic compositions described herein donot comprise nonrespiratory antigens. Thus, one em bodiment provides acomposition as described herein with the proviso that it does not include a nonrespiratory antigen. The nonrespiratory antigens d o not cause respiratory disease ina subject. Nonl imiting examples of such nonrespir atory antigens include rabiesvirus, canine parvovirus, enteric canine coronavirus, Leptospira species, and Borreliaburgdorferi.
Bacteria encompassed by the present invention can be cultured andpropagated using various culture media known to those of ordinary skill in the art,including both broth (liquid) and agar (solid; semis olid) cultivation media. Somebacteria can also be cultured and propagated in mammalian cells or nonm ammaliancells.
The viruses and bacteria encompassed by the present invention can beattenuated or inactivated prior to use in an immunogenic composition or vaccine.
Methods of attenuation and inactivation are well known to those skilled in the art.
Methods for attenuation include, but are not limited to, serial passage in cell cultureon a suitable cell line (viruses and some bacteria), serial passage in broth culture(bacteria), ultraviolet irradiation (viruses and bacteria), and chemical mutagenesis(viruses and bacteria). Methods for viral or bacterial inactivation include, but are notlimited to, treatment with formalin, betapropriolactone (BPL) or binary ethyleneimine(BEI), or other methods known to those skilled in the art.
Inactivation by formalin can be performed by mixing the suspension containingthe microorganism with 37% formaldehyde to a final formaldehyde concentration of0.5%. The microorganismf ormaldehyde mixture is mix ed by constant stirring forapproximately 24 hours at room temperature. The inactivated microorganism mixtureis then tested for residual live organisms by assaying for growth on a suitable cell lineor broth media.
For some antigens, inactivation by BEI can be performed by mixing thesuspension containing the microorganism described herein with 0.1 M BEI (2b romoethylamine in 0.175 N NaOH) to a final BEI concentration of 1 mM. For otherantigens, the final BEI concentration is 2 mM. One skilled in the art would know theappropriate concentration to use. The virusB EI mix ture is mixed by constant stirringfor approximately 48 hours at room temperature, followed by the addition of 1.0 Msodium thiosulfate to a final concentration of 0.1 mM. Mixing is continued for anadditional two hours. The mixture containing the inactivated microorganism is testedfor residual live virus by assaying for growth on a suitable cell line or broth media.
Immunogenic compositions and vaccines encompassed by the presentinvention can include one or more veterinarilya cce ptable carriers. As used herein, a"veterinarilyacceptable carrier" includes any and all solvents, dispersion media,coatings, adjuvants, stabilizing agents, diluents, preservatives, antibacterial andantifungal agents, isotonic agents, adsorption delaying agents, and the like. Diluentscan include water, saline, dextrose, ethanol, glycerol, and the like. Isotonic agentscan include sodium chloride, dextrose, mannitol, sorbitol, and lactose, among othersknown to those skilled in the art. Stabilizers include albumin, among others known tothe skilled artisan. Preservatives include merthiolate, among others known to theskilled artisan.
The adjuvant can be metabolizable, referring to adjuvants consisting ofcomponents that are capable of being metabolized by the target species such asvegetable oil based adjuvants. A metabolizable adjuvant can be a metabolizable oil.
Metabolizable oils are fats and oils that typically occur in plants and animals, andusually consist largely of mixtures of triacylglycerols, also known as triglycerides orneutral fats. These nonpolar, water insoluble substances are fatty acid triesters ofglycerol. Triacylglycerols differ according to the identity and placement of their threefatty acid residues or side chains.
The adjuvant can also be nonm etabolizable, referri ng to adjuvants consistingof components that cannot be metabolized by the body of the animal subject to whichthe emulsion is administered. Nonm etabolizable oi ls suitable for use incompositions of the present invention include alkanes, alkenes, alkynes, and theircorresponding acids and alcohols, the ethers and esters thereof, and mixturesthereof. Preferably, the individual compounds of the oil are light hydrocarboncompounds, i.e., such components have 6 to 30 carbon atoms. The oil can besynthetically prepared or purified from petroleum products. Preferred nonmetabolizable oils for use in compositions described herein include mineral oil,paraffin oil, and cycloparaffins, for example. The term "mineral oil" refers to a nonmetabolizable adjuvant oil that is a mixture of liquid hydrocarbons obtained frompetrolatum via a distillation technique. The term is synonymous with "liquefiedparaffin", "liquid petrolatum" and "white mineral oil." The term is also intended toinclude "light mineral oil," i.e., oil which is similarly obtained by distillation ofpetrolatum, but which has a slightly lower specific gravity than white mineral oil.
Mineral oil can be obtained from various commercial sources, for example, J.T. Baker(Phillipsburg, PA), USB Corporation (Cleveland, OH). Light mineral oil iscommercially available under the name DRAKEOL®.
Adjuvants include, but are not limited to, the Emulsigen® adjuvant system(MVP Laboratories; Ralston, NE), the RIBI adjuvant system (Ribi Inc.; Hamilton, MT),alum, aluminum hydroxide gel, oili n water emulsion s, wateri noil emulsions such as,e.g., Freund's complete and incomplete adjuvants, Block copolymer (CytRx; Atlanta,GA), SAFM (Chiron; Emeryville, CA), AMPHIGEN adjuvant, saponin, Quil A, QS2 1(Cambridge Biotech Inc.; Cambridge, MA), GPI0 100 ( Galenica Pharmaceuticals,Inc.; Birmingham, AL) or other saponin fractions, monophosphoryl lipid A, Avridinelipida mine adjuvant, heatlabile enterotoxin from E. coli (recombinant or otherwise),cholera toxin, muramyl dipeptide, squalene/pluronic block copolymer/surfactant (SPoil), sulpholipobetac yclodextrin (SLC D), liposome s containing an immumodulator(e.g., CpG or poly I:C), muramyl dipeptide (MDP), iscomatrix (Quil A/phosphotidylcholine), CpG/DEAEd extran/mineral oil (TXO), CpG, triterpenoids (e.g., Quil A oranother purified or partially purified saponin preparation), sterols (e.g., cholesterol),immunomodulatory agents (e.g., dimethyl dioctadecyl ammonium bromide DDA),polymers (e.g., polyacrylic acid such as CARBOPOL®), and Th2 stimulants (e.g.,glycolipids such as Bay R1005®), and combinations thereof, among many otheradjuvants known to those skilled in the art.
Nonl imiting examples of various combinations that can be used include atriterpenoid plus a sterol (e.g., Quil A/cholesterol, also known as QAC), a triterpenoidplus a sterol, an immunomodulatory agent, and a polymer (e.g., QuilA/cholesterol/DDA/CARBOPOL®, also known as QCDC), and a triterpenoid plus asterol, an immunomodulatory agent, a polymer, and a Th2 stimulant (e.g., QuilA/cholesterol/DDA/CARBOPOL®, and Bay R1005®, also known as QCDCR).
The amounts and concentrations of adjuvants and additives useful in thecontext of the present invention can readily be determined by the skilled artisan. Inone embodiment, the present invention contemplates immunogenic compositions andvaccines comprising from about 20 μg to about 2000 μg of adjuvant. In anotherembodiment, adjuvant is included in an amount from about 100 μg to about 1500 μg,or from about 250 μg to about 1000 μg, or from about 350 μg to about 750 μg. Inanother embodiment, adjuvant is included in an amount of about 500 μg/2 ml dose ofthe immunogenic composition or vaccine.
