Oral respiratory vaccine

Abstract

We provide a novel oral live canine parainfluenza virus vaccine and related polyvalent vaccines. [Solution] A method for providing effective protection to dogs from upper respiratory tract diseases and infectious tracheobronchitis, comprising orally administering a vaccine containing a modified live canine parainfluenza (CPI) virus to the dogs. Methods are also provided for using the vaccine alone or in combination with one or more protective immunogens in a polyvalent vaccine.

Description

[Technical Field]

[0001] Cross-reference of related applications This application is in accordance with Provisional Application U.S. No. 62 / 949,928, 35U, filed on 18 December 2019. This application claims priority under SC§119(e), and the contents of the said application are as follows: Therefore, the entirety is incorporated herein.

[0002] This invention relates to a novel oral live canine parainfluenza virus vaccine and related polyvalent vaccines. Regarding kuchin. Methods for manufacturing vaccines, and one or more other factors in single or multivalent vaccines. Instructions on how to use the vaccine in combination with protective antigens will also be provided. [Background technology]

[0003] Canine parainfluenza (CPI) virus causes upper respiratory tract diseases and infectious bronchitis (kennels). It is a highly contagious virus that causes respiratory illnesses, which contribute to the development of cough (also known as flu cough). Respiratory symptoms may resemble those of canine influenza, but they are unrelated viruses. It is a virus and requires different vaccines for protection. The CPI virus lasts for a long time after infection. The virus is excreted from the respiratory tract of infected animals for two weeks and is usually transmitted through airborne transmission. CPI virus The disease spreads rapidly in kennels and defensive facilities where many dogs are kept together. Clinical symptoms Symptoms include a dry or wet cough, a slight fever, a runny nose, lethargy, and loss of appetite.

[0004] Currently, canine parainfluenza virus, canine distemper virus, canine adenovirus To prevent diseases caused by canine parvovirus and other viruses, healthy dogs Nobivac (registered trademark) is a modified live virus vaccine for vaccinating the Nu. Canine 1-DAPPv, a modified live CPI virus, can be administered subcutaneously or intramuscularly. There are several commercially available dog vaccines available. Furthermore, there are vaccines for canine parainfluenza virus, Canine adenovirus type 2, and Bordetella bronchoseptica (Bor Detella bronchiseptica (B. bronchiseptica) against pathogens suspected to cause tracheobronchitis, including nchiseptica. Commercially available products like Nobivac® Intra-Trac3 offer triple protection. There is a nasal vaccine available. [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] However, oral vaccination has advantages in terms of ease of use and the condition of the animals during and after vaccination. Despite having advantages such as causing less discomfort, commercially available products containing the CPI virus have not been available to date. There was no oral vaccine for upper respiratory tract diseases and / or infectious tracheobronchitis. An oral vaccine for canine parainfluenza virus that helps protect dogs has been available for many years. They are truly needed.

[0006] References cited herein are considered to be "prior art" of this application. It should not be interpreted as an acknowledgment that it is available for use. [Means for solving the problem]

[0007] This invention helps to induce protective immunity in animal subjects receiving vaccination. Immunologically effective doses of modified live inuparaline (which provides effective protection against and / or the same) Vaccine for oral administration to animal subjects such as dogs containing influenza (CPI) virus A composition is provided. In certain embodiments, the potency of the modified live CPI virus in the vaccine is 6.0 log 10 HAID 50 / mL or higher. In other embodiments, the potency of the modified live CPI virus in the vaccine is 6.3 log 10 HAID 50 / mL or higher There is. In still other embodiments, the potency of the modified live CPI virus in the vaccine is 6. 5 log 10 HAID 50 / mL or higher. In still other embodiments, the vaccine The potency of the modified live CPI virus in is 6.7 log 10 HAID 50 / mL or higher There is. In still other embodiments, the potency of the modified live CPI virus in the vaccine is 6.9 log 10 HAID 50 / mL or higher. In still other embodiments, the Potency of the modified live CPI virus in the vaccine is 7.1 log 10 HAID 50 / mL or higher. So In still other embodiments, the potency of the modified live CPI virus in the vaccine is 7.3 lo g 10 HAID 50 / mL or higher. In still other embodiments, the modified Potency of the live CPI virus in the vaccine is 7.6 log 10 HAID 50 / mL or higher. Further In other embodiments, the potency of the modified live CPI virus in the vaccine is 7.8 log1 0HAID 50 / mL or higher. In still other embodiments, the modified live C Potency of the PI virus in the vaccine is 8.0 log10 HAID 50 It is 1 / mL or more. In one embodiment, the titer of the modified live CPI virus in the vaccine is 8.3 log 10 H AID 50 In other embodiments, the modified live CPI in the vaccine is greater than or equal to / mL. The virus titer is 8.6 log 10 HAID 50 The value is 1 / mL or more. Morphologically, the titer of the modified live CPI virus in the vaccine is 8.8 log 10 HAI D 50 It is greater than or equal to / mL.

[0008] In the relevant embodiment of the oral vaccine, the modified live CPI virus in the vaccine The titer of S is 6.0 log 10 HAID 50 / mL~9.5 log 10 HAID 50 / mL The titer of virus I is 6.5 log 10 HAID 50 / mL~9.5 log 10 HA ID 50 In another embodiment, the modified CPI in the vaccine The titer of the virus is 6.8 log 10 HAID 50 / mL~9.5 log 10 HAID 50 In another embodiment, the modified live CPI virus in the vaccine is present. The titer of S is 7.0 log 10 HAID 50 / mL~9.5 log 10 HAID 50 In another embodiment, the modified live CPI virus in the vaccine is The titer is 7.3 log 10 HAID 50 / mL~9.5 log 10 HAID 50 / m L. In yet another embodiment, the titer of the modified live CPI virus in the vaccine. 7.3 log 10 HAID 50 / mL~8.6 log 10 HAID 50 / mL be.

[0009] In certain embodiments of vaccines containing modified live CPI virus, the CPI virus is Unique / identifiable characteristics of the modified live CPI virus with ATCC accession number PTA-126273 To share. In a more specific embodiment, the modified live CPI virus in the vaccine is ATCC It has accession number PTA-126273.

[0010] The present invention further comprises a modified live canine parainfluenza (CPI) virus, and further does not contain Vaccine injection containing toxic B. bronchiseptica Helps (and / or induces) protective immunity in animal subjects receiving the seed (and / To provide an oral vaccine for animal subjects (or to provide effective protection). In the application form, in addition to containing one of the above-mentioned quantities of modified live CPI viruses, The vaccine is also 1 x 10 7 cfu / mL ~ 1 × 10 12 potency equal to cfu / mL Includes non-toxic live B. bronchiseptica. Other fruits In terms of administration, the vaccine is 1 × 10 8 cfu / mL ~ 1 × 1012 cfu / mL Non-toxic live B. bronchiseptica with a titer equal to that of Includes. In another embodiment, the vaccine is 5 × 10 8 cfu / mL ~ 1 × 1 0 12 Non-toxic raw B. bronchiseptica (B. bronchi) with a titer equal to cfu / mL It contains septica. In yet another embodiment, the vaccine is 1 × 10 9 c fu / mL ~ 5 × 10 11 Non-toxic B. bronchiseptica (Bb) Includes (ronchiseptica).

[0011] The oral vaccine of the present invention is a non-toxic live B. bronchisep Modified live canine parasite, containing or not containing tica, along with one or more other immunogens. It may contain the influenza virus. In certain embodiments, the vaccine is Furthermore, it contains a weakened live canine influenza virus. In other embodiments, the vaccine The vaccine further contains attenuated live canine parvovirus. In yet another embodiment, the vaccine Chin further contains attenuated live canine distemper virus. In yet another embodiment, The vaccine further contains attenuated live canine adenovirus type 2. Furthermore, the vaccine also contains attenuated live respiratory canine coronavirus. In terms of form, the vaccine further contains attenuated live canine pneumonia virus. Morphologically, the vaccine further contains canine influenza virus and canine parvovirus. Canine distemper virus, canine adenovirus (type 1 or type 2), respiratory canine cholera Canine pneumonia virus, Streptococcus equii subspecies zoepidemicus (S treptococcus equi zooepidemicus) and / or Myco One or more proteins from Mycoplasma cynos It contains a viral vector encoding the original. In yet another embodiment, the vaccine is Furthermore, the less venomous Streptococcus equii subspecies zoepidemicus (Streptoco Includes ccus equi zooepidemicus. In other embodiments, Furthermore, this vaccine contains attenuated live Mycoplasma sinus (Mycoplasma c It contains (ynos). Furthermore, it contains non-toxic live B. bronchisep Modified live canine parainfluenza virus or modified live canine parainfluenza (tica) The vaccine containing the influenza virus further comprises two or more of these other immunogens. Vaccines can be administered with or without adjuvants.

