ORAL RESPIRATORY VACCINE

Abstract

The present invention is drawn to new oral live canine parainfluenza virus vaccines and related multivalent vaccines. Methods of using the vaccine alone or in combination with one or more other protective immunogens in multivalent vaccines are also provided.

Claims

1. A method of providing effective protection to a canine from upper respiratory diseases and infectious tracheobronchitis comprising orally administering to the canine a vaccine comprising a modified live canine parainfluenza (CPI) virus.

2. The method of claim 1, wherein the vaccine further comprises an avirulent live Bordetella bronchiseptica (B. bronchiseptica).

3. The method of claim 1, wherein the titer of the modified live CPI virus administered is greater than 6.0 log.sub.10HAID.sub.50/dose.

4. (canceled)

5. (canceled)

6. The method of claim 3, wherein the titer of the modified live CPI virus administered is 6.8 log.sub.10HAID.sub.50/dose to 9.5 log.sub.10HAID.sub.50/dose.

7. (canceled)

8. (canceled)

9. The method of claim 2, wherein the titer of the avirulent live B. bronchiseptica administered is equal to or greater than 1 × 10.sup.7 cfu/dose.

10. (canceled)

11. (canceled)

12. The method of claim 1, wherein the modified live CPI virus, has the ATCC accession No. PTA-126273.

13. The method of claim 2, wherein the avirulent live B. bronchiseptica has the ATCC accession No. PTA-126272.

14. The method of claim 1, wherein the vaccine is a single-dose vaccine.

15. (canceled)

16. The method of claim 1, wherein the vaccine is a non-adjuvanted vaccine.

17. (canceled)

18. The method of claim 2, wherein the vaccine further comprises a live attenuated immunogen selected from the group consisting of a live attenuated canine influenza virus, a live attenuated canine parvovirus, a live attenuated canine distemper virus, a live attenuated canine adenovirus type 2, a live attenuated respiratory canine coronavirus, a live attenuated canine pneumovirus, a live attenuated Mycoplasma cynos, a live attenuated Steptococcus equi zooepidemicus, and any combination thereof.

19. The method of claim 2, wherein the vaccine further comprises a live attenuated canine parvovirus, a live attenuated canine distemper virus, and a live attenuated canine adenovirus type 2.

20. The method of claim 1, wherein the vaccine is orally administered in a dose of 0.2 mL to 5 mL.

21. (canceled)

22. (canceled)

23. A vaccine for providing effective protection to a canine from upper respiratory diseases and infectious tracheobronchitis by oral administration comprising a titer of the modified live CPI virus equal to or greater than 6.0 log.sub.10RAID.sub.50/mL.

24. The vaccine of claim 23, further comprising an avirulent live Bordetella bronchiseptica (B. bronchiseptica).

25. The vaccine of claim 24, wherein the titer of the avirulent live B. bronchiseptica in the vaccine is 1 × 10.sup.7 cfu/mL to 1 × 10.sup.12 cfu/mL.

26.-28. (canceled)

29. The vaccine of claim 25 wherein the titer of the modified live CPI virus in the vaccine is 6.0 log.sub.10HAID.sub.50/mL to 9.5 log.sub.10HAID.sub.50/mL.

30. The vaccine of claim 29 wherein the modified live CPI virus has the ATCC accession No. PTA-126273.

31. The vaccine of claim 30, wherein the avirulent live B. bronchiseptica has the ATCC accession No. PTA-126272.

32.-35. (canceled)

36. The vaccine of claim 29, wherein the vaccine further comprises a live attenuated immunogen selected from the group consisting of a live attenuated canine influenza virus, a live attenuated canine parvovirus, a live attenuated canine distemper virus, a live attenuated canine adenovirus type 2, a live attenuated respiratory canine coronavirus, a live attenuated canine pneumovirus, a live attenuated Mycoplasma cynos, a live attenuated Steptococcus equi zooepidemicus, and any combination thereof.

37. The vaccine of claim 29, wherein the vaccine further comprises a live attenuated canine parvovirus, a live attenuated canine distemper virus, and a live attenuated canine adenovirus type 2.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0029] Accordingly, the present invention provides modified live canine parainfluenza virus vaccines, including multivalent vaccines useful as oral vaccines. The present invention also provides methods of orally immunizing a canine against canine parainfluenza virus, comprising oral administration to a canine of a vaccine comprising a modified live canine parainfluenza (CPI) virus. The present invention further provides single-dose vaccines that comprise a modified live canine parainfluenza virus. Such vaccines aid in the protection of (and/or provide effective protection to) the vaccinated canine for at least 6 months against upper respiratory diseases and infectious tracheobronchitis, without the need of a booster vaccine. In certain embodiments, the single-dose vaccine is administered in a 1 mL dose to the canine.

[0030] The present invention further provides vaccines comprising a modified live canine parainfluenza virus in combination with one or more other canine pathogens and/or immunogens that further elicit immunity to canine influenza virus (e.g., H3N2 and/or H3N8), canine parvovirus, canine distemper virus, canine adenovirus, canine respiratory coronavirus, canine pneumovirus, Mycoplasma species (e.g., Mycoplasma cynos), and Steptococcus equi zooepidemicus.