The immunogenic compositions and vaccines can also include antibiotics.
Such antibiotics include, but are not limited to, those from the classes ofaminoglycosides, carbapenems, cephalosporins, glycopeptides, macrolides,penicillins, polypeptides, quinolones, sulfonamides, and tetracyclines. In oneembodiment, the present invention contemplates immunogenic compositions andvaccines comprising from about 1 μg/ml to about 60 μg/ml of antibiotic. In anotherembodiment, the immunogenic compositions and vaccines comprise from about 5μg/ml to about 55 μg/ml of antibiotic, or from about 10 μg/ml to about 50 μg/ml ofantibiotic, or from about 15 μg/ml to about 45 μg/ml of antibiotic, or from about 20μg/ml to about 40 μg/ml of antibiotic, or from about 25 μg/ml to about 35 μg/ml ofantibiotic. In yet another embodiment, the immunogenic compositions and vaccinescomprise less than about 30 μg/ml of antibiotic.
Immunogenic compositions and vaccines encompassed by the presentinvention can include one or more polynucleotide molecules encoding for a virus orbacteria, or viral or bacterial protein. DNA or RNA molecules can be used inimmunogenic compositions or vaccines. The DNA or RNA molecule can beadministered absent other agents, or it can be administered together with an agentfacilitating cellular uptake (e.g., liposomes or cationic lipids). Total polynucleotide inthe immunogenic composition or vaccine will generally be between about 0.1 µg/mland about 5.0 mg/ml. In another embodiment, the total polynucleotide in theimmunogenic composition or vaccine can be from about 1 µg/ml and about 4.0mg/ml, or from about 10 µg/ml and about 3.0 mg/ml, or from about 100 µg/ml andabout 2.0 mg/ml. Vaccines and vaccination procedures that utilize nucleic acids (DNAor mRNA) have been well described in the art, for example, U. S. Pat. No. 5,703,055,U.S. Pat. No. 5,580,859, and U.S. Pat. No. 5,589,466, all of which are incorporatedherein by reference.
In addition to the viruses or bacteria described above, immunogeniccompositions and vaccines encompassed by the present invention can include otheradditional antigens. Antigens can be in the form of an inactivated whole or partialpreparation of the microorganism, or in the form of antigenic molecules obtained bygenetic engineering techniques or chemical synthesis. Other antigens appropriatefor use in accordance with the present invention include, but are not limited to, thosederived from pathogenic viruses such as canine distemper virus, canine herpesvirus,canine influenza virus, rabies virus, pathogenic bacteria such as Bordetellabronchiseptica, Leptospira bratislava, Leptospira canicola, Leptospira grippotyphosa,Leptospira icterohaemorrhagiae, Leptospira pomona, Leptospira hardjobovis,Porphyromonas spp., Bacteriodes spp., Borrelia spp., Streptococcus spp., includingStreptococcus equi subspecies zooepidemicus, Ehrlichia spp., Mycoplasma spp.,including Mycoplasma cynos, and Microsporum canis. Antigens can also be derivedfrom pathogenic fungi such as Candida, protozoa such as Cryptosporidium parvum,Neospora caninum, Toxoplasma gondii, Eimeria spp., Babesia spp., Giardia spp.,Leishmania spp., or helminths such as Taenia, Cuterebra, Echinococcus, andParagonimus spp.
Forms, Dosages, Routes of AdministrationImmunogenic compositions and vaccines encompassed by the presentinvention can be administered to animals to induce an effective immune responseagainst CIRDC. Accordingly, the present invention provides methods of stimulatingan effective immune response by administering to an animal a therapeuticallyeffective amount of an immunogenic composition or vaccine described herein.
Immunogenic compositions and vaccines described herein can beadministered to an animal to vaccinate the animal subject against CIRDC. Theimmunogenic compositions and vaccines can be administered to the animal toprevent or treat CIRDC in the animal. Accordingly, described herein are methods ofvaccinating an animal against CIRDC, and preventing or treating CIRDC, comprisingadministering to the animal a therapeutically effective amount of an immunogeniccomposition or vaccine described herein.
Immunogenic compositions and vaccines encompassed by the presentinvention can be made in various forms depending upon the route of administration.
For example, the immunogenic compositions and vaccines can be made in the formof sterile aqueous solutions or dispersions suitable for injectable use, or made inlyophilized forms using freezedrying techniques. L yophilized immunogeniccompositions and vaccines are typically maintained at about 4°C, and can bereconstituted in a stabilizing solution, e.g., saline or HEPES, with or without adjuvant.
Immunogenic compositions and vaccines can also be made in the form ofsuspensions or emulsions.
Immunogenic compositions and vaccines of the present invention include atherapeutically effective amount of one or more of the abovedescribedmicroorganisms. Purified viruses and/or bacteria can be used directly in animmunogenic composition or vaccine, or can be further attenuated, or inactivated.
Typically, an immunogenic composition or vaccine contains between about 1×1012 3and about 1×10 viral or bacterial particles, or between about 1×10 and about11 4 101×10 particles, or between about 1×10 and about 1×10 particles, or between9 6 8about 1×10 and about 1×10 particles, or between about 1×10 and about 1×10particles. The precise amount of a microorganism in an immunogenic composition orvaccine effective to provide a protective effect can be determined by a skilled artisan.
The pertactin antigen is present at between about 1 μg and about 30 μg. Moreparticularly, said pertactin is present at between about 5 μg and about 20 μg, moreparticular still, at between about 7 μg and about 15 μg, and even more particularly, atabout 5 μg, 10 μg, 15 μg or 20 μg.
The immunogenic compositions and vaccines generally comprise aveterinarilya cceptable carrier, in a volume of bet ween about 0.5 ml and about 5 ml.
In another embodiment the volume of the carrier is between about 1 ml and about 4ml, or between about 2 ml and about 3 ml. In another embodiment, the volume of thecarrier is about 1 ml, or is about 2 ml, or is about 5 ml. Veterinarilya cceptablecarriers suitable for use in immunogenic compositions and vaccines can be any ofthose described hereinabove.
Those skilled in the art can readily determine whether a virus or bacterianeeds to be attenuated or inactivated before administration. In another embodimentof the present invention, a virus or bacterium can be administered directly to ananimal without additional attenuation. The amount of a microorganism that istherapeutically effective can vary, depending on the particular microorganism used,the condition of the animal and/or the degree of infection, and can be determined bya skilled artisan.
In accordance with the methods of the present invention, a single dose can beadministered to animals, or, alternatively, two or more inoculations can take placewith intervals of from about two to about ten weeks. Boosting regimens can berequired, and the dosage regimen can be adjusted to provide optimal immunization.
Those skilled in the art can readily determine the optimal administration regimen.
Immunogenic compositions and vaccines can be administered directly into thebloodstream, into muscle, into an internal organ, or under the skin. Suitable meansfor parenteral administration include intravenous, intraarterial, intramuscular, andsubcutaneous administration. Suitable devices for parenteral administration includeneedle (including microneedle) injectors and needlef ree injectors.