[0012] The present invention further relates to the oral administration of a vaccine containing a modified live canine parainfluenza virus to dogs. To protect dogs from upper respiratory tract diseases and infectious tracheobronchitis, including by administering (and To provide a method to help provide effective defense to dogs. In this embodiment, the titer of the modified live CPI virus administered is 6.0 log1 0HAID 50 / Dose or more. In other embodiments of this method, the modified The titer of the mutated CPI virus is 6.3 log 10 HAID 50 This is above the recommended dose. In yet another embodiment of the method, the titer of the modified live CPI virus administered is 6 .5 log 10 HAID 50 / The dose is greater than or equal to. In yet another embodiment of this method The titer of the modified live CPI virus administered is 6.7 log 10 HAID 50 / The dose is greater than or equal to the dose. In yet another embodiment of this method, the modified raw CPI is administered. The titer is 7.0 log 10 HAID 50 / This is above the dosage. In the administration method, the titer of the modified live CPI virus administered is 7.3 log 10 H AID 50 / Dose or more. In yet another embodiment of this method, the dose administered The titer of the modified raw CPI is 7.6 log 10 HAID 50 / This is above the dosage. In yet another embodiment, the titer of the modified live CPI virus administered is 7.8 Log 10 HAID 50 / The dose is greater than or equal to the dose. In yet another embodiment of this method, The titer of the modified raw CPI given is 8.0 log 10 HAID 50 / Dose is greater than / In yet another embodiment of this method, the titer of the modified live CPI virus administered 8.3 log 10 HAID 50 / Dosage or greater. Further embodiments of this method In this case, the titer of the modified raw CPI administered is 8.6 log 10 HAID 50 / for The amount is greater than the amount. In yet another embodiment of this method, the modified raw CPI administered The titer of Virus is 8.8 log 10 HAID 50 / This is above the recommended dosage.

[0013] In related embodiments of this method, the titer of the modified live CPI virus to be administered is , 6.0 log 10 HAID 50 / dose ~ 9.5 log 10 HAID 50 / dose. In other embodiments of this method, the titer of the modified live CPI virus to be administered is . In yet other embodiments of this method, the titer of the modified live CPI virus to be administered is 6.3 log 10 HAID 50 / dose ~ 9.5 log 10 HAID 50 / dose. In still other embodiments of this method, the titer of the modified live CPI virus to be administered is 6.5 log 10 HAID 50 / dose ~ 9.5 log 10 HAID 50 / dose. In still further embodiments of this method, the titer of the modified live CPI virus to be administered is 6.8 log 10 HAID 50 / dose ~ 9.5 log 10 HAID 50 / dose. In still further embodiments of this method, the titer of the modified live CPI virus to be administered is 7.0 log 10 HAID 50 / dose ~ 9.5 log 10 HAID 50 / dose. In yet further other embodiments of this method, the titer of the modified live CPI virus to be administered is 7.3 log 10 HAID 50 / dose ~ 9.5 log 10 HAI D 50 / dose. In still other embodiments of this method, the modified live C to be administered The titer of the PI virus is 7.3 log 10 HAID 50 / dose~9.0 log 10 H AID 50 / Dosage. A method of orally administering a vaccine containing a modified live CPI virus. In one embodiment, the CPI virus has ATCC accession number PTA-126273. The modified live CPI virus shares its unique / identifiable characteristics. More specific implementations of the method of the present invention. Morphologically, the modified live CPI virus has ATCC accession number PTA-126273.

[0014] In one embodiment of the present invention, to protect dogs from upper respiratory tract diseases and infectious tracheobronchitis. The method that assists (and / or provides effective protection to dogs) is a non-toxic B. b. b. Modified CPI will further include Ronxeptica (B. bronchiseptica) Vaccines containing (i.e., modified live CPI virus and non-toxic live B. bronchiseptica) (Vaccines containing both B. bronchiseptica) are for animal subjects, for example This includes oral administration to dogs.

[0015] In certain embodiments of this method, the amount of one of the modified live CPI viruses is In addition, this vaccine is 1 x 10 7 Non-toxic B. bronchi with a potency exceeding cfu / dose This also includes B. bronchiseptica. In certain embodiments of this method The titer of the non-toxic B. bronchiseptica is 5 x 10 7 The cfu / dose is greater than or equal to the above. In a more specific embodiment of this method, the above The titer of non-toxic live B. bronchiseptica is 1 × 1 08 The cfu / dose is greater than or equal to the specified amount. In a more specific embodiment of this method, the non-toxic The titer of sexually active B. bronchiseptica is 5 × 10⁻⁶. 8 The cfu / dose is greater than or equal to the specified dose. In certain embodiments, the non-toxic B. bronchuseptica The titer of (B. bronchiseptica) is 1 × 10⁻⁶ 9 It is above cfu / dose. In a related embodiment of this method, the non-toxic live B. bronchiseptica (B. bro The titer of nchiseptica is 1 × 10⁻⁶ 7 cfu / dose~1×10 12 CFU / for The quantity. In yet another embodiment of this method, the non-toxic B. bronchiseptica The titer of (B. bronchiseptica) is 1 × 10⁻⁶ 9 cfu / dose~1×10 1 2 The value is cfu / dose. In yet another embodiment of this method, the non-toxic raw B. blotch. The titer of B. bronchiseptica is 1 × 10⁻⁶ 8 cfu / dose ~5×10 11 In yet another embodiment of this method, the non-toxic The titer of sexually active B. bronchiseptica is 5 × 10⁻⁶. 8 cfu / dose~5×10 11 In yet another embodiment of this method, The titer of the aforementioned non-toxic live B. bronchiseptica is , 1 x 10 9 cfu / dose~5×10 11 This is cfu / dose. In the application method, the non-toxic B. bronchiseptic a) The titer is 5 × 10 9 cfu / dose~5×10 11 The dosage is CFU / dose.

[0016] In a more specific embodiment of the method, the titer of the modified live CPI virus is 6.0 Log 10 HAID 50 / The dose is greater than or equal to the aforementioned non-toxic raw B. bronchiseptica (B. The titer of bronchiseptica is 1 × 10⁻⁶ 7 The cfu / dose is greater than the stated amount. In a more specific embodiment of the law, the titer of the modified live CPI virus is 6.5 Log 10 HAID 50 / The dose is greater than or equal to the aforementioned non-toxic raw B. bronchiseptica (Bb The titer of ronchiseptica is 1 × 10⁻⁶ 7 The amount is greater than or equal to the cfu / dose. In a more specific embodiment, the titer of the modified live CPI virus is 6.5 l og 10 HAID 50 / The dose is greater than or equal to the aforementioned non-toxic raw B. bronkiseptica (B. br The titer of *onchiseptica* is 5 × 10 8 It is above cfu / dose.

[0017] In certain embodiments of the method, the titer of the modified live CPI virus is 6.0 Log 10 HAID 50 / dose or more ~9.5 log 10 HAID 50 / Dose, before The titer of the non-toxic live B. bronchiseptica is 1× 10 7 It is cfu / dose or more. In other embodiments of the said method, the modified CPI The titer of the virus is 6.5 log 10HAID 50 / dose or more ~9.5 log 10 HA ID 50 / Dose, and the non-toxic raw B. bronchisep The titer of tica is 1 × 10⁻⁶ 7 It is greater than or equal to cfu / dose. Further implementations of this method In this state, the titer of the modified live CPI virus is 6.8 log 10 HAID 50 / for Amount~9.5 log 10 HAID 50 / Dose, and the aforementioned non-toxic raw B. bronchuseptica The titer of (B. bronchiseptica) is 1 × 10⁻⁶ 7 It is above cfu / dose. In yet another embodiment of this method, the titer of the modified live CPI virus is 6.8 Log 10 HAID 50 / dose~9.5 log 10 HAID 50 / Dose, the above-mentioned no The titer of toxic B. bronchiseptica is 1 × 10⁻⁶. 9 In another embodiment, the modified live CPI virus The titer is 7.0 log 10 HAID 50 / dose~9.0 log 10 HAID 50 / for This is the quantity of the non-toxic live B. bronchiseptica. The potency is 1 × 10⁻⁶ 7 In other embodiments, the modified The titer of the CPI virus is 7.0 log 10 HAID 50 / dose~9.0 log 10 HAID 50 / Dose, and the non-toxic raw B. bronchis The titer of eptica is 1 × 10⁻⁶ 9 It is above cfu / dose.

[0018] In yet another embodiment of this method, the titer of the modified live CPI virus is 6.0 Log 10 HAID 50 / The dose is greater than or equal to the aforementioned non-toxic raw B. bronchiseptica (B. The titer of bronchiseptica is 5 × 10 8 cfu / dose~5×10 11 cf u / dose. In yet another embodiment of this method, the modified live CPI virus The titer is 6.5 log 10 HAID 50 / The dose is greater than or equal to the aforementioned non-toxic raw B. bronki The titer of B. bronchiseptica is 5 × 10 8 cfu / dose~5 ×10 11 The value is cfu / dose. In yet another embodiment of this method, the modified raw C The titer of the PI virus is 6.5 log 10 HAID 50 / The dose is greater than or equal to the above, and is non-toxic. The titer of live B. bronchiseptica is 5 × 10⁻⁶. 9 c fu / dose~5×10 11 The dosage is CFU / dose.