[0031] More specifically, the present invention also includes multivalent oral vaccines comprising modified live canine parainfluenza virus together with an avirulent live B. bronchiseptica. The present invention further provides methods of immunizing a canine against canine parainfluenza virus and B. bronchiseptica, comprising oral administration to a canine of a vaccine comprising a modified live canine parainfluenza virus and an avirulent live B. bronchiseptica. In specific embodiments of the present invention, a canine receiving only one dose of a single-dose vaccine (or multivalent vaccine) comprising a modified live canine parainfluenza virus and an avirulent live B. bronchiseptica is protected for at least 6 months against upper respiratory diseases and infectious tracheobronchitis caused by a wild type CPI virus and a wild type avirulent live B. bronchiseptica, without the need of a booster vaccine. In more specific embodiments, the single-dose vaccine is administered in a 1 mL dose to the canine.

[0032] In particular embodiments, the vaccines comprise an immunologically effective amount of a modified live CPI virus and the avirulent live B. bronchiseptica, as determined by the amount of CPI virus serum neutralizing antibodies and B. bronchiseptica agglutinating and/or IgA antibodies induced in a vaccinated canine. In a related aspect, the present invention provides a vaccine that comprises a specific, minimum amount of each antigen that is effective against virulent CPI virus and virulent B. bronchiseptica. In yet another aspect, a vaccine of the present invention is both safe and effective and aids in the protection of (and/or provides effective protection to) a canine from upper respiratory diseases and infectious tracheobronchitis due to CPI virus and B. bronchiseptica infections in dogs.

[0033] The present invention also provides multivalent vaccines comprising a live attenuated CPI virus and an avirulent live B. bronchiseptica in further combination with one or more other canine pathogens and/or immunogens that further elicit immunity to canine influenza virus (e.g., H3N2 and H3N8), canine parvovirus, canine adenovirus, canine distemper virus, canine adenovirus, canine respiratory coronavirus, canine pneumovirus, Mycoplasma species (e.g., Mycoplasma cynos) and Steptococcus equi zooepidemicus.

[0034] As used herein the term “approximately” is used interchangeably with the term “about” and signifies that a value is within twenty-five percent of the indicated value i.e., a dose of a vaccine comprising “approximately” 4.0 mL can contain 3.0 to 5.0 mL.

[0035] As used herein the term, “canine” is used interchangeably with the term “dog” and includes all domestic dogs, i.e., Canis lupus familiaris or Canis familiaris, unless otherwise indicated.

[0036] As used herein, the term “feline” refers to any member of the Felidae family. Domestic cats, pure-bred and/or mongrel companion cats, and wild or feral cats are all felines.

[0037] As used herein, a “vaccine” is a composition that is suitable for application to an animal, e.g., a canine, and comprises one or more antigens, i.e., one or more immunogens, typically combined with a pharmaceutically acceptable carrier such as a liquid containing sterile water, which upon administration to the animal induces an immune response strong enough to minimally aid in the protection (and/or provide effective protection) from a disease arising from an infection with a wild-type micro-organism (e.g., virus or bacterium), i.e., strong enough for aiding in the prevention of the disease, providing effective protection, preventing, or ameliorating the disease. In preferred embodiments, the vaccine is strong enough to provide effective protection from a disease arising from an infection with a wild-type micro-organism (e.g., virus or bacterium). The use here, of the term “vaccine” encompasses both monovalent vaccines and multivalent vaccines. A vaccine for oral administration to an animal subject also can be termed an “oral vaccine”.

[0038] As used herein, a “multivalent vaccine” is a vaccine that comprises two or more different antigens. In particular embodiments of this type, the multivalent vaccine stimulates the immune system of the recipient against two or more different pathogens.

[0039] As used herein, the terms “protect”, “protecting”, “provides effective protection” “providing effective protection”, “aids in the protection”, “aiding in the protection” and “aids in eliciting protective immunity” do not require complete protection from any indication of infection. For example, “aids in the protection” can mean that the protection is sufficient such that, after challenge, symptoms of the underlying infection are at least reduced, and/or that one or more of the underlying cellular, physiological, or biochemical causes or mechanisms causing the symptoms are reduced and/or eliminated. It is understood that “reduced,” as used in this context, means relative to the state of the infection, including the molecular state of the infection, not just the physiological state of the infection.

[0040] As used herein, the terms “providing effective protection” and “provides effective protection” are used interchangeably. “Providing effective protection” to a canine from upper respiratory diseases and infectious tracheobronchitis, with respect to a modified live canine parainfluenza virus comprised by a vaccine of the present invention is determined by finding a statistically significant decrease in the duration of CPI virus shedding and/or clinical signs of disease between vaccinated dogs and placebo-vaccinated control dogs following a challenge with live wild type CPI virus. “Providing effective protection” to a canine from upper respiratory diseases and infectious tracheobronchitis, with respect to an avirulent live B. bronchiseptica comprised by a vaccine of the present invention is determined by finding a statistically significant decrease in the number of affected dogs between vaccinated dogs and placebo-vaccinated control dogs following a challenge with live wild type B. bronchiseptica. An affected dog is defined as having spontaneous coughing or spontaneous coughing with retching on two or more consecutive days during the post-challenge observation period.

[0041] As used herein, the “duration of viral shedding” is the number of days from the first to last occurrence that the virus was shed from the nare of a canine as determined by virus titration from nasal swabs. In particular embodiments of the present invention, the duration of viral shedding is determined for the CPI virus.