Parenteral formulations are typically aqueous solutions which can containexcipients such as salts, carbohydrates, proteins, and buffering agents (preferably toa pH of from about 3 to about 9, or from about 4 to about 8, or from about 5 to about7.5, or from about 6 to about 7.5, or about 7 to about 7.5), but, for some applications,they can be more suitably formulated as a sterile nonaqueous solution or as a driedform to be used in conjunction with a suitable vehicle such as sterile, pyrogenf reewater or saline.
The preparation of parenteral formulations under sterile conditions, forexample, by lyophilization, can readily be accomplished using standardpharmaceutical techniques well known to those skilled in the art.
The solubility of materials used in the preparation of parenteral solutions canbe increased by the use of appropriate formulation techniques known to the skilledartisan, such as the incorporation of solubilityen hancing agents, including buffers,salts, surfactants, liposomes, cyclodextrins, and the like.
Compositions for parenteral administration can be formulated to be immediateor modified release. Modified release formulations include delayed, sustained,pulsed, controlled, targeted and programmed release. Thus, immunogeniccompositions and vaccines can be formulated as a solid, semis olid, or thixotropicliquid for administration as an implanted depot, providing modified release of theimmunogenic compositions and vaccines.
Other means of immunogenic composition or vaccine administration includedelivery by microneedle or needlef ree ( e.g. Powderject™, Bioject™, etc.) injection.
In cases where subcutaneous or intramuscular injection is used, an isotonicformulation is preferred. Generally, additives for isotonicity can include sodiumchloride, dextrose, mannitol, sorbitol, and lactose. In particular cases, isotonicsolutions such as phosphate buffered saline are used. The formulations can furtherencompass stabilizers such as gelatin and albumin. In some embodiments, a vasoconstrictive agent is added to the formulation. The pharmaceutical preparationsaccording to the present invention are generally provided sterile and pyrogenf ree.
However, it is well known by those skilled in the art that the formulations for thepharmaceutically accepted carrier are those pharmaceutical carriers approved in theregulations promulgated by the United States Department of Agriculture, orequivalent government agency in a foreign country such as Canada or Mexico, orany one of the European nations, for any canine vaccine, polypeptide (antigen)subunit immunogenic compositions and vaccines, recombinant virus vector vaccines,and DNA vaccines. Therefore, the pharmaceutically accepted carrier for commercialproduction of the immunogenic compositions or vaccines is a carrier that is alreadyapproved or will be approved by the appropriate government agency in the UnitedStates of America or foreign country. The immunogenic compositions and vaccinescan further be mixed with an adjuvant that is pharmaceutically acceptable. In certainformulations of the immunogenic compositions and vaccines, the immunogeniccomposition or vaccine is combined with other canine immunogenic compositions orvaccines to produce a polyvalent product that can protect canine against a widevariety of diseases caused by other canine pathogens.
The immunogenic compositions described herein can prevent infection from acanine respiratory pathogen or can prevent CIRDC in a canine for a period of aboutthree months or more. The compositions can prevent infection from said caninerespiratory pathogen or can prevent CIRDC in said canine for a period of about sixmonths or more. The compositions can prevent infection from said canine respiratorypathogen or can prevent CIRDC in said canine for a period of about one year.
Detection and Diagnostic MethodsThe extent and nature of the immune responses induced in the animal can beassessed by using a variety of techniques. For example, sera can be collected fromthe inoculated animals, and tested for the presence or absence of antibodies specificfor the immunogens. Detection of responding cytotoxic Tl ymphocytes (CTLs) inlymphoid tissues, indicative of the induction of a cellular immune response, can beachieved by assays such as T cell proliferation. The relevant techniques are welldescribed in the art.
KitsInasmuch as it may be desirable to administer an immunogenic composition orvaccine in combination with additional compositions or compounds for example, forthe purpose of treating a particular disease or condition it is within the scope of thepresent invention that an immunogenic composition or vaccine can conveniently beincluded in, or combined in, the form of a kit suitable for administration or coadministration of the compositions.
Thus, kits encompassed by the present invention can comprise one or moreseparate pharmaceutical compositions, at least one of which is an immunogeniccomposition or vaccine in accordance with the present invention, and a means forseparately retaining said compositions, such as a container, divided bottle, or dividedfoil packet. An example of such a kit is a syringe and needle, and the like. A kit of thepresent invention is particularly suitable for administering different dosage forms, forexample, oral or parenteral, for administering the separate compositions at differentdosage intervals, or for titrating the separate compositions against one another. Toassist one administering a composition encompassed by the present invention, the kittypically comprises directions for administration.
Another kit contemplated herein can comprise one or more reagents usefulfor the detection of an infected animal. The kit can include reagents for analyzing asample for the presence of whole microorganisms, polypeptides, epitopes orpolynucleotide sequences. The presence of virus, bacteria, polypeptides, orpolynucleotide sequences can be determined using antibodies, PCR, hybridization,and other detection methods known to those of skill in the art.
Another kit contemplated herein can provide reagents for the detection ofantibodies against particular epitopes. Such reagents are useful for analyzing asample for the presence of antibodies, and are readily known and available to one ofordinary skill in the art. The presence of antibodies can be determined usingstandard detection methods known to those of skill in the art.
In certain embodiments, the kits can include a set of printed instructions, or alabel indicating that the kit is useful for the detection of infected animals.
AntibodiesAntibodies can either be monoclonal, polyclonal, or recombinant. Theantibodies can be prepared against the immunogen or a portion thereof. Forexample, a synthetic peptide based on the amino acid sequence of the immunogen,or prepared recombinantly by cloning techniques, or the natural gene product and/orportions thereof can be isolated and used as the immunogen. Immunogens can beused to produce antibodies by standard antibody production technology well knownto those skilled in the art. Antibody fragments can also be prepared from theantibodies by methods known to those skilled in the art, and include Fab, F(ab') , andFv fragments.
In the production of antibodies, screening for the desired antibody can beaccomplished by standard methods in immunology known in the art. In general,ELISAs and Western blotting are the preferred types of immunoassays. Both assaysare well known to those skilled in the art. Both polyclonal and monoclonal antibodiescan be used in the assays. The antibody can be bound to a solid support substrate,conjugated with a detectable moiety, or be both bound and conjugated as is wellknown in the art. The binding of antibodies to a solid support substrate is also wellknown in the art. The detectable moieties contemplated for use in the presentinvention can include, but are not limited to, fluorescent, metallic, enzymatic andradioactive markers such as biotin, gold, ferritin, alkaline phosphatase, bgalactosidase, peroxidase, urease, fluorescein, rhodamine, tritium, C, andiodination.
The present invention is further illustrated by, but by no means limited to, thefollowing examples.
ExamplesExample 1. Evaluation of CRCoV-Containing VaccinesSixty 8 to 9weeko ld beagle dogs in good general h ealth were used in thestudy. All animals received physical examination upon arrival and again on studyday 2 or 1 . Animals were observed once daily for general health status from arrivalstudy day 8 to study day 39. Tympanic temperatures were collected starting onstudy day 1 prior to vaccination. Blood samples ( approximately 5 mL) for serologywere collected in SST tubes on study days 0 and 21 prior to each vaccination.