[0019] In another embodiment of this method, the titer of the modified live CPI virus is 6.0 lo g 10 HAID 50 / dose~9.5 log 10 HAID 50 / Dose, and the above non-toxic The titer of live B. bronchiseptica is 5 × 10⁻⁶. 8 c fu / dose~5×10 11 In yet another embodiment of this method, the value is cfu / dose. The titer of the modified live CPI virus is 6.5 log 10 HAID 50 / dose~9.5 Log 10 HAID 50 / Dose, and the non-toxic raw B. bronkiseptica (B. br The titer of *onchiseptica* is 5 × 10 8 cfu / dose~5×10 11 cfu / This is the dosage. In another embodiment, the titer of the modified live CPI virus is 6.5 Log 10 HAID 50 / dose~9.5 log 10 HAID 50 / Dose, the above The titer of non-toxic live B. bronchiseptica is 5 × 1 0 9 cfu / dose~5×10 11 In yet another embodiment of this method... In this case, the titer of the modified live CPI virus is 6.8 log 10 HAID 50 / dose~ 9.0 log 10 HAID 50 / Dose, and the non-toxic raw B. bronchiseptica (B. The titer of *.bronchiseptica* is 5 × 10 8 cfu / dose~5×10 11 c The dosage is fu / dose. In yet another embodiment of this method, the modified live CPI virus The titer is 6.8 log 10 HAID 50 / dose~9.5 log 10 HAID 50 / This is the dosage, and refers to the non-toxic live B. bronchiseptica. The titer is 5 × 10 9 cfu / dose~5×10 11 It is cfu / dose. Morphologically, the titer of the modified live CPI virus is 7.0 log 10 HAID 50 / Dose ~9.0 log 10 HAID 50 / Dose, and the non-toxic raw B. bronchusepti The titer of the mosquito (B. bronchiseptica) is 5 × 10 8 cfu / dose~5×10 11 The cfu / dose is also used in other embodiments. The value is 7.0 log 10 HAID 50 / dose~9.0 log 10 HAID 50 / dose And the power of the non-toxic live B. bronchiseptica The value is 5 × 10 9 cfu / dose~5×10 11 The dosage is CFU / dose.

[0020] Modified live CPI virus and non-toxic live B. bronchisep In a specific embodiment of a method for oral administration of a vaccine containing tica, the non-toxic live B. The Bronchiseptica virus is an ATCC accession number Non-toxic live B. bronchise (B. bronchise) containing PTA-126272 It shares the unique / identification characteristics of ptica. In a more specific embodiment of the method, The non-toxic B. bronchiseptica is a species that has been treated by ATCC. It has the registration number PTA-126272. In a more specific embodiment, the modified The CPI virus has ATCC accession number PTA-126273 and is the non-toxic B. b. Ronxeptica (B. bronchiseptica) is ATCC accession number PTA-1 It has 26272.

[0021] In other embodiments, it helps protect dogs from upper respiratory tract diseases and infectious tracheobronchitis. The method (and / or the method of providing effective protection to dogs) is a modified canine parainfluenza. This includes orally administering a vaccine containing the virus and one or more additional immunogens. In certain embodiments of the law, the vaccine further contains attenuated live canine influenza virus Includes Rus. In other embodiments of the method, the vaccine further includes attenuated live dogs. Contains parvovirus. In yet another embodiment of the method, the vaccine is Furthermore, it contains attenuated live canine distemper virus. In yet other embodiments of the method... Furthermore, the vaccine contains attenuated live canine adenovirus type 2. In embodiments of other species, the vaccine further contains attenuated live respiratory canine coronavirus Includes. In yet another embodiment of the method, the vaccine further includes attenuated live dogs. Contains pneumonia virus. In yet another embodiment of the method, the vaccine further In addition, canine influenza virus, canine parvovirus, canine distemper virus, Nuadenovirus (type 1 or 2), respiratory canine coronavirus, canine pneumonia virus, s Treptococcus equii subspecies zoepidemicus i zooepidemicus) and / or Mycoplasma sinus (Mycopl One or more viruses encoding one or more protein immunogens from (asma cynos) Includes a vector. In yet another embodiment of the method, the vaccine further includes Low-venomous Streptococcus equii subspecies zoepidemicus Includes equi zooepidemicus). Further other species embodiments of the method. In this vaccine, the attenuated live Mycoplasma sinus (Mycoplasma Includes a cynos. Furthermore, it protects dogs from upper respiratory tract diseases and infectious tracheobronchitis. Methods of support in (and / or methods of providing effective protection against dogs) are these immune A vaccine containing modified live canine parainfluenza virus in combination with two or more original vaccines is administered orally. It may include administering the drug.

[0022] In related embodiments, the protection of dogs from upper respiratory tract diseases and infectious tracheobronchitis is supported. A method of providing support (and / or a method of providing effective defense to dogs) is a modified live canine parainfluenza. Enzavirus, non-toxic live B. bronchiseptica This includes, and orally administering to dogs a vaccine containing one or more additional attenuated live immunogens. In certain embodiments of the method, the vaccine further comprises live attenuated canine influenza. Contains a virus. In other embodiments of the method, the vaccine further contains a weakened live virus. Contains canine parvovirus. In yet other embodiments of the method, the vaccine It further contains attenuated live canine distemper virus. Further embodiments of the method. In this method, the vaccine further contains attenuated live canine adenovirus type 2. In other embodiments, the vaccine further contains attenuated live respiratory canine coronavirus Includes. In yet another embodiment of the method, the vaccine further includes attenuated live venom. Contains canine pneumonia virus. In yet another embodiment of the method, the oral vaccine Furthermore, canine influenza virus, canine parvovirus, canine distemper virus Canine adenovirus (type 1 or 2), respiratory canine coronavirus, canine pneumonia virus Rus, Streptococcus equii subspecies zoepidemicus equi zooepidemicus) and / or Mycoplasma sinus (Myc Virusbes encoding one or more protein immunogens from Oplasma cynos Including a cytoplasm. In yet another embodiment of the method, the vaccine is further weakened. Poisonous Streptococcus equii subspecies zoepidemicus Includes equi zooepidemicus). Further embodiments of the method include Furthermore, the vaccine contains attenuated live Mycoplasma sinus (Mycoplasma Includes cynos. Furthermore, dogs with the above-mentioned upper respiratory tract disease and infectious tracheobronchitis Methods to support defense (and / or provide effective defense to dogs) are these immunogens Combined with 2 or more, modified live canine parainfluenza virus and non-toxic B. bronchi This includes oral administration of a vaccine containing Septica (B. bronchiseptica). It is possible.

[0023] In certain embodiments, it helps protect dogs from upper respiratory tract diseases and infectious tracheobronchitis. The method (and / or the method of providing effective protection to dogs) is a modified live canine paraphlebe Canine parvovirus, attenuated live canine parvovirus, attenuated live canine distempervirus, attenuated live canine distempervirus Nuadenovirus type 2, and non-toxic live B. bronchisep This includes oral administration of a vaccine containing tica.

[0024] In certain embodiments, the present invention provides a method for treating upper respiratory tract diseases and infectious tracheobronchitis in dogs. Methods to support defense (and / or methods to provide effective defense to dogs) are simple for dogs. This may include administering a single dose of the vaccine orally. Therefore, in certain embodiments, The method involves administering a single dose containing modified live canine parainfluenza virus to dogs. This includes administering the vaccine orally. In a related embodiment, the method is used to treat dogs. Modified live canine parainfluenza virus and non-toxic live B. bronchiseptica (B. This includes orally administering a single-dose vaccine containing both bronchiseptica. nothing.

[0025] In another embodiment, the present invention provides protection for dogs from upper respiratory tract diseases and infectious tracheobronchitis. Methods to support (and / or provide effective protection to dogs) are 2 or more for dogs. This may include orally administering a dose of the vaccine. Therefore, in certain embodiments, The method involves administering two or more modified live canine parainfluenza viruses to dogs. This includes orally administering a dose of the vaccine. In a related embodiment, the method is For dogs, modified live canine parainfluenza virus and non-toxic live B. bronchusepti Oral administration of two or more doses of vaccine containing both B. bronchiseptica. This includes giving in to the agreement.

[0026] In certain embodiments, it helps protect dogs from upper respiratory tract diseases and infectious tracheobronchitis. A method (and / or a method that provides effective protection to dogs) is an adjuvant against dogs. This includes orally administering an unprocessed vaccine. Specific embodiments of these methods include: A vaccine containing a modified live canine parainfluenza virus, which is adjuvanted. This includes orally administering a vaccine, which is not available, to dogs. Specific embodiments include modified live canine parainfluenza virus and non-toxic live B. bron. A vaccine containing Xeptica (B. bronchiseptica), and an adjuvant. This includes orally administering a vaccine that is not pre-treated to dogs. In certain embodiments, the vaccine is administered as a single-dose vaccine.

[0027] Methods to support the defense of dogs against upper respiratory tract diseases and infectious tracheobronchitis (and / or dogs Another embodiment of a method that provides effective protection is to administer an adjuvanted vaccine to dogs. This includes oral administration. Specific embodiments of these methods include modified live canine parainfluenza. A vaccine containing the influenza virus, and that vaccine contains an adjuvant. This includes oral administration to dogs. Related embodiments of these methods are modified live dogs. Parainfluenza virus and non-toxic live B. bronchis A vaccine containing both eptica and adjuvant, administered to dogs. This includes oral administration. In certain embodiments of this type, the vaccine is a single dose. It is administered as a vaccine.

