Injectable vaccines against canine respiratory diseases

Abstract

Provided is an injectable vaccine comprising: a heat treated purified whole cell extract of B bronchiseptica , wherein this extract has been heated for 10 to 60 minutes at 45°C to 65°C; and p68 pertactin protein. Methods of making and methods of using the vaccine are also provided.

Description

ZP000502 INJECTABLE VACCINES AGAINST CANINE RESPIRATORY DISEASES FIELD OF INVENTION

[0001] This invention is generally in the field of injectable canine vaccines. BACKGROUND

[0002] Canine infectious respiratory disease complex (CIRDC) is a highly contagious disease that is common in dogs housed in crowded conditions, such as re-homing centers and boarding or training kennels. Many dogs suffer only from a mild cough and recover after a short time. However in some cases, a severe bronchopneumonia can develop.

[0003] The pathogenesis of CIRDC is considered to be multifactorial, involving several viruses and bacteria. Infectious agents known to be causative agents of CIRDC include canine influenza virus (CIV) (Crawford et al., Science, 310(5747):482-485, 2005), canine parainfluenzavirus (CPIV) (Binn et al., Exp. Biol. Med., 126:140-145, 1967), canine adenovirus serotype 2 (CAV-2) (Ditchfield et al., Can. Vet. J., 3:238-247, 1962), and the bacterium Bordetella bronchiseptica (Bemis et al., Lab. Anim. Sci., 29:48-52, 1977).

[0004] Regarding CIV, equine influenza virus has been recognized as a major respiratory pathogen in horses since about 1956. Disease symptoms caused by equine influenza virus can be severe and are often followed by secondary bacterial infections. An influenza virus, H3N8 equine influenza virus, is able to infect canines, with fatalities in some cases as high as 36 percent. More recently, H3N2 virus, previously found in poultry, evolved to infect canines.

[0005] Disease caused by CPIV is common in the upper respiratory tract. Disease caused by CPIV alone can be mild or subclinical, with signs becoming more severe if concurrent infection with other respiratory pathogens occurs.

[0006] CAV-2 causes respiratory disease which, in severe cases, can include pneumonia and bronchopneumonia.

[0007] Bordetella bronchiseptica (B bronchiseptica) has been reported as being a primary etiological agent in the respiratory disease tracheobronchitis or "kennel cough". It predisposes dogs to the influence of other respiratory agents, and frequently exists concurrently with them. Subunit vaccines, such as those involving the use of p68 protein of B bronchiseptica (pertactin),have been explored but to date have not been included in any commercial canine vaccines, possibly due to insufficient immunogenicity, adverse reactions, and / or formulation stability.

[0008] The pathology of CIRDC indicates that it is involved in lung damage and, in some cases, bronchopneumonia, but it is distinct from kennel cough (primary etiological agent: B. bronchiseptica) which mainly involves upper respiratory tract changes. Kennel cough is a milder syndrome than CIRDC, and does not have the wide range of pathology noted in CIRDC. CIRDC is also distinguished by an increased severity and mortality.

[0009] CIRDC is rarely fatal, but it delays re-homing of dogs at rescue centers, disrupts schedules in training kennels, and results in considerable treatment costs and welfare concerns. Vaccines are available against some of the infectious agents associated with CIRDC. However, despite the use of these vaccines, CIRDC is still prevalent world-wide, possibly due to the lack of efficacious vaccines against all the infectious agents involved in CIRDC.

[0010] Oral vaccines against B bronchiseptica or against a combination of CPIV and B bronchiseptica have been commercially available in the U.S. However, due to aggressive nature of some dogs, a need exists for an injectable vaccine that protects dogs against CIRDC.

[0011] Accordingly, there remains a need for an immunogenic compositions and vaccines, capable of being safely administered to a canine, which provides efficient immunoprotection against the agents that cause CIRDC without deleterious side effects or interference with other antigens in a combination vaccine. The present disclosure fulfils these and other related needs. SUMMARY OF INVENTION

[0012] The instant invention addresses these and other needs by providing, in the first aspect, an injectable vaccine comprising: a heat treated purified whole cell extract of B bronchiseptica, wherein this extract has been heated for 10 to 60 minutes at 45°C to 65°C; and p68 pertactin protein, wherein the amount of said p68 pertactin protein is about 1 µg to about 30 µg per dose and the amount of Bsp22 in said B bronchiseptica extract is about 0.1 to about 5 µg per dose.

[0013] In certain embodiments of this first aspect, the whole cell extract of B bronchiseptica is prepared by the method comprising the steps of: culturing B bronchiseptica cells in liquid media; subjecting said cultured B bronchiseptica cells to extraction comprising incubating said cultured B bronchiseptica cells at pH of 9.5 to 10.5 and in 1M salt solution for 16-96 hours, therebyproducing a whole cell extract of B bronchiseptica; separating the treated media comprising said whole cell B bronchiseptica extract from cells and cell debris, thereby producing a purified whole cell extract of B bronchiseptica; and heating said whole cells B bronchiseptica extract for 10 to 60 minutes at 45°C to 65°C, thereby producing the heat treated purified whole cell extract of B bronchiseptica.

[0014] In certain embodiments, the extraction is conducted at temperature of 2-10 °C and said whole cell bacterial extract is heat treated at about 30 to about 35 minutes for about 56 to 58 °C.

[0015] In certain embodiments, the 1M salt solution further comprises 0.05M to about 0.2 M glycine.

[0016] In certain embodiments, said vaccine comprises about 6 to about 10 µg of p68 pertactin per dose. More preferably, vaccine also comprises at least 0.3 µg of purified whole cell extract of B bronchiseptica per dose.

[0017] The vaccine according to any of the embodiments of the first aspect as described above, further comprises a Canine Influenza antigen, a Canine Parainfluenza antigen, or both. In certain embodiments, the Canine Influenza antigen comprises an inactivated canine influenza virus of H3N2 type, H3N8 type, or a combination thereof. In further embodiments, the canine parainfluenza antigen is a modified live canine parainfluenza virus.

