WO2023230479A1

WO2023230479A1 - Combined vaccine containing infectious bronchitis virus attenuated massachusetts and recombinant lasota virus expressing arkansas spike

Info

Publication number: WO2023230479A1
Application number: PCT/US2023/067361
Authority: WO
Inventors: Haroldo E. TORO GUZMAN; Leticia FRIZZO DA SILVA
Original assignee: Auburn University; Zoetis Services Llc
Priority date: 2022-05-23
Filing date: 2023-05-23
Publication date: 2023-11-30

Abstract

Disclosed are compositions comprising rLS/ArkSe.GMCSF and a live attenuated IBV vaccine, and methods of using the same for inducing an immune response against IBV or vaccinating against IBV.

Description

COMBINED VACCINE CONTAINING INFECTIOUS BRONCHITIS VIRUS ATTENUATED MASSACHUSETTS AND RECOMBINANT LASOTA VIRUS EXPRESSING ARKANSAS SPIKE

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 63/460,691 filed on April 20, 2023, and U.S. Provisional Application No. 63/344,785 filed on May 23, 2022, the contents of which are incorporated by reference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant 2015-68004-23131 awarded by the National Institute of Food Agriculture. The government has certain rights in the invention.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing (169996.00477.xml; Size: 24,851 bytes; and Date of Creation: May 23, 2023) is herein incorporated by reference in its entirety.

