Patent Publication Number: US-10772953-B2

Title: Recombinant spike ectodomain proteins, compositions, vectors, kits, and methods for immunizing against avian infectious bronchitis virus

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application claims the benefit under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 62/543,142, filed on Aug. 9, 2017, the content of which is incorporated herein by reference in its entirety. 
    
    
     STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT 
     This invention was made with government support under Grant/Contract No. 2015-68004-23131 awarded by the National Institute of Food &amp; Agriculture (NIFA). The government has certain rights in the invention. 
    
    
     BACKGROUND 
     The present invention relates generally to the field of recombinant proteins, compositions, vectors, kits, and methods for immunizing against coronaviruses. In particular, the invention relates to recombinant infectious bronchitis virus (IBV) spike ectodomain proteins, compositions, vectors, kits, and methods for immunizing avian species against infection by IBV. 
     In the poultry industry avian infectious bronchitis virus (IBV) continues to be the most common contributor to respiratory disease in chicken populations despite worldwide extensive vaccination with a multiplicity of type-specific vaccines. IBV replicates primarily in the upper respiratory tract causing respiratory disease in large chicken populations. IBV&#39;s surface spike (S) glycoprotein is post-translationally cleaved into an S1 subunit (˜550 amino acids) and an S2 subunit (˜600 amino acids) (Lai and Holmes, 2001). Like other coronaviruses, the S1 subunit of the S glycoprotein is responsible for viral attachment to host cells and is important for host protective immune responses as it induces virus neutralizing-antibodies (Cavanagh, 1981, 1983, 1984; Cavanagh and Davis, 1986; Koch et al., 1990; Koch and Kant, 1990; Mockett et al., 1984). Because of the relevance of S1 for the first step of replication (i.e., attachment to cells) and immunological escape, the extensive variation exhibited by The S1 glycoprotein among IBV coronaviruses (Kusters et al., 1987; Kusters et al., 1989b) is likely the most relevant phenotypic characteristic for this virus&#39;s “adaptation” and evolutionary success (Toro et al., 2012b). Genetic diversity among coronaviruses is achieved by high mutation frequency and recombination events (Enjuanes et al., 2000a; Enjuanes et al., 2000b; Lai and Cavanagh, 1997; Stadler et al., 2003). Selection acting on diverse populations results in rapid evolution of the virus and the emergence of antigenically different strains (Toro et al., 2012b). More than 30 different IBV types have been identified during the last 5 decades in the U.S. alone. According to a 2012 review, over 30 different genotypes of IBV are currently affecting chicken populations worldwide (Jackwood, 2012). These have recently been grouped into 6 genotypes divided into 32 lineages (Valastro, V., E. C. Holmes, P. Britton, A. Fusaro, M. W. Jackwood, G. Cattoli, and I. Monne. S1 gene-based phylogeny of infectious bronchitis virus: An attempt to harmonize virus classification. Infect Genet Evol 39:349-364. 2016). Multiple surveillance studies performed in the U.S. have demonstrated that serotypes/genotypes Arkansas (Ark), Massachusetts (Mass), Connecticut (Conn), DE072, and Georgia variants GAV and GA98 are the most prevalent (Jackwood et al., 2005; Nix et al., 2000; Toro et al., 2006). The scientific literature states that the amino acid sequences of the S1 subunit of different IBVs can have as little as 50% amino acid sequence identity (Valastro et al., 2016). However IBV S1 amino acid sequences with even less (˜45%) amino acid sequence identity can be found in GenBank. The amino acid sequences of IBV S2 subunits are more conserved than those of S1 subunits; nevertheless considerable variation is found. One report indicated that IBV S2 subunits exhibited as little as 74% amino acid identity (Toro et al., 2014). A more recent analysis of IBV S2 ectodomain amino acid sequences in GenBank showed some with as little as 70% amino acid sequence identity. 