[0018] In the second aspect, the disclosure provides a method of preparing the vaccine according to any embodiment of the first aspect, the method comprising: culturing B bronchiseptica cells in liquid media; subjecting said cultured B bronchiseptica to extraction comprising incubating said cultured B bronchiseptica cells at pH of 9.5 to 10.5 and in 1M salt solution for 16-96 hours, wherein further said solution further comprises about 0.05M to about 0.2 M glycine, thereby producing the whole cell extract of B bronchiseptica; thereby producing the whole cell extract of B bronchiseptica; separating the treated media comprising said whole cell bacterial extract from B bronchiseptica cells and cell debris, thereby producing the purified whole cell extract of B bronchiseptica; heating the purified whole cell extract of B bronchiseptica for 10 to 60 minutes at 45°C to 65°C thereby producing the heat-treated purified whole cell extract of B bronchiseptica; and combining said heat treated purified whole cell extract of B bronchiseptica and p68 pertactin.

[0019] In the third aspect, the disclosure provides a method of eliciting protective immune response against B bronchiseptica in a dog, the method comprising injecting the vaccine according to any of the embodiments of the first aspect of the invention to said dog. DETAILED DESCRIPTION

[0020] For a better understanding of the invention, the following non-limiting definitions are provided:

[0021] The term “about” as applied to a reference number refers to the reference number plus or minus 10 percent of said value.

[0022] The term "culture", as used herein, means a population of cells or microorganisms growing in the absence of other species or types.

[0023] The phrase that a vaccine “does not induce self-trauma compared to placebo” refers to non-statistically-significant differences in the frequency of self-trauma among dogs vaccinated with the vaccine according to the invention (prepared using a method comprising the step of heating as described herein) versus dogs vaccinated with a placebo formulation.

[0024] The phrase that a vaccine “does not induce vocalization compared to placebo” refers to non-statistically-significant differences in vocalization frequencies among dogs vaccinated with the vaccine according to the invention (prepared using a method comprising the step of heating as described herein) versus dogs vaccinated with a placebo formulation.

[0025] “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 such composition given subsequent to the first dose, which can be but is not required to be the same vaccine or immunogenic composition as the first dose.

[0026] The term “Pertactin” or “p68 Pertactin” refers to B bronchiseptica p68 pertactin protein.

[0027] "Protection", "protecting", "protective immunity", and the like, as used herein with respect to a vaccine or other composition, means that the vaccine or composition prevents or reduces the symptoms of the disease caused by the organism from which the antigen(s) used in the vaccine or composition is derived. The terms "protection", "protecting", and the like, alsomean that the vaccine or composition can be used to "treat" the disease, or one or more symptoms of the disease that already exists in a subject.

[0028] "Subject", as used herein, refers to any animal having an immune system, which includes mammals, such as dogs.

[0029] "Therapeutically effective amount" refers to an amount of an antigen or vaccine that would induce an immune response in a subject receiving the antigen or vaccine which is adequate to prevent or reduce signs or symptoms of disease, including adverse health effects or complications thereof, caused by infection with a pathogen, such as a virus or a bacterium. Humoral immunity or cell-mediated immunity or both humoral and cell-mediated immunity may be induced. The immune response of an animal to a vaccine may be evaluated, e.g., indirectly through measurement of antibody titers, lymphocyte proliferation assays, or directly through monitoring signs and symptoms after challenge with wild type strain. The protective immunity conferred by a vaccine can be evaluated by measuring, e.g., reduction in clinical signs such as mortality, morbidity, temperature number, overall physical condition, and overall health and performance of the subject. The amount of a vaccine that is therapeutically effective may vary depending on the particular adjuvant used, the particular antigen used, or the condition of the subject, and can be determined by one skilled in the art.

[0030] "Treating" refers to preventing a disorder, condition, or disease to which such term applies, or to preventing or reducing one or more symptoms of such disorder, condition, or disease.

[0031] "Vaccine" or "vaccine composition," as used herein, refers to an immunogenic composition containing the antigens and capable of eliciting specific immune response against the antigen. Administration of the vaccine to a subject results in a protective immune response, which can be completely protective or partially protective. The vaccine can be introduced directly into the subject by any known route of administration, including parenterally, perorally, and the like. The terms mean a composition which prevents or reduces an infection, or which prevents or reduces one or more signs or symptoms of infection. The protective effects of a vaccine composition against a pathogen are normally achieved by inducing in the subject an immune response. Generally speaking, abolished or reduced incidences of infection, amelioration of thesigns or symptoms, or accelerated elimination of the microorganism from the infected subjects are indicative of the protective effects of a vaccine composition.

[0032] "Veterinarily acceptable", as used herein, refers to substances which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of veterinary subjects without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit-to-risk ratio, and effective for their intended use.

[0033] "Veterinarily acceptable carrier", as used herein, refers to a carrier medium that does not interfere with the effectiveness of the biological activity of the active ingredient, and is not toxic to the veterinary subject to whom it is administered.

[0034] In a broad aspect, this disclosure provides an injectable vaccine comprising: a) a heat treated purified whole cell extract of Bordetella bronchiseptica (B bronchiseptica); b) p68 pertactin protein, wherein the amount of said p68 pertactin protein is about 5 µg to about 30 µg per dose, preferably, at least 8 µg per dose, or at least 12 µg per dose or at least 15 µg per dose, or at least 20 µg per dose or at least 25 µg per dose, and the amount of bsp22 in said heat treated purified whole cell extract of Bordetella bronchiseptica is about 0.3 to about 5 µg per dose. B bronchiseptica extract

[0035] The methods of culturing B bronchiseptica in liquid medium are well known in the art. For example, B bronchiseptica can be cultured in basal medium supplemented with cysteine, nicotinic acid an iron source, maintained at pH 7.0-7.6, preferably, about 7.2-7.4.