[0028] Methods to support the defense of dogs against upper respiratory tract diseases and infectious tracheobronchitis (and / or dogs In certain embodiments of the method of providing effective protection against, the vaccine of the present invention is 0. It is administered orally in doses of 2 mL to 5 mL. In related embodiments, the vaccine of the present invention It is administered orally in doses of 0.2 mL to 4 mL. In other specific embodiments, the present invention The vaccine is administered orally in doses of 0.3 to 1.5 mL. Furthermore, in other embodiments... The vaccine of the present invention is administered orally in doses of 0.2 mL to 3.0 mL. In embodiments, the vaccine of the present invention is administered orally in a dose of 0.2 mL to 2.5 mL. In further embodiments, the vaccine of the present invention is administered in doses of 0.2 mL to 2.0 mL. It is administered orally. In further embodiments, the vaccine of the present invention is 0.5 to 2.5 It is administered orally in a dose of mL. In yet another embodiment, the vaccine of the present invention is 0. It is administered orally in doses of 75 mL to 2.0 mL. In further embodiments, the present invention The vaccine is administered orally in doses of 0.5 mL to 2.0 mL. In other embodiments... Furthermore, the vaccine of the present invention is administered orally in a dose of 0.5 mL to 1.5 mL. In this embodiment, the vaccine of the present invention is administered orally in a dose of 0.75 mL to 1.5 mL. In certain embodiments, the vaccine of the present invention is administered orally in a dose of 1.0 mL. It can be done.

[0029] For these and other aspects of the present invention, refer to the following detailed description, including the following examples. This will deepen understanding. [Modes for carrying out the invention]

[0030] Therefore, the present invention relates to a modified live canine paralysis vaccine, which includes a polyvalent vaccine useful as an oral vaccine. This invention provides a canine parainfluenza virus vaccine. A method for orally immunizing dogs against Rus, using modified live canine parainfluenza (CP) I) The present invention also provides a method comprising orally administering a virus-containing vaccine to dogs. Furthermore, a single-dose vaccine containing a modified live canine parainfluenza virus is provided. Such vaccines do not require a booster vaccine and effectively treat upper respiratory tract diseases and infectious tracheal diseases. Supports the protection of vaccinated dogs against bronchitis for at least 6 months (and / or provides effective protection against dogs. In certain embodiments, the single The vaccine is administered to dogs in a dose of 1 mL.

[0031] The present invention further relates to canine influenza viruses (e.g., H3N2 and / or H3N8) ), canine parvovirus, canine distempervirus, canine adenovirus, canine respiratory tract Coronavirus, canine pneumonia virus, Mycoplasma species (e.g., Mycoplasma sicca Mycoplasma cynos, and Streptococcus equii subspecies - Epidemicus (Streptococcus equi zooepidemicus) ) combined with one or more other canine pathogens and / or immunogens that further induce immunity against ) We provide a vaccine containing a modified live canine parainfluenza virus.

[0032] More specifically, the present invention also relates to non-toxic live B. bronxeptica (B. bronchi A polyvalent oral vaccine containing modified live canine parainfluenza virus (septica) This invention further includes canine parainfluenza virus and B. bronchiseptica. A method for immunizing dogs against (B. bronchiseptica), and modified Canine parainfluenza virus and non-toxic live B. bronchus This invention provides a method for orally administering a vaccine containing iseptica to dogs. In certain embodiments of the invention, modified live canine parainfluenza virus and non-toxic live B A single-dose vaccine containing Bronchiseptica (or Dogs given only one dose of the multivalent vaccine do not require a booster vaccine and can survive in the wild. CPI virus and wild-type non-toxic B. bronchisep For upper respiratory tract diseases and infectious tracheobronchitis caused by tica, Both provide protection for 6 months. In a more specific embodiment, the single-dose vaccine is administered to dogs. It is administered at a dose of 1 mL.

[0033] In certain embodiments, the vaccine contains CPI virus serum neutralizing antibodies and B. blotch. B. bronchiseptica agglutination and / or vaccination The immunologically effective amount of modified CPI is determined by the amount of IgA antibody induced in dogs. Contains viruses and non-toxic live B. bronchiseptica. In a related embodiment, the present invention relates to the toxic CPI virus and the toxic B. bronchosepti Contains specific minimum amounts of each antigen that are effective against B. bronchiseptica. To provide a vaccine. In yet another embodiment, the vaccine of the present invention is safe and effective. Yes, CPI virus and B. bronchise in dogs. Supports the protection of dogs from upper respiratory tract diseases and infectious tracheobronchitis caused by ptica infections. (and / or provides effective protection against dogs).

[0034] The present invention also relates to canine influenza viruses (e.g., H3N2 and H3N8), dogs Parvovirus, canine adenovirus, canine distemper virus, canine adenovirus Canine respiratory coronavirus, canine pneumonia virus, Mycoplasma species (for example, Myco Plasma cynos (Mycoplasma cynos) and Streptococcus e Streptococcus equi zooepidemicus (a subspecies of quail) One or more other canine pathogens and / or immunogens that further induce immunity against micus) Furthermore, in combination, attenuated live CPI virus and non-toxic live B. bronchiseptica (Bb We offer a multivalent vaccine containing ronchiseptica.

[0035] The term "approximately" as used herein is interchangeable with the term "about". This means the value is within 25% of the specified value, i.e., "approximately" 4.0 mL. A single dose of the vaccine containing [ingredient] can contain 3.0 to 5.0 mL.

[0036] As used herein, the term "canine" is equivalent to "dog." This term is used interchangeably with the term Can, and unless otherwise specified, it refers to all pet dogs, i.e., Can This includes is lupus familiaris or Canis familiaris. .

[0037] As used herein, the term “cat” refers to any member of the Felidae family. Domestic cats, purebred and / or mixed-breed pet cats, and wild or feral cats are all considered cats. .

[0038] As used herein, “vaccine” is suitable for application to animals, for example, dogs. This involves one or more antigens, i.e., one or more immunogens, typically in a liquid containing sterile water. It contains a pharmaceutically acceptable carrier and, when administered to animals, it can cause wild-type microorganisms (for example) (and) to at least support protection against diseases resulting from infections caused by viruses or bacteria. Strong enough to provide (and / or effective protection), i.e., to support and effectively prevent disease. It provides adequate protection and induces a sufficiently strong immune response to prevent or improve the disease. In a more accurate embodiment, the vaccine is administered by wild-type microorganisms (e.g., viruses or bacteria). It is strong enough to provide effective protection against diseases resulting from infection. Here, "wa The use of the term "kuching" encompasses both monovalent and polyvalent vaccines. Vaccines administered orally to elephants can also be called "oral vaccines."

[0039] As used herein, "polyvalent vaccine" refers to a vaccine containing two or more different antigens. In this particular embodiment, the polyvalent vaccine is effective against two or more different pathogens. It stimulates the immune system of the recipient.

[0040] As used herein, "protect" and "protect (prot)" are used to mean "to defend (protect) (prot) (ecting), "provides effective p Rotation), providing effective defense "Supportive protection" and "Supporting defense (aids in the public)" (Action), "Aiding in the Protect tion) and "aids in eliciting protective immunity" The term "protective immunity" refers to the ability to avoid any signs of infection. It does not require complete defense. For example, "defensive support" is a foundational feeling after the challenge. The symptoms of the infection are at least reduced, and / or the underlying cellular, biological factors that cause the symptoms are eliminated. Protection such that one or more physical or biochemical causes or mechanisms are mitigated and / or eliminated. It can mean that it is sufficient. The word "mitigation" used in this context simply means the infection This is understood to mean a relationship with the infectious state, including the molecular state of infection, rather than just the physiological state. .

[0041] When used in this specification, the phrases "provides an effective defense" and "provides an effective defense" are used in this specification. The term "ru" is used interchangeably. Modified biological components composed of the vaccine of the present invention Regarding the Nupara Influenza virus, it causes upper respiratory tract diseases and infectious tracheobronchitis in dogs. "Providing effective protection against fire" is a challenge against live wild-type CPI virus The following is the CPI between vaccinated dogs and placebo-vaccinated control dogs. To find a statistically significant reduction in the duration of viral shedding and / or clinical signs of the disease. Therefore, it is determined. Non-toxic live B. bronchus punctata composed of the vaccine of the present invention. Regarding (B. bronchiseptica), upper respiratory tract disease and infectious tracheal tract disease in dogs. "Providing effective protection" from bronchitis is the role of wild-type B. bronchiseptica (B. br Following the challenge by onchiseptica, vaccinated dogs and plasters A statistically significant reduction in the number of affected dogs was found between dogs vaccinated against -vo and dogs vaccinated against -vo. This is determined by the following: Infected dogs must be observed for at least two consecutive days during the observation period after the challenge. This is defined as having spontaneous coughing or spontaneous coughing accompanied by nausea.

