RU2844767C1 - Attenuated isolate of strain dmv1639 of infectious bronchitis virus - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: described is an infectious bronchitis virus (IBV) isolate for producing an immune response to IBV, wherein the IBV isolate provides a heat-attenuated IBV PDRC DMV/1639 isolate deposited in the ATCC under patent deposit registration number PTA-126757. What is disclosed is a composition for producing an immune response to IBV containing the described IBV isolate and a pharmaceutically acceptable carrier. Described is a vaccine for producing an immune response on IBV containing the recovered described IBV isolate. Also disclosed is a vaccine for birds of the order Galliformes, containing an amount of a heat attenuated isolate of IBV PDRC DMV/1639 deposited in the ATCC under patent deposit registration number PTA-126757, sufficient to protect birds from one or more clinical signs induced by infection caused by infectious bronchitis virus (IBV), in farm birds. Disclosed is a method of producing an immune response to infectious bronchitis virus (IBV) in poultry, wherein method comprises administering to farm birds described IBV isolate, disclosed composition or vaccine. Disclosed is a method of producing anti-IBV antibodies in poultry, wherein method comprises administering to farm birds described IBV isolate, disclosed composition or vaccine. Also disclosed is a method for reducing the severity of one or more clinical signs and/or viral load induced by an infection caused by infectious bronchitis virus (IBV) in farm birds, wherein the method comprises administering to farm birds an effective amount of the described IBV isolate, the disclosed composition or vaccine. Also disclosed is a method for reducing the susceptibility of chicken birds to infection caused by infectious bronchitis virus (IBV), wherein the method comprises administering to the bird an effective amount of the described IBV isolate, the disclosed composition or vaccine. Invention also describes a method of protecting birds of order Galliformes from infection caused by infectious bronchitis virus (IBV), wherein the method comprises administering to the bird an effective amount of the described IBV isolate, the disclosed composition or vaccine.

EFFECT: invention widens the range of agents for protecting birds from infectious bronchitis virus.

14 cl, 11 dwg, 5 tbl, 6 ex

Description

CONTINUING APPLICATION DETAILS

This application claims priority to U.S. Provisional Patent Application Ser. No. 63/081,392, filed September 22, 2020, which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

Avian infectious bronchitis virus (IBV) causes an economically significant upper respiratory disease in chickens (EconomicData section of US Poultry & Egg Association, 2016). Because of its prevalence and infectivity, nearly all commercial poultry in the United States are vaccinated against IBV in a serotype-specific manner (Cavanagh, 2007, Veterinary Research ; 38:281-297). IBV is an enveloped, positive-sense, single-stranded ribonucleic acid (RNA) virus that belongs to the genus Gammacoronavirus in the family Coronaviridae . Like most RNA viruses, IBV is genetically diverse due to a high rate of mutation and recombination events. New IBV variants continually emerge, complicating vaccination-based control of infectious bronchitis (IB). The Delmarva/1639 IBV strain (DMV/1639) was first isolated during the Delaware/Maryland/Virginia (DELMARVA) IB outbreak in the United States in 2011 (Gelbe et al., 2012, Avian Dis ; 57(1):65-70). It currently represents a major economic problem in the poultry industry that continues to spread. Although cross-protection against the DMV/1639 strain by vaccination with one or more other IBV serotypes may be partially effective in limiting the clinical signs associated with DMV/1639 infection, there is a need for improved vaccines against DMV/1639-type viruses.

ESSENCE OF THE INVENTION

The present invention provides an infectious bronchitis virus (IBV) isolate, wherein the IBV isolate comprises heat-attenuated IBV isolate PDRC DMV/1639, deposited with the ATCC under Patent Deposit Accession Number PTA-126757, or a progeny or derivative thereof, wherein the progeny or derivative thereof has substantially the same biological and serological characteristics as the heat-attenuated IBV isolate PDRC DMV/1639, deposited with the ATCC under Patent Deposit Accession Number PTA-126757. In some aspects, the IBV isolate is lyophilized, freeze-dried, or frozen.

The present invention also provides compositions comprising an IBV isolate, progeny or derivative as described herein. In some aspects, the composition further comprises a pharmaceutically acceptable carrier. In some aspects, the composition further comprises an adjuvant. In some aspects, the composition further comprises another viral material. In some aspects, the composition is formulated for intranasal, intraocular, oral, mucosal, intramuscular, subcutaneous or in ovo administration. In some aspects, the composition is formulated for spraying or nebulizing as an aerosol.

The present invention also provides vaccines comprising an IBV isolate or progeny or derivative thereof as described herein, or a composition as described herein. In some aspects, the vaccine provides a reduction in the severity of one or more clinical signs and/or viral load induced by IBV infection in poultry.

The present invention provides a vaccine for birds of the order Galliformes , the vaccine comprising an amount of the heat-attenuated IBV isolate PDRC DMV/1639, deposited with the ATCC under Patent Deposit Registration Number PTA-126757, or a progeny or derivative thereof, sufficient to protect birds from one or more clinical signs induced by infection with infectious bronchitis virus (IBV) in poultry.

The present invention also provides an effervescent tablet comprising an IBV isolate, progeny or derivative thereof, composition or vaccine as described herein.

The present invention also provides methods for producing an immune response to infectious bronchitis virus (IBV) in poultry, the method comprising administering an IBV isolate, progeny or derivative thereof, composition or vaccine as described herein.

The present invention also provides methods for producing antibodies to IBV in poultry, the method comprising administering to the poultry an IBV isolate or progeny or derivative thereof, a composition or vaccine as described herein.

The present invention also provides methods for reducing the severity of one or more clinical signs and/or viral load induced by infection with infectious bronchitis virus (IBV) in poultry, the method comprising administering an effective amount of an IBV isolate or progeny or derivative thereof, a composition or vaccine as described herein.

The present invention also provides methods for reducing the susceptibility of Galliformes to infection with infectious bronchitis virus (IBV), the method comprising administering to the bird an effective amount of an IBV isolate or progeny or derivative thereof, a composition, or a vaccine as described herein.

The present invention also provides methods for protecting Galliformes from infection by infectious bronchitis virus (IBV), the method comprising administering to the bird an effective amount of an IBV isolate, progeny or derivative thereof, composition or vaccine as described herein.

In some aspects of the method described herein, administration is intranasal, intraocular, oral, transmucosal, intramuscular, or subcutaneous.

In some aspects of the method described herein, the administration involves in ovo administration.

In some aspects of the method described herein, the IBV isolate or progeny or derivative thereof, composition or vaccine is administered via an aerosol.

In some aspects of the method described herein, the IBV isolate, or progeny or derivative, composition or vaccine is administered by spraying.

In some aspects of the method described herein, the IBV isolate, progeny, or derivative, composition, or vaccine is administered via drinking water.

In some aspects of the method described herein, the administration comprises administration to a breeding female bird.

In some aspects of the method described herein, the farm bird comprises a bird of the order Galliformes .

In some aspects of the method described herein, the bird is a chicken or a turkey.

BRIEF DESCRIPTION OF THE FIGURES

Fig. 1A and 1B. Infection rates at day seven post-vaccination. Fig. 1A shows the viral load (inverted Ct value) and percent positivity for vaccination with De1639, PDRC DMV1639, and MA DMV/1639 + Mass. Fig. 1B shows the viral load and percent positivity for vaccination with DE1639 + iBron, iBron, and iBron + Mass.

Fig. 2. Infection levels at day 14 for chickens vaccinated with autogenous DE1639.

Fig. 3A and 3B. Viral loads for chickens vaccinated with autogenous DE1639 vaccine alone at day 28. Fig. 3A shows the inverted Ct values. Fig. 3B shows the percentage of positive results.

Fig. 4. Clinical signs after challenge depending on vaccine/challenge group.

Fig. 5. Viral load after infection based on detection data using DMV-specific PCR.

Fig. 6. Vaccine virus infection levels at day seven for chickens vaccinated with DE1639 and PDRC DMV/1639.

Fig. 7. Infection levels at day 14 for chickens vaccinated with DE1639.

Fig. 8. Viral loads for chickens vaccinated with DE1639 alone at day 28 pre-challenge.

Fig. 9. Clinical signs after challenge depending on vaccine/challenge group.

Fig. 10. Viral load after infection based on detection data using DMV-specific PCR.