[0036] After B bronchiseptica reach the desired cell density, the cells are subjected to extraction, thereby producing a whole cell B bronchiseptica extract. Preferably, the extraction process comprises high-salt, high pH treatment, preferably in the presence of glycine, at final concentration of about 0.05 M to about 0.5 M, e.g., about 0.1M to about 0.2 M. The high salt component of the treatment may comprise addition of salt to the final concentration of about 1.0 M sodium chloride. The high pH component may comprise adjustment of pH to about 9.0 to about 11.0, more preferably, from about 9.5 to about 10.5, even more preferably, from about9.8 to about 10.2. The extraction may last about 16 to about 96 hours (e.g., about 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, about 84 hours, or 96 hours) at temperature of about 2 to about 10 °C, more preferably, at 2 °C to 8 °C. After the extraction step, the pH is adjusted to neutral (7.0 to 7.6, preferably 7.2 to 7.4) and the cellular debris are separated from the fluids and discarded.

[0037] The whole cell B bronchiseptica extract may be purified, e.g., by centrifugation, to separate the liquid from cells and cell debris. The preferred method comprises cell harvesting followed by extraction in high pH / high salt solution in the presence of glycine, as described above, adjustment of pH to 7.4, followed by centrifugation to remove cell debris, followed by sterile filtering and then followed by heat treatment. Th whole cell B bronchiseptica extract that has undergone the step of purification is referred to as a purified whole cell B bronchiseptica extract.

[0038] The purified whole cell B bronchiseptica extract is heat treated. Preferably, the heat treatment is for the time period of 10-60 minutes at 45-65°C, or 37°C for about 48 hours. One of the ordinary skill in the art should recognize that the duration of the heat treatment would decrease if the temperature at which the extract is treated is increased. Thus, if the heat treatment temperature is 65°C then the duration of the heat treatment should be 10-20 minutes. In contrast, if the heat treatment temperature is 45°C, then the duration of the heat treatment can be longer, e.g., 50-60 minutes.

[0039] In certain preferred embodiments, the heat treatment entails heating the whole cell B bronchiseptica extract for about 20 minutes at about 60°C, or for about 30 minutes at about 55°C, or for about 40 minutes at about 50°C. In the most preferred embodiment, the whole cell B bronchiseptica extract is heated for about 30 minutes at about 56°C.

[0040] The whole cell B bronchiseptica extract that has undergone the steps of extraction, purification, and heat treatment is referred to as the purified heat treated purified whole cell extract of B bronchiseptica.

[0041] The amount of the purified heat treated purified whole cell extract of B bronchiseptica per dose is measured by the amount of a marker protein such as, for example, Bsp22. Thus, a reference to the amount of the heat treated purified whole cell extract of B bronchiseptica perdose refers to the amount of Bsp22 protein. In certain embodiments, the amount of the heat treated purified whole cell extract of B bronchiseptica per dose (or, more precisely the amount of Bsp22 protein per dose) is at least 0.3 µg per dose to about 1.5 µg per dose, preferably from about 0.32 µg per dose to about 1.5 µg per dose, or from about 0.4 µg per dose to about 1 µg per dose of from about 0.4 to about 0.75 µg per dose or from about 0.45 to about 0.55 µg per dose or at about 0.48 µg per dose. Pertactin

[0042] In the vaccines according to the invention, the heat treated purified whole cell extract of B bronchiseptica is supplemented by p68 pertactin. In certain embodiments, p68 pertactin is the recombinant B bronchiseptica p68 antigen which is recognized by the p68-specific monoclonal antibody clone 2-7 (described in US 7,736,658, which is incorporated herein by reference) and in one preferred embodiment, has an amino acid sequence as set forth in US 7,736,658 or having homology thereto. The recombinant p68 pertactin antigen is preferably prepared in a soluble form, such that native-like structure is preserved or restored during processing.

[0043] Accordingly, one aspect of the invention provides a recombinant p68 that is substantially free (less than about 80 percent, 90 percent, 95 percent or even 99 percent) of aggregates. In 25 another embodiment the recombinant p68 is solubilized with urea, preferably about 0.1 M, 0.5 M, 1 M, 2 M, 3 M, or 6 M solution of urea. Thereafter, the p68 antigen can be purified, such as through column chromatography. One such solubilization process is described in Surinder et al., J. Bioscience and Bioengineering, v. 99(4), pgs 303-310 (2005). Pertactin antigens used herein also include lipidated forms. Examples of production of lipidated proteins is provided in Erdile et al., Infection and Immunity, (1993) v.61(1), p. 81-90, incorporate by reference. The methods disclosed therein can be used to prepare post-translationally modified pertactin proteins that contain an attached lipid moiety.

[0044] The pertactin antigen is present at between about 5 µg and about 30 µg. More particularly, said pertactin is present at between about 5 µg and about 20 µg, more particular still, at between about 7 µg and about 15 µg, and even more particularly, at about 5 µg, about 10 µg, about 15 µg or about 20 µg.Additional antigens

[0045] Preferably, the vaccines disclosed herein contain antigens against other pathogens. In certain embodiments the additional antigen is a CIV (Canine Influenza Virus) antigen, or a Canine Parainfluenza virus, or both. In certain embodiments, the Canine Influenza virus comprises an inactivated virus of H3N2 type or H3N8 type or both H3N2 and H3N8 Canine Influenza virus. In further embodiments, the vaccine may comprise Canine parainfluenza virus, most preferably in a modified live form. Adjuvants

[0046] In certain embodiments, the vaccines disclosed herein are not adjuvanted. In other embodiments, the vaccines may include a suitable adjuvant. Suitable adjuvants include, without limitations, aluminum, triterpenoid saponins, including Quil A or purified fractions thereof, preferably in combination with sterols such as for example cholesterol, and optionally, in combination with glycolipids, to form so-called ISCOMS.

[0047] In other embodiments, the adjuvants may include CpG containing immunostimulatory oligonucleotides, most preferably, P-class CpG oligonucleotides.

[0048] P-class CpGs are characterized by the presence of one or more TLR-9 activating motif(s) and two palindromes or two complementarity areas. Preferably, the one or more TLR-9 activating motifs are at the 5’ of the oligonucleotide and may be completely or partially be incorporated into the 5’ palindrome or the 5’ complementarity area. TLR-9 activating motifs are known and include, without limitations, TCG, TTCG, TTTCG, TYpR, TTYpR, TTTYpR, UCG, UUCG, UUUCG, TTT, or TTTT. The 5’ palindrome or the 5’ complementary area is at least 6 bases long. The 3’ palindrome or the 3’ complementary area is at least 8 bases long and is generally rich in C and G. These structural features of the P-class CpGs confer the ability to spontaneously self- assemble into concatamers either in vitro and / or in vivo.