The CRCoV vaccine strain was derived from strain CRCoV.669, depositedwith the ATCC as PTA1 1444 in compliance with Budap est Treaty on theInternational Recognition of the Deposit of Microorganisms for the Purposes ofPatent Procedure. The CIV vaccine strain was derived from that deposited with theATCC as PTA7 694. The CPIV and CAV2 isolates were derived from virus seedsused to formulate vaccines in the Vanguard® vaccine line (Pfizer). The antigenswere prepared at the highest passages of virus (Master Seed Virus+5). The vaccinecompositions contained an adjuvant consisting of Quil A (20 ug), cholesterol (20 ug),dimethyl dioctadecyl ammonium bromide (DDA; 10 ug), and Carbopol® (a polyacrylicacid; 0.05% v/v). The CRCoV antigen was formulated to target 1.3 relative antigenunits (RAU) per dose. Experimental vaccines were tested for sterility.
A heterologous CRCoV isolate (“Max” strain; passage 1) was used as thechallenge material. The virus stock material was propagated and titered on HRT18Gcells, and was determined to have a titer of 10 TCID /mL. This challenge materialwas tested and confirmed satisfactory for sterility, and free of mycoplasma orcanine/feline extraneous agents.
One animal was vaccinated on day 21; all remaining animals were vaccinatedon study day 22. Animals were vaccinated subcutaneously with the appropriatevaccine or placebo according to the study design shown in Table 1. The firstvaccination was administered in the right shoulder region (study day 0) and thesecond vaccination was administered in the left shoulder region (study day 22).
Table 1. Study DesignVaccination Challenge NecropsyGroup IVP N Study Study StudyDays Route Day Dose DayAdjuvanted PlaceboT01 QuilA/cholesterol/DDA/Carbopol 10 46(QC/DC)Adjuvanted PlaceboT02 10 56(QC/DC)CRCoV/CIV/CPIV/CAV2T03 10 0 and 10 46(QC/DC)SC 4222 TCIDCRCoV/CIV/CPIV/CAV2T04 10 56(QC/DC)CRCoV monovalent RTUT05 10 46(QC/DC; emulsified)CRCoV monovalent RTUT06 10 56(QC/DC; emulsified)Investigational Veterinary Product (IVP) was administered (SC) subcutaneously.
Challenge dose with CRCoV Max isolate at passage 1 intranasally.
RTU: Readytouse liquid vaccine.
After the first vaccination, animals were observed daily (from study days 1through 8) for post vaccination injection swelling. After the second vaccination,animals were observed daily for post vaccination injection swelling through day 29.
Observations were continued twice weekly for animals that had injection siteswelling/pain beyond the days listed above, until swelling/pain resolved. Tympanictemperatures were collected daily for one week after each vaccination.
Blood samples (approximately 8 mL) for serology were collected in SST tubeson study day 42 prior to challenge. Tympanic temperatures were collected on studydays 40, 41, and 42 prec hallenge. Two types of oro pharyngeal swabs (VTM [VirusTransport Medium] for virus isolation, and Amies for bacterial isolation) werecollected from each dog prior to challenge on study day 42. Animals were observedonce daily prec hallenge on study days 40, 41, and 42, for clinical signs of respiratorydisease to establish baseline values.
On study day 42, all animals were challenged intranasally (IN) with the CRCoVchallenge virus at a target challenge dose of 10 /mL/dog. All animals were sedatedprior to challenge administration by intravenous injection of Domitor®. Aftersedation, each animal received 1.0 mL of challenge virus, given approximately 0.5mL per nostril slowly using a syringe without a needle. After challengeadministration, sedation was reversed by an intramuscular injection of Antisedan®.
Tympanic temperatures, clinical observations, and oropharyngeal swabs werecollected daily postc hallenge from study day 42 to 56. Blood samples(approximately 5 mL) for serology were collected on study day 46 and study day 56(prior to necropsy).
At necropsy, the complete lung and trachea was aseptically removed andplaced on a sterile drape, and the lung lobes were evaluated grossly for lung lesions(consolidation). Each lung lobe was scored for percentage of lung consolidation.
One lung set had insufficient exsanguination, and was not evaluated. The tracheawas transected, the lumen evaluated for gross pathology, and any findings wererecorded.
After the lungs had been scored, the right caudal lung lobe was lavaged byflushing with approximately 30.0 mL of media for bacteriological analysis and virusisolation. A pair of tissue samples was collected from the trachea and the nasalcavity, one for virus isolation and the second for histopathology. The right cranial lunglobes were divided into three samples, including one for bacteriology sampling.
Blood for serology was collected on predetermined study days.
Results. All animals were confirmed by IFA testing to be negative forantibodies (IFA titer <40) against CRCoV before study day 0. Oropharyngeal swabsevaluated for CRCoV virus isolation confirmed that all animals were free of CRCoVon study day 0 prior to vaccination. Placebov accin ated controls remained CRCoVseronegative until study day 42. The group was confirmed CRCoVf ree by virusisolation on study day 42, indicating lack of extraneous CRCoV in the facility. Alldogs were confirmed to be free of Bordetella bronchiseptica on study day 42.
An ELISA assay was used to measure the CRCoV antigen concentration inthe vaccine as relative antigen unit (RAU) against a specific batch of CRCoVdesignated reference antigen. CRCoV antigen was determined to be 0.5 RAU/dose.
Following vaccination, the majority of the vaccinated dogs, including theplacebovaccinated group, developed an injection sw elling at the injection site. Forthe monovalent vaccinated group, the swelling sizes were generally small (2 cm orless in the longest dimension) in the majority of the vaccinates. These swellingsresolved within two weeks for the majority of the dogs. There was no pain or systemicreactions related to the vaccine in any of the vaccinated dogs. No clinical fever(>39.5°C) was observed, except in one dog, although the elevated temperature inthat dog was not related to vaccination (the temperature was collected prior to thesecond vaccination). These findings indicate that the monovalent vaccine causesonly injection swellings within what is expected for an adjuvanted vaccine.
Serum neutralization titers were tabulated and compared between groups. Allmonovalentv accinated dogs (100%) developed SN tite rs (GMT 371) three weeksafter the second vaccination, indicating active immunization. A strong postc hallenge(anamnestic) serumneutralizing response was measur ed on day 56 in thecombination vaccinated dogs (GMT 6,915) compared to the placebovaccinated dogs(GMT 471). These results were statistically significantly different, and indicate thatthe CRCoV vaccine antigen effectively stimulated and primed the immune responsesof dogs against CRCoV infection.
Following challenge, all placebov accinated animals (100%) shed virus in theiroropharyngeal secretions at least for one day between day 1 and day 6 postchallenge, indicating induction of CRCoV infection. The monovalent vaccinesignificantly reduced (p =0.0237) the mean number of days with oropharyngealshedding (2.1 days) when compared to placebo (3.3 days), indicating vaccineefficacy in reducing CRCoV infection.
All placebov accinated dogs (100%) tested positive for virus isolation on day 4postc hallenge in their trachea, nasal cavity, and lungs, indicating CRCoV infection ofthe respiratory organs. There was no virus isolated from any organ on day 14 postchallenge, suggesting a typical respiratory viral infection similar to canine influenza.
By contrast, the monovalent vaccine prevented infection in 90% and 50% of thevaccinated dogs’ lungs (pv alue <0.0001) and trache a (pv alue <0.0237),respectively. This indicates that the monovalent vaccine induced sufficient immunitythat prevented virus infection in these critical organs. There were no significantdifferences in the rate of nasal cavity infection between vaccinates and controls.