Fig. 11. Phylogenetic tree comparing different DMV/1639 virus isolates. The lower rectangle represents isolates collected during the initial outbreak in 2015, with the autogenous vaccine indicated by the arrow. Isolates discovered later in 2019 are shown in the upper rectangle, with the PDRC vaccine indicated by the arrow.

DETAILED DESCRIPTION

The present invention relates to new materials and methods in the field of poultry virology, in particular in the field of infectious bronchitis virus (IBV).

Avian infectious bronchitis virus (IBV) is a gammacoronavirus. The enveloped IBV virus contains a positive-sense, single-stranded RNA genome that encodes the viral RNA-dependent RNA polymerase, three major structural proteins (nucleocapsid, membrane, and spike (S) proteins), and numerous regulatory proteins (Masters, 2006, AdvVirRes ; 66:193–292). The IBV spike glycoprotein is translated as a precursor protein (So) and then cleaved into two subunits, the N-terminal glycoprotein S1 and the C-terminal glycoprotein S2, by host cell serine proteases. Glycoproteins S1 and S2 mediate cell attachment, viral-cellular membrane fusion, and play an important role in host cell specificity by forming club-shaped protrusions on the virus surface. Glycoprotein S1 induces the production of antibodies that neutralize the virus and inhibit hemagglutination.

New variant strains arise from rapid recombination, insertions, deletions, or point mutation events, primarily in the S1 gene. In addition to serologic tests, PCR and partial sequencing of the S1 gene can be used to group and type IBV isolates. Several changes in the S glycoprotein sequence can result in the emergence of a new serotype. It has been documented that differences in the IBV S1 sequence as small as 5% can result in loss of cross-protection between otherwise similar isolates (Cavanagh, 2003, Avian Pathol ; 32:567-582). Based on spike protein 1 (S1) variability, six IBV genotypes have been described, comprising 32 distinct viral lineages (Valastro et al., 2016, InfectGenetEvol ; 39:349-364), which are generally recognized.

IBV Delmarva/1639 (DMV/1639) was first isolated during the 2011 Delmarva Peninsula infectious bronchitis (IB) outbreak (Gelbe et al., 2012, Avian Dis ; 57(1):65-70), and infections with this IBV variant represent an ongoing problem for poultry producers. There is currently no commercial vaccine against this IBV strain. Although cross-protection against DMV/1639 through vaccination with one or more other IBV serotypes may be effective in limiting mortality and morbidity associated with DMV/1639 infection, there is a need for improved vaccines against DMV/1639-type viruses.

The present invention provides a live, heat-attenuated isolate of IBV strain DMV1639, as well as progeny and derivatives thereof. When administered to birds as a live composition, this attenuated IBV isolate is safe and effective in preventing IBV infections and reducing the incidence and severity of IBV infections.

This live, heat-attenuated isolate of IBV strain DMV1639 (also referred to herein as heat-attenuated DMV/1639, attenuated DMV/1639, PDRC DMV/1639, heat-attenuated PDRC DMV1639, and attenuated DMV/1639 Georgia isolate) was deposited with the Patent Depository of the American Type Culture Collection (ATCC®), 10801 University Boulevard, Manassas, VA 20110-2209, USA, on May 15, 2020, under Patent Application Number PTA-126757. This deposit is in accordance with the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Application.

Also included in the present invention are isolated progeny and isolated derivatives of the live heat-attenuated isolate of IBV strain DMV1639, deposited with the ATCC® under Patent Application Number PTA-126757 on May 15, 2020, with equivalent or similar biological, serological, and/or genetic characteristics. As used herein, serological, biological, and genetic characteristics may include one or more of the characteristics described in the data provided in the examples included herein. More particularly, progeny strains or derivatives of strain PTA-126757 may retain the particularly beneficial protective properties related to the present invention, as described in more detail in the examples included herein.

The IBV virus isolate of the present invention can be propagated by conventional methods, including but not limited to any of the methods described in the Examples section included herein. Briefly, a substrate capable of supporting replication of the IBV virus isolate is inoculated with the IBV virus isolate of the present invention, which is propagated until the virus replicates to the desired infectious titer or mass antigen content. The virus-containing material is then collected. Suitable substrates can include embryonated eggs, primary cell cultures (avian), such as, for example, chicken embryonic liver cells, chicken embryonic fibroblasts or chicken kidney cells, mammalian cell lines, such as, for example, the VERO cell line or the BGM-70 cell line, or avian cell lines, such as, for example, QT-35, QM-7 or LMH.

In preferred embodiments, the virus can be propagated on embryonated eggs, including but not limited to embryonated chicken eggs. For example, embryonated chicken eggs between 9 and 11 days of age can be inoculated via the chorioallantoic sac (CAS) route (Dufour-Zavala, "A laboratory manual for the isolation, identification and characterization of avian pathogens," 5th ed. American Association of Avian Pathologists, Jacksonville, Fla. 2008). The inoculated eggs can be incubated at 37°C for 48 hours, after which the chorioallantoic fluid is collected.

The compositions and vaccines of the present invention may have a titer of from about 10 ^1.5 to about 10 ¹⁰ EID ₅₀ (embryo infective dose)/mL. In some aspects, the composition or vaccine of the present invention may have a titer of about 10 ^1.5 EID ₅₀ /mL, about 10 ² EID ₅₀ /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 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 _EID50 /mL, about ¹⁰⁷ _EID50 /mL, about ^107.5 EID50/mL, about 108 _EID50 /mL, about ^108.5 _EID50 /mL, about ¹⁰⁹ _EID50 /mL, about ^109.5 _EID50 /mL, about ¹⁰¹⁰ _EID50 / _mL , or any range ^thereof . For example, in some embodiments, a composition or vaccine of the present invention may have a titer of from about ¹⁰² _EID50 /mL to about ¹⁰⁸ _EID50 /mL. Titers can be measured, for example, in allantoic fluid.

Viruses can be titrated, for example, using the following protocol: 10-fold serial dilutions of virus are made in sterile deionized water and each dilution is inoculated into five 10-day-old embryonated SPF chicken eggs (0.1 ml/egg). The inoculated eggs are incubated at 37°C for 7 days and the embryos are examined for IBV-specific lesions. Embryo mortality within 24 hours of inoculation is considered nonspecific and is not included in the calculation of virus titer. Virus titers are calculated according to the method of Reed and Muench (1938, American Journal of Hygiene 27:493-497) and are expressed as the dose that infects 50% of the embryos (EID ₅₀ ).

The present invention provides compositions and vaccines comprising the isolated virus as described herein. In some embodiments, the vaccine preparations of the present invention may be prepared and tested in accordance with 9 CFR 113.327 for testing IBV vaccines.

In some embodiments, the virus is live. In some embodiments, the virus is inactivated or killed. The viruses, compositions, and vaccines based on them of the present invention can be stored prior to use in any of a variety of forms. For example, such materials can be lyophilized or freeze-dried and can be rehydrated for use. In some embodiments, the virus or composition or vaccine based on it can be frozen.

In some embodiments, the virus, composition or vaccine based on it can be formulated as an effervescent tablet. Such effervescent tablets can be, for example, packaged in lightweight aluminum blisters. The tablet can be dissolved in water and administered, for example, orally, nasally or by means of a spray aerosol, whereby the droplets penetrate the body through the mucous membranes of the birds.

The compositions and vaccines of the present invention may comprise, for example, water or a 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 (such as sorbitol, mannitol, starch, sucrose, dextrin or glucose) or proteins (such as albumin or casein). The stabilizer is particularly preferred if the dry vaccine preparation is obtained by lyophilization. Suitable preservatives include, for example, thimerosal, merthiolate and gentamicin. Diluents include, but are not limited to, water, an aqueous buffer (such as buffered saline), alcohols and polyols (such as glycerol).

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

The composition or vaccine of the present invention may further comprise one or more immunogens derived from other pathogens that cause infection in poultry. Such immunogens may be derived, for example, from Marek's disease virus (MDV), other serotypes of infectious bronchitis virus (IBV), including but not limited to any of those described herein, Newcastle disease virus (NDV), egg drop syndrome virus (EDS), turkey rhinotracheitis virus (TRTV), poxvirus, reovirus, chicken parvovirus, and avian nephritis virus (including but not limited to ANV-1 and ANV-2).

The compositions and vaccines of the present invention may be substantially pure. As used herein, "substantially pure" shall mean a material that is substantially free of macromolecules or other biological particles that are typically found with it in nature.