[0049] In order to increase lipophilicity of the CpG oligonucleotides, at least one lipophilic substituted nucleotide analog may be included, preferably at the 5’ end of the oligonucleotide. The P-class immunostimulatory oligonucleotides may be modified according to techniques known in the art. For example, J-modification refers to iodo-modified nucleotides. E- modification refers to ethyl-modified nucleotide(s). Thus, E-modified P-class immunostimulatoryoligonucleotides are P-class immunostimulatory oligonucleotides, wherein at least one nucleotide (preferably 5’ nucleotide) is ethylated. Additional modifications include attachment of 6-nitro-benzimidazol, O-Methylation, modification with proynyl-dU, inosine modification, 2- bromovinyl attachment (preferably to uridine). The oligonucleotides modified by an addition of a lipophilic moiety are generally described in US 20100166780.

[0050] In certain embodiments, CpGs according to the invention comprise the modified backbone including, without limitations, phosphorothioate modifications, halogenations, alkylation (e.g., ethyl- or methyl-modifications), and phosphodiester modifications.

[0051] Adjuvanting combinations are also envisioned, including without limitations, a combination of Aluminum with the triterpenoid saponins (e.g., Quill A) and / or CpG, including without limitations, P-class CpG. Another preferred combination is triterpenoid saponins (e.g., Quill A), cholesterol and CpG, including without limitations, P-class CpG. Carriers

[0052] Other components of the compositions can include pharmaceutically acceptable excipients, such as carriers, solvents, and diluents, isotonic agents, buffering agents, stabilizers, preservatives, antibacterial agents, antifungal agents, and the like. Typical carriers, solvents, and diluents include water, saline, dextrose, ethanol, glycerol, oil, and the like. Representative isotonic agents include sodium chloride, dextrose, mannitol, sorbitol, lactose, and the like. Useful stabilizers include gelatin, albumin, and the like. The compositions can also contain antibiotics or preservatives, including, for example, gentamicin, merthiolate, or chlorocresol. The various classes of antibiotics or preservatives from which to select are well known to the skilled artisan. Methods of using

[0053] The vaccines comprising the combination of p68 pertactin and the heated purified whole cell extract of B bronchiseptica as described herein are suitable for administration to dogs that are at least 6 weeks old and may be 7 weeks old, 8 weeks old, 10 weeks old, 12, weeks old, or older. Preferably, the dog is 8 weeks old or older.

[0054] The vaccines comprising the combination of p68 pertactin and the heated purified whole cell extract of B bronchiseptica as described herein should be administered to dogs parenterally, preferably intramuscularly or subcutaneously, more preferably, subcutaneously. In certainembodiments, the vaccines comprising the combination of p68 pertactin and the heated purified whole cell extract of B bronchiseptica as described herein are administered in a first parenteral administration and in a second parenteral administration, wherein the second administration is 14-42, more preferably 14-35, more preferably 21-35, or more preferably 21-28 or about 21 days after or about 28 days after the first administration. In the most preferred set of embodiments, the dog is re-vaccinated with an annual dose of the vaccine, wherein the annual dose is administered about 1 year after the first dose or a preceding annual dose.

[0055] An important feature of the vaccines comprising the combination of p68 pertactin and the heated purified whole cell extract of B bronchiseptica as described herein is that said vaccines are safer than vaccines produced by a method the B bronchiseptica was grown in a liquid medium but without the heating step. As is discussed in further detail in the examples, administration of the vaccines disclosed herein and containing the purified whole cell extract of B bronchiseptica that was heated as described above result in the reduction of pain, as measured by vocalization. Preferably, administration of the vaccines containing the combination of p68 pertactin and the heated purified whole cell extract of B bronchiseptica as described herein results in decreased frequency and / or decreased duration of vocalization by the dogs, compared to the administration of a comparison vaccine wherein the purified whole cell extract of B bronchiseptica has not been heated. Preferably, the frequency and / or the duration of vocalization is decreased by 50%, more preferably by 60%, even more preferably by 70%, even more preferably by 80%.

[0056] In addition to the improved safety profiles, the vaccines comprising the combination of p68 pertactin and the heated purified whole cell extract of B bronchiseptica as described herein are highly efficient. There are no specific regulatory requirements for efficacy of B bronchiseptica vaccines. One criterion of efficacy accepted in the industry is that lower bound 95% confidence interval bound for match-pair prevented fraction for coughing on at least two consecutive days should be more than zero. In certain embodiments, the administration of the vaccines comprising the combination of p68 pertactin and the heated purified whole cell extract of B bronchiseptica as described herein result in the lower bound 95% confidence interval being more than 10%, preferably more than 20%, or more than 30% or more than 40% or more than 45%.Even more preferably, the matching pair prevention fraction is at least 60% or at least 70% or at least 80% or at least 90%.

[0057] In further preferred embodiments the vaccines comprising the combination of p68 pertactin and the heated purified whole cell extract of B bronchiseptica as described herein additionally decrease frequency, intensity or duration of at least one additional symptom of B bronchiseptica infection, including without limitations, depression, fever, ocular discharge, nasal discharge, cough on consecutive days, retching, sneezing.

[0058] The invention will now be described in the following examples. EXAMPLES Example 1 (comparative)

[0059] In this comparative example, safety of a vaccine prepared without the heating step is described.

[0060] Previous testing of a vaccine comprising unheated B bronchiseptica extract spiked with p68 pertactin in mice revealed that the administration of the vaccine resulted in unacceptable skin lesions. To exclude the possibility that the effect was species-specific, two vaccine preparations, T01 and T02 (two different preparations of a vaccine comprising B bronchiseptica extract and p68 subunit. The respective doses of the extract are subunit are the same in both preparations) were tested in dogs.