The CRCoV challenge caused only mild clinical signs under experimentalconditions. Ocular and nasal discharges and conjunctivitis were reported in dogsacross treatment groups. There were 5 animals reported with clinical fever (>39.5°C)during the post challenge period two in the placeb o groups, one in a monovalentvaccine group, and two in the combination vaccine groups.
Gross evaluation of the lungs, trachea, and nasal turbinates was performed onday 4 and 14 postc hallenge. There was no remarkabl e gross lesion reported, exceptthat two dogs one in T05, one in T01 had low leve ls of lung consolidation; Twodogs one in T01, one in T03 had focal areas of nec rosis in the nasal turbinates.
For histopathology, lung, trachea, and nasal cavity tissues were examined andscored. Depending on the extent of changes observed, a score (0 to 4) wasassigned. Changes attributable to the challenge were most notable in the nasalturbinates, then the trachea, and finally the lungs. This is consistent with arespiratory challenge virus that has its primary effect on the upper respiratory tract(nasal turbinates and trachea), with a subsequent and lesser effect on the lowerrespiratory tract (lung). This demonstrates that the CRCoV infection caused tissuepathology in the respiratory organs.
Previous studies have shown that ciliary damage in the trachea on day 4 postchallenge is a characteristic pathologic sequel of CRCoV infection. The data showedthat the monovalent vaccine prevented tracheal ciliary damage in 60% of the animalswhen compared to placebo vaccinated (30% normal animals), but the reduction wasnot significant (P= 0.1538). Diagnostic bacteriology performed on lungs and lunglavages confirmed that all animals were negative for Bordetella bronchiseptica,Pasteurella spp., Staphlyococcus intermedius and Streptococcus canis. Lung, lunglavage, or both were positive for Mycoplasma spp. in only 4 animals. This findingsuggests that the lesions were specific for, and resulting from, the virus infection.
In summary, all CRCoVv accinated dogs (100%) in T03 T 06 developed serumneutralizing titers three weeks after the second vaccination, indicating activeimmunization. The monovalent vaccine induced immune responses in the vaccinatesthat reduced virus shedding in oropharyngeal secretions and in respiratory organs. Italso reduced tracheal ciliary damage in vaccinates compared to placebov accinatedcontrols. The histopathological examination showed that the monovalent vaccineprevented tracheal ciliary damage in 60% of the animals when compared to placebovaccinated animals (30%).
Example 2. Efficacy Testing of a Bivalent CRCoV/CIV Vaccine in DogsSixty 7 to 8week old beagle dogs in good general health were used in thestudy. All animals received a physical examination upon arrival on study day 9. Allanimals, with the exception of one dog that was removed on study day 7 , received asecond physical examination on study day 2, and de emed suitable for the study.
Animals were observed once daily for general health status from arrival studyday 7 to study day 39. Blood samples (approximate ly 6 mL) for serology werecollected in serum separation tubes (SST) on study days 0 and 21 prior to eachvaccination. Two sets of nasal swabs one for CRCo V and one for CIV virusisolation were collected from each dog prior to va ccination on Day 0 to confirmfreedom from CRCoV and CIV. Tympanic temperature was collected anddocumented on Days 1 and 0 prior to vaccination, t o establish a baseline prior tovaccination. Tympanic temperatures were collected prior to second vaccination onDay 21. Animals were palpated on the shoulder region on study days 0 and 21 priorto vaccination, to ensure that no preexisting lesi ons were present on the injectionsite area.
One dog in T04 was removed from the study due to respiratory distress onstudy day 7. One dog in T05 was removed from the s tudy due to respiratory distresson study day 0 prior to vaccination. Additionally, two animals were removed from thestudy posti nclusion due to conditions unrelated to the conduct of the study. One dogin T06 was removed from study on study day 21 prior to receiving the secondvaccination due to respiratory distress. One dog in T02 was removed from the studyon day 21 prior to receiving the second vaccination, due to unresolvedkeratoconjunctivitus and prolapsed nictitans.
Two bivalent vaccines were prepared, an inactivated CRCoV/inactivated CIVvaccine adjuvanted with Emulsigen® at 5% v/v, and an inactivatedCRCoV/inactivated CIV vaccine adjuvanted with Rehydragel™ at 5% v/v (Table 2).
The CRCoV vaccine strain was derived from that deposited with the ATCC as PTA11444. The CIV vaccine strain was derived from what was deposited with the ATCCas PTA7 694. Both antigen bulks used to make the v accines were produced atmaximum passage of virus and cells, to meet immunogenicity requirements. TheCRCoV antigen was formulated to target 1.55 RAU/dose. The CIV antigen wasformulated to target 640 HA Units/dose.
Table 3. Study DesignVaccination Challenge NecropsyGroup IVP NStudy StudyDays Route Day Dose Study DayT01 Saline 10 46T02 Saline 9 56CRCoVC IVT03 10 46% AlOHCRCoVC IVT04 9 56% AlOH0 and 21 SC 42TCIDCRCoVC IV 50T05 5% 9 46Emulsigen®CRCoVC IVT06 5% 9 56Emulsigen®Investigational Veterinary Product (IVP) was administered subcutaneously (SC).
Target challenge dose of CRCoV Max isolate (passage 1), administered intranasally.
AlOH: Aluminum hydroxide gelA heterologous CRCoV isolate (“Max” strain; passage 1) was used as thechallenge material. The virus stock material was propagated and titrated on HRT18Gcells and determined to have a titer of 10 TCID /mL. This challenge material wastested and confirmed satisfactory for sterility testing, being free of mycoplasma andcanine/feline extraneous agents.
Animals were vaccinated with the appropriate vaccine or placebo on Days 0and 21 (Table 2.). The first vaccination was administered in the right shoulder regionon Day 0, and the second vaccination was administered in the left shoulder region onDay 21.
Animals were observed daily for injection swelling/pain after first vaccinationfrom study days 0 to 8, and thereafter on study days 12, 15, 19, 21, 22, and 26. Onstudy day 8, swelling observations for 18 animals were inadvertently not recorded.
On study day 21, extra observations for right shoulder (first dose vaccination)observations were recorded for some animals.
After vaccination on study day 21, animals were observed daily for injectionswelling/pain post vaccination on study days 21 to 29, and thereafter on study days33, 36, and 40. All swellings resulting from the second vaccination were resolved bystudy Day 40. Tympanic temperatures were collected on Vaccination Days 0 to 7 and21 to 28, approximately 3 hours following each vaccination.
Blood samples (approximately 6 mL) for serology were collected in SST tubeson study day 42 prior to challenge. Also prior to challenge, tympanic temperatureswere collected on study days 40, 41, and 42, to establish baseline values. Two typesof nasal swabs (VTM for CRCoV virus isolation; Amies for bacterial isolation) werecollected from each dog prior to challenge on study day 42. Animals were observedonce daily prec hallenge on study days 40, 41, and 42 for clinical signs of respiratorydisease, to establish baseline values.
