Attenuated isolate of infectious bronchitis virus strain DMV1639

Abstract

A heat-attenuated infectious bronchitis virus (IBV) isolate of PDRC DMV / 1639, deposited with the ATCC under patent designation PTA-12657, its progeny and derivatives, and compositions thereof are presented. Methods of administering the isolates and compositions as vaccines to prevent pathogenic IBV infection in birds of the order Galliformes are also presented.

Description

[Technical Field]

[0001] Continuous application data This application claims the benefits under U.S. Provisional Patent Application No. 63 / 081,392, filed September 22, 2020 (incorporated herein by reference). [Background technology]

[0002] Avian infectious bronchitis virus (IBV) is an economically significant cause of upper respiratory disease in chickens (Economic Data, in US Poultry & Egg Association, 2016). Due to its prevalence and infectivity, almost all commercial poultry in the United States are serotype-specifically vaccinated against IBV (Cavanagh, 2007, Veterinary Research; 38:281-297). IBV is an enveloped, positive-sense single-stranded ribonucleic acid (RNA) virus belonging to the genus Gammacoronavirus of the family Coronaviridae. Like most RNA viruses, IBV exhibits genetic diversity due to its high mutation rate and recombination events. The continuous emergence of new IBV variants complicates vaccine-based infectious bronchitis (IB) control. The IBV Delmarva / 1639 (DMV / 1639) strain was first isolated in 2011 from an IB outbreak on the Delaware / Maryland / Virginia (DELMARVA) peninsula in the United States (Gelb et al., 2012, Avian Dis; 57(1):65-70). It continues to spread and is now a significant economic problem in the poultry industry. Cross-protection against the DMV / 1639 strain through vaccination with one or more other IBV serotypes may be partially effective in limiting the clinical signs associated with DMV / 1639 infection, but there is a need for an improved vaccine against the DMV / 1639 virus. [Overview of the project] [Means for solving the problem]

[0003] The present invention comprises an infectious bronchitis virus (IBV) isolate, the IBV isolate comprising the thermo-attenuated IBV isolate PDRC DMV / 1639 deposited with ATCC under patent designation PTA-12657, or its offspring or derivatives, the offspring or derivatives having essentially the same biological and serological properties as the thermo-attenuated IBV isolate PDRC DMV / 1639 deposited with ATCC under patent designation PTA-12657. In some embodiments, the IBV isolate is lyophilized, freeze-dried, or frozen.

[0004] The present invention also comprises compositions comprising IBV isolates or progeny or derivatives described herein. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the composition further comprises an adjuvant. In some embodiments, the composition further comprises other viral materials. In some embodiments, the composition is formulated for intranasal, intraocular, oral, mucosal, intramuscular, subcutaneous, or in ovo administration. In some embodiments, the composition is formulated for spray or aerosolization.

[0005] The present invention also includes a vaccine or composition described herein, comprising an IBV isolate or its offspring or derivatives as described herein. In some embodiments, the vaccine reduces one or more clinical signs and / or viral loads induced by IBV infection in poultry.

[0006] The present invention comprises a vaccine for birds of the order Galliformes, the vaccine comprising a sufficient amount to protect birds from one or more clinical signs induced by infectious bronchitis virus (IBV) infection in poultry, the heat-attenuated IBV isolate PDRC DMV / 1639 deposited with ATCC under patent designation PTA-12657 or its offspring or derivatives.

[0007] The present invention also includes effervescent tablets comprising an IBV isolate or offspring or derivative or composition or vaccine described herein.

[0008] The present invention also includes a method for generating an immune response against infectious bronchitis virus (IBV) in poultry, the method comprising administering an IBV isolate or its offspring or derivatives or composition or vaccine as described herein.

[0009] The present invention also includes a method for generating anti-IBV antibodies in poultry, the method comprising administering to poultry an IBV isolate or its offspring or derivatives or composition or vaccine described herein.

[0010] The present invention also includes a method for reducing one or more clinical signs and / or viral loads induced by infectious bronchitis virus (IBV) infection in poultry, the method comprising administering an effective amount of the IBV isolate or its offspring or derivatives or compositions or vaccines described herein.

[0011] The present invention also includes a method for reducing the susceptibility of Galliformes birds to infectious bronchitis virus (IBV) infection, the method comprising administering an effective amount of the IBV isolate or its offspring or derivatives or composition or vaccine described herein to the birds.

[0012] The present invention also includes a method for protecting Galliformes birds from infectious bronchitis virus (IBV) infection, the method comprising administering an effective amount of an IBV isolate or offspring or derivative or composition or vaccine described herein to the birds.

[0013] In some embodiments of the methods described herein, administration is intranasal, intraocular, oral, mucosal, intramuscular, or subcutaneous.

[0014] In some aspects of the methods described herein, the administration includes in ovo administration.

[0015] In some embodiments of the methods described herein, IBV isolates or their offspring or derivatives, compositions, or vaccines are administered by aerosol.

[0016] In some embodiments of the methods described herein, IBV isolates or offspring or derivatives, compositions, or vaccines are administered by spraying.

[0017] In some embodiments of the methods described herein, IBV isolates or offspring or derivatives, compositions, or vaccines are administered by drinking water.

[0018] In some embodiments of the methods described herein, the administration includes administration to breeding hens.

[0019] In some aspects of the methods described herein, poultry includes birds of the order Galliformes.

[0020] In some embodiments of the methods described herein, the bird is a chicken or a turkey. [Brief explanation of the drawing]

[0021] [Figure 1A] Positive response 7 days after vaccination. Figure 1A shows viral load (inverted Ct value) and positive percentage after vaccination with De1639, PDRC DMV1639, and MA DMV / 1639+Mass. [Figure 1B] Positive response 7 days after vaccination. Figure 1B shows viral load and positive percentage after vaccination with DE1639+iBron, iBron, and iBron+Mass. [Figure 2] Good signs on day 14 of chickens vaccinated with our own DE1639 vaccine. [Figure 3A-3B] Viral load at day 28 in chickens vaccinated only with home-bred DE1639. Figure 3A shows inverted Ct values. Figure 3B shows positive percentage. [Figure 4]Clinical signs after the challenge in the vaccine / challenge group. [Figure 5] Viral load after challenge based on PCR detection of DMV-specific virus. [Figure 6] Vaccine response on day 7 in chicks vaccinated with DE1639 and PDRC DMV / 1639. [Figure 7] Goodness on day 14 in chickens vaccinated with DE1639. [Figure 8] The viral load on day 28 in chickens vaccinated only with DE1639 before the challenge. [Figure 9] Clinical signs after the challenge in the vaccine / challenge group. [Figure 10] Viral load after challenge based on PCR detection of DMV-specific virus. [Figure 11] A phylogenetic tree comparing different isolates of the DMV / 1639 virus. The lower box represents isolates from the 2015 outbreak of origin, along with the autologous vaccine indicated by the arrow. More recent isolates from 2019 onwards are in the upper box, along with the PDRC vaccine indicated by the arrow. [Modes for carrying out the invention]

[0022] This invention relates to novel materials and methods in the field of poultry virology, particularly in the field of infectious bronchitis viruses (IBV).

[0023] Avian infectious bronchitis virus (IBV) is a gamma coronavirus. The enveloped IBV virus has a single-stranded positive-sense RNA genome encoding viral RNA-dependent RNA polymerase, three major structural proteins (nucleocapsid, membrane, and spike (S) protein), and numerous regulatory proteins (Masters, 2006, Adv Vir Res; 66:193-292). The IBV spike glycoprotein is translated as a precursor protein and then cleaved by host cell serine proteases into two subunits: the N-terminal S1 glycoprotein and the C-terminal S2 glycoprotein. The S1 and S2 glycoproteins mediate cell attachment and viral cell membrane fusion, play a crucial role in host cell specificity, and form club-shaped protrusions on the viral surface. The S1 glycoprotein induces viral neutralizing antibodies and hemagglutination inhibitory antibodies.

[0024] Novel variant strains arise primarily from rapid recombination, insertion, deletion, or point mutation events in the S1 spike protein gene. Along with the use of serological-based testing, PCR and partial sequencing of the S1 gene can be used to group and type IBV isolates. Even slight variations in the spike glycoprotein sequence can result in new serotypes. It has been established that even small differences of around 5% in the IBV S1 sequence can lead to a loss of cross-protection with other similar isolates (Cavanagh, 2003, Avian Pathol; 32:567-582). Based on spike 1 (S1) protein variability, six genotypes of IBV, including 32 identifiable viral lineages, have been described and are globally recognized (Valastro et al., 2016, Infect Genet Evol; 39:349-364).