[0042] As used herein, the “duration of bacterial shedding” is the number of days from the first to last occurrence that the bacteria were shed from the nare of a canine as determined by bacterial titration from nasal swabs. In particular embodiments of the present invention, the duration of bacterial shedding is determined for B. bronchiseptica.

[0043] As used herein, a canine that “seroconverts” means that the canine’s antibody titer to a specific antigen (e.g., a CPI virus or a B. bronchiseptica) is at least 2-fold greater than the baseline value for that specific antigen.

[0044] As used herein, the terms “live attenuated virus” and “live modified virus” are used interchangeably and are live attenuated viral immunogens that are immunogenic, but not pathogenic.

[0045] As used herein, the terms “live attenuated bacterium”, “avirulent live culture of a bacterium”, and “avirulent live bacterium” are used interchangeably and are live attenuated bacterial immunogens (e.g., an avirulent live B. bronchiseptica) that are immunogenic, but not pathogenic.

[0046] As used herein, when a “dose” of a vaccine to be administered to an animal subject is defined as comprising a specific quantity or range of quantities of antigen e.g., by weight such as 4 .Math.g/dose or 2 to 6 .Math.g/dose, by a titer, such as 7.3 log.sub.10HAID.sub.50/dose or 7.3 - 8.6 log.sub.10HAID.sub.50/dose, administering the entire dose either can be performed in a single administration or alternatively, in multiple administrations over an interval of 3 hours or less. In particular embodiments, a vaccine dose is orally administered to the animal subject in a single administration, all at one time.

[0047] As used herein a “single-dose vaccine” is a vaccine (or multivalent vaccine) comprising at least one immunogen from a pathogen in which the vaccine (or multivalent vaccine) that is administered to an animal in a single administration, or alternatively, in multiple administrations over a short period of time, i.e., over an interval of 3 hours or less, and still aid in the protection of (and/or provide effective protection to) the animal from the pathogen for at least six (6) months, without the need of administering a second dose of the vaccine (e.g., a booster vaccine). In particular embodiments of this type, a single-dose vaccine is orally administered as a single aliquot of the vaccine to an animal subject, e.g., a canine, all in a single administration. In particular embodiments, the single dose vaccine is orally administered in a one (1) mL dose to the animal subject. Accordingly, it is contemplated that a vaccine of the present invention may be orally administered to the animal subject, e.g., a canine, as a single-dose vaccine, for which the duration of immunity is at least 6 months. In certain embodiments, the duration of immunity is at least 9 months. In other embodiments, the duration of immunity is at least 12 months. In still other embodiments, the duration of immunity is at least 18 months.

[0048] In alternative aspects, a second dose of a vaccine (or multivalent vaccine) is administered one (1) week, multiple weeks, or months following the primary administration over a 6 to 18-month duration after the administration of the initial oral dose. A booster vaccine can be administered by injection (e.g., intramuscularly, subcutaneously), intranasally, or orally. Accordingly, in some embodiments, the vaccine is orally administered in at least two (2) doses. In some such embodiments, for example, the vaccine is administered twice, with the second dose (e.g., a booster vaccine) being administered at least about 2 weeks after the first. In some embodiments, the vaccine is administered twice, with the second dose being administered no greater than 8 weeks after the first. In other embodiments, the second dose is administered from about 2 weeks to about 4 months after the first dose, from about 2 to about 8 weeks after the first dose, or from about 3 weeks to about 4 weeks after the first dose. In some embodiments, the second dose is administered about 4 weeks after the first dose. The first and subsequent dosages may vary, such as, for example, in amount and/or form. Often, however, the dosages are the same with respect to the amount and form. Whether the administration is performed as a single dose vaccine or in multiple doses [i.e., booster vaccine(s)], it should be understood that subsequent administrations of the vaccine are likely to be needed to be provided to a given animal subject after the 6 to 18 month duration of immunity (or even longer), such as in a yearly or every other year administration regimen.

[0049] When a single dose of an oral vaccine of the present invention is sufficient to aid in the protection of (and/or provide effective protection to) the animal from the pathogen for at least six (6) months, the quantity of the antigen(s) in that dose generally comprises a therapeutically effective amount of the vaccine for the 6 month or longer duration. On the other hand, when a booster vaccine dose is required to supplement that initial dose, the combined quantity of the initial vaccine and the booster vaccine may constitute the therapeutically effective amount.

[0050] The terms “adjuvant” and “immune stimulant” are used interchangeably herein and are defined as one or more substances that cause stimulation of the immune system.

[0051] As used herein, a “non-adjuvanted vaccine” is a vaccine or a multivalent vaccine that does not contain an adjuvant.

[0052] As used herein, the term “pharmaceutically acceptable” is used adjectivally to mean that the modified noun is appropriate for use in a pharmaceutical product. When it is used, for example, to describe an excipient in a pharmaceutical vaccine, it characterizes the excipient as being compatible with the other ingredients of the composition and not disadvantageously deleterious to the intended recipient animal, e.g., a canine.