Each group of six dogs from all treatment groups was administered thechallenge virus by aerosolization of 19 mL of challenge material in the Plexiglasschamber for approximately 30 minutes. The volume of challenge virus nebulized inthe chamber was adjusted proportionally when less than six dogs were challenged ata time. Virus titration performed on CRCoV challenge samples collected afterchallenge administration confirmed that the amount of live challenge virusaerosolized in the chamber contained 10 TCID / mL.Post challenge, tympanictemperatures, clinical observations, and nasal swabs (Sterile Dacron Swabs, Puritan258 061 PD) for virus isolation (VTM tubes) were co llected daily from dogs fromstudy day 42 to 56. Blood samples (approximately 6 mL) for serology were collectedon study day 46 and study day 56 prior to necropsy.
At necropsy, the complete lung and trachea were aseptically removed andplaced on a sterile drape. The lung lobes were evaluated grossly for lung lesions(consolidation). Each lung lobe was scored for percentage of lung consolidation.
Lung sets from two animals had insufficient exsanguination, and could not beevaluated and scored. The trachea was transected, the lumen evaluated for grosspathology, and any findings were recorded. After the lungs had been scored, eachright caudal lung lobe was lavaged by flushing with approximately 30.0 mL of VTM(no antibiotic) for diagnostic bacteriological analysis and for virus isolation.
After the lungs were scored, tissue samples were collected from the trachea,and nasal cavity, and the whole left middle lung lobe was collected forhistopathology. Tissue samples were collected from the trachea, the nasal cavity,and right cranial lung lobe for virus isolationand for bacteriology.
Blood for serology was collected on predetermined study days.
Nasal swabs (Amies transport medium without charcoal) were collected fromeach dog only on study day 42 (prior to challenge) for diagnostic bacteriology. Theseswabs were tested for the presence of Bordetella spp., Pasteurella spp.,Staphylococcus spp., Mycoplasma spp. and Streptococcus canis.
Results. Fiftynine beagle puppies were confirmed by IFA testing to benegative for antibodies (IFA titer <40) against CRCoV on study day 0 prior tovaccination. Serum samples were also tested by serum neutralization and confirmedto be negative (SN titer <20) for antibodies to CRCoV. Nasal swabs evaluated forCRCoV virus isolation confirmed that all animals were free of CRCoV virus on studyday 0 prior to vaccination. CIV virus and antibody testing on study day 0 confirmedthat the animals to be free of CIV virus and CIV HAI antibodies (HAI titer <8). Basedon these two criteria, the animals were confirmed susceptible, and therefore suitablefor evaluation of the efficacy and safety of CRCoV and CIV vaccines. Salinevaccinated controls remained CRCoV seronegative until study day 42. All animalswere confirmed CRCoVf ree by virus isolation study day 42, indicating lack ofextraneous CRCoV exposure in the facility. All dogs were confirmed to be free ofBordetella bronchiseptica on study day 42 (prec hallenge).
Dogs were vaccinated with two formulations containing inactivated CRCoVand inactivated CIV antigens, adjuvanted with either Emulsigen® or Rehydragel™.
CRCoV antigen potency in the vaccine was measured by a doublea ntibodysandwich ELISA, employing a CRCoVs pecific serum ne utralizing monoclonalantibody 41.1.1. Measured against a designated reference antigen, potency wasdetermined to be 1.14 RAU/dose. The guinea pig HAI titer of CIV was 955. (Passcriterion was an HAI titer >161.)Ten out of the 19 animals that received the Emulsigen® formulation (T05 andT06) developed measureable injection swelling after the first vaccination. There wasscratching reported in the majority of dogs immediately following vaccination. Pain totouch was reported in only 2 dogs. Except for one dog, the swellings in this groupwere all resolved by the next day. There was a slight numerical increase in injectionswelling in size and frequency after the second vaccination, but they were all withinwhat is expected as a typical reaction to an adjuvanted vaccine. There was nosystemic reaction reported in any of the vaccinated dogs, as confirmed by the lack ofclinical fever (<39.5° C). These findings indicate that this vaccine formulation is safeto administer to dogs at this age group, and the safety profile is within what isexpected for an adjuvanted vaccine.
The majority of dogs (T03 and T04) that received the Rehydragel™formulation developed injection swelling after each vaccination. The swellingsappeared three days after the first vaccination, with the majority of swellings resolvedby study day 19. A similar reaction was seen after the second vaccination, where themajority of swellings resolved by study day 36. The injection swellings were generallysmall in size, and typical of Alum adjuvant reactions. There was no pain and no feverreported, confirming the lack of systemic reaction to vaccination. These findingsindicate that this vaccine formulation is safe to administer to dogs at this age group,and the safety profile is within what is expected for an adjuvanted vaccine.
Serum neutralization titers were tabulated, and compared between groups(Figure 1). Both vaccine formulations induced serum neutralizing antibody (SN)responses in all the vaccinated dogs after the first dose, indicating activeimmunization (Figure 1). The geometric mean SN response (GMT for Rehydragel™= 552; GMT for Emulsigen® = 2030) increased after the second vaccination,indicating a booster effect of the second vaccination. Both vaccine formulationsresulted in a robust anamnestic SN response after challenge (GMT for Rehydragel™= 10,725 and Emulsigen® = 11,584 on study day 56 for the remaining dogs in thestudy), indicating an effective immune memory response. It is important to note thatthe antibody response to CRCoV was achieved in the presence of a CIV antigen,indicating lack of interference between the antigens in the bivalent vaccine.
Fiftys ix dogs remaining in the study were challeng ed on study day 42 byaerosolization. Postc hallenge nasal virus isolatio n demonstrated that all salinevaccinated dogs (100%) shed challenge virus for at least three days between days 1and 6 post challenge, indicating the infection of dogs by CRCoV, with a 4.5 meannumber of days of shedding (Figure 2). The two vaccine formulations significantlyreduced the virus shedding to 2.6 days (p<0.0001) and 3.4 days (p=0.0042) forRehydragel™ and Emulsigen®, respectively. These findings indicate that thevaccines induced efficacy that resulted in reduction of virus infection.
Tissue virus isolation data showed that 90100% of the dogs in the salinevaccinated group were positive for virus in their nasal cavity, trachea, and lungtissues on study day 4 postc hallenge, indicating i nfections of the respiratory organs(Figure 3). By contrast, both vaccines significantly reduced the percentage of animalspositive for virus isolation in the lungs (p<0.0001) and in the nasal cavity (p <0.002).
While both vaccines reduced virus isolation in the trachea (virus isolated from 70%for Rehydragel™ group and from 44% for Emulsigen® group), only the Emulsigen®formulation resulted in significant reduction of virus isolation when compared to thesaline controls (p=0.0089). There was no virus isolated from any animals on day 14postc hallenge, indicating that the CRCoV infection is rapid in entering and leavingthe respiratory tissues, a scenario similar to canine influenza. The virus isolation dataindicate that both vaccine formulations significantly reduced virus infection in dogs.
The CRCoV challenge caused only mild respiratory clinical signs underexperimental conditions. Ocular and nasal discharges were reported in dogs acrosstreatment groups.
Except for one animal on study day 41 (one day prior to challenge) in thesaline control group, all animals had normal temperatures prior to challenge. Therewere two animals in the saline control group reported with clinical fever afterchallenge. Both dogs had temperatures of 39.6° C on day 2 postc hallenge (studyday 44). One of those dogs showed fever again (40° C) on day 4 postc hallenge.