Vaccination against IBV is routine for most commercial chickens. Vaccines may be modified live virus vaccines delivered via mass aerosol applications. Serotypes used for vaccination are often selected based on the serotypes to which the birds are likely to be exposed in the field. There is very little cross-protection between the different IBV serotypes. Accordingly, 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 immunological materials of low virulence, immunological materials characterized by a lack of increase in virulence upon reverse passage, and/or immunological materials that prevent infection with virulent wild-type IBV strains.

The composition or vaccine of the present invention can be administered by any suitable known method of inoculating agricultural poultry, including nasally, through the eyes, by injection, with drinking water, with feed, by contact, in ovo , from the mother's body, by inhalation through the respiratory tract, etc. The immunogenic composition or vaccine can be administered by mass administration techniques, such as by placing the vaccine in drinking water or by spraying or nebulizing it as an aerosol. When administered by injection, the immunogenic composition or vaccine can be administered parenterally. Parenteral administration includes, for example, administration by intravenous, subcutaneous, intramuscular or intraperitoneal injection.

The compositions and vaccines of the present invention can be administered to any of a variety of avian species susceptible to infection with IBV, including, but not limited to, farm poultry, birds of the order Galliformes , and exotic avian species. Birds of the order Galliformes include, but are not limited to, chickens, turkeys, grouse, quail, and pheasants. As used herein, farm poultry includes domesticated birds that are kept for the purpose of harvesting their eggs or slaughtering them for meat and/or feathers. Most typical are members of the superorder Galloanserae (poultry), particularly the order Galliformes (which includes, for example, chickens, quail, turkeys, and grouse), and the family Anatidae (in the order Anseriformes ), commonly known as "waterfowl" (including, for example, ducks, geese, and swans). Poultry may also include other birds slaughtered for their meat, such as pigeons or turtle doves, or birds considered game, such as pheasants. Chickens include, but are not limited to, female birds, male birds, broilers, roaster chickens, layers, breeders, offspring of breeders and layers. As used herein, the term "susceptible to" means the potential for or the fact of a deleterious reaction to the referenced microorganism compared to non-susceptible individuals or groups and/or one or more pathological conditions indicative of infection with avian IBV.

The vaccine of the present invention can be administered to poultry before or after hatching. The poultry can receive the vaccine at various ages. For example, broilers can be vaccinated in ovo at one day of age or at 2-3 weeks of age. Layers or breeding stock can be vaccinated, for example, at about 6-12 weeks of age and revaccinated at about 16-20 weeks of age. Such layers or breeding stock can be vaccinated at about 6, about 7, about 8, about 9, about 10, about 11, or about 12 weeks of age. Furthermore, in some embodiments, such layers or breeding stock can be vaccinated at an age that corresponds to about the first two weeks. Such layers or breeding stock can be revaccinated at about 16, about 17, about 18, about 19, or about 20 weeks of age. The offspring of such a laying flock, or breeding flock, may exhibit a titer of antibodies to the polypeptide described herein, which may prevent or mitigate the symptoms of IBV infection in the offspring. In ovo vaccination may be performed, for example, at 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 of these values.

Chickens can be vaccinated at any suitable age, and are usually approximately one to three days old before the first vaccination. Chickens can be vaccinated only once. Alternatively, if two doses of vaccine are used, the first is given, for example, when the chickens are three days to a week old, and the second after an additional one to 10 weeks.

Several doses of the composition can be administered throughout the life of the hen. Since maternal immunity is the main source of protection for broiler offspring, breeding hens are usually vaccinated, although broiler chickens can be vaccinated if desired.

In some embodiments, the live attenuated IBV isolate of the present invention may be administered at a dose of from about 10 ^1.5 to about 10 ¹⁰ EID ₅₀ per bird. In some aspects, the live attenuated IBV isolate of the present invention can be administered at a dose of about 10 ^1.5 EID ₅₀ per bird, about 10 ² EID ₅₀ per bird, about 10 ^2.5 EID ₅₀ per bird, about 10 ³ EID ₅₀ per bird, about 10 ^3.5 EID ₅₀ per bird, about 10 ⁴ EID ₅₀ per bird, about 10 ^4.5 EID ₅₀ per bird, about 10 ⁵ EID ₅₀ per bird, about 10 ^5.5 EID ₅₀ per bird, about 10 ⁶ EID ₅₀ per bird, about 10 ^6.5 EID ₅₀ per bird, about 10 ⁷ EID ₅₀ per bird, about 10 ^7.5 EID ₅₀ per bird, about 10 ⁸ EID ₅₀ per bird, about 10 ^8.5 EID ₅₀ per bird, about 10 ⁹ EID ₅₀ per bird, about 10 ^9.5 EID ₅₀ per bird, about 10 ¹⁰ EID ₅₀ per bird, or any range thereof. For example, in some embodiments, a dose of about 10 ² to about 10 ⁵ EID ₅₀ per bird can be administered.

A virus, composition, or vaccine as described herein can be administered to poultry or other animals to induce an immune response to the IBV virus and/or the IBV S1 polypeptide, including but not limited to the IBV serotype DMV 1639. The immune response can, for example, include one or more of a cell-mediated immune response, which includes the production of lymphocytes in response to exposure to an antigen, and/or a humoral immune response, which includes the production of plasma lymphocytes (B cells) in response to exposure to an antigen, followed by the production of antibodies. A humoral immune response can include an IgG, IgM, IgA, IgD, and/or IgE response. When determining a humoral or cellular immune response, the determination can be made by any of a variety of methods, including but not limited to any of those described herein. The immune response may or may not provide protective immunity. Such an immune response may result in a reduction in the severity or amelioration of symptoms of IBV infection in the future, such as symptoms of infection with IBV serotype DMV/1639. Such an immune response may result in the prevention of future IBV infection in poultry, such as the prevention of infection with IBV serotype DMV/1639. Immunity may include the induction of an increased level of protection in a population of birds following vaccination compared to an unvaccinated group.

The present invention includes a method for producing an immune response against IBV in poultry, the method comprising administering an isolated virus, composition or vaccine as described herein. In some aspects, the immunity comprises humoral and/or cellular immunity. In some aspects, the immunity comprises mucosal immunity.

Administration of an isolated virus, composition, or vaccine as described herein may result in a reduction, inhibition, or prevention of one or more manifestations of an IBV infection, including one or more manifestations of the disease infectious bronchitis (IB). Such symptoms may include one or more of the following: suppression of body weight gain, decreased egg production, mortality, clinical signs (such as, for example, lacrimation, wheezing, wheezing, sinus exudate, conjunctivitis, and/or pulmonary rales during breathing), histopathological signs (such as, for example, tracheal lesions), serum IBV antibody titer (determined, for example, by ELISA), and/or IBV virus shedding (measured, for example, by real-time RT-PCT of tracheal swab samples).

The present invention provides a method for reducing, inhibiting or preventing IBV infection in poultry, the method comprising administering an isolated virus, composition or vaccine as described herein. In some aspects, administering an isolated virus, composition or vaccine as described herein reduces, inhibits or prevents infection with the IBV variant strain DMV/1639. In some aspects, administering an isolated virus, composition or vaccine as described herein results in cross-protection, reduction, inhibition or prevention of one or more disease manifestations upon infection with an IBV strain other than the IBV strain DMV/1639.

In some aspects of the methods of the present invention, the administration comprises injection, spraying, oral administration, or inhalation administration. In some aspects of the methods of the present invention, the administration induces mucosal immunity. In some aspects of the methods of the present invention, the administration comprises in ovo administration. In some aspects, in ovo administration comprises administration at 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 of these values.

The compositions of the present invention may be substantially pure. As used herein, "substantially pure" shall mean a material that is substantially free of any similar macromolecules or other biological particles that are normally found with it in nature. In some embodiments, the organisms used in such formulations are live. In some embodiments, the organisms, compositions, or vaccines may be lyophilized. The present invention provides isolated viruses. As used herein, "isolated" refers to a material that has been removed from its original environment (e.g., the natural environment, if naturally occurring) and thus altered by the "hand of man" from its natural state.

The viruses, compositions and vaccines of the present invention can be administered to birds of any of a variety of bird species susceptible to infection with IBV, including but not limited to farm birds, Galliformes and exotic bird species. Galliformes include but are not limited to chickens, turkeys, grouse, quail and pheasants. As used herein, farm birds include domesticated birds that are kept for the purpose of harvesting their eggs or slaughtering them for meat and/or feathers. The most typical are members of the superorder Galloanserae (fowl), particularly the order Galliformes (which includes, for example, chickens, quail, turkeys, and grouse), and the family Anatidae (in the order Anseriformes ), commonly known as "waterfowl" (which includes, for example, ducks, geese, and swans). Farm poultry may also include other birds that are slaughtered for their meat, such as pigeons or turtle doves, or birds that are regarded as game, such as pheasants.