[0053] In specific embodiments, a vaccine of the present invention also may be administered with a pharmaceutically acceptable immune stimulant and/or adjuvant and/or bioadhesive polymer. In this context, an adjuvant is used to enhance an immune response to one or more vaccine antigens/isolates. Accordingly, “adjuvants” are agents that nonspecifically increase an immune response to a particular antigen, thus reducing the quantity of antigen necessary in any given vaccine, and/or the frequency of injection necessary in order to generate an adequate immune response to the antigen of interest. Suitable adjuvants for the vaccination of animals include, but are not limited to, mineral gels, such as aluminum hydroxide, aluminum phosphate and alum; surfactants, such as pluronic polyols, and oil emulsions. One adjuvant exemplified below, CARBIGEN™, is a terminally sterilized, carbomer-based (CARBOPOLⓇ934P) adjuvant suspension containing an emulsified component and is free of animal origin ingredients obtained from MVP Adjuvants [located on 4805 “G” Street Omaha, NE]. CARBOPOLⓇ934P is an acrylic acid homopolymer crosslinked with allyl sucrose or allyl pentaerythritol. PVP-K60, a bio-adhesive polymer, is a hygroscopic, amorphous polyvinylpyrrolidone linear nonionic polymer, which is soluble in water and organic solvents, and is pH stable (K60 refers to its molecular weight).

[0054] Information concerning adjuvants and various aspects of immunoassays are disclosed, e.g., in the series by P. Tijssen, Practice and Theory of Enzyme Immunoassays, 3rd Edition, 1987, Elsevier, New York, incorporated by reference herein. Pharmaceutically acceptable immune stimulants, include bacterial and/or fungal cell wall components (e.g., lipopolysaccharides, lipoproteins, glycoproteins, muramylpeptides), mucoadhesive polymers, various complex carbohydrates derived from plants (e.g., glycans, acemannan), various proteins and peptides derived from animals (e.g., hormones, cytokines, co-stimulatory factors), and novel nucleic acids derived from viruses and/or other sources (e.g., double stranded RNA, CpG).

[0055] Live attenuated CPI viruses for use in the vaccines of the present invention may be prepared by conventional means. Conventional means generally include, for example, modifying pathogenic strains by in vitro passaging, cold adaptation, modifying the pathogenicity of the organism by genetic manipulation, selecting non-virulent wild type strains, and other methods well known to the skilled artisan. Such attenuated CPI viruses can then be tested to determine whether they are appropriate for oral administration as detailed in the examples below.

[0056] A live modified CPI virus strain can be derived by serial passage of the wild-type virus through cell culture. In alternative embodiments, the live modified CPI virus strain is derived by serial passage of the wild-type virus through a laboratory animal and/or non-host animals. The accumulation of genetic mutation during such passage(s) typically leads to progressive loss of virulence of the organism to the original host. In some embodiments, the live attenuated virus strain is prepared by cold adaptation. A cold-adapted virus has an advantage of replicating only at the temperature found in the upper respiratory tract. A method of generation of a cold-adapted equine influenza virus has been described in U.S. 6,177,082 [hereby incorporated by reference in its entirety]. A desired resulting cold-adapted virus confers one or more of the following phenotypes: cold adaptation, temperature sensitivity, dominant interference, and/or attenuation. In addition, both modified live canine parainfluenza viruses and avirulent live B. bronchiseptica have been previously disclosed and are included in commercially available canine vaccines.

BIOLOGICAL DEPOSIT

[0057] Cultures of the following biological material have been deposited with the following international depository: American Type Culture Collection (ATCC) 10801 University Boulevard, Manassas, Va. 20110-2209, U.S.A., under conditions that satisfy the requirements of the Budapest Treaty. All restrictions imposed by the depositor on the availability to the public of the deposited material will be irrevocably removed upon granting of a patent.

TABLE-US-00001 Organism Accession No. Date of Deposit Modified live Canine Parainfluenza Virus PTA-126273 Dec. 5, 2019 Attenuated live Bordetella bronchiseptica B-C2 PTA-126272 Dec. 5, 2019

[0058] The following examples serve to provide further appreciation of the invention, but are not meant in any way to restrict the effective scope of the invention.

EXAMPLES

Example 1

Efficacy of an Oral Canine Parainfluenza Virus and B. Bronchiseptica Vaccine Administered with and Without a Bio-Adhesive Polymer or Bio-Adhesive Adjuvant

Materials and Methods

Vaccine

[0059] The experimental vaccines contained avirulent live B. bronchiseptica antigen B-C2, ATCC accession No. PTA-126272 and modified live CPI virus, strain Cornell, antigen ATCC accession No. PTA-126273, that was blended with stabilizer solution [hydrolyzed gelatin, N-Z Amine AS (an enzymatic hydrolysate of casein obtained from Millipore Sigma, Burlington, MA), Sorbitol-d, Sodium phosphate dibasic] and then freeze dried. Vaccines that were administered to dogs in Treatment Groups A, C, and D contained 10-15% CARBIGEN™ as an adjuvant. The vaccine administered to the dogs in Treatment Group C also contained the bio-adhesive polymer, PVP-K60.

[0060] The titer of the CPI virus of the vaccines varied from 7.5 - 8.5 log.sub.10HAID.sub.50/mL, whereas the titer of the B. bronchiseptica of the vaccines varied from 9.2 x 10.sup.9 - 1.4 × 10.sup.10 cfu/mL. All vaccines were freeze dried in vials, and on the day of vaccination, the lyophilized vaccine in each vial was rehydrated with 1 mL of sterile water and like preparations were pooled. The titer of the CPI virus is provided in log.sub.10HAID.sub.50/mL, which is a unit commonly used to estimate the concentration of virus in a sample as calculated by the Spearman-Karber method.