[0025] The Delmarva / 1639 (DMV / 1639) strain of IBV was first isolated in 2011 from an infectious bronchitis (IB) outbreak in the Delmarva Peninsula (Gelb et al., 2012, Avian Dis; 57(1):65-70), and infection with this IBV variant presents a continuing challenge for poultry producers. Currently, there is no commercially available vaccine for this strain of IBV. While cross-protection against the DMV / 1639 strain through vaccination with one or more other IBV serotypes may prove effective in limiting mortality and morbidity associated with DMV / 1639 infection, there is a need for an improved vaccine against the DMV / 1639 virus.

[0026] This invention provides a live, heat-attenuated isolate of IBV strain DMV1639, as well as its offspring and derivatives. When administered to birds as a live preparation, the attenuated IBV isolate is safe and effective in preventing IBV infection and reducing the incidence and severity of IBV infection.

[0027] This raw, heat-attenuated isolate of IBV strain DMV1639 (also referred herein as heat-attenuated DMV / 1639, attenuated DMV / 1639, PDRC DMV / 1639, heat-attenuated PDRC DMV1639, and attenuated DMV / 1639-Georgia isolate) was deposited on 15 May 2020 with the American Type Culture Collection (ATCC®) patent depositary (10801 University Boulevard, Manassas, Virginia 20110-2209, USA) under patent deposit number PTA-126757. Such deposit is in accordance with the Budapest Convention on the International Recognition of the Deposit of Microorganisms in Patent Proceedings.

[0028] The present invention also includes isolated offspring and isolated derivatives of the live, heat-attenuated isolate of IBV strain DMV1639, deposited with ATCC® on May 15, 2020, under patent deposit number PTA-126757, which have equivalent or similar biological, serological, and / or genetic properties. As used herein, serological, biological, and genetic properties may include one or more of the properties described in the data of the examples included herein. More specifically, offspring or derivative strains of PTA-126757 may possess particularly advantageous defensive properties belonging to the present invention, which are described in more detail in the examples included herein.

[0029] The IBV virus isolates according to the present invention can be propagated by conventional methods, including, but not limited to, any of those described in the Examples section contained herein. Briefly, the IBV virus isolate of the present invention is inoculated into a substrate capable of supporting the replication of the IBV virus isolate, and the mixture is propagated until the virus replicates to a desired infectivity titer or antigen mass content. The virus-containing material is then collected. Suitable substrates may include embryonic eggs, primary (chicken) cell cultures such as chicken embryonic hepatocytes, chicken embryonic fibroblasts, chicken kidney cells, etc., mammalian cell lines such as the VERO cell line or BGM-70 cell line, or chicken cell lines such as QT-35, QM-7, LMH, etc.

[0030] In preferred embodiments, the virus can be grown in embryonic eggs, including, but not limited to, embryonic chicken eggs. For example, embryonic chicken eggs aged 9-11 days can be inoculated via the cassoallantoic cavity (CAS) pathway (Dufour-Zavala, “A laboratory manual for the isolation, identification and characterization of avian pathogens,” 5th ed. American Association of Avian Pathologists, Jacksonville, Fl. 2008. The inoculated eggs can be incubated at 37°C for 48 hours, and the cassoallantoic fluid can be collected at that time).

[0031] The compositions and vaccines of the present invention are about 10 10 , 7 , 9 , 6 ,

[0032] , 8 , 8 , 50 , 50 , 50 , 50 , 50 , , 50 , 7.5 , 9.5 , 6.5 , 8.5 , <00000!40>, 50 , 50 , 50 , , 50 , 50 ~ about 10 10 EID 50 (embryo infectious dose) / ml may have a titer. In some embodiments, the composition or vaccine of the present invention is about 10 1.5 EID 50 / ml, about 10 2 EID<00000!7> / ml, about 10 2.5 EID 50 / ml, about 10 3 EID 50 / ml, about 10 3.5 EID 50 / ml, about 10 4 EID 50 / ml, about 10 4.5 EID[[ID=3!5]] 50 / ml, about 10 5 EID 50 / ml, about 10 5.5 EID 50 / ml, about 10 6 EID 50 / ml, about 10 6.5 EID 50 / ml, about 10 7 EID 50 / ml, about 10 7.5 EID 50 / ml, about 10 8 EID 50 / ml, about 10 8.5 EID 50 / ml, about 10 9 EID 50 / ml, about 10 9.5 EID[[ID=7!5]] 50 / ml, about 10 10 EID 50 / ml, or may have a titer within any range thereof. For example, in some applications, the composition or vaccine of the present invention is about 10 2 EID 50 / ml to about 10 8 EID 50 / ml may have a titer. The titer can be measured, for example, in allantoic fluid.

[0032] <; The virus can be titrated, for example, using the following protocol: Prepare 10-fold serial dilutions of the virus in sterile deionized water, and inoculate each dilution into five 10-day-old embryo-bearing SPF chicken eggs (0.1 ml / egg). Incubate the inoculated eggs at 37°C for 7 days, and examine the embryos for IBV-specific lesions. Embryo mortality within 24 hours after inoculation is considered nonspecific and is not included in the viral titer calculation. Viral titer is calculated using the Reed and Muench method (Reed and Muench, 1938, American Journal of Hygiene 27:493-497), and the 50% embryo infection dose (EID) is used. 50 It is represented as ).

[0033] The present invention comprises compositions and vaccines containing isolated viruses as described herein. In some applications, vaccine preparations of the present invention may be prepared and tested for IBV vaccine testing in accordance with Section 113.327 of Title 9 of the Code of Federal Regulations (CFR).

[0034] In some embodiments, the virus is live. In some embodiments, the virus is inactivated or killed. The viruses, compositions, and vaccines of the present invention can be stored in any of the following forms until use. For example, such materials can be freeze-dried and rehydrated for use. In some embodiments, the viruses, compositions, or vaccines can be frozen.

[0035] In some embodiments, the virus, its composition, or vaccine may be formulated as an effervescent tablet. Such an effervescent tablet may be packaged, for example, in a lightweight aluminum blister pack. The tablet is dissolved in water and administered, for example, orally, nasally, or by aerosol spray, so that a drop enters the bird's mucous membrane.

[0036] The compositions and vaccines of the present invention may, for example, comprise water or culture medium. Such compositions and vaccines may comprise one or more suitable pharmaceutically acceptable carriers, stabilizers, preservatives, diluents, and / or buffers. Suitable stabilizers include, for example, SPGA, carbohydrates (e.g., sorbitol, mannitol, starch, sucrose, dextrin, or glucose), or proteins (e.g., albumin or casein). Stabilizers are particularly advantageous when the dried vaccine preparation is prepared by lyophilization. Suitable preservatives include, for example, thimerosal, methylthiolate, and gentamicin. Suitable diluents include, but are not limited to, water, aqueous buffers (e.g., buffered saline), alcohols, and polyols (e.g., glycerol).

[0037] The composition or vaccine of the present invention may also contain one or more compounds having adjuvant activity. Suitable compounds or compositions for this purpose include aluminum hydroxide, aluminum phosphate, aluminum oxide, vegetable oils, animal oils, oil-in-water or water-in-oil emulsions, such as mineral oils like Bayol F® and Marcol 52®, complete Freund's adjuvants, incomplete Freund's adjuvants, or vegetable oil-based compounds such as vitamin E acetate, and saponins.

[0038] The compositions or vaccines of the present invention may further comprise one or more immunogens derived from other pathogens that infect poultry. Such immunogens may be derived from, for example, Marek's disease virus (MDV), other serotypes of infectious bronchitis viruses (IBV), Newcastle disease virus (NDV), egg-low syndrome (EDS) virus, turkey rhinotracheitis virus (TRTV), poxvirus, reovirus, chicken parvovirus, and avian nephritis virus (including, but not limited to, ANV-1 and ANV-2).

[0039] The compositions and vaccines of the present invention may be substantially pure. As used herein, “substantially pure” means materials that are essentially free from macromolecules or other biological entities that are normally found together in nature.

[0040] IBV vaccination is common to most commercial chickens. The vaccine may be a modified live virus vaccine delivered via high-volume aerosol application. The serotype used for vaccination is often selected based on which serotypes the birds may be exposed to in the field. There is little cross-protection between different serotypes of IBV. Therefore, the present invention provides immunological materials that, when administered, do not result in significant clinical signs or lesions indicative of IBV disease. The present invention also provides low-pathogenic immunological materials, immunological materials that do not increase in pathogenicity when backpassed, and / or immunological materials that prevent infection by pathogenic wild-type strains of IBV.

[0041] The compositions or vaccines of the present invention may be administered to poultry by any preferred known inoculation method, for example, nasally, ocularly, by injection, in drinking water, in feed, by exposure, in ovo, in the mother, by respiratory inhalation, or otherwise. The immunogenic compositions or vaccines may be administered by high-dose techniques, for example, by adding the vaccine to drinking water, by spraying, or by aerosolization. When administered by injection, the immunogenic compositions or vaccines may be administered parenterally. Parenteral administration may include, for example, intravenous, subcutaneous, intramuscular, or intraperitoneal injection.