The term "farm poultry" is intended to encompass any breed of chicken, pheasant, emu, ostrich, and other types of birds susceptible to infection with IBV. Chickens include, but are not limited to, female birds, male birds, broilers, roaster chickens, layers, breeders, and the offspring of breeders and layers. In some embodiments, the compositions of the present invention and the methods of the present invention are also applicable to animals other than poultry that are susceptible to infection with IBV. As used herein, the term "susceptible to" means the potential for or fact of a detrimental response to the microorganism in question, such as, for example, decreased vitality or lack of weight gain compared to non-susceptible individuals or groups, and/or one or more pathological conditions indicative of infection with IBV, including, but not limited to, any of those described herein.

The compositions and vaccines of the present invention can be formulated for delivery by any of a variety of routes known in the veterinary art, such as, for example, transmucosal, intranasal, intraocular or oral administration. The compositions and vaccines of the present invention can be formulated for delivery to the mucous membrane of the respiratory tract and can be administered in such a way that they immediately or over time come into contact with the mucous membranes of the respiratory tract of the bird. The compositions and vaccines of the present invention can be formulated for delivery by any of a variety of methods known in the veterinary art, such as, for example, spraying or nebulizing as an aerosol.

The immunogenic composition or vaccine of the present invention can be administered by any suitable known method of inoculating birds, including but not limited to nasally, through the eye, by eye drops, by injection, in drinking water, in feed, by contact, in ovo , maternally, etc.

The immunogenic composition or vaccine can be administered by mass administration techniques, such as by placing the vaccine in drinking water or by spraying the animal's environment. The composition can be administered by spraying an individual or herd with a solution, such aerosol delivery can involve administering the composition incorporated into fine liquid particles. Such aerosol particles can have a droplet size in the range of about 10 to about 100 microns, more preferably a droplet size in the range of about <1 to about 50 microns. Conventional nebulizers and aerosol generators, such as commercially available knapsack, incubator spray, and atomic spray aerosol generators, can be used to produce fine particles. Administration via drinking water can be accomplished using a conventional device. When administered by injection, the immunogenic composition or vaccine can be administered parenterally. Parenteral administration includes, for example, administration by intravenous, subcutaneous, intramuscular or intraperitoneal injection.

The composition or vaccine of the present invention can be administered to birds before or after hatching. Birds can receive such a vaccine composition at any age. When delivered after hatching, the materials can 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 range thereof. For inovo administration, the materials can be delivered after about seventeen days of incubation, after about eighteen days of incubation, after about nineteen days of incubation, after about twenty days of incubation, and any range thereof.

The viruses of the present invention can be used in any of the commonly used methods for detecting IBV, such as, for example, hemagglutination (HA) (Lashgarian 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 (Kwone et al., 1993, Avian Dis ; 37:194–202), and real-time RT-PCR (Calisone et al., 2006, J Virological Methods ; 138:60–65).

Illustrative embodiments of the present invention include, but are not limited to, the following.

1. An isolate of infectious bronchitis virus (IBV), wherein the IBV isolate comprises the heat-attenuated IBV isolate PDRC DMV/1639 deposited with the ATCC under Patent Deposit Accession Number PTA-126757, or a progeny or derivative thereof, wherein the progeny or derivative thereof is characterized by substantially the same biological and serological characteristics as the heat-attenuated IBV isolate PDRC DMV/1639 deposited with the ATCC under Patent Deposit Accession Number PTA-126757.

2. The IBV isolate of embodiment 1, wherein the IBV isolate is lyophilized, freeze-dried, or frozen.

3. A composition comprising an IBV isolate according to embodiments 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 progeny or derivative thereof according to embodiments 1 or 2, or a composition according to embodiments 3 or 4.

6. The vaccine of embodiment 5, wherein the vaccine provides a reduction in the severity of one or more clinical signs and/or viral load induced by IBV infection in poultry.

7. A vaccine for birds of the order Galliformes , comprising an amount of the heat-attenuated IBV isolate PDRC DMV/1639, deposited with the ATCC under Patent Deposit Registration Number PTA-126757, or a progeny or derivative thereof, sufficient to protect birds from one or more clinical signs induced by infection with infectious bronchitis virus (IBV) in poultry.

8. The composition or vaccine according to any one of embodiments 3-7, further comprising an adjuvant.

9. The composition or vaccine according to any one of embodiments 3-8, further comprising other viral material.

10. The composition or vaccine of any one of embodiments 3-9, wherein the composition or formulation is formulated for intranasal, intraocular, oral, transmucosal, intramuscular, subcutaneous, or in ovo administration.

11. The composition or vaccine of any one of embodiments 3-10, wherein the composition or vaccine is formulated for spraying or nebulizing as an aerosol.

12. An effervescent tablet comprising an IBV isolate or progeny or derivative thereof, a composition or a vaccine according to any one of embodiments 1-11.

13. A method for producing an immune response to infectious bronchitis virus (IBV) in poultry, the method comprising administering to the poultry an IBV isolate or progeny or derivative thereof, a composition, vaccine or effervescent tablet according to any one of embodiments 1-12.

14. A method for producing antibodies to IBV in poultry, the method comprising administering to the poultry an IBV isolate or its progeny or derivative, a composition, a vaccine or an effervescent tablet according to any one of embodiments 1-12.

15. A method for reducing the severity of one or more clinical signs and/or viral load induced by an infectious bronchitis virus (IBV) infection in poultry, the method comprising administering to the poultry an effective amount of an IBV isolate or progeny or derivative thereof, a composition, a vaccine or an effervescent tablet according to any one of embodiments 1-12.

16. A method for reducing the susceptibility of a Galliformes bird to infection by infectious bronchitis virus (IBV), the method comprising administering to the bird an effective amount of an IBV isolate or progeny or derivative thereof, a composition, vaccine or effervescent tablet according to any one of embodiments 1-11.

17. A method for protecting a Galliformes bird from an infection caused by an infectious bronchitis virus (IBV), wherein the method comprises administering to the bird an effective amount of an IBV isolate or its progeny or derivative, a composition, a vaccine or an effervescent tablet according to any one of embodiments 1-11.

18. The method according to any one of embodiments 13-17, wherein the administration is intranasal, intraocular, oral, transmucosal, intramuscular, or subcutaneous.

19. The method according to any one of embodiments 13-18, wherein the administration comprises administration in ovo .

20. The method according to any one of embodiments 13-19, wherein the IBV isolate or progeny or derivative thereof, composition or vaccine is administered via an aerosol.

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

22. The method according to any one of embodiments 13-19, wherein the IBV isolate, or progeny, or derivative, composition, or vaccine is administered via drinking water.

23. The method according to any one of embodiments 13-22, wherein the administration comprises administration to a breeding female bird.

24. The method according to any of embodiments 13-23, wherein the agricultural bird comprises a bird of the order Galliformes .

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

The term "and/or" means one or all of the listed elements or a combination of any two or more of the listed elements.

The words "preferred" and "preferably" refer to embodiments of the present invention that may provide certain advantages in certain circumstances. However, other embodiments may also be preferred in the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not applicable, nor does it imply the exclusion of other embodiments from the scope of the present invention.

The terms "comprises" and its variants do not have a limiting meaning in cases where these terms appear in the description of the invention and the claims.

Unless otherwise stated, singular forms of nouns and the phrase "at least one" are used interchangeably to mean one or more than one.

Also, listing numeric ranges by endpoints includes all numbers that fall within that range (e.g. 1 through 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

For any method disclosed herein that includes separate steps, these steps may be performed in any convenient order. And, if desired, two or more steps in any combination may be performed simultaneously.

Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, etc., used in the specification and claims are to be understood in all instances as being modified by the term "about." Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending on the desired properties sought to be achieved by the present invention. At the very least, and not as an attempt to limit fundamental principles of equivalents to the scope of the claims, each numerical parameter should at least be interpreted in light of the number of significant digits disclosed and by applying standard rounding techniques.

Although the numerical ranges and parameters representing the broad scope of the present invention are approximate values, the numerical values set forth in the specific examples are presented as precisely as possible. All numerical values, however, inherently include a range inevitably determined by the standard deviation present in the test measurements corresponding to them.