[0042] When used herein, "viral shedding period" refers to the period of viral titration from a nasal swab. Therefore, it is measured from the first outbreak to the last outbreak in which the virus was expelled from the dog's nasal cavity. This is the number of days. In a particular embodiment of the present invention, the duration of virus shedding is defined as the duration of CPI virus shedding. Measure the S.

[0043] As used herein, “bacterial shedding period” refers to the period determined by bacterial titration from a nasal swab. The number of days from the first occurrence of bacteria being expelled from the dog's nasal cavity to the last occurrence is measured. Yes. In certain embodiments of the present invention, the duration of bacterial shedding is controlled to B. bronchiseptica (B Measure the amount of bronchiseptica.

[0044] As used herein, a dog that is "seroconverted to antibodies" is a dog that has tested positive for a specific antigen (e.g., CPI). Dogs against the virus or B. bronchiseptica This means that the antibody titer is at least twice as high as the baseline value for that particular antigen. ru.

[0045] As used herein, the terms “attenuated live virus” and “modified live virus” are used in this specification. It is a reusable, immunogenic, but non-pathogenic, attenuated live viral immunogen.

[0046] As used herein, "attenuated bacteria," "non-toxic bacterial culture," and "non-toxic" are used. The term "live bacteria" is used interchangeably and refers to attenuated live bacteria that are immunogenic but not pathogenic. Epidemics (e.g., non-toxic live B. bronchiseptica) That is the case.

[0047] When used herein, the “dose” of a vaccine administered to an animal subject is, for example, Weight-based, such as 4 μg / dose or 2-6 μg / dose, 7.3 log10HAID50 Antigens based on potency criteria such as / dose or 7.3-8.6 log10HAID50 / dose When defined as including a specific amount or range of amounts, the total dose may be administered as a single dose. It is possible to administer it, or it can be done by administering it multiple times at intervals of 3 hours or less. Specific implementation In terms of form, the vaccine dose is administered orally to the animal subjects in a single dose. .

[0048] As used herein, “single-dose vaccine” means at least one of the pathogens A vaccine (or polyvalent vaccine) containing an immunogen, and that vaccine (or polyvalent vaccine) It can be administered as a single dose, or as multiple doses over a short period, i.e., at intervals of 3 hours or less. It is administered to an object, and a second dose of the vaccine (e.g., a booster vaccine) is also administered. If not necessary, provide support for the animal's defense against pathogens for at least 6 months (and / or provides effective protection to the animal). In certain embodiments of this type, a single dose is used. Kuchin is administered as a single dose to animal subjects, such as dogs, as a single portion of the vaccine. It is administered orally. In certain embodiments, a single-dose vaccine is administered to animal subjects in a 1 mL dose. It is administered orally. Therefore, the vaccine of the present invention provides immunity for at least 6 months. It is intended to be administered orally as a single-dose vaccine to animal subjects, such as dogs. In certain embodiments, the duration of immunity is at least 9 months. In the administration method, the duration of immunity is at least 12 months. Furthermore, in other embodiments... Furthermore, the duration of immunity is at least 18 months.

[0049] In another embodiment, the second dose of the vaccine (or polyvalent vaccine) is the first oral dose. For a period of 6 to 18 months thereafter, the first dose is administered one week, several weeks, or several months later. The booster vaccine is administered by injection (e.g., intramuscular, subcutaneous), nasal, or orally. It is possible. Therefore, in some embodiments, the vaccine is used for at least two purposes. The vaccine is administered orally in doses. In some such embodiments, for example, the vaccine is administered in two doses. The second dose (e.g., booster vaccine) is administered at least approximately the same amount as the initial dose of the first dose. It is administered two weeks later. In some embodiments, the vaccine is administered twice, and the second dose The dose is administered up to 8 weeks after the first dose. In other embodiments, the second dose is Approximately 2 weeks to 4 months after the first dose, approximately 2 weeks to 8 weeks after the first dose, or the first It is administered approximately 3 to 4 weeks after the first dose. In some embodiments, the second dose is the It is administered approximately 4 weeks after the first dose. The first and subsequent doses are, for example, the amount and / or The form may change. However, in many cases, the dosage is determined in terms of quantity and form. It is the same. The administration is given as a single dose vaccine or multiple doses [i.e., Regardless of whether it is administered as a [Superstar vaccine (or similar)], subsequent administration of the vaccine should be once a year. After a period of 6 to 18 months of immunization (or longer), such as a single dose or every other year, the prescribed dosage is administered. It should be understood that it may be necessary to provide this to animal subjects.

[0050] A single dose of the oral vaccine of the present invention provides protection to animals from pathogens for at least 6 months. If sufficient to provide support (and / or effective protection to the animal), the dose The amount of antigen released generally contains a therapeutically effective dose of vaccine for a duration of six months or more. On the other hand, if a booster vaccine dose is needed to supplement the initial dose, the initial vaccine The combined dose of the booster vaccine may constitute a therapeutically effective dose.

[0051] The terms "adjuvant" and "immunostimulant" are used interchangeably in this specification. It is defined as one or more substances that trigger stimulation of the immune system.

[0052] As used herein, “non-adjuvanted vaccine” means a vaccine that does not contain an adjuvant. It is a single vaccine or a polyvalent vaccine.

[0053] As used herein, the term “pharmaceutically acceptable” means that the noun being modified is It is used adjectivally to mean that something is suitable for use in pharmaceutical products. For example, pharmaceutical When using it to describe excipients in a vaccine, it means that the excipient is not part of the composition. It is characterized by being compatible with the ingredients and not adversely harmful to the intended target animal, such as dogs. It is something that is attached.

[0054] In certain embodiments, the vaccine of the present invention also comprises a pharmaceutically acceptable immunostimulant and It can be administered together with an adjuvant and / or a bioadhesive polymer. In this context, adjuvants enhance the immune response to one or more vaccine antigens / isolates. It is used to enhance the immune response to a specific antigen. Therefore, an "adjuvant" is used to strengthen the immune response to a specific antigen. It is a drug that nonspecifically increases the response, and therefore, the antigen required in any vaccine. The amount, and / or the number of injections required to produce a sufficient immune response against the target antigen. It reduces [something]. Suitable adjuvants for animal vaccination include aluminum hydroxide. Mineral gels such as aluminum phosphate and alum; and pluronic polyols. This includes, but is not limited to, surfactants and oil emulsions. See below for examples. One adjuvant shown is CARBIGEN (trademark name), which contains an emulsifying agent. In a microbial carbomer-based (CARBOPOL® 934P) adjuvant suspension Yes, MVP Adjuvants [Location: 4805 "G" Street Oma It does not contain animal-derived components obtained from [ha, NE]. CARBOPOL (Registered (Registered Trademark) 934P is a cross-linked ayl pentaerythritol. It is a homopolymer of acrylic acid.

[0055] PVP-K60, a bioadhesive polymer, is a hygroscopic amorphous polyvinyl pylori polymer. It is a linear nonionic polymer that is soluble in water and organic solvents and has pH stability (K60 This refers to its molecular weight.

[0056] Information regarding various aspects of adjuvants and immunoassays can be found, for example, in P.Tijss. A series by en, Practice and Theory of Enzyme Immunoassays, 3rd Edition, 1987, Elsevie Disclosed in r, New York, which is incorporated herein by reference. Scientifically acceptable immunostimulants include bacterial and / or fungal cell wall components (e.g., lipopolysaccharides). , lipoproteins, glycoproteins, muramyl peptides), mucoadhesive polymers, plant-derived various complex carbohydrates (e.g., glycans, acemannan), various proteins and peptides (e.g., hormones, cytokines, co-stimulatory factors) derived from animals, and novel nucleic acids (e.g., double-stranded RNA, CpG) derived from viruses and / or other sources.

[0057] The live attenuated CPI virus for use in the vaccines of the present invention can be prepared by conventional means. Conventional means include, for example, modification of pathogenic strains by in vitro passage, cold adaptation, modification of the pathogenicity of organisms by genetic manipulation, selection of non-pathogenic wild-type strains, and other methods well known to those skilled in the art. Next, such a live attenuated CPI virus can be tested to determine whether it is suitable for oral administration, as detailed in the following examples. can.

[0058] The modified live CPI virus strain can be induced by serial passage of the wild-type virus in cell culture. In another embodiment, the modified live CPI virus strain is induced by serial passage of the wild-type virus through experimental animals and / or non-host animals. Accumulation of genetic mutations during such passage typically results in a progressive loss of the pathogenicity of the organism for the original host. In some embodiments, the live attenuated virus strain is prepared by cold adaptation. Cold-adapted viruses have the advantage of replicating only at the temperatures found in the upper respiratory tract. A method for generating cold-adapted equine influenza virus is described in U.S. Patent No. 6,177,082 which is incorporated herein by reference in its entirety]. The desired resulting low temperature Adapted viruses exhibit the following phenotypes: cold adaptation, temperature sensitivity, dominant interference, and / or attenuation. 1 or more of the following are added. Furthermore, modified live canine parainfluenza virus and non-toxic live B Both of B. bronchiseptica have been previously disclosed. It is included in commercially available dog vaccines.