[0061] B bronchiceptica was comprised of cell harvesting followed by extraction in high pH / high salt solution in the presence of glycine, as described above, adjustment of pH to 7.4, followed by centrifugation to remove cell debris, sterile filtration and heat treatment. Concentration was measured by Bsp22 amount which was 0.460 µg per ml in each preparation. The vaccine was supplemented with 18 µg / mL p68. One ml of each vaccine was administered to the respective group.

[0062] Dogs received two vaccinations administered subcutaneously 21 days apart. The safety of the vaccine was evaluated by observation of any local or systemic reactions for 10 dayspost-each vaccination unless swelling or sensitivity to palpation persisted beyond the 10-day post-vaccination time period.

[0063] Frequency distributions of injection site swelling and pain were calculated for each treatment, location, and time point. Frequency distributions of injection site swellings (if present at least once) and pain (if present at least once were calculated for each treatment group and vaccination. Frequency distributions of injection site swellings (if present at least once during the study) and pain (if present at least once during the study were calculated for each treatment group.

[0064] The duration of injection site swellings and pain were calculated for each animal at each location. The volume of the injection site reactions, if present, was calculated using the following formula: ^ Volume = 6 x length x width x height

[0065] Injection site pain was observed approximately 24 hours following administration of the first vaccine in 4 dogs in T01 and 2 dogs in T02. The frequency distribution of injection site pain by treatment and vaccination is shown in Table 1. The duration of pain for all dogs that were observed to experience it was one day. No animals were observed to experience pain after the second vaccination. Table 1. Injection Site Pain Group Vaccination Result Total Observations NO Yes Number number % Number % T01 1 6 60.0 4 40.0 10 2 10 100.0 0 0 10 T02 1 8 80.0 2 20.0 10 2 10 100.0 0 0 10

[0066] Injection site swelling was observed in 80% of the dogs in T01 after the first vaccination and 70% of the dogs in T02 on at least one observation period between Days 1-10. One dog (T01) had a single observation on Day 14 of injection site swelling that occurred past Day 10 observations. Following second vaccination, 50% of dogs in T01 and 30% of dogs in T02experienced injection site swelling on at least one observation period between Days 21 (3-6 hour post-vaccination observation) and 31. The incidence and duration of injection site swelling are in Tables 2 and 3. Table 2. Injection Site swelling Group Vaccination Result Total Observations NO Yes Number number % Number % T01 1 2 20.0 8 80.0 10 2 3 30.0 7 70.0 10 T02 1 5 50.0 5 50.0 10 2 7 70.0 3 30.0 10 Table 3. Duration of Injection Site Swelling (days) Group VaccinationNumberof Standard Animals Mean Median Deviation Minimum Maximum T01 110 4.8 4.0 4.4 0 13 210 2.3 0.5 3.4 0 8T02 110 3.4 2.0 3.5 0 9 210 2.2 0.0 3.6 0 9

[0067] Injection site swelling lasted for up to 13 days after the first vaccination and up to 9 days following the second vaccination as described.

[0068] Scratching was observed in 2 dogs in T01 and 1 dog in T02 immediately following the first vaccination. Scratching was observed in 5 dogs in T01 and 4 dogs in T02 immediately following the second vaccination. Injection site pain was observed in 6 dogs (4 dogs in T01 and 2 dogs in T02) approximately 24 hours following the first vaccination. Injection site pain lasted for 1 day. No serious adverse events related to vaccination (anaphylaxis, lethargy, hives, facial swelling / edema, vomiting, depression, hair loss, skin discoloration, or lesions) were recorded during the course of the study.

[0069] Swelling at the injection sites post-vaccination was observed in both treatment groups. Following the first vaccination 8 animals (80%) in T01 and 5 animals (50%) in T02 were observed to have swelling at the vaccination site. Following the second vaccination, 7 animals (70%) in T01 and 3 animals (30%) in T02 were observed with swelling at the vaccination site.

[0070] The data gathered in this study demonstrate pain responses in minimum age purpose- bred dogs that are not compatible with the product concept. Example 2

[0071] The purpose of this study was to evaluate the immunogenicity of a Bordetella Bronchiseptica Bacterial Extract, Subunit Vaccine in 8-week-old puppies. Efficacy was judged by challenge with an aerosolized virulent Bordetella bronchiseptica three weeks after the second vaccination.

[0072] Forty (40) beagles, approximately 8 weeks of age on Day 0, were randomly assigned to two treatment groups, 20 dogs per treatment group, using a randomized complete block design (matched pair), with block based on dam and date of birth. Animals in treatment group T01 served as controls receiving a true placebo vaccine containing 0.063% PBS (LP) – 0.85% NaCl / 0.063% Phosphate Buffer Solution administered subcutaneously on Days 0 and 21 in the right and left shoulder areas, respectively.

[0073] Animals in treatment group T02 served as test vaccinates, receiving the Bordetella Bacterial Extract Subunit vaccine administered subcutaneously on Days 0 and 21 in the right and left shoulder areas, respectively.

[0074] B bronchiceptica was comprised of cell harvesting followed by extraction in high pH / high salt solution in the presence of glycine, as described above, adjustment of pH to 7.4, followed by centrifugation to remove cell debris, sterile filtration and heat treatment. Concentration was measured by Bsp22 amount which was 0.25 µg per ml in preparation. The vaccine was supplemented with 10.0 µg per ml of p68. One ml of vaccine was administered.

[0075] All dogs in both treatment groups were challenged with a virulent B. bronchiseptica (Bihr cat strain) via aerosolization at a target dose per dog of 1 x 108organism on Day 42 (actual dose per dog was 9.49 x 107).

[0076] Blood samples for serology (MAT) were collected from all animals upon arrival, Days 0 and 21 prior to each vaccination, Day 42, and Day 70. Nasal swabs for bacterial isolation of B. bronchiseptica were collected from all animals upon arrival, Day 0 prior to vaccination, Day 42, and every other day starting on Days 43 through 69, and Day 70. Arrival, Day 0 and Day 42 resultswere reported qualitatively, and Days 43-70 results were reported quantitatively. Injection site observations were collected prior to and following each vaccination on Days 0-10 and 21-31. Vaccination phase tympanic temperatures were collected on Day -1, Day 0 (prior to and 3-6 hours post-first vaccination), Days 1-12, Day 20, Day 21 (prior to and 3-6 hours post-second vaccination), and Days 22-31. Post-vaccination clinical observations occurred immediately following IVP administration and 3-6 hours post-each vaccination on Days 0 and 21. Challenge phase tympanic temperatures and clinical observations were collected on Days 41-70.