BACKGROUND

Infectious bronchitis vims (IBV) remains a major cause for economic losses in the poultry industry. IBV’s evolutionary success relies on genetically diverse populations, which allow for quick adaptation to changes in selective pressure. Novel variants of infectious bronchitis (IB) vims (IBV) continue to emerge as a result of naturally occurring recombination events between IB wildtype and vaccine vimses. In addition, distinct IBV vaccine subpopulations continue to be isolated from outbreaks of disease in chickens vaccinated with attenuated IBV vaccines. Indeed, accumulating evidence indicates that the long-term use of attenuated IBV vaccines has complicated the control of the disease and perpetuated IB associated economic losses to the poultry industry. Therefore, the use of recombinant vimses expressing IBV immunodominant epitopes instead of live vaccines of differing types appears to be a better option to reduce emergence of novel IBV. Accordingly, there is a remaining need in the art for improved IBV vaccine compositions. SUMMARY Disclosed herein are compositions comprising rLS/ArkSe.GMCSF and a live attenuated IBV vaccine, and methods of using the same for inducing an immune response against IBV or vaccinating against IBV. The inventors have discovered that the combined composition provided enhanced protection to the subjects. The first aspect of the present invention comprises a composition comprising a live attenuated IBV vaccine and a recombinant Newcastle disease virus LaSota vector (rLS) co- expressing infectious bronchitis virus (IBV) Arkansas (Ark)-type trimeric spike ectodomain (Se) and granulocyte-macrophage colony-stimulating factor (GM-CSF) (rLS/ArkSe.GMCSF). In some embodiments, the live attenuated IVB vaccine comprises a IBV Massachusetts (Mass)-type vaccine. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition comprises a vaccine composition. A second aspect of the present invention provides a method of eliciting an immune response against infectious bronchitis virus (IBV). In some embodiments, the method comprises administering an effective amount of the pharmaceutical composition described herein to a subject to elicit an immune response against IBV. In some embodiments, a method of vaccinating a subject against infectious bronchitis virus (IBV) is provided. The method comprises, administering the pharmaceutical composition described herein to a subject to vaccinate the subject against IBV by generating an immune response to IBV. A third aspect of the present invention provides a method of eliciting an immune response against infectious bronchitis virus (IBV) in a subject comprising administering an effective amount of a live attenuated IBV vaccine and a recombinant Newcastle disease virus LaSota vector co- expressing infectious bronchitis virus (IBV) Arkansas (Ark)-type trimeric spike ectodomain and granulocyte-macrophage colony-stimulating factor (GM-CSF) (rLS/ArkSe.GMCSF), to the subject, wherein, the live attenuated IBV vaccine and rLS/ArkSe.GMCSF are administered at the same time. In some embodiments, the immune response elicited by the method comprises generating antibodies to IBV. In some embodiments, a method of reducing tracheal damage due to IBV infection is provided, the method comprising administering the composition described herein to a subject. In some embodiments, the subject is poultry. In some embodiments, the poultry is chicken. In some embodiments, the method provides administration of the compositions described herein wherein the administration is oral administration, or the administration is via spraying on or over the subject, or the composition is provided in the drinking water. In some embodiments, the method provides for the composition to be administered in the first five days of live. In some embodiments, the method provides for the composition to be administered only one time. In some embodiments the subject administered the composition exhibits greater protection against challenge by virulent IBV relative to subjects not administered the composition. BRIEF DESCRIPTION OF THE DRAWINGS The present technology can be better understood by reference to the following drawings. The drawings are merely exemplary to illustrate certain features that may be used singularly or in combination with other features and the present technology should not be limited to the embodiments shown. Fig.1. Newcastle disease virus (NDV) antibodies determined by commercial ELISA in chickens (n=21/group) collected 20 days after single vaccination with the combined vaccine containing 10³ EID₅₀/per bird of attenuated Mass vaccine and either 10⁶EID₅₀ of recombinant LaSota strain of NDV expressing recombinant infectious bronchitis virus (IBV) surface glycoprotein (spike) ectodomain from the Arkansas strain of IBV and granulocyte-macrophage colony-stimulating factor (rLS/ArkSe.GMCSF) (rLS^6+Mass) or 10⁷ EID₅₀ of rLS/ArkSe.GMCSF (rLS^7+Mass). Controls included chickens vaccinated with 10³ EID₅₀/per bird of attenuated Mass and ArkDPI live vaccines. Data analyzed by ANOVA with Tukey’s multiple comparison post-test. Different letters indicate significant differences at P<0.05. Fig 2. Virus neutralizing ability of sera against IBV Ark from chickens vaccinated with rLS^7+Mass or chickens vaccinated with Mass. Relative IBV RNA detected by qRT-PCR in the allantoic fluids of embryonated eggs inoculated with diluted-virus/constant-serum mixtures compared to Ark virus alone. For simplicity reasons, shown are only Ark virus dilutions (10^-5, 10- ⁶) at which neutralization became evident compared to the virus alone. Values analyzed by ANOVA and Tukey posttest. Different letters indicate significant differences at P<0.05. Fig. 3. Viral load in the tracheas of chickens (n=21/group) vaccinated as described in legend of Fig.1. Chickens were challenged with 10³ EID₅₀/bird of virulent Ark (ARK) at 21 days of age and relative IBV RNA in the tracheas determined by quantitative RT-PCR five days after challenge. NV/ARK = non-vaccinated/Ark-challenged; NV/NC = non-vaccinated/non-challenged. Data analyzed by ANOVA and Tukey posttest (boxes: 25^th percentile, median, 75^th percentile; whiskers: minimum and maximum). Different letters indicate significant differences at P<0.05. Exact P values between distinct groups determined by two-tailed t-test. Fig 4. Tracheal histomorphometry of chickens vaccinated as described in legend of Fig. 1 and challenged with 10⁴ EID₅₀/bird of virulent Ark (ARK) at 21 days of age. Histomorphometry performed 5 days after challenge. NV/NC = non-vaccinated/non-challenged and NV/ARK = non- vaccinated/Ark challenged. Tracheal histomorphometry (A) mucosal thickness and (B) lymphocytic infiltration presented in arbitrary units using ImageJ. Values analyzed by ANOVA and Tukey post-test (boxes: 25^th percentile, median, 75^th percentile; whiskers: minimum and maximum). Different letters indicate significant differences at P<0.05. DETAILED DESCRIPTION Disclosed herein are compositions comprising rLS/ArkSe.GMCSF (SEQ ID NO: 1 or sequences having at least 90%, 92%, 94%, 95%, 97%, 98%, or 99% sequence identity thereto) and a live attenuated IBV vaccine, and methods of using the same for inducing an immune response against IBV, or vaccinating against IBV, which may be described herein using definitions as set forth below and throughout the application. Previously, the inventors disclosed a novel viral vector based on Newcastle disease virus LaSota strain that expressed a recombinant form of infectious bronchitis virus (IBV) surface glycoprotein (spike) ectodomain (rLS/ArkSe) and demonstrated that inoculation of subjects with said novel viral vector improved immune response to IBV challenge. See, U.S. Pat. No. 10,772,953, which is incorporated by reference herein in its entirety. The inventors subsequently discovered that a recombinant Newcastle disease virus vector expressing both IBV spike ectodomain (Se) and granulocyte-macrophage colony-stimulating factor (rLS/ArkSe.GMCSF) elicited a significantly enhanced immune response compared to the same vector lacking the capability of inducing GM-CSF expression (rLS/ArkSe), leading to improved protection of subjects to challenge by IBV (See International Patent Publication WO2022/261554, incorporated herein by reference). The inventors further used the rLS/ArkSe.GMCSF along with a live-attenuated IBV vaccine in a “prime-boost” strategy, wherein a subject is administered a first composition and then after a determined period of time, the subject is administered a second composition, which may be the same or different than the first composition. The first composition (and the second composition) may be administered one or more times. The inventors showed that administering the rLS/ArkSe.GMCSF vaccine, then subsequently administering a live-attenuated IBV vaccine, improved protection compared to administration of IBV live-attenuated vaccine alone, or administration of IBV live-attenuated vaccine and subsequent administration the rLS/ArkSe.GMCSF. This study evaluates protection conferred by administering rLS/ArkSe.GMCSF and an attenuated IBV vaccine in combination. The inventors have discovered that the combined composition provided enhanced protection to the subjects. In particular, the inventors administered rLS/ArkSe.GMCSF together an IBV Massachusetts (Mass)-type vaccine. This combination provided enhanced protection against Ark-type challenge as compared to vaccination with attenuated Mass vaccine alone. These