In several places in this application, guidance is provided in the form of a list of examples, which examples can be used in various combinations. In each case, the set forth list serves only as a representative group and should not be construed as an exclusive list. It should be understood that the specific examples, materials, quantities and procedures are to be construed broadly in accordance with the scope and spirit of the invention as set forth herein.

All headings throughout the document are included for the convenience of the reader and should not be used to limit the meaning of text that follows the heading unless specified.

The present invention is illustrated by the following examples. It should be understood that the specific examples, materials, quantities and procedures are to be construed in a broad sense consistent with the scope and spirit of the present invention as set forth herein.

EXAMPLES

Example 1

Live heat-attenuated IBV strain DMV1639 for use as a vaccine for agricultural poultry

Heat treatment was used to attenuate the avian infectious bronchitis virus (IMBV) strain DMV/1639, a coronavirus. Briefly, the original sample was collected in 2019 from a Georgia broiler farm showing signs of respiratory disease. The virus was isolated and then passaged 3 times in embryonated eggs. This sample (total passage 4) was then heat shock attenuated by 14 heat shock passages following the previously published attenuation method of Jackwood et al. (Jackwood, et al., 2010, Avian Pathology ; 39:227-233). The virus recovered from heat shock passage 14 (hsp14) was then passaged once in embryonated eggs to propagate the virus, resulting in a total of 19 passages, 5 normal passages and 14 heat shock passages.

As described in the following examples, the treated virus was propagated in embryonated eggs and tested for safety and efficacy in poultry. Using the 9 CFR criteria for testing an IBV vaccine, the attenuated virus passed both the safety and efficacy tests. This strain, DMV/1639, is a recently isolated virus from Georgia and is an evolved version of the virus that causes upper respiratory disease in poultry. It is currently a significant economic problem in the poultry industry and continues to spread. There is currently no commercial vaccine available for this strain of IBV.

This live 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, heat-attenuated PDRC DMV/1639, and attenuated DMV/1639 Georgia isolate) was deposited with the Patent Depository of the American Type Culture Collection (ATCC®), 10801 University Boulevard, Manassas, VA 20110, USA, on May 15, 2020, under Patent Application Number PTA-126757.

Example 2

Comparison of heat-attenuated PDRCs DMV1639, DE1639, Ma5 and iBron GA08 for homologous and heterologous protective capacity

The concept of cross-protection for IBV vaccines has been extensively studied by combining several types of IBV vaccines, typically Ma5 and 4/91. Recently, some vaccine manufacturers have suggested that a single IBV vaccine may provide cross-protection against heterologous serotypes upon challenge without the need for additional IBV vaccines. In addition, it appears that some vaccines in use may not provide complete protection against homologous viruses currently circulating in the field. To investigate these issues, four different vaccines were tested against pathogenic DMV/1639 challenge to assess protection.

Materials and methods

Viruses: The vaccines used in this study were Mass type (BoehringerIngelheim), iBron GA08 type (Ceva), DE1639-based vaccine (also referred to herein as autogenous DMV/1639 vaccine and MA DMV/1639) and attenuated (heat-treated) PDRC DMV/1639 vaccine. The challenge virus used was a newly discovered isolate of DMV/1639/11.

Experimental design. The experimental design is shown in Table 1. Day-old maternal antibody-positive broiler chickens were used for this experiment. One hundred chickens from each group were vaccinated by spraying with the full dose of each vaccine at day-old and housed in colonial houses depending on the group. On day 7, all chickens in each group were swabbed and the vaccine virus was detected using real-time RT-PCR. At 28 days of age, 10 birds for each challenge virus and 5 birds for each control group were placed in isolators and challenged with 1× ¹⁰⁴ EID ₅₀ DMV/1639/11. The titer of the challenge virus was confirmed by back titration on embryonated eggs. Necropsy was performed 5 days after challenge. The DE1639-based vaccine group was housed in a colonial house and swabbed until 42 days of age to assess the shedding of the vaccine virus as a field virus.

Infection Vaccinated Infected with DMV/1639/11 Without processing Vaccine DE1639 100 10 5 DE1639 + Mass 100 10 5 iBron 100 10 5 DE1639 + iBron 100 10 5 Mass + iBron 100 10 5 PDRC DMV/1639 100 10 5 Without processing 5 5 5

Table 1. Experimental scheme.

Necropsy/clinical signs. Clinical signs were recorded and scored based on published laboratory scoring methods (Jackwood et al., 2015, Avian Diseases ; 59(3):368-374. https://doi.org/10.1637/11026-012415-Reg.1), where 0 = none, 1 = mild wheezing or wheezing, 2 = more severe wheezing, sinus exudate, conjunctivitis, and 3 = wheezing sounds in the lungs during breathing.

Necropsy/RNA extraction and detection of infectious virus were performed by real-time RT-PCR. At necropsy, intrachoanal swabs from the oropharynx were collected at the palatal cleft and placed in 1 ml of ice-cold PBS for virus detection by real-time RT-PCR. Viral RNA was extracted from 50 μl of PBS using the MagMAX-96 RNA Isolation Kit (AmbionInc., Austin, TX) according to the manufacturer's protocol on a KingFisher Magnetic Particle Processor (ThermoScientific, Waltham, MA). Real-time RT-PCR was performed using the 7500 FastReal-Time PCR System from AppliedBiosystems (LifeTechnologies, Carlsbad, CA) and the AgPath-IDTM One-Step RT-PCR Kit (AmbionInc.) according to the manufacturer's recommendations. The primers and probe for real-time RT-PCR were matched to the specific serotype to be tested. Primers were obtained from Integrated DNA Technologies (Coralville, IA) and the Taqman probe was synthesized in collaboration with BioSearchTechnologies (Novato, CA).

Results

For this study, 100 chickens were spray vaccinated with the vaccines listed in Table 1. All 100 chickens in each group were then placed in colonial-type houses on fresh litter to simulate field environmental conditions. At 7 days post-vaccination (dpv), choanal swabs were taken from all chickens in each group to measure the amount of vaccine virus present, and the data can be seen in Fig. 1A and 1B. As expected from previous sampling to determine vaccine virus infection rates, the DE1639-based vaccine only infected ~60% of chickens when vaccinated, and the resulting viral load was relatively low (mean Ct value of ~33). This contrasts with the PDRC DMV/1639-based vaccine developed at the PDRC, which was nearly 100% infectious, with a mean Ct value of ~26.5. When the DE1639-based vaccine was combined with other vaccines, the infection rate was actually reduced compared to either vaccine alone. When combined with the Mass vaccine virus, the DE1639 vaccine virus only infected ~35% of chickens, while the viral load was similar (Ct value of ~32).

The Mass vaccine virus achieved 100% infection with a mean Ct of ~25, demonstrating that a vaccine application issue was not the cause of DE1639 vaccine virus vaccination failure in this cohort. The same trend was seen with the combination of the DE1639 vaccine with Ceva’s iBron, where only ~10% of the chicks were positive with a Ct of ~33 for autogenous DMV/1639. Chicks were ~95% positive for iBron with a mean Ct of ~26, further demonstrating that an application issue was not present. When iBron was assessed alone, nearly 100% of the chicks were positive with a mean Ct of ~25, demonstrating adequate infection and replication. Interestingly, for the iBron-based vaccine mixed with Mass, the chicks were still 100% positive for iBron with a mean Ct of ~26, but only 60% of the chicks were positive for Mass with a mean Ct of ~27.5. However, there was a "tail" of positive values indicating that not all birds were being uniformly vaccinated with Mass. It appears that some vaccine interference may occur when combining iBron (a strong vaccine) with DE1639 or Mass from BI (weaker vaccines).

Because infection rates at day 7 were not high for the DE1639-vaccinated groups, it was decided to re-swab the DE1639-vaccinated groups at day 14 to assess viral load and potential shedding of the vaccine virus as a field virus. These data can be seen in Fig. 2. Both the positivity rate (~85%) and viral load (median Ct value was 25.5) were increased in the DE1639-only vaccinated group at day 14 compared to day 7, indicating that peak vaccine virus replication occurs much later for this vaccine than for standard IBV vaccines. The percentage of DE1639-based vaccine positivity in the group vaccinated with this vaccine and the Mass-based vaccine also increased (from ~35% positivity at day 7 to ~60% positivity at day 14), but the mean viral load (Ct was ~32) did not change. This may indicate that the interaction of the Mass-based vaccine and the DE1639-based vaccine is still suppressing the vaccine and allowing for a field-type vaccine virus spillover reaction where portions of the population become positive at different times. In the group vaccinated with DE1639 and iBron, only one sample was positive, indicating that the iBron-based vaccine essentially blocks the effect of the DE1639-based vaccine. The group receiving the DE1639-based vaccine alone was also swabbed at 28 dpv to determine the amount of vaccine virus still present. The results are shown in Fig. 3A and 3B.~25% of birds were still positive, with viral load values ranging from 36 to 22 Ct values (average Ct value was ~28). This indicates that the vaccine virus continues to spread in the flock as a field virus.