Animals

[0061] Seven, 10-week old Beagles (Marshall Bioresources) were housed communally in a BSL-2 facility on concrete floors covered with wood shavings. Food and water were available ad libitum.

Vaccination and Collection of Serum

[0062] On study day 0, dogs in Treatment Group D were vaccinated by the oral route, with a 1 mL dose of vaccine consisting of CPI virus, B. bronchiseptica (Bb), and 15% CARBIGEN™. A spray apparatus was attached to a syringe to deliver the vaccine to the back of the throat. On study day 14, all the dogs were vaccinated via the oral route with their respective pooled vaccine [see, Table 1 below]. Whole blood was collected on study day 27 by venipuncture of the jugular vein. The serum was separated by centrifugation and stored at -10° C. or colder until tested.

Detection of CPI Virus Neutralizing Antibodies

[0063] CPI virus neutralizing antibodies were detected using a standard serum neutralization (SN) assay. Serum dilutions were incubated with the CPI virus vaccine strain and inoculated onto dog kidney cells. After 5-7 days, monolayers were fixed and stained with fluorescein-conjugated CPI virus antiserum, and SN antibody titers were calculated as the reciprocal of the serum dilution causing 50% inhibition of virus infection.

TABLE-US-00002 STUDY DESIGN Treatment Group No. of Animals Vaccine Dose Route Vaccination Day A 7 Bb + CPI virus + 15% CARBIGEN™ 1.0 mL Oral 14 B 7 Bb + CPI virus 14 C 7 Bb + CPI virus + 10% CARBIGEN™ + PVP-K60 14 D 7 Bb + CPI virus + 15% CARBIGEN™ 0 and 14 E 7 Placebo-vaccinated controls 14

Challenge

[0064] On study day 28, dogs were challenged with a virulent CPI virus.

Post-Challenge Observations and Samples

[0065] Clinical observations were recorded for 11 days post-challenge, and nasal swabs were collected daily for 11 days post-challenge.

Results

Serology

[0066] Prior to vaccination, all the dogs had CPI virus serum neutralization titers less than 2, indicating the dogs were naïve at the time of vaccination. The placebo-vaccinated control dogs remained seronegative (SN < 2) just prior to challenge. Nearly all the dogs in each treatment group seroconverted (titer > 4) following vaccination [See, Table 2 below].

TABLE-US-00003 SERUM ANTIBODY TITERS TO CPI VIRUS Treatment Group No. of Dogs That Seroconverted on Study Day 27 (Titer Range) A Bb + CPI virus + 15% CARBIGEN™ 6/7 (19 - > 362) B Bb + CPI virus 6/7 (16 - > 256) C Bb + CPI virus + 10% CARBIGEN™ + PVP-K60 6/7 (38 - 152) D Bb + CPI virus + 15% CARBIGEN™ 2 vaccinations 7/7 (32 - 256) E Placebo-vaccinated controls 0/7

CPI Virus Shedding

[0067] Duration of CPI viral shedding was the primary variable to evaluate vaccine efficacy. The duration of viral shedding from first to last occurrence, in days, for each animal was calculated, and the mean duration of shedding was determined for each treatment group. The mean duration of viral shedding for the placebo-vaccinated control group was 7 days. In contrast, the mean duration of shedding for Treatment Groups A and C was 0 days, while the mean duration of shedding for Treatment Groups B and D was 1 day [see, Table 3 below].

TABLE-US-00004 DURATION OF SHEDDING Treatment Group Mean Duration of Viral Shedding in Days A Bb + CPI virus + 15% CARBIGEN™ 1 B Bb + CPI virus 1 C Bb + CPI virus + 10% CARBIGEN™ + PVP-K60 0 D Bb + CPI virus + 15% CARBIGEN™ 2 vaccinations 1 E Placebo-vaccinated controls 7

[0068] These results indicate that the CARBIGEN™ and PVP-K60 had no significant effect on the duration of CPI viral shedding in the vaccine dogs.

Example 2

Dose Response of The CPI Virus of The Multivalent Oral CPI Virus and B. Bronchiseptica Vaccine with and without Adjuvant

Materials and Methods

Vaccine

[0069] The experimental vaccines contained avirulent live B. bronchiseptica antigen B-C2 ATCC accession No. PTA-126272 and modified live CPI virus, strain Cornell, antigen ATCC accession No. PTA-126273, that was blended with stabilizer [hydrolyzed gelatin, N-Z Amine AS, Sorbitol-d, Sodium phosphate dibasic] and then freeze dried. Vaccines that were administered to dogs in Treatment Groups C and D contained 15% CARBIGEN™ as an adjuvant. On the day of vaccination, each vial of lyophilized vaccine was rehydrated with 1 mL of sterile water, and like preparations were pooled. The titer of the CPI virus in the vaccines used in Treatment Groups A - D was 8.6, 7.3, 7.6, and 6.3 log.sub.10HAID.sub.50/mL, respectively, as denoted in Table 5.

Animals

[0070] Fifteen, 17-week old Beagles (Marshall Bioresources) were housed communally in a BSL-2 facility on concrete floors covered with wood shavings. Food and water were available ad libitum.