[0042] The compositions and vaccines of the present invention may be administered to any of the various bird species susceptible to IBV infection, including, but not limited to, poultry, Galliformes birds, and exotic bird species. Galliformes birds include, but not limited to, chickens, turkeys, grouse, quail, and pheasants. As used herein, poultry refers to domestic birds raised for the purpose of collecting eggs or being killed for their meat and / or feathers. These are most typically members of the superorder Galloanserae (food birds), particularly the order Galliformes (e.g., chickens, quail, turkeys, and grouse) and the family Anatidae (Anseriformes), which are widely known as "waterfowl" (e.g., ducks, geese, and swans). Other birds that are killed for their meat may include, for example, the Greater White Dove or Little Dove, or the pheasant, which is considered a game bird. Chickens may include, but are not limited to, hens, roosters, broilers, roasters, laying hens, breeders, offspring of breeding hens, and laying hens. As used herein, the term “susceptible” means the likelihood or reality of one or more pathological conditions that indicate an adverse response to a reference microorganism and / or avian IBV infection, compared to a less susceptible individual or group.

[0043] The vaccine of the present invention may be administered to poultry before or after hatching. Poultry may be vaccinated at various ages. For example, broilers may be vaccinated in ovo at 1 day of age or 2-3 weeks of age. Laying stock or breeding stock may be vaccinated, for example, at about 16-20 weeks of age and grown to about 6-12 weeks of age. Such laying stock or breeding stock may be vaccinated at about 6, about 7, about 8, about 9, about 10, about 11, or about 12 weeks of age. In some embodiments, such laying stock or breeding stock may also be vaccinated within the first about 2 weeks of age. Such laying stock or breeding stock may be grown to about 16, about 17, about 18, about 19, or about 20 weeks of age. Offspring of such laying stock or breeding stock may demonstrate antibody titers to the polypeptides described herein that can prevent or reduce symptoms of IBV infection in the offspring. In ovo vaccination can be administered over a period of approximately 13, 14, 15, 16, 17, 18, 19, 20 days, or any of these periods.

[0044] Chickens can be vaccinated at any suitable age, with the first dose usually given around 1 to 3 days after birth. Chickens may be vaccinated only once, or, if a two-dose vaccine is used, the first dose may be given when the chickens are 3 days to 1 week old, followed by a second dose 1 to 10 weeks later.

[0045] Multiple doses of the composition can be administered throughout the chicken's life. Since maternal immunity is the primary source of protection for broiler offspring, breeding stock are typically vaccinated, but broiler chickens can be vaccinated if desired.

[0046] In some embodiments, the raw attenuated IBV isolate of the present invention is about 10 1.5 ~about 10 10 EID 50 / Can be administered in a dose of 10. In some embodiments, the raw attenuated IBV isolate of the present invention is about 10 1.5 EID 50 / Bird, about 10 2 EID 50 / Bird, about 10 2.5 EID 50 / Bird, about 10 3 EID 50 / Bird, about 10 3.5 EID 50 / Bird, about 10 4 EID 50 / Bird, about 10 4.5 EID 50 / Bird, about 10 5 EID 50 / Bird, about 10 5.5 EID 50 / Bird, about 10 6 EID 50 / Bird, about 10 6.5 EID 50 / Bird, about 10 7 EID 50 / Bird, about 10 7.5 EID 50 / Bird, about 10 8 EID 50 / Bird, about 10 8.5 EID 50 / Bird, about 10 9 EID 50 / Bird, about 10 9.5 EID 50 / Bird, about 10 10 EID 50 / may be administered in doses within the range of either 10 or 10. For example, in some applications, about 10 2 ~about 10 5 EID 50 A dose of / tori may be administered.

[0047] The viruses, compositions, or vaccines described herein, including but not limited to the IBV DMV1639 serotype, may be administered to poultry or other animals to induce an immune response to the IBV virus and / or IBV S1 polypeptide. The immune response may include, for example, one or more cell-mediated immune responses, including the production of lymphocytes in response to exposure to an antigen, and / or humoral immune responses, including the production of plasma lymphocytes (B cells) in response to antigen exposure, accompanied by subsequent antibody production. Humoral immune responses may include IgG, IgM, IgA, IgD, and / or IgE responses. The determination of humoral or cellular immune responses may be made by any of the various methods described herein, including but not limited to those described herein. The immune response may or may not confer protective immunity. Such an immune response may result in a reduction or mitigation of symptoms of future IBV infection, for example, symptoms of infection with the DMV / 1639 serotype IBV virus. Such an immune response may prevent future IBV infections in poultry, for example, infection with the DMV / 1639 serotype of IBV virus. Immunity may include inducing a higher level of protection in a poultry population after vaccination compared to an unvaccinated group.

[0048] The present invention includes a method for generating an anti-IBV immune response in poultry, the method comprising administering an isolated virus, composition, or vaccine as described herein. In some embodiments, the immunity includes humoral and / or cellular immunity. In some embodiments, the immunity includes mucosal immunity.

[0049] Administration of any isolated virus, composition, or vaccine described herein may result in the reduction, inhibition, or prevention of one or more symptoms of IBV infection, including one or more symptoms of infectious bronchitis (IB). Such symptoms may include weight suppression, decreased spawning, mortality, clinical signs (e.g., lacrimation, wheezing, sneezing, sinus exudate, conjunctivitis, and / or rales), histopathological indications (e.g., tracheal lesions), anti-IBV serum antibody titers (e.g., determined by ELISA), and / or IBV virus isolation (e.g., measured by real-time RT-PCR of tracheal swab samples).

[0050] The present invention includes methods for reducing, inhibiting, or preventing IBV infection in poultry, the methods comprising administering an isolated virus, composition, or vaccine described herein. In some embodiments, administration of an isolated virus, composition, or vaccine described herein reduces, inhibits, or prevents infection by an IBV DMV / 1639 variant strain. In some embodiments, administration of an isolated virus, composition, or vaccine described herein provides cross-protection, reduction, inhibition, or prevention of one or more disease symptoms of infection by IBV strains other than the DMV / 1639 strain.

[0051] In some aspects of the method of the present invention, administration includes injection, spraying, oral administration, or respiratory administration. In some aspects of the method of the present invention, administration induces mucosal immunity. In some aspects of the method of the present invention, administration includes in ovo administration. In some aspects, in ovo administration includes administration for about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, or any range thereof.

[0052] The compositions of the substance of the present invention may be substantially pure. As used herein, “substantially pure” means a material that essentially contains no similar macromolecules or other biological entities that would normally be found together in nature. In some embodiments, the organisms used in such formulations are alive. In some embodiments, the organisms, compositions, or vaccines may be freeze-dried. The present invention includes isolated viruses. As used herein, “isolated” means a material that has been taken out of its environment of origin (for example, the natural environment if it exists naturally) and thus “modified by human hands” from its natural state.

[0053] The viruses, compositions, and vaccines of the present invention may be administered to any of the various bird species susceptible to IBV infection, including, but not limited to, poultry, Galliformes birds, and exotic bird species. Galliformes birds include, but not limited to, chickens, turkeys, grouse, quail, and pheasants. As used herein, poultry refers to domestic birds raised for the purpose of collecting eggs or being killed for their meat and / or feathers. These are most typically members of the superorder Galloanserae (food birds), particularly the order Galliformes (e.g., chickens, quail, turkeys, and grouse), and the family Anatidae (Anseriformes), which are widely known as "waterfowl" (e.g., ducks, geese, and swans). Other birds that are killed for their meat may also be considered poultry, such as the Greater White-fronted Dove or the Little Dove, or the pheasant, which is considered a game bird.

[0054] "Poultry" is intended to encompass any breed of chicken, pheasant, emu, ostrich, and any other type of bird susceptible to IBV infection. Chickens include, but are not limited to, hens, roosters, broilers, roasters, laying hens, breeders, offspring of breeding hens, and laying hens. In some embodiments, the compositions and methods of the substance of the present invention are also applicable to animals other than poultry susceptible to IBV infection. As used herein, the term "susceptible" means the likelihood or reality of an adverse reaction to a reference microorganism, such as reduced vitality or stunted growth, and / or one or more pathological conditions that are indicators of IBV infection, including, but not limited to, any of those described herein, compared to a less susceptible individual or group.

[0055] The compositions and vaccines of the present invention can be formulated for delivery by any of the various routes known in veterinary technology, such as mucosal, intranasal, intraocular, or oral administration. The compositions and vaccines of the present invention can be formulated for delivery to the respiratory mucosa and can be administered to come into immediate or final contact with the respiratory mucosa of birds. The compositions and vaccines of the present invention can be formulated for delivery by any of the various modes known in veterinary technology, such as spraying or aerosolization.

[0056] The immunogenic composition or vaccine of the present invention may be administered to birds by any preferred known inoculation method, for example, but not limited to, nasally, ophthalmally, by eye drops, by injection, in drinking water, in feed, by exposure, in ovo, in the mother, or otherwise.