[0077] Agglutinating antibodies to B. bronchiseptica were determined by the Particulate Antigen (Micro) Agglutination Test (MAT) according to site procedures. Briefly, two-fold serial dilutions of test serum, known positive serum, negative serum were made in v bottom microtiter plate, using normal saline containing 0.05% Tween 20 as the diluent. Serum samples were added as 50 µL per well. A 50 µL of B. bronchiseptica antigen was added to each well and mixed for 60 seconds on microtiter plate mixer. The plates were incubated at 37 ± 2°C for 2 hours and then the plates were stored for 20-48 hours at 2-8°C. The plates were read visually on a mirror stand. The titer was expressed as the reciprocal of highest dilution showing complete agglutination.

[0078] Whether or not an animal coughed for two consecutive days post-challenge was calculated for each animal. A frequency distribution of coughing / not coughing for two consecutive days was calculated for each treatment. Coughing / not coughing for two or more consecutive days was analyzed with a generalized linear mixed model with a logit link function and binomial distribution if possible. The fixed effect in the model was treatment and the random effects were room and block within room. Treatment T01 was compared to treatment group T02 using a contrast. The least squares means, standard errors, and 95% confidence intervals were back-transformed to the original scale. The matched pair prevented fraction and its 95% confidence interval were calculated for coughing for at least two consecutive days for T02 relative to T01. Pairs were the blocks.

[0079] The percentage of observation periods post-challenge which an animal coughed and the duration of coughing (first observation day of coughing through the last observation day of coughing) post-challenge were calculated for each animal. Prior to analysis, the percentage of observation periods coughed were transformed with an arcsine square root transformation. Thetransformed percentage of observation periods and duration of coughing were analyzed with a general linear mixed model. The fixed effect in the model was treatment. The random effects in the model were room, block within room, and residual. Treatment T01 was compared to treatment group T02 using a contrast. The least square means, standard errors, and 95% confidence intervals were back-transformed for the transformed variables. Treatment minimums and maximums were calculated.

[0080] Antibody titers were logarithmically transformed prior to analysis. The transformed titers were analyzed using a general linear mixed model for repeated measures. The fixed effects in the model were treatment, time point and treatment by time point interaction. The random effects in the model were room, block within room, treatment by block within room interaction and residual. The treatment least square means, standard errors, and 95% confidence intervals for each time point were back-transformed. Treatment T01 was compared to treatment group T02 at each time point using contrasts. Treatment minimums and maximums were also calculated for each time point. Results

[0081] The study was valid. All animals were free of B. bronchiseptica on Day 0 and 42 (prior to challenge) by bacterial nasal swab isolation. All animals had MAT antibody titers of ≤8 on Day 0, and the control animals (T01) remained negative by MAT pre-challenge. The challenge was valid as 100% of the control animals developed respiratory clinical signs indicative of B. bronchiseptica infection and shed organism in their nasal secretions.

[0082] Sixteen of 20 (80%) dogs administered the placebo control (T01) coughed for 2 consecutive days post-challenge. This is in contrast to only 1 of 20 (5%) dogs administered the Bordetella bronchiseptica killed bacterin vaccine (T02) (Table 1). The matched-pair prevented fraction for coughing for two consecutive days was 93.75% with a 95% lower bound confidence interval of 45.50%. See Table .

[0083] The percentage of animals coughing for two consecutive days was significantly lower in the vaccinate group compared to the control group (p=0.0014).

[0084] There was a significant difference (p<0.0001) in the percent of observation periods of coughing between treatment groups, with the control animals (T01) being observed post-challenge coughing in 17.0% of observations compared to vaccinates (T02) with 0.9% (

[0085] Table 5.). Table 4. Frequency Distribution for Post-challenge Coughing for Two or More Consecutive Days by treatment Group Coughed Two Consecutive Days No Yes Total Treatment No. of Animals % No. of Animals % No. of Animals T01 4 20.0 16 80.0 20 T02 19 95.0 1 5.0 20 Table 5. Back-transformed Least Squares Means for Percentage of Observation Periods with Coughing Observed Post-challenge by Treatment Group Arsin Back-transformed Estimates in (Sqrt(Percentage)) Percentage Least 95% Number Squares Standard Standard Confidence Treatment of Animals Mean Error Mean Error Limits Range T01 20 0.41 0.037 17.0 2.73 (11.2, 23.9) 1.8 to 44.6 T02 20 0.10 0.021 0.9 0.41 (0.3, 2.0) 0 to 5.4

[0086] There was a significant difference (p<0.0001) between treatment groups for the duration of coughing. Control animals (T01) had a least square mean of 16.0 days of coughing compared to vaccinated animals (T02) with a mean duration of coughing of 3.2 days (Table 6). Table 6. Least Squares Means of Duration of Coughing in Days by Treatment Group Number Least 95% of Squares Standard Confidence Treatment Animals Mean Error Limits Range T01 20 16.0 1.78 (12.9, 19.1) 1 to 26Number Least 95% of Squares Standard Confidence Treatment Animals Mean Error Limits Range T02 20 3.2 1.24 (1, 5.3) 0 to 21

[0087] All animals in both treatment groups (100%) shed bacteria post-challenge (Table 7). Table 7. Frequency Distribution for Post-challenge Bacterial Nasal Shedding Post-challenge by Treatment Group Ever Isolated? Yes Total Treatment No. of Animals % No. of Animals T01 20 100.0 20 T02 20 100.0 20

[0088] There was a significant difference (p=0.0031) between treatment groups for the duration of post-challenge bacterial nasal shedding. Treatment group T01 (controls) had a least squares mean of 22.7 days of shedding compared to T02 (vaccinates) with a least squares mean of 17.4 days of shedding post-challenge (Table 8). Table 8. Least Squares Means of Duration of Bacterial Nasal Shedding in Days by Treatment Group Number Least 95% of Squares Standard Confidence Treatment Animals Mean Error Limits Range T01 20 22.7 1.14 (20.7, 24.6) 17 to 26 T02 20 17.4 1.71 (14.4, 20.3) 3 to 28

[0089] Nasal bacterial isolation had a significant difference in geometric means between treatment groups on Days 45 (p=0.0065), 47 (p=0.0003), 61 (p=0.0231), and 63 (p=0.0322).