[0059] biological deposit The cultures of the following biological materials are subject to the following international deposit, provided they meet the requirements of the Budapest Convention. Organization: American Type Culture Collection (ATC) C) 10801 University Boulevard, Manassas, It is deposited in Va. 20110-2209, USA. The deposited items are not available to the public. All restrictions imposed by the depositor to make it available are removed at the time the patent is granted. That cannot be changed. [Table 1]

[0060] The following examples may help to provide a further understanding of the present invention, but in any way this invention may not be presented. This does not mean limiting the effective range of the light. [Examples]

[0061] Example 1 Administered with or without a bioadhesive polymer or bioadhesive adjuvant. Oral canine parainfluenza virus and B. bronchi Septica) Vaccine efficacy Materials and methods vaccine: The experimental vaccine is a stabilizer solution [hydrolyzed gelatin, NZ amine AS (Millipo Enzymatic hydrolyzate of casein obtained from Sigma, Burlington, MA ) was mixed with sorbitol-d, disodium phosphate and then lyophilized, a non-toxic live B. bronchiseptica antigen B-C2, ATCC deposit number PTA-126272 and modified live CPI virus, strain Cornell, antigen ATC deposit number PTA-126273. The vaccines administered to dogs in treatment groups A, C and D contained 10 - 15% CARBIGEN (trademark) as an adjuvant. The vaccine administered to dogs in treatment group C also contained PVP-K60, a bioadhesive polymer.

[0062] The titer of the CPI virus in the vaccine was 7.5 - 8.5 log 10 HAID 50 / mL and varied, while the titer of B. bronchiseptica (B.bronchiseptica ) in the vaccine varied from 9.2×10 9 to 1.4×10 10 cfu / mL. All vaccines were lyophilized in vials and the lyophilized vaccine in each vial was rehydrated with 1 mL of sterile water on the day of inoculation and similar preparations were pooled. The titer of the CPI virus is provided in log HAID / mL units, which is a unit commonly used to estimate the concentration of the virus in the sample calculated by the Spearman-Karber method. 10 HAID 50 / mL units, which is a unit commonly used to estimate the concentration of the virus in the sample calculated by the Spearman-Karber method. Animals:

[0063] Animals: Seven 10-week-old beagles (Marshall Bioresources) were housed together in BSL-2 facilities on a concrete floor covered with wood shavings. Diet and water were available ad libitum and modified live CPI virus, strain Cornell, antigen ATC deposit number PTA-126273. The vaccines administered to dogs in treatment groups A, C and D contained 10 - 15% CARBIGEN (trademark) as an adjuvant. The vaccine administered to dogs in treatment group C also contained PVP-K60, a bioadhesive polymer. The titer of the CPI virus in the vaccine was 7.5 - 8.5 log HAID / mL and varied, while the titer of B. bronchiseptica (B.bronchiseptica) in the vaccine varied from 9.2×10 to 1.4×10 cfu / mL. All vaccines were lyophilized in vials and the lyophilized vaccine in each vial was rehydrated with 1 mL of sterile water on the day of inoculation and similar preparations were pooled. The titer of the CPI virus is provided in log HAID / mL units, which is a unit commonly used to estimate the concentration of the virus in the sample calculated by the Spearman-Karber method. Animals: Seven 10-week-old beagles (Marshall Bioresources) were housed together in BSL-2 facilities on a concrete floor covered with wood shavings. Diet and water were available ad libitum It should be noted that the original text seems to have some formatting and content issues that made the translation a bit challenging to present cleanly. There are some repeated and overlapping parts in the original that were adjusted as best as possible during translation. I made it possible.

[0064] Vaccination and serum collection: On day 0 of the trial, dogs in treatment group D were given CPI virus and B. bronchiseptica (Bb From ronchiseptica (Bb) and 15% CARBIGEN (trademark name) A 1 mL dose of vaccine was administered orally. A spray device was attached to the syringe. The vaccine was delivered to the back of the throat. On the 14th day of the trial, all dogs were given their respective pools. The vaccine was administered orally [see Table 1 below]. On day 27 of the trial, the jugular vein was vaccinated. Whole blood was collected by pulse puncture. Serum was separated by centrifugation and kept at -10°C or below until testing. It was stored at a specific temperature.

[0065] Detection of CPI virus neutralizing antibodies: CPI virus neutralizing antibodies were detected using a standard serum neutralization (SN) assay. The diluted solution was incubated with the CPI virus vaccine strain and inoculated into canine kidney cells. After 5-7 days, the monolayer was fixed and stained with fluorescein-conjugated CPI virus antiserum. The SN antibody titer is then calculated as the reciprocal of the serum dilution that causes 50% inhibition of viral infection. I calculated it.

[0066] Table 1 Test design [Table 2]

[0067] Challenge: On the 28th day of the experiment, the dogs were administered the toxic CPI virus.

[0068] Post-challenge observations and samples: Clinical observations were recorded for 11 days after the challenge, and daily for 11 days after the challenge. A nasal swab sample was collected.

[0069] result Serology: Prior to vaccination, all dogs had a CPI virus serum neutralizing titer of less than 2. This indicated that the dog had not been infected with the virus at the time of vaccination. The control dogs that received the vaccine remained seronegative (SN<2) until immediately before vaccination. Almost all dogs in the group became serotonically positive for antibodies after vaccination (titer > 4) [see Table 2 below].

[0070] Table 2 Serum antibody titer against CPI virus [Table 3]

[0071] CPI virus shedding: The duration of CPI virus shedding was a key variable for evaluating vaccine efficacy. For each animal, the period of viral shedding from the first to the last dose was calculated in days, and each treatment group was assigned... The average shedding period was calculated. The average viral shedding period for the placebo-vaccinated control group was 7 It was 0 days. In contrast, the average elimination period for treatment groups A and C was 0 days, but for treatment groups B and The average discharge period for D was 1 day [see Table 3 below].

[0072] Table 3 Emission period [Table 4]

[0073] These results indicate that CARBIGEN (trademark) and PVP-K60 are effective in vaccinated dogs. This indicates that it had little effect on the CPI virus shedding period.

[0074] Example 2 Polyvalent oral CPI virus and B. bronchiseptica with and without adjuvants ( CPI virus dose response of B. bronchiseptica vaccine Materials and methods vaccine: The experimental vaccine contains stabilizers [hydrolyzed gelatin, NZ amine AS, sorbitol-d, Mix with disodium phosphate, then freeze-dry, non-toxic raw B. bronchiseptica ( B. bronchiseptica) antigen B-C2, ATCC accession number PTA-1262 72, and modified live CPI virus, strain Cornell, antigen ATCC accession number PTA- It contained 126273. The vaccine administered to dogs in treatment groups C and D was adjuvant. It contained 15% CARBIGEN (trademark) as a base. Freeze-dried on the day of vaccination. Each vial of vaccine was rehydrated with 1 mL of sterile water, and the same preparation was pooled. Treatment Group A The titers of the CPI virus in the vaccines used in ~D were 8, as shown in Table 5. .6, 7.3, 7.6, and 6.3 log 10 HAID 50 It was / mL.

[0075] animal: Fifteen 17-week-old Beagles (Marshall Bioresources) were fed wood chips. They were raised together in a BSL-2 facility on a covered concrete floor. Food and water were freely available. I made it possible to take it.

[0076] Vaccination and serum collection: On day 0 of the trial, dogs were given their individual pooled vaccine doses of 1 mL via oral route. Then I got vaccinated [see Table 4 below]. I attached a spray device to the syringe and injected it into the back of my throat. The vaccine was delivered. On the 27th day of the trial, whole blood was collected by jugular vein puncture. Serum The components were separated by centrifugation and stored at a temperature of -10°C or below until testing.

[0077] Detection of CPI virus neutralizing antibodies: CPI virus neutralizing antibodies were detected using a standard SN assay. Serum dilutions were then collected in C The PI virus vaccine strain was incubated with the vaccine strain and inoculated into canine kidney cells. (5-7 days) Next, the monolayer was fixed, stained with fluorescein-conjugated CPI virus antiserum, and the SN antibody titer was measured. This was calculated as the reciprocal of the serum dilution that causes 50% inhibition of viral infection.

[0078] Challenge: On the 28th day of the experiment, the dogs were administered the toxic CPI virus.

[0079] Post-challenge observations and samples: Clinical observations were recorded for 14 consecutive days, and nasal swab samples were collected for 10 consecutive days after the challenge. We collected it.

[0080] Table 4 Test design [Table 5]

[0081] result Serology: Prior to vaccination, all dogs had a CPI virus serum neutralizing titer of less than 2. This indicated that the dog had not been infected with the virus at the time of vaccination. The control dogs that received the vaccine remained seronegative (SN<2) until immediately before vaccination. Almost all dogs in the group became serotonically positive for antibodies after vaccination (titer > 4) [see Table 5 below].