[0057] Immunogenic compositions or vaccines may be administered by high-dose techniques, for example, by adding the vaccine to drinking water or by spraying it into the environment of animals. Compositions may be administered by spraying a solution onto individuals or herds, and such aerosol delivery may involve the administration of compositions incorporated into small liquid particles. Such spray particles may have a droplet size in the range of about 10 to about 100 microns, more preferably about <1 to about 50 microns. Conventional spray devices and aerosol generators, such as commercially available spray generators for napsack sprays, hatchery sprays, and atomist sprays, may be used to generate small particles. Administration via drinking water may be carried out using conventional devices. When administered by injection, immunogenic compositions or vaccines may be administered parenterally. Parenteral administration may include, for example, intravenous, subcutaneous, intramuscular, or intraperitoneal injection.

[0058] The composition or vaccine of the present invention may be administered to birds before or after hatching. Birds may ingest such composition of vaccine at any of the various ages. In post-hatching delivery, the material may be delivered, for example, about one week after hatching, about two weeks after hatching, about three weeks after hatching, about four weeks after hatching, about five weeks after hatching, about six weeks after hatching, or any of these ranges. In in ovo delivery, the material may be delivered during incubation of about 17 days, about 18 days, about 19 days, about 20 days, or any of these ranges.

[0059] The virus of the present invention can be used in any of the methods commonly used for IBV detection, such as hemagglutination (HA) (Lashgari and Newman, 1984, Avian Dis; 28:435-443), hemagglutination inhibition (King and Hopkins, 1983, Avian Dis; 27:100-112), AGPT (Lohr, 1980, Avian Dis; 24:463-467; and Lohr 1981, Avian Dis; 15:1058-1064), RT-PCR (Kwon et al., 1993, Avian Dis; 37:194-202), and real-time RT-PCR (Calison et al. 2006, J Virological Methods; 138:60-65).

[0060] Exemplary embodiments of the present invention include, but are not limited to, the following: 1. An infectious bronchitis virus (IBV) isolate comprising a thermally attenuated IBV isolate PDRC DMV / 1639 deposited with ATCC under patent designation PTA-12657, or its progeny or derivatives, wherein the progeny or derivatives have essentially the same biological and serological characteristics as thermally attenuated IBV isolate PDRC DMV / 1639 deposited with ATCC under patent designation PTA-12657. 2. The IBV isolate according to Embodiment 1, wherein the IBV isolate is freeze-dried, lyophilized, or frozen. 3. A composition comprising the IBV isolate described in Embodiment 1 or 2. 4. The composition according to Embodiment 3, further comprising a pharmaceutically acceptable carrier. 5. A vaccine comprising an isolated IBV isolate or its offspring or derivatives as described in Embodiment 1 or 2, or the composition as described in Embodiment 3 or 4. 6. The vaccine according to Embodiment 5, wherein the vaccine reduces one or more clinical signs and / or viral loads induced by IBV infection in poultry. 7. A vaccine for Galliformes birds comprising a sufficient amount to protect birds from one or more clinical signs induced by infectious bronchitis virus (IBV) infection in poultry, comprising the thermo-attenuated IBV isolate PDRC DMV / 1639 or its offspring or derivatives deposited with ATCC under patent designation PTA-12657. 8. A composition or vaccine according to any one of Embodiments 3 to 7, further comprising an adjuvant. 9. A composition or vaccine according to any one of Embodiments 3 to 8, further comprising other viral materials. 10. The composition or vaccine according to any one of Embodiments 3 to 9, wherein the composition or formulation is formulated for intranasal, intraocular, oral, mucosal, intramuscular, subcutaneous, or in ovo administration. 11. The composition or vaccine according to any one of Embodiments 3 to 10, wherein the composition or vaccine is formulated for spraying or aerosolizing. 12. An effervescent tablet comprising an IBV isolate or its progeny or derivative, composition, or vaccine as described in any one of Embodiments 1 to 11. 13. A method for generating an immune response against infectious bronchitis virus (IBV) in poultry, comprising administering to poultry an IBV isolate or its offspring or derivative, composition, vaccine, or effervescent tablet described in any one of Embodiments 1 to 12. 14. A method for generating anti-IBV antibodies in poultry, comprising administering to poultry an IBV isolate or its offspring or derivative, composition, vaccine, or effervescent tablet described in any one of Embodiments 1 to 12. 15. A method for reducing one or more clinical signs and / or viral load induced by infectious bronchitis virus (IBV) infection in poultry, comprising administering to poultry an effective amount of an IBV isolate or its offspring or derivatives, composition, vaccine, or effervescent tablet described in any one of Embodiments 1 to 12. 16. A method for reducing the susceptibility of Galliformes birds to infectious bronchitis virus (IBV) infection, comprising administering to birds an effective amount of an IBV isolate or its offspring or derivatives, composition, vaccine, or effervescent tablet described in any one of Embodiments 1 to 11. 17. A method for protecting Galliformes birds from infectious bronchitis virus (IBV) infection, comprising administering to the birds an effective amount of an IBV isolate or offspring or derivative, composition, vaccine or effervescent tablet described in any one of Embodiments 1 to 11. 18. The method according to any one of Embodiments 13 to 17, wherein the administration is intranasal, intraocular, oral, mucosal, intramuscular, or subcutaneous. 19. The method according to any one of Embodiments 13 to 18, wherein the administration includes in ovo administration. 20. The method according to any one of Embodiments 13 to 19, wherein an IBV isolate or its progeny or derivatives, composition, or vaccine is administered by aerosol. 21. The method according to any one of Embodiments 13 to 19, wherein an IBV isolate or progeny or derivative, composition, or vaccine is administered by spraying. 22. The method according to any one of Embodiments 13 to 19, wherein an IBV isolate or offspring or derivative, composition, or vaccine is administered by drinking water. 23. The method according to any one of Embodiments 13 to 22, wherein the administration includes administration to breeding hens. 24. The method according to any one of embodiments 13 to 23, wherein the poultry includes birds of the order Galliformes. 25. The method according to any one of embodiments 13 to 24, wherein the bird is a chicken or a turkey.

[0061] The term "and / or" means one or all of the enumerated elements, or any combination of two or more of the enumerated elements.

[0062] The terms "preferred" and "preferably" refer to embodiments of the present invention that may provide a particular benefit under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the enumeration of one or more preferred embodiments does not imply that other embodiments are unhelpful, nor is it intended to exclude other embodiments from the scope of the present invention.

[0063] The term "comprises" and its variations do not have a limited meaning when they appear in this description and the claims.

[0064] Unless otherwise specified, "a," "an," "the," and "at least one" are interchangeable and mean one or more.

[0065] Furthermore, enumerating a numerical range by endpoints includes all numbers contained within that range (for example, 1-5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

[0066] In any method disclosed herein, including individual steps, the steps may be carried out in any feasible order. Furthermore, any combination of two or more steps may be carried out simultaneously, as needed.

[0067] Unless otherwise specified, all numbers representing component amounts, molecular weights, etc., used herein and in the claims should be understood to be modified in all cases by the term "approximately." Therefore, unless otherwise specified, the numerical parameters shown herein and in the claims are approximations that may vary depending on the desired properties to be obtained by the present invention. At the very least, without any intention to limit the doctrine of equivalents to the claims, each numerical parameter should be interpreted by applying at least the usual rounding techniques in light of the reported number of significant figures.

[0068] Although the numerical ranges and parameters representing the broad scope of this invention are approximations, the numerical values ​​shown in the specific examples are reported as accurately as possible. However, all numerical values ​​inherently contain a range that inevitably arises from the standard deviation found in each of their respective test measurements.

[0069] Throughout this application, guidance is provided in several places through lists of examples, which can be used in various combinations. In each case, the enumerated lists serve only as representative groups and should not be construed as exclusive lists. Specific examples, materials, quantities, and procedures should be understood to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.

[0070] Throughout this document, all headings are for the reader's convenience and, unless otherwise specified, should not be used to restrict the meaning of the text that follows them.

[0071] The present invention is illustrated by the following examples. Specific examples, materials, quantities, and procedures should be understood to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein. [Examples]

[0072] Example 1 Live heat-attenuated IBV strain DMV1639 for use as a vaccine in poultry We attenuated the DMV / 1639 strain of avian coronavirus infectious bronchitis virus using heat treatment. Briefly, we collected a source sample in 2019 from a broiler farm in Georgia that exhibited respiratory symptoms. The virus was isolated and then passaged three times in embryo-laden eggs. This sample (total of 4 passages) was then heat-shock attenuated via 14 heat-shock passages according to the previously reported attenuation method by Jackwood et al. (Jackwood, et al., 2010, Avian Pathology; 39:227-233). To expand the virus, the virus recovered after 14 heat-shock passages (hsp14) was then passaged once via embryo-laden eggs, resulting in a total of 19 passages: 5 traditional passages and 14 heat-shock passages.