results were particularly unexpected given the known interference between IBV and NDV replication (Raggi et al. Avian Dis.7:106-122; 1963.). Infectious bronchitis (IB) is an acute, highly contagious viral disease of poultry. IB affects poultry of all ages and based on the organ system affected the disease is manifested in three major clinical forms—respiratory, renal and reproductive. Infectious bronchitis virus (IBV) is a member of the genus Gammacoronavirus in the family Coronaviridae. The viral genome is a single- stranded, positive-sense RNA of about 27.6 Kb in length. The 5′-two-third of the viral genome codes for the non-structural proteins responsible for RNA replication and transcription. The 3′- one-third of the viral genome codes for four structural proteins, namely, spike (S), envelope (E), membrane (M) and nucleocapsid (N) proteins, in addition to several non-structural proteins. The S protein of IBV is heavily glycosylated and plays a major role in eliciting protective immune responses. It is present as trimers on the surface of the virion and contains conformation dependent epitopes. The S protein is cleaved post-translationally by host cell proteases into S1 (N-terminal, globular head domain) and S2 (C-terminal, stalk domain) subunits. Many different IBV serotypes and genotypes circulate worldwide. These serotypes arise due to high frequency of mutations and/or recombination events. Control of IBV is difficult because there is little to no cross-protection between the numerous different serotypes of the virus. Cross-protection between different serotypes is variable or poor. Live-attenuated vaccines have been successful in controlling IB in the field. However, live-attenuated vaccines provide cross- protection against some of the IBV variants but not all. Furthermore, use of live-attenuated IBV vaccines can lead to production of variant IBV strains by mutations and/or recombination. One example of a live attenuated IBV vaccine is the Massachusetts (Mass)-type vaccine. Compositions: In a first aspect, the present invention provides a composition comprising, a live attenuated IBV vaccine and a recombinant Newcastle disease virus LaSota vector (rLS) co-expressing infectious bronchitis virus (IBV) Arkansas (Ark)-type trimeric spike ectodomain (Se) and granulocyte-macrophage colony-stimulating factor (GM-CSF) (rLS/ArkSe.GMCSF). Accordingly, in one aspect of the current disclosure, recombinant Newcastle disease virus vectors are provided. In some embodiments, the recombinant Newcastle disease virus vectors comprise: a nucleic acid encoding an infectious bronchitis virus (IBV) spike ectodomain (Se); and a nucleic acid encoding granulocyte-macrophage colony-stimulating factor (GM-CSF). As used herein, “vector” refers to some means by which DNA or RNA can be introduced into a host. There are various types of vectors including virus, plasmid, bacteriophages, cosmids, and bacteria. As used herein, a “viral vector” or “Newcastle disease virus (NDV) vector” refers to recombinant virus, e.g., Newcastle disease virus, that has been engineered to express a heterologous polypeptide (e.g., a recombinant IBV Se protein and GM-CSF, as disclosed herein) in infected cells. The recombinant virus typically also includes cis-acting elements for expression of the heterologous polypeptide. The term “IBV” is meant to encompass numerous serotypes and strains of IBV that have been isolated and will be isolated in the future throughout the United States and the world and characterized, including but not limited to: B/D207/84; B/D274/84; B/UK167/84; B/UK142/86; E/D3896/84; E/UK123/82; Brazil/BR1/USP-73/09; 793B/4-91/91; FR/CR88121/88; China/Q1/98; China/LDL971/97; LX4; CAV/CAV9437/95; CAV/CAV1686/95; CAV/CAV56b/91; PA/Wolgemuth/98; PA/171/99; CA/557/03 S1; JAA/04 S1 vaccine; HN99 S1; N1/62/S1; GA08; Ark/ArkDPI/81 S1; Ark/Ark99/73; CAL99; CAL99/CAL99/99 S1; CAL99/NE15172/95 S1; Holte/Holte/54; JMK/JMK/64; Gray/Gray/60; Iowa/Iowa609/56; Ca/1737/04; DMA/5642/06 SI; GA07/GA07/07; QX/QXIBV/99; Mass/H52; Mass/H120; Mass/Mass41/41; Conn/Conn46/51 vaccine; FL/FL18288/71; DE/DE072/92 vaccine; Georgia 98; GA98/0470/98; GA-08; GA-13; and Dutch/D1466/81. The complete genomic sequences of many strains of IBV have been reported. (See Ammayappan et al., Virology Journal 2008, 5:157, reporting the genomic sequence of Ark/ArkDPI/81; which is incorporated herein by reference in its entirety). Live attenuated strains of IBV are available commercially as vaccines and may include Mass/Mass41/41 S1 and Ark/ArkDPI/81 S1. In some embodiments, an IBV Massachusetts (Mass)-type vaccine is disclosed. Mass-type is the most prevalent serotype of infectious bronchitis virus worldwide with regional-specific genotypes and serotypes. The Massachusetts type vaccine can be live attenuated, live modified or inactivated. Massachusetts-type IB vaccines include H120, H52, Ma5, W93 and 28/86. In some embodiments, the M41 Mass-type vaccine is used. The M41 type may comprise the sequence of GenBank accession no: DQ834384. Other live attenuated and/or inactivated vaccine strains include, but are not limited to Ma5, D274, H52, CR88121. Other live attenuated vaccine stains include H120, 4/91, 1/96, GA-98, Arkansas, 1212B, Connecticut, B48, VicS and Armidale. Other inactivated vaccine stains include 249G and PL84084. Live modified vaccine strains include Delaware. There are numerous strains of infectious bronchitis virus. However, each strain has the common feature of an S protein that has two subunits that are proteolytically cleaved in a cell. In the disclosed working examples, the inventors disclose a recombinant Newcastle disease virus vector expressing the S protein ectodomain (Se) from the Arkansas strain of IBV and GMCSF as well as a live attenuated Mass-type IBV vaccine. However, it is to be understood that the current disclosure contemplates the use of spike protein ectodomains from other strains of IBV and other attenuated vaccine. Moreover, such approaches may be advantageous to generate viral vectors expressing spike ectodomains from locally significant strains of IBV to better elicit relevant immune responses in subjects. Compositions provided herein may comprise a combination of rLS/ArkSe.GMCSF and a live attenuated IBV vaccine. The term "combination therapy" is used in its broadest sense and means that a subject is administered at least two agents. More particularly, the term "in combination" with respect to therapy administration refers to the concomitant administration of two (or more) active agents for the treatment of a disease state. As used herein, the active agents may be combined and administered in a single dosage form, may be administered as separate dosage forms at the same time, or may be administered as separate dosage forms that are administered alternately or sequentially on the same days. In one embodiment of the presently disclosed subject matter, the active agents are combined and administered in a single dosage form. In another embodiment, the active agents are administered in separate dosage forms. Further, the presently disclosed compositions can be administered alone or in combination with adjuvants that enhance stability of the agents, facilitate administration of pharmaceutical compositions containing them in certain embodiments, provide increased dissolution or dispersion, increase activity, provide adjuvant therapy, and the like, including other active ingredients. In some embodiments, such combination therapies utilize lower dosages of the conventional therapeutics, thus avoiding possible toxicity and adverse side effects incurred when those agents are used as monotherapies. When administered in combination, the effective concentration of each of the agents to elicit a particular biological response may be less than the effective concentration of each agent when administered alone, thereby allowing a reduction in the dose of one or more of the agents relative to the dose that would be needed if the agent was administered as a single agent. The effective concentration of each agent may similarly be adjusted based on the interaction of the agents, whereby the concentration of one or more of the agents are decreased, increased or kept the same. The effects of multiple agents may, but need not be, additive or synergistic. In some embodiments, when administered in combination, the two or more agents can have a synergistic effect. As used herein, the terms "synergy," "synergistic," "synergistically" and derivations thereof, such as in a "synergistic effect" or a "synergistic combination" or a "synergistic composition" refer to circumstances under which the biological activity of a combination of an agent and at least one additional therapeutic agent is greater than the sum of the biological activities of the respective agents when administered individually. In some embodiments, the compositions disclosed herein may be used in an effective amount. As used herein the term “effective amount” refers to the amount or dose of the compound that provides the desired effect. In some embodiments, the effective amount is the amount or dose of the compound, upon single or multiple dose administration to the subject, which provides the desired effect in the subject under diagnosis or treatment. Suitably the desired effect may include, but not be limited to inducing an immune response to IBV, assessment of respiratory rales, tracheal IBV RNA, tracheal histomorphometry, and virus isolation in embryonated