     Because IBV exists as multiple different serotypes that do not provide for cross-protection after host exposure or vaccination, a multiplicity of serotype-specific IBV vaccines have been developed worldwide. For example, vaccination programs in the U.S. currently comprise mono- or polyvalent vaccines including Mass, Conn, GA98, DE072, and Ark serotypes. In Europe, IBV vaccines commonly include strains belonging to serotypes Mass, UK4/91, D274, and D-1466. However, IBV&#39;s high ability to evolve allows it to consistently circulate in commercial poultry and cause outbreaks of disease in spite of extensive vaccination. In addition, accumulating evidence indicates that attenuated IBV vaccines may also be contributing to the emergence and circulation of vaccine-like viruses in host populations (Toro et al., 2012b; Toro et al., 2012c). Indeed, viral sub-populations differing from the predominant live vaccine population have been shown to emerge during a single passage of attenuated IBV vaccine in chickens (McKinley et al., 2008; van Santen and Toro, 2008). 
     In an effort to understand the mechanisms underlying the emergence of vaccine-like viruses, S1 gene sequences of virus populations of all four IBV Ark-serotype attenuated vaccines commercially available in the U.S. were analyzed before and after replication in chickens (Gallardo et al., 2010; van Santen and Toro, 2008). The results from these analyses demonstrated different degrees of genetic heterogeneity among Ark-derived vaccines prior to inoculation into chickens, ranging from no apparent heterogeneity to heterogeneity in 20 positions in the S gene. In all except one position, nucleotide differences resulted in different amino acids encoded and therefore in phenotypic differences among subpopulations present in the vaccines. Significantly, it has been observed that specific minor subpopulations present in each of the vaccines were rapidly “selected” during a single passage in chickens. Indeed, by 3-days post-ocular vaccination, viral populations with S gene sequences distinct from the vaccine major consensus sequence at 5 to 11 codons were found to predominate in chickens (Gallardo et al., 2010; McKinley et al., 2008; van Santen and Toro, 2008). Thus, the use of attenuated coronavirus vaccines may be contributing to the problem of antigenic variation, and the development of a novel vaccine technology to increase the resistance of chicken populations to IBV and reduce economic losses is essential for the poultry industry. 
     The currently most effective and most widely used IBV vaccination program for broilers utilizes live-attenuated vaccines of various serotypes generated by extensive serial passage in embryonated chicken eggs, applied by eyedrop or spray in the hatchery and often a second time by spray or drinking water in chicken houses at 14-18 days of age. Disadvantages of this type of vaccine include the long time required to produce attenuated vaccine strains of emerging serotypes and the risk of increase in virulence as the vaccine strain circulates among inadequately vaccinated chickens in a flock. 
     The vast majority of efforts to generate vectored vaccines or recombinant subunit vaccines against IBV to overcome the disadvantages of live-attenuated vaccines have utilized sequences encoding the amino terminal portion (S1) of the spike protein. However, here, the inventors have shown that immunization with a larger portion of the spike protein, including the entire ectodomain, provides more effective protection against IBV infection than the S1 domain alone. Importantly, the recombinant proteins disclosed herein may be derived and prepared from any IBV strain in order to generate a more effective antigen for inducing a protective immune response. 
     SUMMARY 
     Disclosed are recombinant spike ectodomain proteins, compositions, vectors, kits, and methods for inducing an immune response against avian infectious bronchitis virus (IBV). In particular, the recombinant proteins, compositions, vectors, kits, and methods may be utilized to immunize poultry against disease associated with IBV infection or to protect poultry from IBV infection. 
     One aspect of the subject matter of this disclosure is a recombinant protein of IBV which may be referred to as a recombinant S protein of IBV that includes the ectodomain portion (S ectodomain) of the native S protein from which the recombinant S protein is derived and that typically does not include any of the non-ectodomain portion of the native S protein. The disclosed recombinant protein may include in succession from N-terminus to C-terminus an amino acid sequence represented by the formula “N ter -S1-S2 ecto -MD-C ter ” where “N ter ” represents the N-terminus of the protein, “S1” represents the S1 domain of a spike protein (S) of IBV (or a variant amino acid sequence thereof), “S2 ecto ” represents