Another notable observation was that significant clinical respiratory signs were observed at 7 and 14 days post-vaccination in groups receiving the iBron-based vaccine alone or in combination with another vaccine. Wheezing was observed in 1/4-1/3 of the chickens when swabbed to measure vaccine virus infection levels. No clinical respiratory signs were observed in any of the groups receiving the other vaccines.

Chickens in each vaccine group were challenged 28 days after vaccination with DMV/1639 type IB pathogenic virus at a dose of ~1 x 10 ⁴ EID ₅₀ per bird. 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 sign scores than the unvaccinated DMV/1639 challenged group except the Mass/iBron vaccinated and challenged groups, although all groups had lower scores. The results are shown in Fig. 4. This is not surprising given previous results of vaccine virus challenge studies using DMV/1639 type IBV challenge viruses and multiple IBV vaccines.

The viral load values determined by DMV/1639 type-specific PCR showed that all groups had statistically significantly lower viral load values than the unvaccinated challenged group. The results are shown in Fig. 5. Overall, the mean Ct value for the unvaccinated challenged group was ~23, and all 5 samples were very close to this value. The DE1639-based vaccine-vaccinated group had 8/10 samples positive for the virus after challenge, but the samples were divided into two distinct groups. One group was clustered around the Ct value of 29, and the other group was clustered around the Ct value of 34.

Since this assay cannot differentiate between vaccine virus and challenge virus, it is impossible to say that all positive samples are due to vaccination or challenge, or that none are. However, based on the day 28 samples from this group and the experience with these studies, it is likely that half of these positives are due to challenge virus. The PDRC DMV/1639 vaccinated group had only one positive sample after challenge, with a Ct value of ~33, indicating low virus presence. This test shows that the PDRC DMV/1639 vaccine is completely protective against the challenge virus based on 9-CFR standards (90% or more of the samples were negative for the virus). The group vaccinated with the DE1639 and Mass vaccines had only 2 positive samples after challenge, with a mean Ct of ~32. This is interesting given that the DE1639 vaccine in combination with the Mass vaccine appeared to be somewhat suppressed. However, it also cannot be ruled out that by randomly selecting chickens for challenge, the present inventors inadvertently selected chickens that had received the DMV/1639 vaccine and thus developed some immunity. The group that was vaccinated with the DE1639 and iBron-based vaccine had 8/10 positive samples, with a mean Ct value of ~31. Ct values in this group ranged from ~26 to 38, indicating that only partial protection against challenge was achieved.

It appears that suppression of the DE1639-based vaccine by the iBron-based vaccine did impact the production of specific neutralizing antibodies against DMV/1639 challenge. For the Mass and iBron-based vaccine and iBron-only vaccine groups, 9/10 and 8/10 samples were positive post-challenge, with mean Ct values of ~30 and 32, respectively. These two groups were very similar in mean Ct value and total number of positives post-challenge to the DE1639 and iBron-based vaccine group, indicating that specific neutralizing antibodies were not properly produced in either group.

Discussion

Based on field observations, the experiment worked out as expected. The DE1639 vaccine virus did not establish full infection during the initial vaccination and then persisted in the birds until at least the time point corresponding to the challenge. In addition, the virus was detected at a higher frequency post-challenge than the vaccine virus detected pre-challenge, indicating that at least some of the virus detected post-challenge was the intended-to-challenge virus. This means that either the DE1639 vaccine virus was not a full antigen match to the intended-to-challenge virus or birds that were not vaccinated due to low infection levels were not protected from challenge. In either case, the vaccine was not effective in stopping the intended-to-challenge virus.

The same was true for the groups vaccinated with DE1639 and iBron, Mass and iBron, and iBron alone. In these groups, almost all birds received the iBron-based vaccine, and subgroups of birds received the other vaccine in combination. This indicates that the iBron-based vaccine suppressed the effect of the other vaccines to some extent, and that the iBron-based vaccine alone did not provide complete protection.

The only group that was fully protected (90% or more negative) was the group vaccinated with the PDRC DMV/1639-based vaccine. This was developed from a newly discovered isolate in 2019 and appears to be an adequate antigen match to the field virus. In addition, this vaccine virus was administered at a lower titer than the other vaccine viruses (~1×10 ^3.5 EID ₅₀ ), but still had near-perfect infection and replication dynamics.

The DE1639 vaccine does not provide high protection against challenge and the vaccine persists in the herd, creating a field-type vaccine virus spillover scenario and obscuring the diagnostic picture. The Mass and iBron combination was as protective as the DE1639 vaccine when assessing post-challenge virus detection rates, and other experiments with this combination showed similar results. This vaccine combination may provide a reduction in clinical signs and potentially reduce the effects of challenge on productivity and culling, but it does not completely stop infection and replication of the DMV/1639 challenge virus.

Example 3

Comparison of the currently used DE1639-based vaccine and the DMV/1639-based vaccine in terms of homologous protective capacity

The DMV/1639 IB virus has been continuously circulating in poultry flocks since at least 2014, despite the use of a live attenuated autogenous vaccine in some production companies. Apparently, the current vaccine may not provide complete protection against homologous viruses currently circulating in the field. For this reason, the new heat-attenuated DMV/1639 vaccine of the present invention was developed to improve protection against currently circulating field viruses of this variant serotype.

Materials and methods

Viruses. The University of Delaware heat-attenuated live DMV/1639 virus vaccine (DE1639) and the PDRC attenuated DMV/1639 vaccine as described herein (deposited with the ATCC under patent deposition number PTA-126757) were used in this study. The challenge virus was a newly discovered isolate of DMV/1639/11 from a broiler flock on the Delmarva Peninsula.

Experimental design. The experimental design is presented in Table 2 below. One day old MDA positive broiler chickens were used for this experiment. One hundred chickens from each group were vaccinated by spraying with the full dose of each vaccine at 1 day of age and housed in colonial houses depending on the group. On day 7, all chickens in each group were swabbed and the vaccine virus was detected using real-time PCR. At 28 days of age, 10 birds for each challenge virus and 5 birds for each control group were placed in isolators and challenged with 1×10 ⁴ EID ₅₀ DMV/1639/11. The challenge virus titer was confirmed by back titration on embryonated eggs. Necropsies were performed 5 days after challenge.

Infection Vaccinated Infected with DMV/1639/11 Without processing Vaccine DE1639 100 10 5 PDRC DMV/1639 100 10 5 Without processing 5 5 5

Table 2. Experimental scheme.

Necropsy/clinical signs. Clinical signs were recorded and scored based on published laboratory scoring methods (Jackwood et al., 2015, Avian Diseases ; 59(3):368-374. https://doi.org/10.1637/11026-012415-Reg.1), where 0 = none, 1 = mild wheezing or wheezing, 2 = more severe wheezing, sinus exudate, conjunctivitis, and 3 = wheezing sounds in the lungs during breathing.

Necropsy/RNA extraction and detection of infectious virus were performed by real-time RT-PCR. At necropsy, intrachoanal swabs from the oropharynx were collected at the palatal cleft and placed in 1 ml ice-cold PBS for virus detection by real-time RT-PCR. Viral RNA was extracted from 50 μl PBS using the MagMAX-96 RNA Isolation Kit (AmbionInc., Austin, TX) according to the manufacturer's protocol on a KingFisher Magnetic Particle Processor (Thermo Scientific, Waltham, MA). Real-time RT-PCR was performed using the 7500 Fast Real-Time PCR System from Applied Biosystems (Life Technologies, Carlsbad, CA) and the AGPATH-ID ^™ One-Step RT-PCR Kit (AmbionInc.) according to the manufacturer's recommendations. Real-time RT-PCR primers and probe were tailored to the specific serotype to be tested. Primers were obtained from Integrated DNA Technologies (Coralville, IA) and the TAQMAN ^® probe was synthesized with assistance from Bio Search Technologies (Novato, CA).