That dog received treatment for concurrent gastroenteritis. This may explain the feverresponse following CRCoV challenge in this dog, since this virus has not been shownpreviously to cause fever under experimental condition. There was no clinical feverreported in any of the vaccinated dogs.
Gross necropsy evaluation of the lungs, trachea, and nasal turbinates wasperformed on day 4 and 14 postc hallenge. There was no remarkable gross lesionreported, except for lung consolidation in two dogs from T05, two dogs from T01, andone dog from T02. The cause of these lesions was unclear, but unlikely due toCRCoV, since the lesions were not consistent, and CRCoV has not been shown tocause lung consolidation. Examination of the diagnostic bacteriology of the tissuesdid not suggest the involvement of any other pathogen.
The lung, trachea, and nasal cavity tissue sections were examined andscored. Depending on the extent of changes observed, a score (0 to 4) wasassigned. Previous studies conducted have shown that the ciliary damage in thetracheal epithelia on day 4 postc hallenge is a cha racteristic pathologic effectassociated with CRCoV infection. (Priestnall et al 2009)The histopathology datarevealed that 70% of salinev accinated dogs experie nced some degree of trachealciliatede pithelial damage on day 4 postc hallenge. By contrast, both vaccinesreduced the number of affected dogs to 40% for the Rehydragel™ (p=0.1184) and0% for the Emulsigen® (p=0.0003). This indicates that the vaccines induced efficacythat protected against or reduced the tracheal mucociliary damage, an importantinnate defense mechanism, in infected dogs.
To assess potential involvement of other respiratory pathogens in the study,animals were tested for diagnostic bacteriology prior to challenge (nasal swabs) andafter challenge (lung tissue/lavage). Results obtained demonstrated that the animalswere mostly free of other respiratory pathogens, indicating that the clinical outcomemeasured after challenge was due specifically to CRCoV infection.
In summary, all CRCoVC IVv accinated dogs (100%) de veloped CRCoVserum neutralizing antibody titers three weeks after the second vaccination,indicating active immunization followed by strong postchallenge anamnesticresponse, indicating good priming of the immune system. The two vaccineformulations significantly reduced viral shedding. Both vaccine formulationssignificantly reduced the percentage of animals positive for virus isolation in the lungs(p<0.0001) and in the nasal cavity (p<0.002). Both vaccines reduced virus isolation inthe trachea, albeit only the Emulsigen® formulation resulted in significant reduction ofvirus isolation when compared to the saline controls (p 0.0089). Both of the vaccinesalso reduced the number of tracheal ciliatedepithe lial affected dogs. Efficacy of theCRCoV antigen in these vaccines was achieved in the presence of CIV antigen,indicating lack of interference on the CRCoV by CIV fraction.
Example 3. Safety and Efficacy of Bordetella bronchiseptica-ContainingVaccines in DogsFifty (50) dogs, divided into 5 treatment groups, were selected for the study.
Animals were determined to be fit for the study based on a physical examination onDay 4,Blood samples (approximately 8 mL) for serology were collected in SST tubesfrom all animals on Study Days 2, 21 and 28 prior to each vaccination. The serumsamples collected on Day 2 were used to confirm an imals were free of B.bronchiseptica. Nasal swabs were collected prior to vaccination on Day 0, and testedfor the presence of B. bronchiseptica. Tympanic temperatures were collectedstarting on Day 4 , to establish a baseline prior t o vaccination.
Animals were vaccinated with the appropriate vaccine on Days 0, 21, and 28according to the study design shown in Table 4. The vaccines were administeredsubcutaneously to each dog in the right shoulder region for the first vaccination, andin the left shoulder region for the second vaccination.
Table 4. Study DesignVaccination ChallengeGroup IVP N Vol Study Study Target(mL) Days Route Day Dose/Dog Routebronschiseptica(inactivated) 0 andT01 10 1.0+ Pertactin 28(10µg)No Adjuvant0 andT02 Saline 10 1.0bronschiseptica(inactivated) 0 andT03 10 1.0+ Pertactin 21(10µg) IntranasalNo Adjuvant SC 56 10 (aerosol;CRCoV/CIV/CPIV chamber)/ CAV2rehydrated with0 andT04 bronschiseptica 10 1.0(inactivated)+ Pertactin(10µg)No AdjuvantCRCoV/CIV/CPIV/ CAV2 0 andT05 10 1.0rehydrated with 28water (diluent)Investigational Veterinary Product (IVP) was administered (SC) subcutaneously.
Target challenge dose of 10^9 organisms of Bordetella bronchiseptica strain.
All animals were observed on vaccination Days 0, 21, and 28 for injection sitereactions following vaccination. They were observed daily for injection reactions postvaccination from Days 1 to 7 and 2235. Tympanic t emperatures were collected onDays 0 to 7 and 21 to 35.
Blood samples (approximately 6 mL) for serology were collected on Day 55,one day prior to challenge. Tympanic temperatures were collected on Days 54, 55,and 56 prior to challenge. Nasal swabs were collected on Day 55, one day prior tochallenge, and tested for the presence of B. bronchiseptica. Animals were observedtwice daily (a.m. and p.m.), approximately 30 minutes each session on Days 54 and55, and in the a.m. on Day 56, for clinical signs of respiratory disease, in order toestablish baseline values.
Bordetella bronchiseptica challenge strain was used to prepare a targetchallenge dose of 10 CFU/4 mL/dog. On Day 56, dogs from all treatment groupswere challenged intranasally with B. bronchiseptica by aerosolization in a Plexiglaschamber for a total of 30 minutes for each pen challenged. Five dogs from the samepen (one from each treatment group) were challenged at a time.
Tympanic temperatures was recorded once daily after challenge from Days 56to 77. Clinical observations were performed twice daily (a.m. and p.m.), forapproximately 30 minutes in each room per each session, from Day 56 and until Day76 and once (a.m.) on Day 77. Briefly, cough, nasal discharge, sneeze, oculardischarge, retch, and depression were observed using the following scoring system:Absent (0), Mild (1), Moderate (2), and Severe (3). Nasal swabs were collected onDays 59, 62, 66, 69, 74, 76 and 77, to determine shedding of challenge organisms.
Blood samples (approximately 6 mL) for serology were collected on Day 77.
Nasal swabs for isolation of B. bronchiseptica were collected using swabs andtransport media.
Agglutinating antibodies to B. bronchiseptica were determined by the MicroAgglutination Test (MAT). Serum samples from treatment groups T04 and T05 fromDays 0, 28, 55, and 77 were titrated for CRCoV antibodies by serum neutralizationand IFA, and for CIV by HAI. B. bronchiseptica isolation from nasal swabs wasperformed according to standard procedure. Each sample was tested qualitativelyfor the presence or absence of bacteria.
Results. Fifty (50) healthy approximately 8weeko ld beagle puppies wereconfirmed by nasal swab culture isolation to be free of B. bronchiseptica organismson Day 0. Serum samples evaluated for B. bronchiseptica agglutinating antibodiesby the MAT confirmed that all puppies were susceptible with MAT titers of ≤8 onDay 2.
All experimental vaccines evaluated in this study produced mild to no injectionswellings after the first vaccination. Injection swellings were limited to study day 0 forthe majority of vaccinates. Mild to no injection swellings were also reported after thesecond vaccination. The injection site swellings when they occurred, resolvedbetween one to three days after the second vaccination. Scratching was reportedpredominantly in the 5way combination group (T04). There was no clinical feverreported after vaccinations. There were no injection swellings reported in the salinegroup. The data confirmed the safety of the vaccines.