[0090] There was also a significant difference (p=0.0234) between treatment groups for area under the curve for nasal bacterial isolation. The back-transformed least squares mean summary for area under the curve (AUC) for nasal bacterial isolation is listed in Table 9.Table 9. Back-transformed Least Squares Means of AUC for Nasal Bacterial Isolation Back-transformed No. of 95% 95% Geometric Standard Lower Upper Treatment Animals Mean Error CI CI Minimum Maximum T01 20 4669 1997.8 1979 11015 190 100605 T02 20 1312 561.6 556 3097 40 32740

[0091] There was a significant difference in antibody titers (p<0.0001) on Day 42 pre-challenge showing that the vaccinated animals produced titers against Bordetella bronchiseptica while the control animals did not. By the conclusion of the post-challenge period all animals (including the control animals) were positive for antibodies to B. bronchiseptica indicating exposure to challenge. A summary of the geometric mean titers by treatment group is listed in Table 10. Table 10. Geometric Mean Antibody Titers by Treatment Group Day of Study Treatment -10 0 21 42 70 T01 4.1 7.7 8.6 9.5 304.4 T02 4.1 7.0 10.2 50.2 247.3

[0092] No animals in any treatment group (0%) experienced anaphylaxis, convulsions, stinging, vomiting, wheals, or other clinical signs following either vaccination timepoint. One animal (5.00%) in T01 (controls) and one animal in T02 (vaccinates) was noted with self-trauma following first vaccination only. Vocalization at administration of IVP was observed in four animals (20.0%) in T01 (controls) following first vaccination and no animals (0%) following second vaccination. Three animals (15.0%) in T02 (vaccinates) vocalized following first and second vaccination. Frequency distributions of post-vaccination clinical signs by treatment group and vaccination timepoint are summarized in Table 11.Table 11. Frequency Distributions of Post-vaccination Clinical Signs by Treatment Group and Vaccination Time Point Ever Present? No Yes Total No. of No. of No. of Clinical Sign Treatment Vaccination Animals % Animals % Animals 1 19 95.00 1 5.00 20 T01 2 20 100.00 0 0.00 20 Self-Trauma 1 19 95.00 1 5.00 20 T02 2 20 100.00 0 0.00 20 1 16 80.00 4 20.00 20 T01 Vocalization at 2 20 100.00 0 0.00 20 administration 1 17 85.00 3 15.00 20 T02 2 17 85.00 3 15.00 20

[0093] The vaccine was considered to be safe and comparable to the placebo. Challenge Phase

[0094] Fever was the most prevalent clinical sign in both treatment groups post-challenge with 19 out of 20 animals (95.0%) in T01 (controls) and 15 out of 20 animals (75.0%) in T02 (vaccinates) exhibiting tympanic temperatures of ≥ 39.5°C. All animals (100%) in T01 were observed retching post-challenge compared to 8 animals (40.0%) in T02. Post-challenge changes in respiration were noted in 14 animals (70.0%) in T01 compared to 1 animal (5.0%) in T02. Sneezing post-challenge was observed in 12 animals (60.0%) in T01 compared to 4 animals (20.0%) in T02. Ocular discharge, scored 2 (moderate) or higher, was observed in 8 animals (40.0%) in T01 compared to 4 animals (20.0%) in T02 post-challenge. Nasal discharge, scored 2 (moderate) or higher, was observed in 9 animals (45.0%) in T01 compared to 1 animal (5.0%) in T02. No animals (0%) in either treatment group were noted with depression post-challenge. Frequency distributions of post-challenge clinical signs by treatment group are listed in Table 12. The frequency of post-challenge clinical signs of respiratory disease was decreased in the vaccinated animals.Table 12. Frequency distributions of post-challenge clinical signs by treatment group Ever Present Post-challenge? No Yes Total No. of No. of No. of Clinical Sign Treatment Animals % Animals % Animals T01 20 100.00 0 0.00 20 Depression T02 20 100.00 0 0.00 20 T01 1 5.00 19 95.00 20 Fever T02 5 25.00 15 75.00 20 T01 11 55.00 9 45.00 20 Nasal Discharge T02 19 95.00 1 5.00 20 T01 12 60.00 8 40.00 20 Ocular Discharge T02 16 80.00 4 20.00 20 T01 6 30.00 14 70.00 20 Respiration T02 19 95.00 1 5.00 20 T01 0 0.00 20 100.00 20 Retch T02 12 60.00 8 40.00 20 T01 8 40.00 12 60.00 20 Sneeze T02 16 80.00 4 20.00 20 Injection Sites

[0095] No animals (0%) in either treatment group were noted with pain with palpation following either vaccination timepoint. Following first vaccination, no animals (0%) in T01 (controls) had injection site swelling compared to three animals (15.0%) in T02 (vaccinates). Following second vaccination, one animal (5.0%) in T01 was observed with injection site swelling compared to two animals (10.0%) in T02. Frequency distributions of post-vaccination injection site observations by treatment group and vaccination time point are listed in Table 13.

[0096] The vaccine did not cause pain at the injection sites and produced minimal swelling. The vaccine is considered safe.Table 13. Frequency Distributions of Post-vaccination Injection Site Observations by Treatment Group and Vaccination Time Point Ever Present? No Yes Total Injection Site No. of No. of No. of % % Observation Treatment Vaccination Animals Animals Animals 1 20 100.00 0 0.00 20 T01 Pain with 2 20 100.00 0 0.00 20 Palpation 1 20 100.00 0 0.00 20 T02 2 20 100.00 0 0.00 20 1 20 100.00 0 0.00 20 T01 2 19 95.00 1 5.00 20 Swelling 1 17 85.00 3 15.00 20 T02 2 18 90.00 2 10.00 20 Conclusions

[0097] The study was valid. All animals were free of B. bronchiseptica on Day 0 and 42 (prior to challenge) by bacterial nasal swab isolation. All animals had MAT antibody titers of ≤16 on Day 0, and the control animals (T01) remained negative by MAT pre-challenge. All of the control animals developed respiratory clinical signs indicative of B. bronchiseptica infection and shed organism in their nasal secretions indicating robust challenge.