Results

Purity Testing. The PDRC DMV/1639 vaccine was tested for purity by quantitative real-time PCR using primers and probes that detect all known IBV serotypes in the United States. Table 3 below shows that the vaccine tested positive for DMV/1639 only, indicating no contaminants from other IBV variants. The vaccine was also streaked onto a bacterial blood agar plate and no growth was detected after 24 or 48 hours of incubation.

vaccine based on PDRC DMV/1639 Target Ct Target Ct Target Ct Target Ct Generic IBV 18.0197 Arkansas Negative GA08 Negative Mass Negative Target Ct Target Ct Target Ct Target Ct DMV/1639 14.9809 Del072/GA98 Negative GA13 Negative Conn Negative

Table 3. qRT-PCR of PDRC DMV/1639-based vaccine.

Safety study. The virus used in the vaccine experiment was used for the safety study according to USDA 9CFR recommendations. The titer of the vaccine actually administered to the chickens in the efficacy study was 1×10 ^3.4 EID ₅₀ , and at least a 10X dose was required for the safety study. For this purpose, a dose of 3.16×10 ⁵ EID ₅₀ was administered by the oculonasal route individually to 26 SPF chickens on the day of hatching. Four SPF chickens were left unvaccinated as a negative control. The 26 vaccinated chickens were divided into two isolators, one with 15 chickens and the other with 11 chickens. All chickens were monitored daily for 21 days for any significant clinical signs or deaths associated with IBV vaccine-induced infection. Two chickens were euthanized during the study due to hip dysplasia, but they showed no signs of IBV infection and necropsy revealed no lesions typical of IBV. None of the chickens in the study showed any signs of IBV during the 21-day experiment. The results of the daily monitoring can be found in Table 4 below.

Table 4. Safety study.

Efficacy Study. For this study, 100 chickens were spray vaccinated at a time with the vaccines listed in Table 2. All 100 chickens in each group were then placed in colonial-type houses on fresh litter to simulate field environmental conditions. At 7 days post-vaccination (dpv), choanal swabs were taken from all chickens in each group to measure the amount of vaccine virus present. All data can be found in Fig. 6. As expected from previous sampling to determine vaccine virus infection rates, the DE1639-based vaccine only achieved infection in ~60% of chickens upon vaccination, and the resulting viral load was relatively low (mean Ct value of ~33). This is in contrast to the DMV/1639-based vaccine described herein (PDRC DMV/1639), which achieved infection of nearly 100%, with a mean Ct value of ~26.5 (Fig. 6).

Because infection rates on day 7 were not adequate for the DE1639-vaccinated group, this group was swabbed again on day 14 to assess viral load and potential shedding of the vaccine virus as a field virus. These data can be seen in Fig. 7.Both the positivity rate (~85%) and viral load (mean Ct value was 25.5) were increased in the DE1639-vaccinated group at day 14 compared to day 7, indicating that peak vaccine virus replication occurs much later for this vaccine than for standard IBV vaccines. The DE1639-vaccinated group was also swabbed at 28 dpv to determine the amount of vaccine virus still present (Fig. 8). ~25% of the birds were still positive, with viral loads ranging from 36 to 22 Ct values (mean Ct value was ~28). This indicates that the vaccine virus continues to be shed in the flock as a field virus.

Ten chickens were randomly selected from each vaccine group and challenged 28 days after vaccination with DMV/1639 type IB pathogenic virus at a dose of ~1 x 10 ⁴ EID ₅₀ per bird. 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 sign values than the unvaccinated DMV/1639 challenged group (Fig. 9).

Viral load values determined by DMV/1639 type-specific PCR showed that all groups had statistically significantly lower viral load values than the unvaccinated challenge group (Fig. 10). Overall, the mean Ct value for the unvaccinated challenge group was ~23, and all 5 samples were very close to this value. The DE1639-based vaccine group had 8/10 samples positive for the virus after challenge, but the samples fell into two distinct groups. One group clustered around the Ct value of 29, and the other group clustered around the Ct value of 34. Since this assay cannot differentiate between vaccine virus and challenge virus, it is impossible to say that all positive samples are due to vaccination or challenge, or that none are. However, based on the day 28 samples from this group and the experience with these studies, it is likely that half of these positive values are due to challenge virus. The PDRC DMV/1639 vaccinated group had only one positive sample after challenge, with a Ct value of ~33, indicating low levels of virus. This test indicates that the PDRC vaccine is fully protective against this challenge virus based on 9-CFR standards (90% or more samples are negative for virus).

Discussion

The experiment concluded as expected. The DE1639 vaccine virus failed to establish full infection during the initial vaccination and then persisted in the birds until at least the time point corresponding to the challenge. In addition, the virus was detected at a higher frequency post-challenge than the vaccine virus detected pre-challenge, indicating that at least some of the virus detected post-challenge was the intended-to-challenge virus. This means that either the DE1639 vaccine virus was not a full antigen match to the intended-to-challenge virus or that birds that were not vaccinated due to low infection levels were not protected from challenge. In either case, the vaccine was not effective in stopping the intended-to-challenge virus. This is also consistent with the situation in the field, where DMV/1639 is consistently detected in flocks vaccinated with an autogenous DMV-based vaccine. The only group that was fully protected (90% or more negative) was the group vaccinated with the heat-attenuated PDRC DMV/1639 vaccine described herein (deposited in the ATCC® Patent Depository under Patent Application Number PTA-126757). The PDRC DMV/1639 vaccine was developed from a newly discovered isolate in 2019 and appears to be an adequate antigen match to the field virus. In addition, this vaccine virus was administered at a lower titer than the other vaccine viruses (1×100 ^3.4 EID ₅₀ ), but still showed near-perfect infection and replication dynamics.

One possible explanation for the increased efficacy of the newer PDRC vaccine is that it is more antigenically consistent with currently circulating field viruses. The phylogenetic data table shown in Figure 11 compares different isolates of DMV/1639 virus. The bottom rectangle shows isolates collected during the initial outbreak in 2015, with the autogenous vaccine indicated by the arrow. Isolates discovered later in 2019 are shown in the top rectangle, with the PDRC vaccine indicated by the arrow. The sequence of the challenge virus used in this study, isolated from a broiler flock on Delmarva, also falls into the clade shown in the top rectangle. The virus appears to have evolved as it moved across the country but has begun to "settle" as most isolates now cluster together.

Example 4

Reverse passage of heat-attenuated PDRC DMV/1639 vaccine virus to assess attenuation stability

In this example, the stability of the heat-attenuated PDRC DMV/1639 vaccine virus, as described herein, was assessed by back-passaging the vaccine virus into susceptible chickens.

Materials and methods

Virus: The vaccine used in this study was a heat-attenuated PDRC DMV/1639 virus, deposited with the ATCC under patent deposition number PTA-126757.

Experimental design. Specific pathogen free (SPF) 1-day-old chicks were used for this experiment. Five 1-day-old chicks were initially given 50% embryonic infection ( _EID50 ) doses of vaccine of 3.16 x ¹⁰³ intranasally and intraocularly in 0.1 ml. Two or three days after vaccination, a choanal slit was swabbed from each bird, pooled in 3 ml PBS (pH 7.4) and 0.1 ml of this pooled sample was given to each bird in another group of five 1-day-old SPF chicks. This was repeated 10 times. All birds were examined for clinical signs throughout the study and at the last return passage (return passage number 10) five individual birds were necropsied 5 days after vaccination. At this necropsy, intrachoanal swabs were collected and not pooled, and birds were examined for clinical signs and lesions.

Clinical signs. Clinical signs were recorded and scored based on published laboratory scoring methods (Jackwood et al., 2015, Avian Diseases ; 59(3):368–374. https://doi.org/10.1637/11026-012415-Reg.1), where 0 = none, 1 = mild wheezing or wheezing, 2 = more severe wheezing, sinus exudate, conjunctivitis, and 3 = wheezing sounds in the lungs during breathing.

RNA extraction and virus detection were performed by real-time RT-PCR. Intrachoanal swabs from the oropharynx in the palatal cleft were collected and pooled in 3 ml ice-cold PBS for reverse passage groups 1–9 for virus detection by real-time RT-PCR. Intrachoanal swabs from five birds in reverse passage group 10 were retained as individual samples and placed in 1 ml ice-cold PBS for virus detection by real-time RT-PCR.