the ecto-subdomain portion of the S2 domain of the S protein (or a variant amino acid sequence thereof), “MD” represents a multimerization domain (e.g., a trimerization domain) which is not present in the ectodomain of the S protein, and “C ter ” represents the C-terminus of the protein. Optionally, the disclosed recombinant protein may include in succession from N-terminus to C-terminus an amino acid sequence represented by the formula “N ter -S1-Spacer-S2 ecto -MD-C ter ” where “Spacer” represents a spacer sequence of amino acids between S1 and S2 ecto  which typically does not comprise the amino acid sequence Arg-X-(Arg/Lys)-Arg, which is the consensus cleavage recognition sequence for the furin endoprotease. 
     Optionally, the disclosed recombinant proteins may include an N-terminal leader sequence that facilitates translocation of the protein into the endoplasmic reticulum and may otherwise be referred to as a “signal peptide” (SP). For example, the recombinant proteins may be described as having a formula N ter -SP-S1-Spacer-S2 ecto -MD-C t —, where SP is a signal peptide. 
     Optionally, the disclosed recombinant proteins further may comprise a tag for identifying and/or purifying the recombinant proteins. In some embodiments, the tag is present at the C-terminus and the recombinant protein may be described as having a formula N ter -SP-S1-Spacer-S2 ecto -MD-Tag-C ter . 
     Optionally, the recombinant protein may include one or more additional spacer sequences. In some embodiments, the recombinant protein includes an additional spacer between S2 ecto  and MD and the recombinant protein may be described as having a formula N ter -SP-S1-Spacer1-S2 ecto -Spacer2-MD-C ter . 
     Also disclosed herein are vectors that express the disclosed recombinant protein. For example, the disclosed vectors may comprise a polynucleotide encoding the amino acid sequence of the disclosed recombinant proteins operably linked to a promoter for expressing the polynucleotide and recombinant protein in a suitable cell line or in an animal (e.g. a chicken). Suitable vectors may include, but are not limited to, viral vectors. 
     Also disclosed herein are methods for making the disclosed recombinant proteins. The disclosed methods may include expressing the recombinant proteins in a cell line, for example, via a vector that optionally is a viral vector. The recombinant proteins thus made optionally may be purified, for example, via a protein tag, and further processed or formulated for use (e.g., as part of a vaccine composition). 
     Also disclosed herein are methods for vaccinating a subject against infection by IBV. In some embodiments, the methods include administering to the subject (e.g., a chicken) a composition comprising the recombinant proteins as disclosed herein and a suitable carrier. In other embodiments, the methods include administering to the subject (e.g., a chicken) a composition comprising a vector that expresses the recombinant proteins as disclosed herein and a suitable carrier. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1 . Protein histochemistry demonstrating recombinant IBV Ark-type S1 and S-ectodomain binding in various chicken tissues. AEC+ chromogenic substrate was used to identify bound spike protein as indicated by red staining (arrows). (A) S1 binds to the apical surface of the tracheal epithelium, while S-ectodomain (B) binds to the cilia, tracheal epithelium, and the mucin-containing goblet cell secretory vesicles. (C) S1 protein binding was not identified in the lung. (D) S-ectodomain binding was recognized in the epithelium lining the pulmonary parabronchi and atria. (E) Minimal staining at the apical surface of the nasal mucosal epithelium and mucus glands with S1 protein was observed, whereas S-ectodomain (F) exhibited enhanced staining of the nasal mucosal epithelium and mucus glands. (G) Sparse punctate S1 binding occurred along the apical surface of the choanal submucosal glandular epithelium. (H) Intense S-ectodomain binding of the epithelial apical surface and secretory product in the choanal glands was recognized. (I and J) S-ectodomain binding was detected on the epithelial apex of scattered renal tubules; however, S1 binding was not observed. (K) Multifocal, weak staining at the apical surface of the cecal tonsil intestinal epithelium with S1 protein was observed, whereas S-ectodomain (L) exhibited diffuse enhanced staining of the epithelium. (M and N) There was diffuse, strong staining of the cloacal glands with S-ectodomain, and only scattered, weak staining with the S1 protein. Although recombinant S-ectodomain is twice the molecular weight of recombinant S1 protein, it was used at half the μg/ml concentration. Thus the molar concentration of S-ectodomain was one-fourth that of S1. Thus, the increased binding affinity of S-ectodomain compared to S1 is even greater than it appears. 