[0098] The vaccine was both safe and efficacious. No animals in either treatment group had pain with palpation post-vaccination. There were five animals in T02 (vaccinates) and one animal in T01 (controls) of post-vaccination injection site swelling. The matched-pair prevented fraction for coughing on at least two consecutive days was 93.75% with a lower bound of the 95% confidence interval of 45.50%. The efficacy criteria were met.

[0099] There was a significant difference in both the duration of nasal shedding (p=0.0031) and the area under the curve (p=0.0234) post-challenge. T02 also had significantly smaller proportion of animals with two consecutive days of coughing, mean percentage of observation periods coughing and shorter duration of coughing than T01. Animals in T02 had a serological response to vaccination and showed an increase in antibody titers in response to challenge.

[0100] All publications cited in the specification, both patent publications and non-patent publications, are indicative of the level of skill of those skilled in the art to which this invention pertains. All these publications are herein fully incorporated by reference to the same extent as if each individual publication were specifically and individually indicated as being incorporated by reference.

[0101] Although the invention herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present invention. It is therefore to be understood that numerous modifications may be made to the illustrative embodiments and that other arrangements may be devised without departing from the spirit and scope of the present invention as defined by the following claims.

Claims

CLAIMS 1. An injectable vaccine comprising: c) a heat treated purified whole cell extract of B bronchiseptica, wherein this extract has been heated for 10 to 60 minutes at 45°C to 65°C; d) p68 pertactin protein, wherein the amount of said p68 pertactin protein is about 1 µg to about 30 µg per dose and the amount of Bsp22 in said B bronchiseptica extract is about 0.1 to about 5 µg per dose.

2. The vaccine according to claim 1, wherein the whole cell extract of B bronchiseptica is prepared by the method comprising the steps of: a. culturing B bronchiseptica cells in liquid media; b. subjecting said cultured B bronchiseptica cells to extraction comprising incubating said cultured B bronchiseptica cells at pH of 9.5 to 10.5 and in 1M salt solution for 16-96 hours, thereby producing a whole cell extract of B bronchiseptica; c. separating the treated media comprising said whole cell B bronchiseptica extract from cells and cell debris, thereby producing a purified whole cell extract of B bronchiseptica; d. heating said whole cells B bronchiseptica extract for 10 to 60 minutes at 45°C to 65°C, thereby producing the heat treated purified whole cell extract of B bronchiseptica.

3. The vaccine of claim 2, wherein said shock treatment is at temperature of 2-10 °C.

4. The vaccine according to any one of claims 1-3, wherein said whole cell bacterial extract has been heated at about 30 to about 35 minutes for about 56 to 58 °C.

5. The vaccine according to any one of claims 1-4 wherein the step of heating follows the step of separation.

6. The vaccine according to any one of claims 1-5 wherein said 1M salt solution further comprises 0.05M to about 0.2 M glycine.

7. The vaccine according to any one of claims 1-6, wherein said vaccine does not induce self- trauma compared to placebo.

8. The vaccine according to any one of claims 1-7, wherein said vaccine does not induce vocalization compared to placebo.

9. The vaccine according to any one of claims 1-8, wherein said vaccine comprises about 6 to about 10 µg of p68 pertactin per dose.

10. The vaccine according to any one of claims 1-9, wherein said vaccine comprises at least 0.3 µg of purified whole cell extract of B bronchiseptica per dose.

11. The vaccine according to any one of claims 1-10, wherein administration of said vaccine to dogs results in lower bound 95% confidence interval bound greater than 10% for match-pair prevented fraction for coughing on at least two consecutive days.

12. The vaccine according to any one of claims 1-11, which, upon administration to dogs, results in lower bound 95% confidence interval bound greater than 30% for match-pair prevented fraction for coughing on at least two consecutive days.

13. The vaccine according to any one of claims 1-12, which, upon administration to dogs, results the matching pair prevention fraction is at least 60% for coughing on at least two consecutive days.

14. The vaccine according to any one of claims 1-13, which, upon administration to dogs, results the matching pair prevention fraction is at least 90% for coughing on at least two consecutive days.

15. The vaccine according to any one of claims 1-14 wherein said vaccine is not adjuvanted.

16. The vaccine according to any one of claims 1-15, wherein the vaccine further comprises a Canine Influenza antigen, a Canine Parainfluenza antigen, or both.

17. The vaccine according to claim 16 wherein the Canine Influenza antigen comprises an inactivated canine influenza virus of H3N2 type, H3N8 type, or a combination thereof.

18. The vaccine according to claim 17, wherein the canine parainfluenza antigen is a modified live canine parainfluenza virus.

19. A method of preparing the vaccine according to any one of claims 1-18, the method comprising: a. culturing B bronchiseptica cells in liquid media; b. subjecting said cultured B bronchiseptica to extraction comprising incubating said cultured B bronchiseptica cells at pH of 9.5 to 10.5 and in 1M salt solution for 16- 96 hours, wherein further said solution further comprises about 0.05M to about 0.2 M glycine, thereby producing the whole cell extract of B bronchiseptica; thereby producing the whole cell extract of B bronchiseptica; c. separating the treated media comprising said whole cell bacterial extract from B bronchiseptica cells and cell debris, thereby producing the purified whole cell extract of B bronchiseptica; d. heating the purified whole cell extract of B bronchiseptica for 10 to 60 minutes at 45°C to 65°C thereby producing the heat-treated purified whole cell extract of B bronchiseptica;e. combining said heat treated purified whole cell extract of B bronchiseptica and p68 pertactin.

20. A method of eliciting protective immune response against B bronchiseptica in a dog, the method comprising injecting the vaccine according to any one of claims 1-18 to said dog.