Vaccination and Collection of Serum

[0071] On study day 0, the dogs were vaccinated by the oral route with a 1 mL dose of their respective pooled vaccine [see, Table 4 below]. A spray apparatus was attached to a syringe to deliver the vaccine to the back of the throat. Whole blood was collected on study day 27 by venipuncture of the jugular vein. The serum was separated by centrifugation and stored at - 10° C. or colder until tested.

Detection of CPI Virus Neutralizing Antibodies

[0072] CPI virus neutralizing antibodies were detected using a standard SN assay. Serum dilutions were incubated with CPI virus vaccine strain and inoculated onto dog kidney cells. After 5-7 days, monolayers were fixed and stained with fluorescein-conjugated CPI virus antiserum, and SN antibody titers were calculated as the reciprocal of the serum dilution causing 50% inhibition of virus infection.

Challenge

[0073] On study day 28, dogs were challenged with virulent CPI virus.

Post-Challenge Observations and Samples

[0074] Clinical observations were recorded for 14 consecutive days, and nasal swabs were collected for 10 consecutive days post-challenge.

TABLE-US-00005 STUDY DESIGN Treatment Group No. of Animals Vaccine Dose Route Vaccination Day A 6 CPI virus (8.0 log.sub.10HAID.sub.50/mL) + B. bronchiseptica 1.0 mL Oral Study Day 0 B 6 CPI virus (6.5 log.sub.10HAID.sub.50/mL) + B. bronchiseptica C 6 CPI virus (8.0 log.sub.10HAID.sub.50/mL) + B. bronchiseptica with 15% CARBIGEN™ D 6 CPI virus (6.5 log.sub.10HAID.sub.50/mL) + B. bronchiseptica with 15% CARBIGEN™ E 6 Placebo-vaccinated controls

Results

Serology

[0075] Prior to vaccination, all dogs had CPI virus serum neutralization titers less than 2, indicating that the dogs were naïve at the time of vaccination. The placebo-vaccinated control dogs remained seronegative (SN < 2) just prior to challenge. Nearly all the dogs in each treatment group seroconverted (titer > 4) following vaccination [see, Table 5 below].

TABLE-US-00006 SERUM ANTIBODY TITERS TO CPI VIRUS Treatment Group No. of Dogs That Seroconverted on Study Day 27 (Titer Range) A CPI virus (8.6 log.sub.10HAID.sub.50/mL) + B. bronchiseptica 6/6 (19 - 431) B CPI virus (7.3 log.sub.10HAID.sub.50/mL) + B. bronchiseptica 6/6 (7 - 91) C CPI virus (7.6 log.sub.10HAID.sub.50/mL) + B. bronchiseptica with 15% CARBIGEN™ 6/6 (7 - 215) D CPI virus (6.3 log.sub.10HAID.sub.50/mL) + B. bronchiseptica with 15% CARBIGEN™ 2/6 (7 - 256) E Placebo-vaccinated controls 0/7

CPI Virus Shedding

[0076] Duration of CPI viral shedding was the primary variable to evaluate vaccine efficacy. The duration of viral shedding from first to last occurrence, in days, for each animal was calculated, and the mean duration of shedding was determined for each treatment group. All the placebo-vaccinated control dogs (Treatment Group E) shed virus, and the mean duration of shedding was 6 days. In contrast, the mean duration of shedding for Treatment Groups A-D was 1, 0, 1, and 5, respectively [see, Table 6 below].

TABLE-US-00007 DURATION OF SHEDDING Treatment Group Mean Duration of Viral Shedding in Days A CPI virus (8.6 log.sub.10HAID.sub.50/mL) + B. bronchiseptica 1 B CPI virus (7.3 log.sub.10HAID.sub.50/mL) + B. bronchiseptica 2 C CPI virus (7.6 log.sub.10HAID.sub.50/mL) + B. bronchiseptica with 15% CARBIGEN™ 1 D CPI virus (6.3 log.sub.10HAID.sub.50/mL) + B. bronchiseptica with 15% CARBIGEN™ 5 E Placebo-vaccinated controls 6

[0077] These results suggest that ≥ 6.3 log.sub.10HAID.sub.50/mL of the modified live CPI virus in a 1 mL dose is needed for the oral modified live CPI virus vaccine to be efficacious, as evaluated as a function of the duration of viral shedding.

Example 3

Immunogenicity Study in Dogs to Demonstrate Efficacy of the CPI Fraction of an Oral Canine Parainfluenza and Bordetella Bronchiseptica Combination Vaccine

Materials and Methods

Vaccine

[0078] The test vaccine consisted of modified live CPI virus strain Cornell, ATCC accession No. PTA-126273 and avirulent live B. bronchiseptica antigen B-C2, ATCC accession No. PTA-126272 that was blended with stabilizer [hydrolyzed gelatin, N-Z Amine AS, Sorbitol-d, Sodium phosphate dibasic] and then freeze dried. The placebo vaccine consisted of all the components in the test vaccine except for the CPI antigen. On the day of vaccination, each vial of lyophilized vaccine was rehydrated with 1 mL of sterile water, and like preparations were pooled.

Animals

[0079] Twenty, 7-week old Beagles (Marshall Bioresources) were housed communally in a BSL-2 facility on concrete floors covered with wood shavings, and 19, 7-week old Beagles (Marshall Bioresources) were housed similarly in another room. Food and water were available ad libitum.