         FIG. 2 . Relative IBV RNA in (A) tears and (B) trachea of chickens primed at day 12 of age with adjuvanted trimeric recombinant S1, or S-ectodomain (Se), boosted 21 days later, and challenged with virulent Ark-type IBV 21 days post-boost. Nv/C=non-vaccinated (chickens primed and boosted with the adjuvant with PBS)/challenged. Nv/Nc=non-vaccinated/non-challenged. Relative IBV RNA levels determined 5 days post-challenge by qRT-PCR. Lines indicate median log 10  relative RNA copy numbers, boxes indicate 25 th  to 75 th  percentile, and whiskers indicate minimum and maximum values. Different letters indicate significant differences (P&lt;0.05). Nv/Nc were assigned log 10  values of 0 to be included in the graphs with log scale Y axes. 
         FIG. 3 . Tracheal histomorphometry and histopathology 5 days after virulent IBV Ark challenge in chickens primed with adjuvanted trimeric recombinant S1, or S-ectodomain (Se), boosted 21 days later, and challenged with virulent Ark-type IBV 21 days post-boost. (A) Mucosal thickness and (B) thickness of lymphocytic infiltration by tracheal histomorphometry. (C) Severity of tracheal mucosal necrosis and deciliation scored blindly (1=normal, 2=mild, 3=moderate, 4=marked, 5=severe) for each chicken. In box and whisker plots (A and B), lines indicate the median thickness, the boxes indicate the 25 th  and 75 th  percentiles, and the whiskers indicate minimum and maximum values. In the scatter plot (C), each point indicates the lesion score for an individual chicken and the lines indicate mean scores for each group. Nv/C=non-vaccinated (chickens primed and boosted with the adjuvant with PBS)/challenged. Nv/Nc=non-vaccinated/non-challenged. Different letters indicate significant differences (P&lt;0.05). 
         FIG. 4 . IBV antibodies in chicken sera before prime [11 days of age (DOA)], 3 weeks after prime (32 DOA), 2 weeks after boost (45 DOA) and 5 days post-challenge (59 DOA) determined by ELISA. IBV-specific ELISA (A), and S-ectodomain protein-specific ELISA (B) of chickens primed with recombinant S1, S-ectodomain (Se), boosted 21 days later, and challenged 21 days post-boost. Nv/C=non-vaccinated (chickens primed and boosted with the adjuvant with PBS)/challenged. Nv/Nc=non-vaccinated/non-challenged. Mean absorbance values and SEM are shown. In (B) the error bars are so small that they are obscured by the symbols. The S-ectodomain-immunized group showed significantly higher antibody levels (as measured by optical density) than the S1 protein-immunized, adjuvant only, and non-vaccinated non-challenged groups (P&lt;0.05). Statistically significant differences for each time-point post-vaccination indicated by letters. 
         FIG. 5 . Incidence of respiratory signs 5 days-post challenge with virulent Ark-type IBV. Chickens (NDV.Se/C; N=12/group) were vaccinated with recombinant LaSota NDV expressing IBV Ark-type S-ectodomain protein at 1 and 14 days of age and challenged with virulent Ark-type IBV 18 days later. A non-vaccinated/challenged (NvC) control group was mock-vaccinated with LaSota NDV and challenged. A non-vaccinated/non-challenged (Nv/Nc) control group was mock-vaccinated with LaSota NDV and not challenged. Statistically significant differences (Fisher&#39;s exact test; P&lt;0.05) are indicated by different letters over the bars. 
         FIG. 6 . Relative IBV RNA levels in tears 5 days-post challenge with virulent Ark-type IBV. Chickens (NDV.Se/C; N=12/group) were vaccinated with recombinant LaSota NDV expressing IBV Ark-type S-ectodomain protein at 1 and 14 days of age and challenged with virulent Ark-type IBV 18 days later. A non-vaccinated/challenged (NvC) control group was mock-vaccinated with LaSota NDV and challenged. A non-vaccinated/non-challenged (Nv/Nc) control group was mock-vaccinated with LaSota NDV and not challenged. Lines indicate median log 10  relative RNA copy numbers, boxes indicate 25th to 75th percentile, and whiskers indicate minimum and maximum values. Nv/Nc samples, which had no detectable IBV RNA, were assigned log 10  values of 0. Different letters indicate significant differences (One-way ANOVA and Tukey&#39;s multiple comparison test; P&lt;0.05). 
     