[0073] As described in the examples below, the treated virus was grown in embryonic eggs and tested for safety and efficacy in poultry. Using the criteria of Title 9 of the Code of Federal Regulations for IBV Vaccine Testing (CFR), the attenuated virus passed both safety and efficacy tests. This strain of DMV / 1639 is a recent virus isolated from Georgia and represents an evolved form of this virus that causes upper respiratory tract disease in poultry. It is currently a significant economic problem in the poultry industry and continues to spread. Currently, there is no commercially available vaccine for this strain of IBV.

[0074] This raw, heat-attenuated isolate of IBV strain DMV / 1639 (also referred to herein as heat-attenuated DMV / 1639, attenuated DMV / 1639, PDRC DMV / 1639, heat-attenuated PDRC DMV / 1639, and attenuated DMV / 1639-Georgia isolate) was deposited on 15 May 2020 with the American Type Culture Collection (ATCC®) patent depositary (10801 University Boulevard, Manassas, Virginia 20110, USA) under patent deposit number PTA-126757.

[0075] Example 2 Comparison of heat-attenuated PDRC DMV1639, DE1639, Ma5, and iBron GA08 regarding their homogeneous and heterogeneous defense capabilities. The concept of cross-protection against IBV vaccines has been extensively tested when multiple IBV vaccine types are combined (typically Ma5 and 4 / 91). Recently, several vaccine manufacturers have suggested that mono-IBV vaccines may be cross-protective against heterologous challenging serotypes without the need for additional IBV vaccines. In addition, some vaccines used do not appear to provide complete protection against the current congenital virus circulating in the field. To investigate these questions, we tested four different vaccines against pathogenic DMV / 1639 challenge and evaluated their protection.

[0076] Materials and methods The following viruses were used in this study: Mass type (Boehringer Ingelheim), iBron GA08 type (Ceva), DE1639 (also referred to herein as autologous DMV / 1639 and MA DMV / 1639), and attenuated (heat-treated) PDRC DMV / 1639 vaccine. The challenge virus used was a recent isolate of DMV / 1639 / 11.

[0077] Experimental Design. The experimental design is shown in Table 1. One-day-old broiler chicks positive for maternal antibodies were used in this experiment. 100 chicks in each group were vaccinated with the full dose of each vaccine by spray at one day of age and placed in colony houses in separate groups. On day 7, all chicks in each group were swabded and the vaccine virus was detected using real-time RT-PCR. At 28 days of age, 10 chicks for each challenge virus and 5 chicks for each control group were transferred to isolators, and 1 × 10⁶ samples were taken. 4 EID 50 The challenge virus was administered using DMV / 1639 / 11. The challenge virus titer was verified in embryo-bearing eggs by back titration. Necropsy was performed 5 days after challenge. The DE1639 vaccine group was kept in the colony house, and swabs were taken until 42 days of age for vaccine rolling evaluation.

[0078] [Table 1]

[0079] Autopsy / Clinical Signs. Clinical signs were recorded and scored based on the previously reported laboratory scoring method (Jackwood et al., 2015, Avian Diseases; 59(3):368-374. https: / / doi.org / 10.1637 / 11026-012415-Reg.1). The scale was as follows: 0 = asymptomatic, 1 = slight wheezing or sneezing, 2 = more pronounced wheezing, sinus exudate, conjunctivitis, and 3 = rales.

[0080] Autopsy / RNA extraction and real-time RT-PCR for challenge virus detection. At autopsy, nasal swab samples were collected from the oral-pharyngeal region within the cleft palate and placed in 1 ml of ice-cold PBS for real-time RT-PCR for viral detection. Viral RNA was extracted from 50 μl of PBS using a MagMAX-96 RNA Isolation Kit (Ambion Inc., Austin TX) according to the manufacturer's protocol for the KingFisher magnetic particle processor (Thermo Scientific, Waltham, MA). Real-time RT-PCR was performed using an Applied Biosystems 7500 Fast Real-Time PCR System (Life Technologies, Carlsbad, CA) and an AgPath-ID™ One-Step RT-PCR Kit (Ambion Inc.) according to the manufacturer's recommendations. Primers and probes for real-time RT-PCR correspond to the specific serotype being tested. Primers were obtained from Integrated DNA Technologies (Coralville, IA), and Taqman probes were synthesized by BioSearch Technologies (Novato, CA).

[0081] result In this trial, 100 chicks were spray-vaccinated with the vaccines shown in Table 1. Then, to mimic field environmental conditions, all 100 chicks from each group were placed on clean bedding in a colony-type house. Seven days after vaccination (dpv), all chicks from each group were swabded in the posterior nasal cleft, and the present vaccine virus was measured. The data can also be seen in Figures 1A and 1B. As expected, based on previous vaccine-sensing sampling, the DE1639 vaccine infected only about 60% of the chicks at the time of vaccination, and the resulting viral load was relatively low (average Ct value of approximately 33). This is in contrast to the PDRC DMV / 1639 vaccine developed in PDRC, which infected nearly 100% with an average Ct value of approximately 26.5. When the DE1639 vaccine was combined with other vaccines, the infection rate was actually reduced compared to the use of a single vaccine. When combined with the Mass vaccine, the DE1639 vaccine infected approximately 35% of chicks, while the viral load was the same (Ct value of approximately 32).

[0082] The Mass vaccine resulted in 100% infection at an average Ct value of approximately 25, demonstrating that the DE1639 vaccine in this group did not have vaccine application issues causing vaccination failure. The same trend was observed when the DE1639 vaccine was combined with Ceva's iBron, in which case only about 10% of chicks were positive with autologous DMV / 1639 at an average Ct value of approximately 33. With iBron, approximately 95% of chicks were positive at an average Ct value of approximately 26, again demonstrating that there were no application issues. When iBron alone was evaluated, nearly 100% of chicks were positive at an average Ct of approximately 25, suggesting good efficacy and replication. Interestingly, when iBron was mixed with the Mass vaccine, 100% of chicks were still positive with iBron at an average Ct of approximately 26, while only 60% of chicks were positive with Mass at an average Ct of approximately 27.5. However, a positive "tail" exists, and in Mass, not all birds were uniformly vaccinated. Combining iBron (a strong vaccine) with DE1639 or BI-based Mass (a weaker vaccine) may result in some vaccine interference.

[0083] In the DE1639 vaccinated group, the susceptibility on day 7 was not good. Therefore, it was decided to resample the group that had received the vaccine on the 14th day to evaluate the viral load and potential vaccine rolling. This data can be seen in Figure 2. On the 14th day compared to the 7th day, both the positive percentage (about 85%) and the viral load (average Ct of 25.5) increased only in the DE1639 vaccinated group. This suggests that the peak of viral vaccine replication occurs considerably later with this vaccine than with traditional IBV vaccines. In the DE1639 vaccine in the groups vaccinated with this vaccine and Mass, the positive percentage increased similarly (from about 35% positive on day 7 to about 60% positive on day 14), but the average viral load (Ct of about 32) did not change. This suggests that there is still vaccine interference between the Mass vaccine and the DE1639 vaccine, and that the conditions for a rolling response are established with a portion of the population becoming positive at different time points. In the DE1639 and iBron vaccinated groups, only one sample was positive, suggesting that the iBron vaccine essentially inactivates the DE1639 vaccine. Also, on 28 dpv, only the DE1639 vaccine group was resampled to examine how much of the vaccine still remained. The results are shown in Figures 3A and 3B. Approximately 25% of the chickens still had a viral load within the Ct value range of 36 - 22 (average Ct of about 28) and were still positive. This indicates that the vaccine continues to roll within the flock.

[0084] Another notable observation was that the groups given the iBron vaccine alone or in combination with another vaccine showed significant clinical respiratory signs 7 and 14 days after vaccination. When sampled to measure vaccine susceptibility, rales were observed in 1 / 4 to 1 / 3 of the chicks. Clinical respiratory signs were not observed in any of the other vaccine groups.

[0085] Twenty-eight days after vaccination, chickens in each vaccine group were challenged with approximately 1×10 4 EID 50The birds were challenged with pathogenic DMV / 1639 IB virus from birds. Five days after challenge, clinical respiratory signs were recorded, and swabs were collected for IBV load analysis by PCR. With the exception of the Mass / iBron-vaccinated challenge group, all groups had statistically significantly lower clinical signs than the unvaccinated DMV / 1639 challenge group, and all groups scored lower numerically. The results are shown in Figure 4. This is not surprising, given previous results from vaccine challenge trials using DMV / 1639 challenge virus and numerous IBV vaccines.

[0086] Viral loads determined by DMV / 1639-specific PCR showed that all groups had statistically significantly lower viral loads than the unvaccinated challenge group. The results are shown in Figure 5. Overall, the mean Ct value in the unvaccinated challenge group was approximately 23, and all five samples were very close to this value. In the DE1639 vaccine-vaccinated group, 8 / 10 of the samples were virus-positive after the challenge, but the samples were divided into two identifiable groups. One group clustered near the 29 Ct value mark, and the other group clustered near the 34 Ct value mark.