Vaccination and Collection of Serum

[0080] On study day 0, the dogs were vaccinated by the oral route with a 1 mL dose of their respective pooled vaccine [see, Table 7 below]. A spray apparatus was attached to a syringe to deliver the vaccine to the back of the oral cavity (oropharynx). Whole blood was collected on study day 21 by venipuncture of the jugular vein. The serum was separated by centrifugation and stored at -10° C. or colder until tested.

Detection of CPI Virus Neutralizing Antibodies

[0081] CPI virus neutralizing antibodies were detected using a standard SN assay. Serum dilutions were incubated with CPI virus vaccine strain and inoculated onto dog kidney cells. After 5-7 days, monolayers were fixed and stained with fluorescein-conjugated CPI virus antiserum, and SN antibody titers were calculated as the reciprocal of the serum dilution causing 50% inhibition of virus infection.

Challenge

[0082] On study day 21, dogs were challenged with virulent CPI virus.

Post-Challenge Observations and Samples

[0083] Clinical observations were recorded for 14 consecutive days, and nasal swabs were collected for 10 consecutive days post-challenge.

TABLE-US-00008 STUDY DESIGN Treatment Group No. of Animals Vaccine Dose Route Vaccination Day A 20 Test Vaccine CPI + B. bronchiseptica 1.0 mL Oral Study Day 0 B 19 Placebo Vaccine B. bronchiseptica

Results

Serology

[0084] Prior to vaccination, all dogs had CPI virus serum neutralization titers less than 2, indicating that the dogs were naïve at the time of vaccination. Vaccination with the placebo vaccine did not induce antibodies specific to CPI, whereas, vaccination with the test vaccine induced CPI serum neutralization titers of 4 or greater in 15 of 20 (75%) vaccinates, with titers ranging from 6 to 861 (GMT = 31) 3 weeks after vaccination [see, Table 8 below].

TABLE-US-00009 CPI SERUM ANTIBODY TITERS Treatment Group No. of Dogs That Seroconverted on Study Day 21 (Titer Range) A Vaccinates 15/20 (6 - 861) B Placebo-vaccinated Controls 0/20 (<2)

CPI Virus Shedding

[0085] Duration of CPI viral shedding was the primary variable to evaluate vaccine efficacy. The duration of viral shedding from first to last occurrence, in days, for each animal was calculated, and the mean duration of shedding was determined for each treatment group. All but one of the placebo-vaccinated control dogs (Treatment Group B) shed CPI virus (i.e., 95%); whereas, only 11 of the vaccinates shed virus (i.e., 55%). The median duration of CPI viral shedding was 6 days for the placebo-vaccinated control dogs, compared to only a median duration of shedding of 1.5 days for the vaccinated dogs [see, Table 9 below].

TABLE-US-00010 DURATION OF SHEDDING Treatment Group Mean Duration of Viral Shedding in Days A Vaccinates 1.5* B Placebo-vaccinated Controls 6 * p-value< 0.0001

[0086] These results show that 7.1 log.sub.10HAID.sub.50/mL of CPI virus in a 1 mL dose is sufficient for the oral modified live CPI virus vaccine to be efficacious, as evaluated as a function of the duration of viral shedding.

Example 4

Immunogenicity Study to Demonstrate Efficacy of the Bordetella Bronchiseptica Fraction of an Oral Canine Parainfluenza and Bordetella Bronchiseptica Combination Vaccine

Materials and Methods

Vaccine

[0087] The test vaccine consisted of modified live CPI virus strain Cornell, ATCC accession No. PTA-126273 and avirulent live B. bronchiseptica antigen B-C2, ATCC accession No. PTA-126272 that was blended with stabilizer [hydrolyzed gelatin, N-Z Amine AS, Sorbitol-d, Sodium phosphate dibasic] and then freeze dried. The placebo vaccine consisted of all the components in the test vaccine except for the B. bronchiseptica antigen. On the day of vaccination, each vial of lyophilized vaccine was rehydrated with sterile water, and like preparations were pooled.

Animals

[0088] Twenty-one, 7-week old Beagles were housed communally in a BSL-2 facility on concrete floors covered with wood shavings, and twenty-one, 7-week old Beagles were housed similarly in another room. Food and water were available ad libitum.

Vaccination

[0089] On study day 0, the dogs were vaccinated by the oral route with a 1 mL dose of their respective pooled vaccine [see, Table 10 below]. A spray apparatus was attached to a syringe to deliver the vaccine to the back of the oral cavity (oropharynx).

Sample Collection

[0090] On study day 34, whole blood was collected by venipuncture of the jugular vein. The serum was separated by centrifugation and stored at -10° C. or colder until tested. Nasal swabs were also collected on study day 34 to test for the presence of B. bronchiseptica. Once collected, swabs were placed in whirl pack bags and tested immediately.

Detection of B. Bronchiseptica Agglutinating Antibodies

[0091] B. bronchiseptica antibodies were detected using a standard microagglutination test. Briefly, 2-fold serial dilutions of test serum, known positive serum, and known negative serum were performed in a U-bottom microtiter plate, using normal saline containing 0.1% gelatin as the diluent. B. bronchiseptica antigen (100 .Math.L) was added to each well and mixed for 15-30 seconds on a microtiter plate mixer. The plates were incubated at 36 ± 2° C. for 1-3 hours and then incubated at 2-7° C. for 36-72 hours. The plates were read visually for agglutination, and the titers were expressed as the reciprocal of the highest dilution showing complete agglutination.