[0082] Table 5 Serum antibody titer against CPI virus [Table 6]

[0083] CPI virus shedding: The duration of CPI virus shedding was a key variable for evaluating vaccine efficacy. For each animal, the period of viral shedding from the first to the last dose was calculated in days, and each treatment group was assigned... The average shedding period was calculated. All placebo-vaccinated control dogs (treatment group E) were able to Rus was excreted, with an average excretion period of 6 days. In contrast, the average excretion period for treatment groups A-D was... The values ​​were 1, 0, 1, and 5, respectively [see Table 6 below].

[0084] Table 6 Emission period [Table 7]

[0085] These results, evaluated as a function of viral shedding period, were ≥6.3 per 1 mL dose. Log 10 HAID 50 Modified live CPI virus at / mL, oral modified live CPI virus This suggests that kuchin is necessary for the substance to be effective.

[0086] Example 3 Oral canine parainfluenza and Bordetella bronchiseptica To demonstrate the efficacy of the CPI fraction of a bronchiseptica (combined vaccine) Immunogenicity testing in dogs Materials and methods vaccine: The experimental vaccine is a modified live CPI virus strain, Cornell, ATCC accession number PTA- 126273 and the non-toxic form of B. bronchiseptica It consists of antigen B-C2 (ATCC accession number PTA-126272), and is stable to it. Consolidating agent [hydrolyzed gelatin, NZ amine AS, sorbitol-d, disodium phosphate] It was a mixture of [ ] and freeze-dried. The placebo vaccine was tested by removing the CPI antigen. It consisted of all the components of the experimental vaccine. On the day of vaccination, the freeze-dried vaccine was prepared. Each vial was rehydrated with 1 mL of sterile water, and the same preparation was pooled.

[0087] animal: Twenty 7-week-old beagles (Marshall Bioresources) were covered with wood shavings. They were raised together in a BSL-2 facility on a cracked concrete floor, and a 7-week-old Beagle (Mars) Nineteen individuals (hall Bioresources) were similarly kept in a separate room. Diet and Water was made freely available.

[0088] Vaccination and serum collection: On day 0 of the trial, dogs were given their individual pooled vaccine doses of 1 mL via oral route. Then I got vaccinated [see Table 7 below]. I attached a spray device to the syringe and applied it to my mouth ( The vaccine was delivered to the back of the oropharynx. On the 21st day of the trial, whole blood was collected by venipuncture of the jugular vein. The serum was collected. It was separated by centrifugation and stored at a temperature of -10°C or below until testing.

[0089] Detection of CPI virus neutralizing antibodies: CPI virus neutralizing antibodies were detected using a standard SN assay. Serum dilutions were then collected in C The PI virus vaccine strain was incubated with the vaccine strain and inoculated into canine kidney cells. (5-7 days) Next, the monolayer was fixed, stained with fluorescein-conjugated CPI virus antiserum, and the SN antibody titer was measured. This was calculated as the reciprocal of the serum dilution that causes 50% inhibition of viral infection.

[0090] Challenge: On the 21st day of the experiment, the dogs were administered the toxic CPI virus.

[0091] Post-challenge observations and samples: Clinical observations were recorded for 14 consecutive days, and nasal swab samples were collected for 10 consecutive days after the challenge. We collected it.

[0092] Table 7 Test design [Table 8]

[0093] result Serology: Prior to vaccination, all dogs had a CPI virus serum neutralizing titer of less than 2. This indicated that the dog had not been infected with the virus at the time of vaccination. Vaccination with the bovaccine did not induce antibodies specific to CPI, but the test vaccine... In contrast, 15 out of 20 vaccinated animals (75%) had a CPI serum neutralizing titer of 4 or higher. It induced a reaction, and the titer ranged from 6 to 861 (GMT=31) three weeks after vaccination [see table below]. See 8.

[0094] Table 8 CPI serum antibody titer [Table 9]

[0095] CPI virus shedding: The duration of CPI virus shedding was a key variable for evaluating vaccine efficacy.

[0096] For each animal, the period of viral shedding from the first to the last dose was calculated in days, and each treatment group was assigned to... The average shedding period was determined. 1 of the placebo-vaccinated control dogs (treatment group B) All dogs except the head shed the CPI virus (i.e., 95%), while vaccinated dogs... Only 11 of the species shed the virus (i.e., 55%). CPI virus shedding. The median duration of symptoms was 6 days in placebo-vaccinated control dogs, compared to the vaccine-vaccinated dogs. In vaccinated dogs, the median shedding period was only 1.5 days [see Table 9 below].

[0097] Table 9 Emission period [Table 10]

[0098] These results, evaluated as a function of viral shedding period, were 7.1 lo per 1 mL dose. g 10 HAID 50 The oral modified live CPI virus vaccine contains CPI virus at a concentration of / mL. This indicates that it is sufficient to be effective.

[0099] Example 4 Oral canine parainfluenza and Bordetella bronchiseptica combined vaccine Bordetella Immunogenicity study to demonstrate the efficacy of the Terra bronchiseptica fraction Materials and methods vaccine: The experimental vaccine is a modified live CPI virus strain, Cornell, ATCC accession number PTA- 126273 and the non-toxic form of B. bronchiseptica It consists of antigen B-C2 (ATCC accession number PTA-126272), and is stable to it. Consolidating agent [hydrolyzed gelatin, NZ amine AS, sorbitol-d, disodium phosphate] The placebo vaccine was a mixture of [ ] and freeze-dried. (B. bronchiseptica) consists of all components of the test vaccine, excluding the antigen. On the day of vaccination, each freeze-dried vaccine vial was rehydrated with sterile water. They pooled similar preparations.

[0100] animal: 21 seven-week-old beagles were kept together in a BSL-2 facility on a concrete floor covered with wood chips. They were raised in the same way, with 21 seven-week-old beagles being kept in a separate room. Food and water were freely available to them. I made it possible.

[0101] Vaccination: On day 0 of the trial, dogs were given their individual pooled vaccine doses of 1 mL via oral route. Then I got vaccinated [see Table 10 below]. I attached a spray device to the syringe and then... The vaccine was delivered deep into the oropharynx.

[0102] Sample collection: On the 34th day of the experiment, whole blood was collected by venipuncture of the jugular vein. The serum was separated by centrifugation. Separate the samples and store them at a temperature below -10°C until the test. B. bronc To check for the presence of hiseptica, a nasal swab was also collected on day 34 of the experiment. The swab was then placed in a wheel pack bag and immediately inspected.

[0103] Detection of agglutinating antibodies against B. bronchiseptica: Using a standard micro-aggregation test, B. bronchiseptica ICA antibodies were detected. That is, in the test serum, known positive serum, and known negative serum... For a 2x serial dilution, use regular physiological saline containing 0.1% gelatin as a diluent, and apply a U-bottom m The procedure was performed using an ichrotator plate. (B. bronchisept) Add 100 μL of antigen to each well and mix in a microtiter plate mixer. Mix for 5-30 seconds. Incubate the plate at 36±2°C for 1-3 hours, then 2 The plates were incubated at ~7°C for 36-72 hours. The titer was read visually from the plates for aggregation. This was expressed as the reciprocal of the highest dilution that exhibited complete aggregation.

[0104] Challenge: On the 35th day of the experiment, dogs were given the toxic B. bronchiseptic. a) was administered.

[0105] Post-challenge observations and samples: Clinical signs including (but not limited to) runny nose, shortness of breath, depression, and cough. Regarding this, observe the dog twice a day for at least 30 minutes for 28 days after the challenge. Nasal swabs were collected on days 42, 45, 49, 52, 58, and 63 of the experiment. The excretion of administered microorganisms was measured.

[0106] Table 10 Test design [Table 11]

[0107] result Serology: Before vaccination, all dogs were infected with B. bronchisep The dog had a low antibody titer (≤4) against tica, which indicates that the dog had not been vaccinated. This indicates that the individual had not been infected with the virus at the time of inoculation. Of the 21 dogs, 20 were infected with B. bronchiseptica. ) Cohesive titer was induced, ranging from 16 to 128 on day 34 of the experiment; GMT = < 39. In contrast, antibody titers in placebo-vaccinated control dogs were low, ranging from <2 to 8. The temperature remained constant, and GMT = < 1 [see Table 11 below].

[0108] Table 11 B. bronchiseptica serum antibody titer [Table 12]

[0109] Clinical signs after the challenge: After the challenge, all placebo-vaccinated control dogs were tested for B. bronchiseptica ( Clinical signs associated with B. bronchiseptica infection, particularly the development of a spontaneous cough. Infected dogs exhibit spontaneous coughing or vomiting accompanied by spontaneous coughing on two or more consecutive days during the observation period after administration. It was defined as having the condition. All 20 placebo-vaccinated control dogs were affected. In contrast, only 9 out of 21 vaccinated control dogs (43%) developed the disease; p-value < 0.00 01 [See Table 12 below]. Furthermore, during the observation period after administration, if a patient coughed at least once on two consecutive days, No dogs were vaccinated with Kuching, but 18 out of 20 control dogs (90%) received the placebo vaccine. ) coughed 2 to 13 times for two consecutive days.

[0110] Table 12 Summary of dogs that have contracted the disease [Table 13]

[0111] In addition, the number of days on which dogs spontaneously coughed during the 28-day post-treatment observation period was analyzed. Treatment group The average number of days that dogs in group B spontaneously coughed was 16.6 days, compared to the dogs in treatment group A. The response time was only 3.5 days; p-value < 0.0001 [see Table 13 below].