chicken eggs. In some embodiments the composition may comprise a combination of rLS/ArkSe.GMCSF and a live attenuated IBV vaccine, wherein the embryo infectious dose (EID₅₀) of rLS/ArkSe.GMCSF is 1000 to 10000 times more than the EID₅₀ of the live attenuated IBV vaccine in the composition. In some embodiments, the EID₅₀ of rLS/ArkSe.GMCSF is at least 10⁶ and the EID₅₀ of the live attenuated IBV vaccine is 10³ per dose of the composition. In some embodiments, the EID₅₀ of rLS/ArkSe.GMCSF is in the range of 10⁶ to 10⁷ and the EID₅₀ of the live attenuated IBV vaccine is 10³ +/- 10% per dose of the composition The compositions disclosed herein may include pharmaceutical compositions such as vaccine compositions comprising the presently disclosed recombinant vectors, which are formulated for administration to a subject in need thereof. Such compositions can be formulated and/or administered in dosages and by techniques well known to those skilled in the medical arts taking into consideration such factors as the age of the particular subjects and the route of administration. Accordingly, in another aspect of the current disclosure, pharmaceutical compositions are provided. In some embodiments, the pharmaceutical compositions comprise recombinant Newcastle disease virus vector comprising: a nucleic acid encoding an infectious bronchitis virus (IBV) spike ectodomain (Se); and granulocyte-macrophage colony-stimulating factor (GM-CSF), a live attenuated IBV vaccine and a pharmaceutically acceptable carrier. As used herein "vaccine" refers to a composition that includes an antigen. Vaccine may also include a biological preparation that improves immunity or the immune response to a particular disease. A vaccine may typically contain an agent, referred to as an antigen, that resembles or is a part of a disease-causing microorganism, in this case IBV, and the agent may be nucleic acids that are homologous to a portion of IBV, or often be made from weakened or killed forms of the virus, its toxins or one of its surface proteins. The antigen may stimulate the body's immune system to recognize the agent as foreign, destroy it, and "remember" it, so that the immune system can more easily recognize and destroy any of these microorganisms that it later encounters. Vaccines may be prophylactic, e.g., to prevent or ameliorate the effects of a future infection by any natural or "wild" pathogen, or therapeutic, e.g., to treat the disease. Administration of the vaccine to a subject results in an immune response, generally against one or more specific diseases. The amount of a vaccine that is therapeutically effective may vary depending on the particular virus used, or the condition of the patient, and may be determined by a physician. The vaccine may be introduced directly into the subject by the intramuscular, intravenous, subcutaneous, oral, oronasal, or intranasal routes of administration. The vaccine compositions described herein also include a suitable carrier or vehicle for delivery. As used herein, the term “carrier” refers to a pharmaceutically acceptable solid or liquid filler, diluent or encapsulating material. A water-containing liquid carrier can contain pharmaceutically acceptable additives such as acidifying agents, alkalizing agents, antimicrobial preservatives, antioxidants, buffering agents, chelating agents, complexing agents, solubilizing agents, humectants, solvents, suspending and/or viscosity-increasing agents, tonicity agents, wetting agents or other biocompatible materials. A tabulation of ingredients listed by the above categories, may be found in the U.S. Pharmacopeia National Formulary, 1857-1859, (1990). Some examples of the materials which can serve as pharmaceutically acceptable carriers are sugars, such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen free water; isotonic saline; Ringer's solution, ethyl alcohol and phosphate buffer solutions, as well as other nontoxic compatible substances used in pharmaceutical formulations. Wetting agents, emulsifiers and lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions, according to the desires of the formulator. Examples of pharmaceutically acceptable antioxidants include water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfite, sodium metabisulfite, sodium sulfite and the like; oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol and the like; and metal-chelating agents such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid and the like. In another embodiment, the present formulation may also comprise other suitable agents such as a stabilizing delivery vehicle, carrier, support or complex-forming species. The coordinate administration methods and combinatorial formulations of the instant invention may optionally incorporate effective carriers, processing agents, or delivery vehicles, to provide improved formulations for delivery of the IBV antigens described herein. Suitable adjuvants are known in the art and include, but are not limited to, threonyl muramyl dipeptide (MDP) (Byars et al., 1987), Ribi adjuvant system components (Corixa Corp., Seattle, Wash.) such as the cell wall skeleton (CWS) component, Freund's complete adjuvants, Freund's incomplete adjuvants, bacterial lipopolysaccharide (LPS; e.g., from E. coli), or a combination thereof. A variety of other well-known adjuvants may also be used with the methods and vaccines of the invention, such as aluminum hydroxide, saponin, amorphous aluminum hydroxyphosphate sulfate (AAHS), aluminum hydroxide, aluminum phosphate, potassium aluminum sulfate (Alum), and combinations thereof. Cytokines (γ-IFN, GM-CSF, CSF, etc.), lymphokines, and interleukins (IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8. IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, 11-18, 11-19, IL-20, IL-21, and 11-22) have also been used as adjuvants and/or supplements within vaccine compositions and are contemplated to be within the scope of the present invention. For example, one or more different cytokines and/or lymphokines can be included in a composition comprising one or more peptides or a vaccine of the invention. In a preferred embodiment, the adjuvant is an aluminum salt, AS04, MF59, AS01B, CpG 1018, or another adjuvant that is considered to be safe for use in humans by the Centers for Disease Control and Prevention. To aid in administration, vaccines may be mixed with a suitable carrier or diluent such as water, oil (e.g., a vegetable oil), ethanol, saline solution (e.g., phosphate buffer saline or saline), aqueous dextrose (glucose) and related sugar solutions, glycerol, or a glycol such as propylene glycol or polyethylene glycol. Stabilizing agents, antioxidant agents and preservatives may also be added. Suitable antioxidant agents include sulfite, ascorbic acid, citric acid and its salts, and sodium EDTA. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben, and chlorbutanol. The composition for parenteral administration may take the form of an aqueous or nonaqueous solution, dispersion, suspension or emulsion. The vaccine formulation may additionally include a biologically acceptable buffer to maintain a pH close to neutral (7.0-7.3). Such buffers preferably used are typically phosphates, carboxylates, and bicarbonates. More preferred buffering agents are sodium phosphate, potassium phosphate, sodium citrate, calcium lactate, sodium succinate, sodium glutamate, sodium bicarbonate, and potassium bicarbonate. The buffer may comprise about 0.0001-5% (w/v) of the vaccine formulation, more preferably about 0.001-1% (w/v). Other excipients, if desired, may be included as part of the final vaccine formulation. The remainder of the vaccine formulation may be an acceptable diluent, to 100%, including water. The vaccine formulation may also be formulated as part of a water-in-oil, or oil-in-water emulsion. The vaccine formulation may be separated into vials or other suitable containers. The vaccine formulation herein described may then be packaged in individual or multi-dose ampoules or be subsequently lyophilized (freeze-dried) before packaging in individual or multi-dose ampoules. The vaccine formulation herein contemplated also includes the lyophilized version. The lyophilized vaccine formulation may be stored for extended periods of time without loss of viability at ambient temperatures. The lyophilized vaccine may be reconstituted by the end user and administered to a patient. The disclosed compositions may be administered as a vaccine in an amount sufficient to induce an immune response for protecting against infection. Inducing a protective response may include inducing sterilizing immunity against a pathogen (e.g., against IBV), reducing the effects of the pathogen or reducing vial load. The compositions disclosed herein may be delivered via a variety of routes. Typical delivery routes include parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, or subcutaneous delivery), intranasal, oral, and ocular (via eyedrop). Another route of administration may include in ovo administration (e.g., in ovo vaccination). Formulations of the pharmaceutical compositions may include liquids (e.g., solutions and emulsions), sprays, and aerosols. The compositions may also be administered by addition to the drinking water. The pharmaceutical compositions disclosed herein may be delivered to subjects at risk for acquiring an infection by IBV. In order to assess the efficacy of an administered immunogenic composition or vaccine, the immune response can be assessed by