    
    
     DETAILED DESCRIPTION 
     Disclosed are recombinant IBV spike ectodomain proteins, compositions, vectors, kits, and methods for inducing an immune response against avian infectious bronchitis virus (IBV) which may be described herein using definitions as set forth below and throughout the application. 
     Unless otherwise specified or indicated by context, the terms “a,” “an,” and “the,” mean “one or more.” For example, “protein” or “domain” should be interpreted to mean “one or more proteins” and “one or more domains,” respectively. 
     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 depending 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 &gt;10% of the particular term. 
     As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising” in that these latter terms are “open” transitional terms that do not limit claims only to the recited elements succeeding these transitional terms. The term “consisting of,” while encompassed by the term “comprising,” should be interpreted as a “closed” transitional term that limits claims only to the recited elements succeeding this transitional term. The term “consisting essentially of,” while encompassed by the term “comprising,” should be interpreted as a “partially closed” transitional term which permits additional elements succeeding this transitional term, but only if those additional elements do not materially affect the basic and novel characteristics of the claim. 
     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. 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.    
     Infectious Bronchitis Virus (IBV) 
     As used herein infectious bronchitis virus (IBV) refers to an avian bronchitis virus, which is a coronavirus that infects chicken among other avian species and causes the associated disease infectious bronchitis “IB.” As a coronavirus, IBV is an enveloped virus with a positive-sense, single-strand RNA genome having a size of −27.6 kb. The name “coronavirus” is derived from the Latin word “corona” meaning crown or halo, and refers to the characteristic appearance of the virus under electron microscopy, where the virus includes a fringe of large, bulbous surface projections creating an image reminiscent of a crown or halo. This coronal morphology is created by the viral spike protein (S), which is present on the surface of the virus and determines the host tropism for the virus. The S protein comprises an S1 domain and an S2 domain. The S protein of IBV is expressed and cleaved by the endopeptidase furin to generate the S1 subunit and S2 subunit. The S2 subunit transverses the viral membrane and includes an N-terminal ectodomain, a transmembrane domain, and a cytosolic C-terminus. The S1 subunit associates non-covalently with the ectodomain of the S2 subunit. Preferably, the recombinant S proteins disclosed herein include the ectodomain portion of the S protein (i.e., the S1 subunit and the ectodomain portion of the S2 subunit) and do not include the non-ectodomain portion of the S protein (i.e., the non-ectodomain portion of the S2 subunit). 
     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 S1; 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. 
     Polypeptide, Proteins, and Peptides 
     As used herein, polypeptide, proteins, and peptides comprise polymers of amino acids, otherwise referred to as “amino acid sequences.” A polypeptide or protein is typically of length &gt;100 amino acids (Garrett &amp; Grisham, Biochemistry, 2 nd  edition, 1999, Brooks/Cole, 110). A peptide is defined as a short polymer of amino acids, of a length typically of 20 or less amino acids, and more typically of a length of 12 or less amino acids (Garrett &amp; Grisham, Biochemistry, 2 nd  edition, 1999, Brooks/Cole, 110). However, the terms “polypeptide,” “protein,” and “peptide” may be used interchangeably herein. 
     As contemplated herein, a polypeptide, protein, or peptide may be further modified to include non-amino acid moieties. Modifications may include but are not limited to acylation (e.g., O-acylation (esters), N-acylation (amides), S-acylation (thioesters)), acetylation (e.g., the addition of an acetyl group, either at the N-terminus of the protein or at lysine residues), formylation lipoylation (e.g., attachment of a lipoate, a C8 functional group), myristoylation (e.g., attachment of myristate, a C14 saturated acid), palmitoylation (e.g., attachment of palmitate, a C16 saturated acid), alkylation (e.g., the addition of an alkyl group, such as an methyl at a lysine or arginine residue), isoprenylation or prenylation (e.g., the addition of an isoprenoid group such as farnesol or geranylgeraniol), amidation at C-terminus, or glycosylation (e.g., the addition of a glycosyl group to either asparagine, hydroxylysine, serine, or threonine, resulting in a glycoprotein). Additional modifications may include, but are not limited to polysialylation (e.g., the addition of polysialic acid), glypiation (e.g., glycosylphosphatidylinositol (GPI) anchor formation, hydroxylation, iodination (e.g., of thyroid hormones), and phosphorylation (e.g., the addition of a phosphate group, usually to serine, tyrosine, threonine or histidine). In particular, the S protein of IBV generated in eukaryotic cells is heavily glycosylated. In some embodiments, the disclosed recombinant proteins of IBV may be modified to include a non-naturally occurring N-terminal modification such as an acetylation. In some embodiments, the disclosed recombinant proteins of IBV may be modified to include a non-naturally occurring C-terminal modification such as an amidation. 
     The amino acid sequences contemplated herein may include one or more amino acid substitutions relative to a reference amino acid sequence (e.g., relative to any of SEQ ID NOs:1-119). In some cases, these substitutions may be conservative amino acid substitutions relative to the reference amino acid sequence. For example, a variant, mutant, or derivative polypeptide may include conservative amino acid substitutions and/or non-conservative amino acid substitutions relative to a reference polypeptide, which may include but is not limited to any of SEQ ID NOs:1-119. “Conservative amino acid substitutions” are those substitutions that are predicted to interfere least with the properties of the reference polypeptide. In other words, conservative amino acid substitutions substantially conserve the structure and the function of the reference protein. Table 1 provides a list of exemplary conservative amino acid substitutions. 
     