The colony count performed before and after challenge inoculation confirmedthat an average of 1.45 x 10 CFU Bordetella per dog were aerosolized in thechamber. Challenge inoculation induced cough in all saline control dogs (T02) with amean percentage observation coughed of 43.5% and 12.2 days coughed. Treatmentgroup T05, vaccinated with 4way viral only (CRCoV/ CIV/CPIV/CAV2) withoutBordetella antigen developed cough similar to the saline control with a meanpercentage observation coughed of 43.4% and 12.2 days coughed. These findingsindicate that the challenge was adequate and consistent to evaluate the testvaccines.
Dogs in treatment group T01 vaccinated with the Bordetella vaccine weresignificantly protected against challenge (3.6 days coughed, p<0.0001) whencompared to the control group (12.2 days coughed). The same vaccine alsosignificantly protected dogs in T03 when given at 3weeks interval regimen (5.8 dayscoughed, p=0.0004). The reduction in cough scores in these two groups (T01 vs T03)was not significantly different (pv alue=0.1883) su ggesting that the level of protectionfor the vaccine given with a 3 or 4 weeks interval, is similar.
Dogs in T04 that received the nonadjuvanted 5way combination vaccinewere significantly (p=0.0016) protected against Bordetella challenge (6.6 dayscoughed) when compared to the saline controls (12.2 days coughed), and whencompared to T05 receiving the 4way viral (CRCoV/CI V/CPIV/CAV2) combination(12.1 days coughed, p=0.0019) indicating efficacy of the Bordetella fraction in thecombination vaccine lacking adjuvant.
Serological evaluation of the viral fractions in the 5way combination vaccinewas possible for only two fractions, the CIV and CRCoV, where dogs were confirmedseronegative on study day 2. CIV HAI response in the 4way vaccine group (T04)on study day 56 were numerically similar to that in the 5way vaccine group (T05)and indicate lack of interference by the Bordetella fraction on the CIV antigen.
CRCoV SN responses on study day 56 were numerically higher in the 4way vaccinegroup (T04) than in the 5way vaccine group (T05), indicating possible interferenceby the Bordetella on the CRCoV fraction. However, these findings are not conclusivesince these vaccines were not adjuvanted and the formulation was not optimized andCRCoV challenge was not conducted to test efficacy.
The monovalent Bordetella vaccine was confirmed to be safe and efficacious.
The efficacy of the monovalent vaccine was demonstrated when the vaccine wasgiven at 21 or 28d ay intervals. The Bordetella fraction was also shown to beefficacious when given in a 5way nona djuvanted co mbination vaccine.
Example 4. Multivalent serology studyForty dogs, approximately 8 weeks of age and in good general health, werepres creened for Bordetella bronchiseptica by Micro Agglutination Test (MAT), andfor canine respiratory coronavirus (CRCoV) by indirect fluorescent antibody assay(IFA). Serum neutralization (SN) was also used to evaluate antibody levels. On Day0, all dogs were negative for antibodies to Bordetella bronchiseptica as determinedby MAT (<16), and negative for antibodies to CRCoV as determined by IFA (<40). Alldogs were also free of Bordetella bronchiseptica and CRCoV, as determined bynasal swab isolation test prior to first vaccination (Day 0).
Dogs were divided into 5 treatment groups of 8 dogs each, and vaccinatedaccording to the study design shown in Table 1. The vaccines were administered toeach dog in the right shoulder region for the first vaccination, and in the left shoulderregion for the second vaccination.
Table 2. Study DesignInvestigational VaccinationTreatmentVeterinary ProductAdjuvant NGroup(IVP) Study Days RouteT01 CAV2/CPIV/CPV/L4 5% Rehydragel 8CAV2, CPI, CRCoV+T02 QCDC 8Bordetella, CIV1% EMA /CAV2, CPI, CRCoV+T03 3% Neocryl/ 8Bordetella, CIV Subcutaneously0 and 21% Emulsigen SA(SC)CAV2, CPI, CRCoV+T04 QCDC 8Bordetella, CIVCAV2, CPI, CRCoV+T05 QCDC 8Bordetella, CIVEMA= ethylene maleic anhydrideFollowing the second vaccination, due to complications, groups T04 and T05were removed from the study. Dogs in the remaining groups (T01, T02, and T03)were observed daily for post vaccination reactions, and monitored for body(tympanic) temperature for 7 days after each vaccination. Blood samples werecollected from dogs on Days 0, 21, 42 and 56 to measure antibody responses.
Serum samples from Day 0, 21, 42 and 56 were tested for agglutinatingantibodies to Bordetella bronchiseptica by the MAT assay. Serum samples from thesame days were also titrated for CRCoV antibodies by serum neutralization, for CIVby HAI, and for CAV2 and CPI antibodies by serum n eutralization. Geometic meanantibody titers were obtained for each treatment group.
The test vaccines in groups T02 and T03 induced antibody responses in all(100%) the vaccinated dogs after the second dose, indicating active immunizationagainst the viral antigens. The antibody response increased after the secondvaccination in the majority of vaccinated dogs, indicating a booster effect of thesecond vaccination. It is important to note that the antibody responses among theviral fractions was achieved in the presence of multiple viral and bacterial (B.bronchiseptica) antigens, indicating lack of immunological interference. The MATserology is not correlative to protection against Bordetella, but is rather a valuablescreening tool to enroll suitable study animals. In conclusion, based on theimmunological response in vaccinated dogs, efficacy of the viral antigens is predictedin the 5 way multivalent vaccine.
Example 5. Duration of Immunity StudyThe purpose of this study is to demonstrate the duration of immunity of amultivalent respiratory combination vaccine in dogs. The vaccine contains thefollowing antigenic components: modifiedlive CAV 2, modifiedlive CPIV, inactivatedCIV, inactivated CRCoV and a Bordetella bronchiseptica extract supplemented with arecombinant antigen, either pertactin, Bsp22, or both.
All animals are in good general health, and have not received any vaccinationsfor any of the pathogens for which the vaccine is designed to protect against. Dogsare divided into multiple sets of treatment groups. Each set consists of two treatmentgroups, a control group receiving a placebo vaccine, and a vaccinate group receivingthe test vaccine. Animals are vaccinated twice, approximately 24 weeks apart. Theyare observed for injection site reactions following each vaccination.
Approximately 312 months following vaccination, ea ch set of two treatmentgroups (vaccinates and controls) are challenged with one of the pathogens for whichthe vaccine is designed to protect against. Clinical observations are performedleading up to and following challenge. Nasal swabs for isolation of the challengepathogen are collected during the post challenge period. Blood from each animal iscollected for obtaining serum, which is used for subsequent analytical analysis.
Clinical signs of respiratory disease, pathogen shedding post challenge, andserological responses are used as criteria to judge the efficacy of vaccines.
All of the foregoing references are hereby incorporated by reference as if setforth fully herein.
Having thus described in detail various embodiments of the present invention,it is to be understood that the invention defined by the appended claims is not to belimited to particular details set forth in the above description as many apparentvariations thereof are possible without departing from the spirit or scope of thepresent invention.