measuring the induction of antibodies to particular epitopes of IBV and/or cell-mediated responses against IBV. Antibody responses may be measured by assays known in the art such as ELISA. Immune responses also may be characterized by physiological responses. (See Li et al., Vaccine 28 (2010) 1598-1605; and Stemke-Hale et al., Vaccine 2005 Apr 27;23(23):3016-25, the content of which are incorporated herein by reference in their entireties.) Immune response also may be measured by reduction in pathological responses such as respiratory signs after challenge with IBV, or reduction in titer or load as measured using methods in the art including methods that detect nucleic acid of the pathogen. (See, e.g., U.S. Patent No. 7,252,937, the content of which is incorporated by reference in its entirety). Immune response also may be measured by reduction in pathological responses such as a pathological response for an organ of the animal (e.g., the trachea) after challenge with IBV. As used herein, “eliciting an immune response” refers to generation of an immune response against a particular antigen to which a subject has been exposed or administered, e.g., IBV spike ectodomain (Se), for example, by way of compositions described herein. As used herein, an “immune response” may include an antibody response (i.e., a humoral response), where an immunized individual is induced to produce antibodies against an administered antigen (e.g., IgG (IgY), IgA, IgM, or other antibody isotypes). As used herein, an “immune response” also may include a cell-mediated response, for example, a cytotoxic T-cell response against cells expressing foreign peptides derived from an administered antigen in the context of a major histocompatibility complex (MHC) class I molecule. As used herein, “potentiating” or “enhancing” an immune response means increasing the magnitude and/or the breadth of the immune response. For example, the number of cells that recognize a particular epitope may be increased (“magnitude”) and/or the numbers of epitopes that are recognized may be increased (“breadth”). As used herein, “viral load” is the amount of virus present in a sample from a subject infected with the virus. Viral load is also referred to as viral titer. Viral load can be measured in variety of standard ways including copy Equivalents of the viral RNA (vRNA) genome per milliliter individual sample (vRNA copy Eq/ml). This quantity may be determined by standard methods that include RT-PCR. As used herein, the terms “subject,” “host,” or “individual” typically refer to an animal at risk for acquiring an infection by infectious bronchitis virus (IBV), such as an avian species or poultry. The terms “subject,” “host,” or “individual” may be used interchangeably. Suitable avian species for the disclosed vaccine may include poultry such as members of the order Galliformes, and in particular the species Gallus gallus or the subspecies Gallus gallus domesticus. Methods: In a second aspect, the present invention provides a method of eliciting an immune response against infectious bronchitis virus (IBV) in a subject, the method comprising administering an effective amount of a live attenuated IBV vaccine and a recombinant Newcastle disease virus LaSota vector co-expressing infectious bronchitis virus (IBV) Arkansas (Ark)-type trimeric spike ectodomain and granulocyte-macrophage colony-stimulating factor (GM-CSF) (abbreviated rLS/ArkSe.GMCSF), to the subject, wherein, rLS/ArkSe.GMCSF and the live attenuated IBV vaccine are administered at the same time. In some embodiments, eliciting an immune response against IBV comprises generating antibodies to IBV. The antibodies may be of any heavy chain isotype, including IgG, IgM, IgA, IgD or IgE. The antibodies may neutralizing or non-neutralizing antibodies. In some embodiments, the method comprises reducing tracheal damage due to IBV infection. Tracheal damage may be assessed using histomorphometric analysis, or any other method known in the art for assessing changes in tissue structure and function. Tracheal damage may include changes in tracheal mucosal thickness and or changes in lymphocyte infiltration or tracheal inflammation. Tracheal damage may also include other signs of respiratory distress, including coughing, sneezing and or respiratory rales. In some embodiments, the methods described herein may comprise administering the compositions described herein to the subject prior to exposure to IBV. In some embodiments, the methods comprise administering the composition in the first 5 days of life, or at some time within the first 5 days of hatching from an egg. In some embodiments the composition is administered one day after hatch. The methods described herein include administering the compositions by any means known in the art. In some embodiments, the methods comprise administering the composition in the drinking water, or via spraying on or over the subject or via oral administration. In some embodiments, the methods comprise administering the composition only once. In some embodiments, the methods provided herein provide the subject with greater protection against challenge by virulent IBV relative to subject not administered the composition. Greater protection may comprise increases in antibody titer, decreases in viral load or decreased tracheal damage in the subject administered the compositions described herein. Additional definitions The present disclosure is not limited to the specific details of construction, arrangement of components, or method steps set forth herein. The compositions and methods disclosed herein are capable of being made, practiced, used, carried out and/or formed in various ways that will be apparent to one of skill in the art in light of the disclosure that follows. The phraseology and terminology used herein is for the purpose of description only and should not be regarded as limiting to the scope of the claims. Ordinal indicators, such as first, second, and third, as used in the description and the claims to refer to various structures or method steps, are not meant to be construed to indicate any specific structures or steps, or any particular order or configuration to such structures or steps. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to facilitate the disclosure and does not imply any limitation on the scope of the disclosure unless otherwise claimed. No language in the specification, and no structures shown in the drawings, should be construed as indicating that any non-claimed element is essential to the practice of the disclosed subject matter. Unless otherwise specified or indicated by context, the terms “a”, “an”, and “the” mean “one or more.” For example, “a molecule” should be interpreted to mean “one or more molecules.” As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean plus or minus ≤10% of the particular term and “substantially” and “significantly” will mean plus or minus >10% of the particular term. As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of” should be interpreted as being “closed” transitional terms that do not permit the inclusion additional components other than the components recited in the claims. The term “consisting essentially of” should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure. Use of the word “about” to describe a particular recited amount or range of amounts is meant to indicate that values very near to the recited amount are included in that amount, such as values that could or naturally would be accounted for due to manufacturing tolerances, instrument and human error in forming measurements, and the like. All percentages referring to amounts are by weight unless indicated otherwise. In those instances where a convention analogous to “at least one of A, B and C, etc.” is used, in general such a construction is intended in the sense of one having ordinary skill in the art would understand the convention (e.g., “a system having at least one of A, B and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together.). It will be further understood by those within the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description or figures, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or ‘B or “A and B.” No admission is made that any reference, including any non-patent or patent document cited in this specification, constitutes prior art. In particular, it will be understood that, unless otherwise stated, reference to any document herein does not constitute an admission that any of these documents forms part of the common general knowledge in the art in the United States or in any other country. Any discussion of the references states what their authors assert, and the applicant reserves the right to challenge the accuracy and pertinence of any of the documents cited herein. All references cited herein are fully incorporated by reference, unless explicitly indicated otherwise. The present disclosure shall control in the event there are any disparities between any definitions and/or description found in the cited references. Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect a person having ordinary skill in the art to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. The following examples are meant only to be illustrative and are not meant as limitations on the scope of the invention or of the appended claims. EXAMPLES Example 1: Cross-Protection Conferred by Combined Vaccine Containing Infectious Bronchitis Virus Attenuated Massachusetts and Recombinant LaSota Virus Expressing Arkansas Spike Reference is made to the manuscript: Espejo et al., "Cross-Protection Conferred by Combined Vaccine Containing Infectious Bronchitis Virus Attenuated Massachusetts and Recombinant LaSota Virus Expressing Arkansas Spike," the content of which is incorporated herein by reference in its entirety Materials and Methods Chickens. White leghorn chickens were hatched from specific pathogen free (SPF) embryonated eggs (Charles River, North Franklin, CT) and maintained in Horsfall-type isolators in biosafety level 2 facilities with water and food ad libitum. Experimental procedures and animal care were performed in compliance with applicable federal and institutional animal care and use guidelines. Auburn University College of Veterinary Medicine is an Association for Assessment and Accreditation of Laboratory Animal Care -accredited institution. Challenge virus. The previously characterized virulent Ark-type IBV strain with GenBank accession # DQ458217 (18) was used for challenge. The virus was titrated in SPF embryonated chicken eggs as accepted (19) with minor modifications previously described (20). Vaccine viruses. The previously developed rLS/ArkSe.GMCSF virus was used (17). As reported before, this recombinant LaSota vaccine effectively expresses the IBV Ark-type Se in cell culture and is stable after passages in embryonated eggs (17). The rLS/ArkSe.GMCSF virus stock was titrated by 50% egg infective dose (EID₅₀) in 9-day-old SPF chicken embryos. Embryos were considered positive by detection of hemagglutinating activity in the allantoic fluid of inoculated eggs as accepted for NDV (21). In addition, commercially available live attenuated IBV vaccines belonging to the Mass and ArkDPI –types were used. The live attenuated IBV vaccines were titrated in SPF embryonated chicken eggs as described for the challenge virus (above). Due to well-known interference between IBV and NDV (22), two combined vaccines were prepared which differed in the dosage of the recombinant LaSota virus content. Specifically, we used 10⁶ EID₅₀ or 10⁷ EID₅₀ of rLS/ArkSe.GMCSF, while the dose of attenuated Mass vaccine was adjusted to 10³ EID₅₀. For simplicity reasons, these groups have been identified in the figures as rLS^6+Mass and rLS^7+Mass. All vaccinations in chickens were performed with a total volume of 100µl (25µl in each nostril and each eye) of appropriately diluted virus stock. Experimental design. Four groups of chickens (n=21 chickens/group) were vaccinated at 1 day of age (DOA) and additional two unvaccinated groups (n=15 chickens/group) served as controls. Groups 1 was vaccinated with the combined vaccine containing 10⁶EID₅₀ of rLS/ArkSe.GMCSF (rLS^6+Mass) and chickens of group 2 with 10⁷ EID₅₀ of rLS/ArkSe.GMCSF (rLS^7+Mass). Chickens of groups 3 and 4 were vaccinated with 10³ EID₅₀/per bird of the Mass and ArkDPI live vaccines, respectively. Groups 5 and 6 were age-matched non- vaccinated/challenged and non-vaccinated/non-challenged controls. Sera were collected 20 days after vaccination for IBV and NDV serum antibody determinations. NDV antibodies were determined by ELISA (IDEXX Laboratories Inc., Westbrook, ME) following the kit’s manufacturer recommendations. The neutralization ability of systemic IBV antibodies elicited by the combined vaccine (rLS/ArkSe.GMCSF + Mass) versus those elicited by vaccination with Mass alone against Ark was determined by a virus neutralization test (VNT). The VNT was conducted in embryonated chicken eggs by α method (constant-serum, diluted-virus) using pools of five sera and tenfold dilutions of the IBV Ark challenge strain. Instead of determining embryos positive for virus replication based on embryo lesions, we determined relative IBV RNA in the allantoic fluid of each egg by TaqMan© quantitative reverse transcription PCR (qRT-PCR) as described (23). Viral RNA data were analyzed by one-way analysis of variance (ANOVA) followed by Tukey multiple comparisons post-test. Differences were considered significant with P<0.05. At 21 DOA, all chickens in groups 1 through 5 were challenged ocularly with 100µl containing 10³ EID₅₀/chicken of the virulent IBV Ark strain. Protection was evaluated 5 days after challenge by individual assessment of respiratory rales, tracheal IBV RNA, tracheal histomorphometry, and virus isolation in embryonated chicken eggs (14). In brief, nasal and/or tracheal rales, were evaluated blindly by close listening to each bird and scored as 0 (absent), 1 (mild), 2 (moderate), or 3 (severe). Scoring data were compared by Kruskal-Wallis test followed by Dunn’s post-test. Tracheal viral load was determined by qRT-PCR. Briefly, IBV RNA was extracted from homogenized tracheal samples with TriReagent RNA/DNA/ Protein Isolation Reagent (Molecular Research Center, Cincinnati, OH) following the manufacturer’s recommendations. Relative viral copies were determined by qRT-PCR as described above. Viral RNA data were analyzed by one-way ANOVA followed by Tukey’s post-test. Differences were considered significant at P<0.05. Tracheal histomorphometry was performed as previously described (18). In brief, formalin-fixed sections of trachea were processed, embedded in paraffin, sectioned at 4–6 μm and stained with hematoxylin and eosin for histopathological examination. The tracheal mucosal thickness and the thickness of lymphocytic infiltration were measured using ImageJ and the average of five measurements for each chicken calculated. Data were analyzed by ANOVA followed by Tukeys’ post-test. IBV isolation from tracheal samples was performed as accepted (24). Briefly, tracheal samples were collected 5 days post-challenge into tubes containing sterile tryptose broth and antibiotics, and stored frozen at -80 C. Samples were thawed, vortexed, and 0.2 ml of each supernatant inoculated into five 10-day embryonated SPF chicken eggs via the allantoic route. Eggs were incubated at 37 C for 7 days. Embryos dying prior to day four of incubation were eliminated. Embryo deaths occurring after day four of incubation were considered positive. On day 7, eggs containing a live embryo were examined for IBV typical lesions including stunting, curling, and presence of urates in the kidneys. The sample was considered positive for virus isolation if any of the five embryos showed IBV lesions. Normal embryos were scored as negative. Based on results of viral RNA quantitation and because of labor and other costs consideration, virus isolation was restricted to chicken groups vaccinated with rLS^7+Mass, Mass, and ArkDPI. Results NDV antibody by ELISA. As seen in Fig. 1, chicken groups vaccinated with the combination of rLS/ArkSe.GM-CSF and live Mass IBV developed relatively low NDV antibody levels with S/P ratios less or equal to 0.2. Only group 2, vaccinated with 10⁷ EID₅₀ of rLS/ArkSe.GM-CSF showed NDV antibody titers significantly higher (P<0.05) than groups receiving only IBV. Thus, the recombinant LaSota virus showed limited replication in the vaccinated chickens. IBV virus neutralization. As indicated above, instead of determining whether embryos were positive or negative to IBV by classical lesions in each of the virus dilution mixtures with serum, we determined relative IBV RNA in the allantoic fluid of each embryonated egg. Thus, the data obtained were not transformed into a neutralization index but directly analyzed based on the detected viral loads in the eggs inoculated with increasing dilutions of the virus and constant serum mixtures. Figure 2 shows relative IBV RNA detected by qRT-PCR in the allantoic fluid of embryonated eggs inoculated with virus-serum mixtures at virus dilutions 10^-4, 10^-5 and 10^-6. Only at relatively high dilutions of the virus+constant serum mixtures neutralization became evident compared to the virus alone. This result shows that limited systemic antibody levels were elicited by vaccination with either rLS/ArkSe.GM-CSF+Mass or Mass alone. In addition, no significant differences were detected in relative amounts of IBV RNA in embryos inoculated with the mixtures of virus with serum from either vaccinated group, which indicates no difference in the quality (e.g. avidity) of the generated systemic antibodies. Signs and viral load. High incidence of respiratory rales was only observed in the unvaccinated/challenged control group. The absence of respiratory signs in vaccinated chickens was not consistent with viral loads. While significantly lower levels of IBV RNA were detected in he tracheas of all vaccinated groups in comparison with unvaccinated/challenged control chickens (P<0.05), vaccination did not reduce viral loads significantly except in chickens vaccinated with the attenuated ArkDPI vaccine (Fig. 3). Indeed, the lowest IBV RNA concentration following challenge was attained by vaccination with the homologous live attenuated Ark vaccine. Virus isolation. As anticipated, the attenuated Ark vaccine elicited “sterile immunity” against homologous challenge. Consistent with viral RNA quantitation results, chickens vaccinated with the Ark attenuated vaccine strain showed lowest virus isolation from the trachea five days after challenge (Table 1). In contrast, chickens vaccinated with rLS/ArkSe.GM-CSF (rLS^7)+Mass as well as chickens vaccinated with Mass exhibited extensive viral shedding. Although challenge virus was isolated from 49.5% of rLS^7+Mass vaccinated chickens compared to 56.2% of Mass vaccinated chickens, the difference was not significant (P<0.05) as determined by Fisher’s exact test.