[0087] This assay cannot distinguish between vaccine and challenge virus, so it is not possible to say whether all of these positive samples are vaccine or challenge virus. Based on the 28-day samples from this group and the experience when these tests were conducted, it would be estimated that half of these positive values ​​are from the challenge virus. The PDRC DMV / 1639 vaccinated group had only one positive after the challenge with a Ct value of approximately 33, suggesting that the virus was almost nonexistent. This test shows that the PDRC DMV / 1639 vaccine adequately protected against this challenge virus according to the 9-CFR standard (more than 90% of samples were virus-negative). The DE1639 and Mass vaccine vaccinated groups had only two positive samples after the challenge with an average Ct value of approximately 32. It is interesting to consider that the DE1639 vaccine appeared to suppress the virus to the same extent as when combined with the Mass vaccine. However, it cannot be ruled out that some DMV / 1639 vaccinated chickens were inadvertently selected when randomly selecting chickens for the challenge, resulting in some immunization. In the group vaccinated with DE1639 vaccine and iBron, 8 out of 10 samples were positive with an average Ct value of approximately 31. Since the Ct values ​​in this group ranged from approximately 26 to 38, this suggests that only partial protection against the challenge was achieved.

[0088] The suppression of DE1639 vaccine by the iBron vaccine appears to have affected the development of specific neutralizing antibodies against DMV / 1639 challenge. In the Mass, iBron, and iBron-only vaccine groups, 9 / 10 and 8 / 10 samples were positive after challenge, with mean Ct values ​​of approximately 30 and 32, respectively. Since these two groups were very similar to the DE1639 and iBron vaccine groups in terms of mean Ct and total number of positives after challenge, it is suggested that specific neutralizing antibodies were not adequately developed in either of these groups.

[0089] Consideration Based on field observations, the experiment concluded as expected. The DE1639 vaccine did not sufficiently infect the birds at the time of initial vaccination and persisted in the birds at least until the challenge. Furthermore, the virus was detected at a higher rate after the challenge than before the challenge, suggesting that at least some of the viruses detected after the challenge were the challenge virus. This means either the DE1639 vaccine was not a perfect antigenic match for the challenge, or the birds that were not vaccinated were not protected from the challenge due to insufficient infection rates. In either case, the vaccine was not effective in suppressing the challenge virus.

[0090] The same can be said for the groups vaccinated with DE1639 and iBron, Mass and iBron, and iBron alone. In those groups, almost all birds received the iBron vaccine, while a subset of birds received other vaccines in combination. This suggests that the iBron vaccine suppressed other vaccines to some extent, and that the iBron vaccine alone does not provide sufficient protection.

[0091] The only group that was adequately protected (over 90% negative) was the group vaccinated with the PDRC DMV / 1639 vaccine. This vaccine was developed from a recent 2019 isolate and appears to be a good antigenic match against the field virus. Furthermore, this vaccine was administered at a lower titer than other vaccines (approximately 1 × 10⁻⁶). 3.5 EID 50 ), the infection and replication dynamics remained almost complete.

[0092] The DE1639 vaccine does not provide adequate protection from challenge, and the vaccine persists in setting up rolling response scenarios and clouding the diagnostic picture in herds. The combination of Mass and iBron provided similar protection to the DE1639 vaccine when evaluating post-challenge viral detection, and other experiments with this combination showed similar results. While this vaccine combination can reduce clinical signs and potentially reduce the impact of challenge on performance and disposal, it does not adequately inhibit DMV / 1639 challenge virus infection and replication.

[0093] Example 3 Comparison of the alloprotective capabilities of the currently used DE1639 vaccine and the DMV / 1639 vaccine. Despite the use of live attenuated autovaccines by several manufacturers, DMV / 1639 IB virus has consistently circulated in poultry flocks since at least 2014. The vaccines used do not appear to provide complete protection against the currently circulating congenital virus in the field. For these reasons, the novel thermo-attenuated DMV / 1639 vaccine of the present invention was developed in an attempt to increase protection against the currently circulating field virus of this variant serotype.

[0094] Materials and methods Virus. This study used the thermo-attenuated live virus DMV / 1639 vaccine (DE1639) developed by the University of Delaware and the attenuated PDRC DMV / 1639 vaccine described herein (deposited with ATCC under patent designation PTA-12657). The challenge virus used was a recent isolate of DMV / 1639 / 11 from a herd of broiler chickens on the DelMarVa peninsula.

[0095] Experimental Design. The experimental design is shown in Table 2 below. One-day-old broiler chicks positive for maternal antibodies were used in this experiment. 100 chicks in each group were vaccinated with the full dose of each vaccine by spray at one day of age and placed in separate colony houses. On day 7, all chicks in each group were swabded and the vaccine virus was detected using real-time PCR. At 28 days of age, 10 chicks for each challenge virus and 5 chicks for each control group were transferred to isolators and challenged with 1 × 10⁴ EID50 DMV / 1639 / 11. The titer of the challenge virus was verified by back titration in embryonic eggs. Necropsy was performed 5 days after challenge.

[0096] [Table 2]

[0097] Autopsy / Clinical Signs. Clinical signs were recorded and scored based on the previously reported laboratory scoring method (Jackwood et al., 2015, Avian Diseases; 59(3):368-374. https: / / doi.org / 10.1637 / 11026-012415-Reg.1). The scale was as follows: 0 = asymptomatic, 1 = slight wheezing or sneezing, 2 = more pronounced wheezing, sinus exudate, conjunctivitis, and 3 = rales.

[0098] Autopsy / RNA extraction and real-time RT-PCR for challenge virus detection. During autopsy, nasal swab samples were collected from the oral-pharyngeal region within the cleft palate and placed in 1 ml of ice-cold PBS for real-time RT-PCR for viral detection. Viral RNA was extracted from 50 μl of PBS using a MagMAX-96 RNA Isolation Kit (Ambion Inc., Austin TX) according to the manufacturer's protocol for the KingFisher magnetic particle processor (Thermo Scientific, Waltham, MA). Real-time RT-PCR was performed using an Applied Biosystems 7500 Fast Real-Time PCR System (Life Technologies, Carlsbad, CA) and an AGPATH-ID® One-Step RT-PCR Kit (Ambion Inc.) according to the manufacturer's recommendations. Primers and probes for real-time RT-PCR correspond to the specific serotype being tested. Primers were obtained from Integrated DNA Technologies (Coralville, IA), and TAQMAN® probes were synthesized by BioSearch Technologies (Novato, CA).

[0099] result Purity testing. The PDRC DMV / 1639 vaccine was tested for purity by quantitative real-time PCR using primers and probes that detect all known serotypes of IBV in the United States. Table 3 below shows that only DMV / 1639 was positive in the tested vaccine, indicating the absence of other IBV contaminants. Furthermore, when the vaccine was streaked onto blood agar bacterial plates, no growth was detected after 24 or 48 hours of incubation.

[0100] [Table 3]

[0101] Safety testing. The virus used in the vaccine experiment was used in safety testing in accordance with USDA 9CFR guidelines. The titer of the vaccine actually administered to chicks in efficacy testing was 1 × 10⁻⁶. 3.4 EID 50 Therefore, safety testing required at least 10 × doses. For that purpose, 3.16 × 10 5 EID 50 The dose was individually administered to 26 SPF chicks via the oculo-nasal route on the hatching day. Four SPF chicks were raised unvaccinated as a comparative negative control. The 26 vaccinated chicks were divided into two isolators, with 15 chicks in one and 11 in the other. All chicks were monitored daily for a total of 21 days for any worsening clinical signs associated with IBV vaccine infection or mortality. Two chicks were euthanized during the trial due to hind limb dislocation, but showed no signs of IBV infection, and necropsy revealed no lesions typical of IBV. None of the tested chicks showed any signs of IBV over the 21-day experiment. The results of the daily monitoring can be seen in Table 4 below.

[0102] [Table 4]

[0103] Efficacy test. In this trial, 100 chicks were spray-vaccinated with the vaccines shown in Table 2. Then, all 100 chicks in each group were placed on clean dressings in a colony-type house to mimic field environmental conditions. Seven days post-vaccination (dpv), all chicks in each group were swabbed in the posterior choanal cleft to measure the vaccine virus present. All data can be seen in Figure 6. As expected, based on previous vaccine susceptibility sampling, the DE1639 vaccine only infected approximately 60% of the chicks at the time of vaccination, and the amount of virus obtained was relatively low (average Ct value of approximately 33). This is in contrast to the DMV / 1639 vaccine described herein (PDRC DMV / 1639), which infected almost 100% of the chicks with an average Ct value of approximately 26.5 (Figure 6).