Challenge

[0092] On study day 35, dogs were challenged with virulent B. bronchiseptica.

Post-Challenge Observations and Samples

[0093] Dogs were observed for at least 30 minutes twice daily for 28 days post-challenge for clinical signs including, but not limited to, nasal discharge, dyspnea, depression, and coughing. Nasal swabs were collected on study days 42, 45, 49, 52, 58, and 63 to determine shedding of challenge organisms.

TABLE-US-00011 STUDY DESIGN Treatment Group No. of Animals Vaccine Dose Route Vaccination Day A 20 Test Vaccine B. bronchiseptica + CPI 1.0 mL Oral Study Day 0 B 19 Placebo Vaccine CPI

Results

Serology

[0094] Prior to vaccination, all dogs had low antibody titers (≤ 4) to B. bronchiseptica, indicating that the dogs were naïve at the time of vaccination. Vaccination with the test vaccine induced B. bronchiseptica agglutination titers in 20 of the 21 dogs, with a range of 16 to 128 on study day 34; GMT = < 39. In contrast, the antibody titers in all the placebo-vaccinated control dogs remained low with a range of < 2 to 8; GMT = < 1 [see, Table 11 below].

TABLE-US-00012 B. BRONCHISEPTICA SERUM ANTIBODY TITERS Treatment Group No. of Dogs That Seroconverted on Study Day 34 (Titer Range) A Vaccinates 20/21 (16 - 128) B Placebo-vaccinated Controls 0/21 (<2 - 8)

Clinical Signs Post-Challenge

[0095] Following challenge, all the placebo-vaccinated control dogs developed clinical signs associated with a B. bronchiseptica infection, specifically spontaneous coughing. An affected dog was defined as having spontaneous coughing or spontaneous coughing with retching on 2 or more consecutive days during the post-challenge observation period. All 20 placebo-vaccinated control dogs were affected compared to only 9 of the 21 (43%) vaccinated dogs; p-value < 0.0001 [see, Table 12 below]. In addition, none of the vaccinates coughed for two consecutive days more than once during the post-challenge observation period, whereas 18 of the 20 (90%) placebo-vaccinated controls coughed on two consecutive days 2-13 times.

TABLE-US-00013 SUMMARY OF AFFECTED DOGS Treatment Group Total Number of Dogs A Vaccinates 9/21 (43%)* B Placebo-Vaccinated Controls 20/20 (100%) *p-value < 0.0001

[0096] The number of days that a dog spontaneously coughed during the 28-day post-challenge observation period also was analyzed. The mean number of days that dogs in Treatment Group B spontaneously coughed was 16.6 days compared to only 3.5 days for dogs in Treatment Group A; p-value < 0.0001 [see, Table 13 below].

TABLE-US-00014 SUMMARY OF COUGHING Treatment Group Total Number of Dogs A Vaccinates (3.5%)* B Placebo-Vaccinated Controls (16.6%) *p-value < 0.0001

B. Bronchiseptica Shedding

[0097] Nasal swabs were collected twice a week for 4 weeks post-challenge to determine shedding of challenge organisms. On study day 42, there was no difference in bacterial shedding between the placebo-vaccinated control group and the vaccine group. However, by study day 45, the placebo vaccinated control group was shedding 38,141 cfu/mL of B. bronchiseptica, whereas the vaccinate group was shedding only 1,523 cfu/mL. The bacterial shedding in the placebo-vaccinated control group peaked at 16 days post-challenge (study day 52) with 75,293 cfu/mL. In contrast, bacterial shedding in the vaccinated group peaked at 6 days post-challenge (study day 42) with only 4,173 cfu/mL. Bacterial counts continued to decline in the vaccinated dogs until the end of the study. By study day 63, the bacterial shedding in the vaccinated group was only 6 cfu/mL, whereas, the bacterial shedding remained elevated within the placebo-vaccinated control group at 35,400 cfu/mL.

[0098] These data demonstrate a dramatic reduction in the ability of B. bronchiseptica to colonize within the nasal mucosa of the vaccinate group. The overall mean bacterial shedding for the placebo-vaccinated controls and vaccinates was 37,035 cfu/mL and 849 cfu/mL, respectively [see, Table 14 below], and the amount of B. bronchiseptica shed on each collection day was statistically lower in the vaccinated dogs than in the placebo-vaccinated control dogs; p-value ≤ 0.0001.

TABLE-US-00015 B. BRONCHISEPTICA ORGANISMS ISOLATED FROM NASAL SWABS POST-CHALLENGE (CFU/ML) Treatment Group Day 34* Day 42 Day 45 Day 49 Day 52 Day 55 Day 58 Day 63 Overall Mean Vaccinates 0 4,173* 1,523** 253** 751** 33** 50** 6** 849 Controls 0 5,235 38,141 70,390 75,293 35,638 36,183 35,400 37,035 *p-value = 0.0001 **p-value < 0.0001

[0099] These results show that 3.9 x 10.sup.8 cfu/mL of B. bronchiseptica avirulent strain B-C2, ATCC accession No. PTA-126272, in a 1 mL dose is sufficient for the oral vaccine to be efficacious, as evaluated by the number of dogs that had spontaneous coughing or spontaneous coughing with retching on two or more consecutive days during the post-challenge observation period.

[0100] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.