Tracheal histomorphometry. As seen in Fig.4, significantly increased tracheal mucosal thickness and lymphocyte infiltration (P<0.05) was detected in unvaccinated chickens challenged with virulent Ark compared to non-challenged controls. The tracheal mucosa was protected in groups vaccinated with the combination of the recombinant virus and the Mass strain and mucosal histology did not differ (P<0.05) from chickens vaccinated with the homologous attenuated ArkDPI vaccine. Significantly higher values for mucosal thickness and lymphocyte infiltration (P<0.05), i.e., increased mucosal damage, was detected in chickens vaccinated with Mass alone compared to combined vaccinated chickens. Discussion We previously showed that vaccination with rLS expressing Ark-type Se in chickens induces a relatively low antibody NDV response with average ELISA S/P ratios barely reaching levels considered positive by the guidelines of the ELISA kit (16). In the current study, although chickens vaccinated with the Mass + rLS/ArkSe.GM-CSF combined vaccine elicited significantly higher (P<0.05) NDV antibody responses than controls not exposed to NDV, these antibody levels were extremely low indicating limited replication of the recombinant NDV in the chickens. The fact that IBV interferes with the replication of NDV has been known since the early 60s (25). Thus, to avoid IBV outcompeting NDV, the dosages of each component were adjusted. However, the current results indicate that the dosage adjustment will require further refinement. The low IBV systemic antibody response detected by virus neutralization test is consistent with this result. The results of protection against heterologous challenge were inconsistent. On the one hand, the combined vaccine neither reduced the viral load in the trachea (based on both IBV RNA quantitation and virus isolation) nor enhanced the cross-neutralizing ability of systemic antibodies. The lack of reduction of viral shedding is most likely the result of limited replication of the recombinant LaSota virus in the vaccinees. In addition to further dosage adjustments in the combined vaccine discussed above, the virulence of the LaSota strain is significantly weakened as result of the insertion of two foreign genes. Indeed, we previously reported that the insertion of only the IBV Se gene already reduced the ICPI and MDT of the LaSota strain to the level of the B1 strain. In addition, in this experiment we vaccinated chickens at 1 DOA, i.e., chickens with an immature immune system. Finally, in this experiment we did not use a booster vaccination. Although the lack of reduction of viral shedding after challenge is unsatisfactory, the results showing significant reduction of tracheal damage are promising. Indeed, the combined vaccine significantly reduced tracheal damage (as determined by histomorphometry) compared to Mass vaccination alone. Indeed, regardless of the considerations described above, it seems that even a limited replication of the recombinant virus was enough to enhanced the protective ability of the attenuated Mass against Ark challenge. The results showed no difference in virus neutralizing capacity of systemic antibodies in vaccinated chickens. Thus, we speculate that the protection of the tracheal mucosa observed is likely the result of mucosal immunity. Further investigation into quality and quantity of tracheal IgA elicited by vaccination with the combined vaccine is needed. The current USDA APHIS’ 9 CFR regulation for licensing IBV vaccines indicates that if less than 90 percent of vaccinees are negative for virus recovery following challenge, the master seed virus is unsatisfactory. This expected level of protection (sterilizing immunity) can currently only be achieved by attenuated live IBV vaccines. In the current experiment, only the control using attenuated ArkDPI was able to reduce viral shedding after virulent Ark challenge at a level that meets the 9 CFR regulation. In contrast, recombinant IBV vaccines have in general failed to meet this level of protection (15, 16, 17). Use of a prime-boost regime with attenuated Mass followed by rLS/ArkSe.GM-CSF showed encouraging results as heterologous protection was enhanced; i.e. the prime and boost strategy protected against Ark challenge significantly better than Mass only (17). However, this recombinant virus does not meet the regulatory requirements for IBV vaccines. One option to use rLS expressing tailored IBV Se and comply with the licensing requirement would be to use it as a combined vaccine preparation with attenuated Mass. The Mass component of the combined vaccine would comply with the requirement as attenuated Mass provides excellent immunity against homologous (Mass) challenge. The addition of the tailored rLS would enhance the cross-protection provided by Mass. Finally, in the field the combined vaccine would likely be used as a booster after priming with Mass, which as we know from previous work, does enhance cross-protection (17). Ultimately, this strategy should reduce the need for attenuated vaccines of multiple serotypes and therefore reduce the risk of emergence of novel recombinants. References 1. Kusters JG, Jager EJ, Niesters HG, van der Zeijst BA. Sequence evidence for RNA recombination in field isolates of avian coronavirus infectious bronchitis virus. Vaccine. 8:605- 608; 1990. 2. Cavanagh D, Davis PJ, Cook JK. Infectious bronchitis virus: evidence for recombination within the Massachusetts serotype. Avian Pathol.21:401-408; 1992. 3. Wang L, Junker D, Collisson EW. Evidence of natural recombination within the S1 gene of infectious bronchitis virus. Virology.192:710-716; 1993. 4. Jia W, Karaca K, Parrish CR, Naqi SA. A novel variant of avian infectious bronchitis virus resulting from recombination among three different strains. Arch Virol.140:259-271; 1995. 5. Kottier SA, Cavanagh D, Britton P. Experimental evidence of recombination in coronavirus infectious bronchitis virus. Virology.213:569-580; 1995. 6. Jackwood MW, Lee D-H. Different evolutionary trajectories of vaccine-controlled and non-controlled avian infectious bronchitis viruses in commercial poultry. PLoS One.122017 2017. 7. Chen L, Zhang T, Han Z, Liang S, Xu Y, Xu Q, Chen Y, Zhao Y, Shao Y, Li H et al. Molecular and antigenic characteristics of Massachusetts genotype infectious bronchitis coronavirus in China. Vet Microbiol.181:241-251; 2015. 8. Lian J, Wang Z, Xu Z, Chen T, Shao G, Zhang X, Qin J, Xie Q, Lin W. Distribution and molecular characterization of avian infectious bronchitis virus in southern China. Poult Sci. 100:101169; 2021. 9. Xu L, Han Z, Jiang L, Sun J, Zhao Y, Liu S. Genetic diversity of avian infectious bronchitis virus in China in recent years. Infect Genet Evol.66:82-94; 2018. 10. Gallardo RA, van Santen VL, Toro H. Host intraspatial selection of infectious bronchitis virus populations. Avian Dis.54:807-813; 2010. 11. McKinley ET, Hilt DA, Jackwood MW. Avian coronavirus infectious bronchitis attenuated live vaccines undergo selection of subpopulations and mutations following vaccination. Vaccine.26:1274-1284; 2008. 12. van Santen VL, Toro H. Rapid selection in chickens of subpopulations within ArkDPI- derived infectious bronchitis virus vaccines. Avian Pathol.37:293-306; 2008. 13. Toro H. Global Control of Infectious Bronchitis Requires Replacing Live Attenuated Vaccines by Alternative Technologies. Avian Dis.65:635-640; 2021. 14. Abozeid HH, Paldurai A, Varghese BP, Khattar SK, Afifi MA, Zouelfakkar S, El‑Deeb AH, El‑Kady MF, Samal SK. Development of a recombinant Newcastle disease virus‑vectored vaccine for infectious bronchitis virus variant strains circulating in Egypt. Vet Res.50:12; 2019. 15. Shirvani E, Paldurai A, Manoharan VK, Varghese BP, Samal SK. A Recombinant Newcastle Disease Virus (NDV) Expressing S Protein of Infectious Bronchitis Virus (IBV) Protects Chickens against IBV and NDV. Sci Rep.8:11951; 2018. 16. Zegpi RA, He L, Yu Q, K.S. J, Santen VLv, Toro H. Limited protection conferred by recombinant Newcastle disease virus expressing infectious bronchitis spike protein. Avian Dis. 64:53-59; 2020. 17. Khalid Z, He L, Yu Q, Breedlove C, Joiner K, Toro H. Enhanced Protection by Recombinant Newcastle Disease Virus Expressing Infectious Bronchitis Virus Spike-Ectodomain and Chicken Granulocyte-Macrophage Colony-Stimulating Factor. Avian Dis.65:364-372; 2021. 18. Toro H, van Santen VL, Li L, Lockaby SB, van Santen E, Hoerr FJ. Epidemiological and experimental evidence for immunodeficiency affecting avian infectious bronchitis. Avian Pathol.35:1-10; 2006. 19. Villegas P. Titration of biological suspensions. In: Dufour-Zavala L, Jackwood MW, Lee MD, Lupiani B, Reed WM, Spackman E, Woolcock PR, editors. A laboratory manual for the isolation, identification and characterization of avian pathogens.6th ed. Athens, GA: Am Assoc Avian Pathol. p.355-360; 2016. 20. Ghetas AM, van Santen VL, Joiner K, Toro H. Kidney cell-adapted infectious bronchitis ArkDPI vaccine confers effective protection against challenge. Avian Dis.60:418-423; 2016. 21. Alexander DJ, Senne DA. Newcastle disease virus and other avian paramyxoviruses. In: Dufour-Zavala L, Swayne DE, Glisson JR, Pearson JE, Reed WM, Jackwood MW, Woolcock PR, editors. A laboratory manual for the isolation, identification and characterization of avian pathogens. Fifth ed. Athens, GA: Am Assoc Avian Pathol. p.135-141; 2008. 22. Raggi LG, Lee GG. Infectious bronchitis virus interference with growth of Newcastle disease virus. I. Study of interference in chicken embryos. Avian Dis.7:106-122; 1963. 23. Callison SA, Hilt DA, Boynton TO, Sample BF, Robison R, Swayne DE, Jackwood MW. Development and evaluation of a real-time Taqman rt-PCR assay for the detection of infectious bronchitis virus from infected chickens. J Virol Methods.138:60-65; 2006. 24. Gelb J, Jr., Jackwood MW. Infectious bronchitis. In: Dufour-Zavala L, Jackwood MW, Lee MD, Lupiani B, Reed WM, Spackman E, Woolcock PR, editors. A laboratory manual for the isolation, identification and characterization of avian pathogens.6th ed. Am Assoc Avian Pathol. p.233-238; 2016. 25. Raggi LG, Lee GG. Infectious bronchitis virus interference with growth of Newcastle disease virus. II. Interference in chickens. Avian Dis.8:471-480; 1964. Example 2: A New Protectotype Approach to Prevent and Control Infections Bronchitis Virus in Poultry Novel strains of infectious bronchitis coronavirus (IBV) continue to emerge from mutation and recombination events. Live attenuated IBV vaccines significantly contribute to perpetuate the problem worldwide. We have developed recombinant vaccine technology to eliminate type- specific live vaccination. We have focused on IBV Arkansas (Ark)-type spike (S) trimeric ectodomain (Se), i.e., S1 subunit + S2 subunit without transmembrane domain and cytoplasmic tail, as both a subunit vaccine and expressed from a recombinant virus vaccine. After having provided proof of principle that vaccination with trimeric Se protein provides effective protection against challenge, we are advancing this technology for use in the poultry industry. We previously reported that Newcastle disease virus (NDV) recombinant LaSota strain (rLS) expressing IBV Ark-type trimeric spike ectodomain (Se) (rLS/ArkSe) provides suboptimal protection against IBV challenge. Then we developed rLS expressing chicken granulocyte-macrophage colony- stimulating factor (GMCSF) and IBV Ark Se in an attempt to enhance vaccine effectiveness. Our results showed that co-expression of GMCSF and the Se from rLS significantly reduced tracheal viral load and tracheal lesions compared to chickens vaccinated with rLS/ArkSe. We further evaluated enhancement of cross-protection of a Massachusetts (Mass) attenuated vaccine by priming or boosting with rLS/ArkSe.GMCSF. Vaccinated chickens were challenged with Ark and protection was evaluated. Results showed that priming or boosting with the recombinant virus significantly increased cross-protection conferred by Mass against Ark virulent challenge. Greatest reductions of viral loads in both trachea and lachrymal fluids were observed in chickens primed with rLS/ArkSe.GMCSF and boosted with Mass. Consistently, Ark Se antibody levels measured with recombinant Ark Se-protein coated ELISA plates 14 days after boost were significantly higher in these chickens. We concluded that a prime and boost strategy using rLS/ArkSe.GMCSF and the world ubiquitous Mass attenuated vaccine provides enhanced cross-protection. Thus, rLS/GMCSF co-expressing the Se of regionally relevant IBV serotypes can be used in combination with live Mass to protect against regionally circulating IBV variant strains. We now demonstrate the option of vaccinating using the Mass vaccine and the rLS/ArkSe.GMCSF simultaneously. Because of well-known interference between IBV and Newcastle disease virus, we increased the dose of the recombinant NDV and reduced the dose of the attenuated Mass vaccine. Experimental design Specific pathogen free (SPF) chickens (n=20/group) were vaccinated ocularly at day 1 of age. Challenge was performed at 21 days of age. Groups and dosages 1) rLS/ArkSe.GMCSF 10⁶ EID₅₀ + Mass (Pfizer) 10³ EID₅₀; challenge with Ark 10³ 2) rLS/ArkSe.GMCSF 10⁷ EID₅₀ + Mass 10³ EID₅₀; challenge with Ark 10³ EID₅₀ 3) Ark (Merck) 10³ EID₅₀; challenge with Ark 10³ EID₅₀ 4) Mass10³ EID₅₀; challenge with Ark 10³ EID₅₀ 5) + Ctr (unvaccinated –challenged) challenge with Ark 10³ EID₅₀ 6) – Ctr (unvaccinated –non-challenged) Results Simultaneous administration of Mass and rLS/ArkSe.GMCSF will provide protection against IBV and enhanced cross-protection against heterologous challenge (Ark type challenge). Protection is being measured at a minimum by viral load in the trachea (by quantitative RT/PCR and virus isolation in embryonated eggs) and by histomorphometry (tracheal mucosal thickness after challenge). Specifically, chickens vaccinated with the combination of rLS/ArkSe.GMCSF and Mass show increased protection compared to Mass only vaccinated chickens. Protection measures in vaccinated groups 1 and 2 are observed to reach the following: NDV antibody production of HI (log2) level of at least 4-6 or higher. Respiratory signs severity scores about 10% or less compared to non-vaccinated challenged controls. Viral load in the trachea (Log10 relative IBV RNA) of about 4 or less, preferably about 2 or less. Viral load in tears (Log₁₀ relative IBV RNA) of about 4 or less, preferably about 2 or less. Mucosal thickness of about 75% or less compared to non-vaccinated challenged controls. Lymphocytic infiltration of about 75% or less compared to non-vaccinated challenged controls. Necrosis of about 75% or less compared to non-vaccinated challenged controls. Antibody against the IBV S ectodomain (Absorbance 650 nm) of about 2-3 or higher at least 14 days after vaccination. Overall protection of at least 50% or higher compared to non-vaccinated challenged controls, preferably 75% or higher. Methods and measurements of protection described herein are measured and performed as described in AVIAN DISEASES 65:364–372, 2021 “Enhanced Protection by Recombinant Newcastle Disease Virus Expressing Infectious Bronchitis Virus Spike Ectodomain and Chicken Granulocyte-Macrophage Colony-Stimulating Factor” Impact Statement The proposed vectored vaccine technology allows rapid development of effective vaccines against divergent IBV strains at low cost. Results demonstrated that adding a cytokine to the construct enhances protection of the recombinant vaccine. We evaluated simultaneous vaccination with live Mass followed and rLS/Ark.Se–GM-CSF to provide adequate cross-protection against Ark challenge. Simultaneous vaccination provide protection against both NDV and IBV and The industry would benefit significantly if vaccination with live Ark-type viruses and possibly other IBV serotypes could be replaced by priming or boosting with tailored rLS.