[0112] Table 13 Summary of Coughs [Table 14]

[0113] B. bronchiseptica excretion: Nasal swabs are collected twice a week for four weeks after administration to remove the administered microorganisms. The following was measured. On day 42 of the trial, bacterial levels were observed between the placebo control group and the vaccine group. There was no difference in excretion. However, by the 45th day of the trial, the placebo vaccine control group had 3 B. bronchiseptica at 8,141 cfu / mL The vaccine group excreted 1,523 cfu / mL, but the vaccinated group excreted only 1,523 cfu / mL. In the control group vaccinated against the vovirus, bacterial shedding was 75,293 at 16 days post-vaccination (day 52 of the trial). The peak cfu / mL was reached. In contrast, bacterial shedding in the vaccinated group was observed 6 days after vaccination. The peak occurred on day 42, but it was only 4,173 cfu / mL. Bacterial consultation The number of cases continued to decrease in the vaccinated group until the end of the trial. By day 63 of the trial, the number of cases was... Bacterial shedding in the inoculated group was only 6 cfu / mL, compared to the placebo-inoculated control group. Within the system, bacterial excretion remained high at 35,400 cfu / mL.

[0114] These data suggest that B. bronchiseptica is a species of [unclear]. The group inoculated with kuchin showed a significant decrease in its ability to form colonies in the nasal mucosa. The overall mean bacterial shedding in the SEV1-vaccinated control group and the SEV1-vaccinated group was 37, respectively. The levels were 035 cfu / mL and 849 cfu / mL [see Table 14 below], and on each collection day The amount of B. bronchiseptica excreted is determined by the vaccine. The group that received the vaccine had statistically lower levels than the placebo control group (p-value ≤ 0.00). 01).

[0115] Table 14 Bronchiseptica bacteria (cfu / mL) isolated from nasal swabs after administration [Table 15]

[0116] These results indicate that spontaneous coughing or spontaneous coughing accompanied by nausea occurred for two or more consecutive days during the post-administration observation period. Evaluated by the number of dogs that performed the test, 3.9 × 10 per 1 mL dose 8 B. blots of cfu / mL B. bronchiseptica non-toxic strain B-C2, ATCC contract number PTA-126272 indicates that it is sufficient to make the oral vaccine effective. Yes, they are.

[0117] The present invention is not limited in scope by the specific embodiments described herein. In fact, in addition to those described herein, various modifications of the present invention may be made as described above. This will become clear to the contractors. Such modifications are not included in the scope of the attached claims. It is something that is done.

Claims

1. A method that provides effective protection to dogs from upper respiratory tract diseases and infectious tracheobronchitis. The aforementioned dogs were given a vaccine containing a modified live canine parainfluenza (CPI) virus. A method that includes oral administration.

2. The aforementioned vaccine further contains non-toxic vivipathogenic bronchoseptic bacteria (Bordetella bronchiseptica (B Ordetella bronchiseptica (B. bronchiseptica) The method according to claim 1, including ronchiseptica.

3. The titer of the modified live CPI virus administered is 6.0 log １０ HAID ５０ / for The method according to claim 1 or 2, which is greater than the specified amount.

4. The titer of the modified live CPI virus administered is 6.3 log １０ HAID ５０ / for The method according to claim 3, wherein the amount is greater than or equal to the amount.

5. The titer of the modified live CPI virus administered is 6.7 log １０ HAID ５０ / for The method according to claim 4, wherein the amount is greater than or equal to the amount.

6. The titer of the modified live CPI virus administered is 6.8 log １０ HAID ５０ / for Quantity ~ 9.5 log １０ HAID ５０ / The method according to claim 4, which is the dosage.

7. The titer of the modified live CPI virus administered is 7.0 log １０ HAID ５０ / for Amount ~ 9.5 log １０ HAID ５０ The method according to claim 6, which is the dosage.

8. The titer of the modified live CPI virus administered is 7.3 log １０ HAID ５０ / for Amount ~ 8.6 log １０ HAID ５０ The method according to claim 7, which is the dosage.

9. The non-toxic live B. bronchiseptica administered The titer is 1 × 10 ７ The person according to any one of claims 2 to 8, wherein the dose is greater than or equal to cfu / dose. Law.

10. The non-toxic live B. bronchiseptica administered The titer is 1 × 10 ８ cfu / dose~1×10 １２ The amount is cfu / dose, as described in claim 9. The method.

11. The non-toxic live B. bronchiseptica administered The titer is 5 × 10 ８ cfu / dose ~5×10 １１ The value is cfu / dose, as described in claim 10. Method of loading.

12. The modified raw CPI virus has ATCC accession number PTA-126273, The method described in any one of items 1 to 11.

13. The aforementioned non-toxic live B. bronchiseptica is ATC The method according to any one of claims 2 to 12, having accession number PTA-126272. 。

14. The method according to any one of claims 1 to 13, wherein the vaccine is a single-dose vaccine. 。

15. The method further includes administering a booster vaccine, any of claims 1 to 13 The method described in item 1.

16. The vaccine is an adjuvant-free vaccine, according to any of claims 1 to 15. The method described in item 1.

17. The vaccine further comprises an adjuvant, according to any one of claims 1 to 15. method.

18. This vaccine also contains attenuated live canine influenza virus and attenuated live canine parvovirus. S, attenuated live canine distemper virus, attenuated live canine adenovirus type 2, attenuated live respiratory virus Nucocoronavirus, attenuated live canine pneumonia virus, attenuated live Mycoplasma sinus (Myco Plasma cynos), a less venomous live Streptococcus equii subspecies zoepidemicus (Streptococcus equi zooepidemicus) and these Claims 1 to 17, comprising an attenuated bioimmunogen selected from the group consisting of any combination The method described in any one of the items.

19. This vaccine further contains attenuated live canine parvovirus and attenuated live canine distempervirus. , and the method according to any one of claims 1 to 17, comprising attenuated live canine adenovirus type 2. Law.

20. The vaccine is administered orally in a dose of 0.2 mL to 5 mL, according to any of claims 1 to 19. The method described in item 1.

21. The method according to claim 20, wherein the dose is 0.5 mL to 2.0 mL.

22. The method according to claim 20, wherein the dose is 0.75 mL to 1.5 mL.

23. Oral administration to dogs provides effective protection against upper respiratory tract diseases and infectious tracheobronchitis. A vaccine to be provided, 6.0 log １０ HAID ５０ / mL or higher titer A vaccine containing the aforementioned modified live CPI virus.

24. Non-toxic Bordetella bronxeptica B. bronchiseptica (B. bronchiseptica) The vaccine according to claim 23, further comprising (a).

25. The non-toxic live B. bronchiseptica in the vaccine The titer of ca) is 1 × 10 ７ cfu / mL~1×10 １２ Claim 2, which is cfu / mL The vaccine described in section 4.

26. The non-toxic live B. bronchiseptica in the vaccine The titer of ca) is 5 × 10 ８ cfu / mL~5×10 １１ Claim 2, which is cfu / mL The vaccine described in item 5.

27. The titer of the modified live CPI virus in the vaccine is 6.8 log １０ HAID ５０ / mL The vaccine according to any one of claims 23 to 26.

28. The titer of the modified live CPI virus in the vaccine was 7.3 log １０ HAID ５０ / mL The vaccine according to any one of claims 23 to 27.

29. The titer of the modified live CPI virus in the aforementioned vaccine is 6.0 log １０ HAID ５０ / mL to 9.5 log １０ HAID ５０ Any one of claims 23 to 26, which is / mL The vaccines listed.

30. The modified raw CPI virus has ATCC accession number PTA-126273, A vaccine as described in any one of items 23 to 29.

31. The aforementioned non-toxic live B. bronchiseptica is ATC A W according to any one of claims 24 to 30, having accession number PTA-126272. Kuching.

32. The vaccine is for single-dose vaccines, according to any one of claims 23 to 31. The vaccines listed.

33. The vaccine is specifically for use as a booster vaccine, according to any of claims 23 to 31. The vaccine listed in item 1.

34. The vaccine is an adjuvant-free vaccine, according to any of claims 23 to 33. The vaccine listed in item 1.

35. The vaccine further comprises an adjuvant, as described in any one of claims 23 to 33. The vaccine.

36. This vaccine also contains attenuated live canine influenza virus and attenuated live canine parvovirus. S, attenuated live canine distemper virus, attenuated live canine adenovirus type 2, attenuated live respiratory virus Nucocoronavirus, attenuated live canine pneumonia virus, attenuated live Mycoplasma sinus (Myco Plasma cynos), a less venomous live Streptococcus equii subspecies zoepidemicus (Streptococcus equi zooepidemicus) and these Claims 23 to 35 include a weakened bioimmunogen selected from the group consisting of any combination. A vaccine as described in any one of the items.

37. This vaccine further contains attenuated live canine parvovirus and attenuated live canine distempervirus. , and the method according to any one of claims 23 to 35, comprising attenuated live canine adenovirus type 2. vaccine.

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