[0104] Since susceptibility was not good at 7 days in the DE1639 vaccine group, that group was swabbed again on day 14 to evaluate the virus amount and potential vaccine rolling. This data can be seen in Figure 7. On day 14 compared to day 7, both the positive percentage (approximately 85%) and the virus amount (average Ct of 25.5) increased in the DE1639 vaccine group. This suggests that the peak of virus vaccine replication occurs considerably later with this vaccine than with traditional IBV vaccines. Also, the DE1639 vaccine group was swabbed at 28 dpv to examine how much vaccine was still present (Figure 8). Approximately 25% of the chickens still had a positive virus amount within the Ct value range of between 36 and 22 (average Ct of approximately 28), indicating that the vaccine was still rolling within the flock.

[0105] Twenty-eight days post-vaccination, 10 chickens from each vaccine group were randomly selected and challenged with a pathogenic DMV / 1639 type IB virus at a dose of approximately 1×10 4 EID 50 / chicken. Five days after challenge, clinical respiratory signs were recorded and swabs were taken for IBV load analysis by PCR. All groups had statistically significantly lower clinical signs than the non-vaccinated DMV / 1639 challenge group (Figure 9).

[0106] Viral loads determined by DMV / 1639-specific PCR showed that all groups had statistically significantly lower viral loads than the unvaccinated challenge group (Figure 10). Overall, the mean Ct value in the unvaccinated challenge group was approximately 23, and all five samples were very close to this value. In the DE1639 vaccine-vaccinated group, 8 / 10 of the samples were virus-positive after challenge, but the samples were divided into two distinguishable groups. One group clustered near the 29 Ct value mark, and the other clustered near the 34 Ct value mark. Since this assay cannot distinguish between vaccine and challenge virus, it is not possible to say whether all of these positive samples are vaccine or challenge. Based on the 28-day samples from this group and the experience when these tests were performed, it can be estimated that half of these positive values ​​are from the challenge virus. In the PDRC DMV / 1639-vaccinated group, only one sample was positive after challenge, with a Ct value of approximately 33, suggesting that the virus was almost nonexistent. This trial demonstrates that the PDRC vaccine adequately protected against this challenge virus according to the 9-CFR standard (more than 90% of samples were virus-negative).

[0107] Consideration The experiment concluded as expected. The DE1639 vaccine did not sufficiently infect the birds at the time of initial vaccination and subsequently persisted in the birds at least until the challenge. Furthermore, the virus was detected at a higher rate after the challenge than before the challenge, suggesting that at least some of the viruses detected after the challenge were the challenge virus. This means either the DE1639 vaccine was not a perfect antigenic match for the challenge, or the birds that were not vaccinated were not protected from the challenge due to insufficient infection rates. In either case, the vaccine was not effective in suppressing the challenge virus. This is similar to the field conditions in which DMV / 1639 is consistently detected in flocks vaccinated with the autologous DMV vaccine. The only flock that was adequately protected (more than 90% negative) was the flock vaccinated with the thermo-attenuated PDRC DMV / 1639 vaccine described herein (deposited with the ATCC® patent depositary as patent depositary number PTA-126757). The DMV / 1639 vaccine was developed from a recent 2019 isolate and appears to be a good antigenic match against the field virus. Furthermore, although this vaccine was administered at a lower titer than other vaccines (1 × 100), 3.4 EID 50 ), the infection and replication dynamics remained almost complete.

[0108] One possible explanation for the improved efficacy of the newer PDRC vaccine is that it is a better antigenic match against the currently circulating field virus. The phylogenetic table shown in Figure 11 compares isolates of different DMV / 1639 viruses. The lower box represents isolates from the 2015 outbreak of origin, along with the autologous vaccine, indicated by the arrows. More recent isolates from 2019 onwards are in the upper box, along with the PDRC vaccine, indicated by the arrows. The challenge virus sequence isolated from a herd of DelMarVa broilers and used in this study is also included in the clade indicated by the upper box. The virus appears to have evolved as it moves around the country, but it has begun to "confirm" as most isolates are now grouped together into one group.

[0109] Example 4 Backpassing of heat-attenuated PDRC DMV / 1639 vaccine to evaluate attenuation stability In this example, the stability of the heat-attenuated PDRC DMV / 1639 vaccine described herein was evaluated by backpassing of the vaccine in susceptible chickens.

[0110] Materials and methods Virus. This study used a heat-treated (attenuated) PDRC DMV / 1639 virus vaccine deposited with ATCC under patent designation PTA-12657.

[0111] Experimental design. For this experiment, we used 1-day-old SPF (Specific Pathogen Free) chicks. Five 1-day-old chicks were given 3.16 × 10⁻¹⁰ eggs. 3The 50% embryo-infective dose (EID50) of the vaccine was initially administered intranasally and intraocularly in a volume of 0.1 ml. Either two or three days after vaccination, the posterior nasal cleft of each chick was swabded and pooled in 3 ml of PBS (pH 7.4), and 0.1 ml of the pooled sample was given to each of five one-day-old SPF chicks from a separate group. This was repeated 10 times. All chicks were examined for clinical signs throughout the study, and in the final back passage (back passage number 10), five individual chicks were necropsed five days after vaccination. At this necropsy, the chicks were examined for clinical signs and lesions without taking and pooling posterior nasal swabs.

[0112] Clinical signs. Clinical signs were recorded and scored based on the previously reported laboratory scoring method (Jackwood et al., 2015, Avian Diseases; 59(3):368-374. https: / / doi.org / 10.1637 / 11026-012415-Reg.1). The scale was as follows: 0 = asymptomatic, 1 = slight wheezing or sneezing, 2 = more pronounced wheezing, sinus exudate, conjunctivitis, and 3 = rales.

[0113] RNA extraction and virus detection by real-time RT-PCR. Intranasal swabs were collected from the oral-pharyngeal region of cleft palate patients. In re-passage groups 1-9, these were pooled in 3 ml of ice-cold PBS for virus detection by real-time RT-PCR. In re-passage group 10, intranasal swabs from 5 birds were retained as individual samples and placed in 1 ml of ice-cold PBS for virus detection by real-time RT-PCR.

[0114] Viral RNA was extracted from 50 µl of PBS using a MagMAX-96 RNA Isolation Kit (Ambion Inc., Austin TX) according to the manufacturer's protocol for the KingFisher magnetic particle processor (Thermo Scientific, Waltham, MA). Real-time RT-PCR was performed using an Applied Biosystems 7500 Fast Real-Time PCR System (Life Technologies, Carlsbad, CA) and an AGPATH-ID® One-Step RT-PCR Kit (Ambion Inc.) according to the manufacturer's recommendations. Primers and probes for real-time RT-PCR correspond to specific DMV / 1639 serotypes. Primers were obtained from Integrated DNA Technologies (Coralville, IA), and Taqman probes were synthesized by BioSearch Technologies (Novato, CA).

[0115] result None of the birds given the heat-attenuated PDRC DMV / 1639 vaccine developed any clinical signs. In addition, all five birds in the 10 back passages were free of clinical signs and lesions.

[0116] The results of real-time RT-PCR are shown in Table 5. The vaccine virus was detected in all pooled samples from backpassing groups 1-9 and in each of the individual samples taken from five birds in backpassing group 10.

[0117] [Table 5]

[0118] Consideration Based on the data from this experiment, the heat-attenuated PDRC DMV / 1639 vaccine was stable after 10 backpassages in susceptible SPF chickens. The vaccine virus was detected in chicks from each backpassage group, and no clinical signs or lesions were detected in any of the birds (only in backpassage group 10). The CT values ​​were within the range of 22.97 to 17.06, suggesting that a significant amount of vaccine was passaged into the chicks in each group. Considering the data and facts that no clinical signs or lesions were observed in the birds, it is clearly suggested that the attenuation of the PDRC DMV / 1639 vaccine is stable and should be safe for field use.

[0119] Example 5 purity Analysis of samples of the PDRC DMV / 1639 vaccine deposited with ATCC under patent designation PTA-12657 by the University of Delaware's Institute of Agriculture and Natural Resources, concerning infectious coryza, infectious laryngotracheitis virus, Mycoplasma gallisepticum, Mycoplasma synoviae, NAHLN avian influenza (AIV), NAHLN triparamyxovirus, infectious bronchitis virus (IBV), IBV-DE072 / GA99, IBV-Ark, IBV-Mass / Conn, IBV-DMV / 1639, and IBV-GA08, detected only IBV and IBV-DMV1639, suggesting purity. This analysis was performed to ensure that the isolate did not contain any pathogens commonly found in commercial poultry or any pathogens that could potentially devastate commercial poultry (e.g., AI or NDV). This purity is paramount and should be thoroughly evaluated when considering vaccine production. This analysis was also performed to ensure that any results obtained from the tests were not affected positively or negatively by confounding variables such as contamination by other pathogens (see previous examples). These data ensure that the utmost care was taken in quality control during the isolation, propagation, and preparation of the vaccine seeds, and that the presented data is a true reflection of this DMV / 1639 / 11 virus vaccine alone.