Claims

CLAIMS What is claimed: 1. A composition comprising: a) a live attenuated IBV vaccine and b) a recombinant Newcastle disease virus LaSota vector (rLS) co-expressing infectious bronchitis virus (IBV) Arkansas (Ark)-type trimeric spike ectodomain (Se) and granulocyte-macrophage colony-stimulating factor (GM-CSF) (rLS/ArkSe.GMCSF).

2. The composition of claim 1, wherein the live attenuated IVB vaccine comprises a IBV Massachusetts (Mass)-type vaccine.

3. The composition of any one of claims 1 or 2, wherein the embryo infectious dose (EID₅₀) of rLS/ArkSe.GMCSF is 1000 to 10000 times more than the EID₅₀ of the live attenuated IBV vaccine in the composition.

4. The composition of claim 3, wherein the EID₅₀ of rLS/ArkSe.GMCSF is at least 10⁶ and the EID₅₀ of the EID₅₀ of the live attenuated IBV vaccine is 10³ per dose of the composition

5. The composition of claim 3, wherein the EID₅₀ of rLS/ArkSe.GMCSF is in the range of 10⁶ to 10⁷ and the EID₅₀ of the live attenuated IBV vaccine is 10³ +/- 10% per dose of the composition

6. A pharmaceutical composition comprising the composition of any of the previous claims and a pharmaceutically acceptable carrier.

7. A vaccine composition comprising the composition of any one of claims 1-5 and a pharmaceutically acceptable carrier.

8. A method of eliciting an immune response against infectious bronchitis virus (IBV), the method comprising: administering an effective amount of the pharmaceutical composition of claim 6 to a subject to elicit an immune response against IBV.

9. A method of vaccinating a subject against infectious bronchitis virus (IBV), the method comprising: administering the pharmaceutical composition of claim 7 to a subject to vaccinate the subject against IBV by generating an immune response to IBV.

10. A method of eliciting an immune response against infectious bronchitis virus (IBV) in a subject, the method comprising administering an effective amount of: a) a live attenuated IBV vaccine and b) a recombinant Newcastle disease virus LaSota vector co-expressing infectious bronchitis virus (IBV) Arkansas (Ark)-type trimeric spike ectodomain and granulocyte- macrophage colony-stimulating factor (GM-CSF) (rLS/ArkSe.GMCSF), to the subject, wherein, a) and b) are administered at the same time.

11. The method of any one of claims 8-10, wherein the immune response comprises generating antibodies to IBV.

12. A method of reducing tracheal damage due to IBV infection, the method comprising administering the composition of any one of claims 1-5 to a subject.

13. The method of claim 12, wherein the composition is administered to the subject prior to exposure to IBV.

14. The method of any one of claims 8-13, wherein the subject is poultry.

15. The method of claim 14, where in the poultry is chicken.

16. The method of any one of claims 8-15, wherein the administration is oral administration of the composition.

17. The method of claim 16, wherein the composition is administered via spraying on or over the subject.

18. The method of claim 16, wherein the composition is provided in the drinking water.

19. The method of any one of claims 8-18, wherein the subject is administered the composition in the first 5 days of life.

20. The method of any one of claims 8-18, wherein the subject is administered the composition only one time.

21. A method of any one of claims 8-20, wherein the subject administered the composition exhibits greater protection against challenge by virulent IBV relative to subjects not administered the composition.