Viral RNA was extracted from 50 μl of PBS using the MagMAX-96 RNA Isolation Kit (AmbionInc., Austin, TX) according to the manufacturer's protocol on a King Fisher Magnetic Particle Processor (Thermo Scientific, Waltham, MA). Real-time RT-PCR was performed using an Applied Biosystems 7500 FastReal-Time PCR System (LifeTechnologies, Carlsbad, CA) and the AGPATH-ID ^™ One-Step RT-PCR Kit (AmbionInc.) according to the manufacturer's recommendations. Real-time RT-PCR primers and probe were serotype-specific for DMV/1639. Primers were obtained from Integrated DNA Technologies (Coralville, IA), and the Taqman probe was synthesized by Bio Search Technologies (Novato, CA).

Results

None of the birds receiving the heat-attenuated PDRC DMV/1639 vaccine developed clinical signs. In addition, all five birds in the reverse passage 10 group were free of clinical signs and lesions.

The real-time RT-PCR results are presented in Table 5. Vaccine virus was detected in all pooled samples from reverse passage groups 1–9 and in each of the individual samples taken from five birds in passage group 10.

Reverse Passage Group ^a CT ^b meaning 1 17.06 2 20.08 3 21.99 4 21.53 5 20.42 6 22.90 7 22.97 8 20.75 9 22.20 10-1 20.80 10-2 21.97 10-3 20.45 10-4 21,32 10-5 21.51

^a Groups 1–9 were pooled samples from 5 swabs in 3 ml PBS, and groups 10-1–10-5 were 5 individual samples in 1 ml PBS from five birds in the last back-passage group.

^b CT= cycle threshold

Table 5. Real-time RT-PCR data for pooled (groups 1–9) and individual (group 10) intrachoanal swab samples.

Discussion

Based on the data from this experiment, the heat-attenuated PDRC DMV/1639 vaccine was stable in susceptible SPF chickens after 10 back passages. Vaccine virus was detected in chickens from each back passage group and no clinical signs or lesions were observed in any of the birds (back passage group 10 only). The CT values ranged from 22.97 to 17.06, indicating that a significant amount of vaccine was passaged into each group of chickens. Given these data and the fact that no clinical signs or lesions were observed in the birds, there is a strong indication that the attenuated state of the PDRC DMV/1639 vaccine is stable and should be safe for use in the field.

Example 5

Purity

Analysis of a sample of the PDRC DMV/1639-based vaccine deposited with the ATCC under Patent Deposit Registration Number PTA-126757 by the Agricultural and Natural Resources Laboratory of the University of Delaware against infectious coryza, infectious laryngotracheitis virus, Mycoplasma gallisepticum, Mycoplasma synoviae, NAHLN avian influenza virus (AIV), NAHLN avian paramyxovirus, infectious bronchitis virus (IBV), IBV-DE072/GA99, IBV-Ark, IBV-Mass/Conn, IBV-DMV/1639, and IBV-GA08 detected the presence of only IBV and IBV-DMV1639, indicating its purity. This analysis was performed to ensure that the isolate was free of pathogens commonly found in commercial poultry or any pathogens that could be detrimental to commercial poultry (e.g. AI or NDV). When planning vaccine production, this measure of purity is of paramount importance and should be carefully assessed. This analysis was also performed to ensure that any results obtained from testing (see previous examples) were not affected, either positively or negatively, by a confounding variable such as contamination with another pathogen. These data ensure that the most rigorous quality control was exercised in the isolation, propagation and preparation of the vaccine seed and that the data presented are a true reflection of this DMV/1639/11 virus vaccine only.

Example 6

Field research

The PDRC DMV/1639-based vaccine (deposited with the ATCC under Patent Deposit Registration Number PTA-126757) described herein has been administered to over 50 million commercial chickens. Analysis of vaccine virus infection rates indicates that the vaccine is highly effective in infecting and replicating under commercial conditions. This analysis is critical to ensuring the success of the vaccine because most poultry vaccines against infectious bronchitis virus (e.g., the DMV/1639-based vaccine described herein) are tested and validated using the eye drop method. Although this represents the gold standard for laboratory experiments, it is not possible to vaccinate every commercial chicken with eye drops. For this reason, commercial chickens are vaccinated en masse , using a misting cabinet that sprays the vaccine as an aerosol onto the chickens. There are many potential failure points in this process that have affected the effectiveness of other IBV vaccines in the past. These data demonstrate that even when applied on a mass spray basis, the DMV/1639-based vaccine described herein is highly effective in infecting chickens and replicating within them, which is the primary step in inducing an appropriate immune response. These data also aid in determining the most effective vaccine dose, as adverse reactions to the vaccine and the actual dose administered to chickens can be recorded and evaluated in the commercial setting in which the vaccine will be used.

The entire disclosure of all patents, patent applications and publications, and electronically available materials (including, for example, nucleotide sequences provided in, for example, GenBank and RefSeq, and amino acid sequences provided in, for example, SwissProt, PIR, PRF, PDB, and translation products of annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference. In the event of any inconsistency between the disclosure of the present application and the disclosure(s) of any document incorporated herein by reference, the disclosure of the present application shall control. The foregoing detailed description and examples have been presented solely for clarity of understanding. No unnecessary limitations should be construed therefrom. The present invention is not limited to the detailed details set forth and described, and variations obvious to those skilled in the art shall be included within the present invention as defined by the claims.

Claims (14)

1. An isolate of infectious bronchitis virus (IBV) for eliciting an immune response to IBV, wherein the IBV isolate comprises heat-attenuated IBV isolate PDRC DMV/1639, deposited with the ATCC under Patent Deposit Registration Number PTA-126757. 2. The IBV isolate of claim 1, wherein the IBV isolate is lyophilized, freeze-dried, or frozen. 3. A composition for obtaining an immune response to IBV, comprising an IBV isolate according to claim 1 and a pharmaceutically acceptable carrier. 4. The composition of claim 3, further comprising an adjuvant and/or other viral material; optionally wherein the composition is formulated for intranasal, intraocular, oral, transmucosal, intramuscular, subcutaneous or in ovo administration or for spraying or nebulising as an aerosol. 5. A vaccine for obtaining an immune response to IBV, containing an isolated IBV isolate according to claim 1. 6. A vaccine for birds of the order Galliformes, comprising an amount of the heat-attenuated IBV isolate PDRC DMV/1639, deposited with the ATCC under Patent Deposit Registration Number PTA-126757, sufficient to protect birds against one or more clinical signs induced by infection with infectious bronchitis virus (IBV) in poultry. 7. A vaccine according to claim 5 or 6, further comprising an adjuvant and/or other viral material; possibly where the vaccine is formulated for intranasal, intraocular, oral, transmucosal, intramuscular, subcutaneous or in ovo administration or for spraying or nebulization as an aerosol. 8. A method for producing an immune response to infectious bronchitis virus (IBV) in poultry, the method comprising administering to the poultry an IBV isolate according to claim 1 or 2, a composition according to claim 3 or 4, or a vaccine according to any one of claims 5-7. 9. A method for producing antibodies to IBV in poultry, wherein the method comprises administering to the poultry an IBV isolate according to claim 1 or 2, a composition according to claim 3 or 4, or a vaccine according to any of claims 5-7. 10. A method for reducing the severity of one or more clinical signs and/or viral load induced by an infection caused by infectious bronchitis virus (IBV) in poultry, the method comprising administering to the poultry an effective amount of an IBV isolate according to claim 1 or 2, a composition according to claim 3 or 4, or a vaccine according to any of claims 5-7. 11. A method for reducing the susceptibility of a Galliformes bird to an infection caused by the infectious bronchitis virus (IBV), wherein the method comprises administering to the bird an effective amount of an IBV isolate according to claim 1 or 2, a composition according to claim 3 or 4, or a vaccine according to any of claims 5-7. 12. A method for protecting poultry of the order Galliformes from infection caused by the infectious bronchitis virus (IBV), wherein the method comprises administering to the bird an effective amount of an IBV isolate according to claim 1 or 2, a composition according to claim 3 or 4, or a vaccine according to any of claims 5-7. 13. The method according to any one of claims 8 to 12, wherein the administration is intranasal, intraocular, oral, transmucosal, intramuscular or subcutaneous, or wherein the administration comprises administration in ovo , or administration by aerosol or by spraying; optionally wherein the administration comprises administration to a breeding female bird. 14. The method according to any one of claims 8 to 13, wherein the agricultural bird comprises a bird of the order Galliformes, preferably a chicken or a turkey.