[0120] Example 6 Field test The PDRC DMV / 1639 vaccine described herein (deposited with ATCC under patent designation PTA-12657) has been administered to over 50 million chickens in a commercial setting. Vaccine efficacy analysis demonstrates that the vaccine is highly effective in infection and replication under commercial conditions. This analysis is crucial to ensuring vaccine success, as most poultry vaccines against infectious bronchitis viruses (such as the DMV / 1639 vaccine described herein) are tested and validated using eye drop administration. Although the gold standard for laboratory experiments, it is impossible to administer eye drop vaccine to every commercially raised chicken. For this reason, commercial chickens are vaccinated in a mass manner using spray cabinets that aerosolize the vaccine onto chicks. This process has many potential drawbacks that have previously affected the efficacy of other IBV vaccines. This data demonstrates that the DMV / 1639 vaccine described herein is highly effective in infection and replication in chickens—a key step in inducing a proper immune response—even when applied in a mass manner by spray. This data also helps determine most effective doses of vaccines, as it allows for the recording and evaluation of adverse vaccine reactions and actual doses given to chicks in the commercial environment in which the vaccine is used.

[0121] All patents, patent applications, and publications and electronically available materials cited herein (including, for example, nucleotide sequence submissions to GenBank and RefSeq, and amino acid sequence submissions to SwissProt, PIR, PRF, PDB, and translations from the annotated coding regions of GenBank and RefSeq) are incorporated by reference. In the event of any inconsistency between the disclosures of this application and the disclosures of any of the documents incorporated by reference herein, the disclosures of this application shall prevail. The above detailed description and examples are given solely for the purpose of clarifying understanding and should not be construed as unnecessary limitations. The present invention is not limited to the exact details shown and described, and variations that are obvious to those skilled in the art are included within the scope of the invention as defined by the claims. This disclosure includes the following embodiments. [1] Infectious bronchitis virus (IBV) isolates comprising a thermal attenuated IBV isolate PDRC DMV / 1639 deposited with ATCC under patent designation PTA-126757 or its progeny or derivatives, wherein the progeny or derivatives have essentially the same biological and serological characteristics as the thermal attenuated IBV isolate PDRC DMV / 1639 deposited with ATCC under patent designation PTA-126757. [2] The IBV isolate according to Embodiment 1, wherein the IBV isolate is freeze-dried, freeze-dried or frozen. [3] A composition comprising the IBV isolate described in Embodiment 1. [4] The composition according to Embodiment 3, further comprising a pharmaceutically acceptable carrier. [5] A vaccine comprising the isolated IBV isolate or its progeny or derivatives as described in Embodiment 1. [6] The vaccine according to Embodiment 5, wherein the vaccine reduces one or more clinical signs and / or viral loads induced by IBV infection in poultry. [7] A vaccine for Galliformes birds comprising a sufficient amount to protect birds from one or more clinical signs induced by infectious bronchitis virus (IBV) infection in poultry, comprising the heat-attenuated IBV isolate PDRC DMV / 1639 deposited with ATCC under patent designation PTA-126757 or its offspring or derivatives. [8] The composition or vaccine according to any one of Embodiments 3 to 7, further comprising an adjuvant. [9] A composition or vaccine according to any one of Embodiments 3 to 8, further comprising other viral materials.

[10] The composition or vaccine according to any one of Embodiments 3 to 9, wherein the composition or formulation is formulated for intranasal, intraocular, oral, mucosal, intramuscular, subcutaneous, or in ovo administration.

[11] The composition or vaccine according to any one of embodiments 3 to 10, wherein the composition or vaccine is formulated for spraying or aerosolizing.

[12] An effervescent tablet comprising an IBV isolate or its offspring or derivatives, composition, or vaccine as described in any of Embodiments 1 to 11.

[13] A method for generating an immune response against infectious bronchitis virus (IBV) in poultry, comprising administering to the poultry an IBV isolate or its offspring or derivatives, composition or vaccine described in any of Embodiments 1 to 11.

[14] A method for generating anti-IBV antibodies in poultry, comprising administering to the poultry an IBV isolate or its offspring or derivatives, composition, or vaccine described in any of Embodiments 1 to 11.

[15] A method for reducing one or more clinical signs and / or viral load induced by infectious bronchitis virus (IBV) infection in poultry, comprising administering an effective amount of an IBV isolate or its offspring or derivatives, composition or vaccine described in any of Embodiments 1 to 11 to the poultry.

[16] A method for reducing the susceptibility of Galliformes birds to infectious bronchitis virus (IBV) infection, comprising administering to the birds an effective amount of an IBV isolate or its offspring or derivatives, composition or vaccine described in any of Embodiments 1 to 11.

[17] A method for protecting Galliformes birds from infectious bronchitis virus (IBV) infection, comprising administering to the birds an effective amount of an IBV isolate or offspring or derivative, composition or vaccine described in any of Embodiments 1 to 11.

[18] The method according to any one of Embodiments 13 to 17, wherein the administration is intranasal, intraocular, oral, mucosal, intramuscular, or subcutaneous.

[19] The method according to any one of embodiments 13 to 18, wherein the administration includes in ovo administration.

[20] The method according to any one of Embodiments 13 to 19, wherein the IBV isolate or its progeny or derivatives, composition, or vaccine is administered by aerosol.

[21] The method according to any one of Embodiments 13 to 19, wherein the IBV isolate or progeny or derivatives, composition or vaccine is administered by spraying.

[22] The method according to any one of embodiments 13 to 21, wherein the administration includes administration to breeding hens.

[23] The method according to any one of embodiments 13 to 22, wherein the poultry includes birds of the order Galliformes.

[24] The method according to any one of embodiments 13 to 23, wherein the bird is a chicken or a turkey.

Claims

1. Infectious bronchitis virus (IBV) isolates, including thermally attenuated IBV isolate PDRC DMV / 1639 deposited with ATCC under patent designation PTA-126757.

2. The IBV isolate according to claim 1, wherein the IBV isolate is freeze-dried, lyophilized, or frozen.

3. A composition comprising the IBV isolate described in claim 1.

4. The composition according to claim 3, further comprising a pharmaceutically acceptable carrier.

5. A vaccine comprising the isolated IBV isolate described in claim 1.

6. The vaccine according to claim 5, wherein the vaccine reduces one or more clinical signs and / or viral loads induced by IBV infection in poultry.

7. A vaccine for Galliformes birds containing the heat-attenuated IBV isolate PDRC DMV / 1639, deposited with the ATCC under patent designation PTA-126757.

8. A composition or vaccine according to any one of claims 3 to 7, further comprising an adjuvant.

9. The composition or vaccine according to any one of claims 3 to 8, further comprising other viral materials.

10. The composition or vaccine according to any one of claims 3 to 9, wherein the composition or formulation is formulated for intranasal, intraocular, oral, mucosal, intramuscular, subcutaneous, or in ovo administration.

11. The composition or vaccine according to any one of claims 3 to 10, wherein the composition or vaccine is formulated for spraying or aerosolizing.

12. A foaming tablet comprising an IBV isolate, composition, or vaccine according to any one of claims 1 to 11.

13. A method for generating an immune response against infectious bronchitis virus (IBV) in poultry, comprising administering an IBV isolate, composition, or vaccine according to any one of claims 1 to 11 to the poultry.

14. A method for generating anti-IBV antibodies in poultry, comprising administering an IBV isolate, composition, or vaccine according to any one of claims 1 to 11 to the poultry.

15. A method for reducing one or more clinical signs and / or viral load induced by infectious bronchitis virus (IBV) infection in poultry, comprising administering an effective amount of an IBV isolate, composition, or vaccine according to any one of claims 1 to 11 to the poultry.

16. A method for reducing the susceptibility of Galliformes birds to infectious bronchitis virus (IBV) infection, comprising administering an effective amount of an IBV isolate, composition, or vaccine according to any one of claims 1 to 11 to the birds.

17. A method for protecting Galliformes birds from infectious bronchitis virus (IBV) infection, comprising administering an effective amount of an IBV isolate, composition, or vaccine according to any one of claims 1 to 11 to the birds.

18. The method according to any one of claims 13 to 17, wherein the administration is intranasally, intraocularly, orally, mucosally, intramuscularly, or subcutaneously.

19. The method according to any one of claims 13 to 18, wherein the administration includes in oval administration.

20. The method according to any one of claims 13 to 19, wherein the IBV isolate, composition, or vaccine is administered by aerosol.

21. The method according to any one of claims 13 to 19, wherein the IBV isolate, composition, or vaccine is administered by spraying.

22. The method according to any one of claims 13 to 21, wherein the administration includes administration to breeding hens.

23. The method according to claim 13, 14, or 15, wherein the poultry includes birds of the order Galliformes.

24. The method according to claim 16, 17, or 23, wherein the bird is a chicken or a turkey.

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