       
         
           
               
               
               
             
               
                   
                 TABLE 1 
               
               
                   
                   
               
               
                   
                 Original 
                 Conservative 
               
               
                   
                 Residue 
                 Substitution 
               
               
                   
                   
               
             
            
               
                   
                 Ala 
                 Gly, Ser 
               
               
                   
                 Arg 
                 His, Lys 
               
               
                   
                 Asn 
                 Asp, Gln, His 
               
               
                   
                 Asp 
                 Asn, Glu 
               
               
                   
                 Cys 
                 Ala, Ser 
               
               
                   
                 Gln 
                 Asn, Glu, His 
               
               
                   
                 Glu 
                 Asp, Gln, His 
               
               
                   
                 Gly 
                 Ala 
               
               
                   
                 His 
                 Asn, Arg, Gln, Glu 
               
               
                   
                 Ile 
                 Leu, Val 
               
               
                   
                 Leu 
                 Ile, Val 
               
               
                   
                 Lys 
                 Arg, Gln, Glu 
               
               
                   
                 Met 
                 Leu, Ile 
               
               
                   
                 Phe 
                 His, Met, Leu, Trp, Tyr 
               
               
                   
                 Ser 
                 Cys, Thr 
               
               
                   
                 Thr 
                 Ser, Val 
               
               
                   
                 Trp 
                 Phe, Tyr 
               
               
                   
                 Tyr 
                 His, Phe, Trp 
               
               
                   
                 Val 
                 Ile, Leu, Thr 
               
               
                   
                   
               
            
           
         
       
     
     Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge or hydrophobicity of the molecule at the site of the substitution, and/or (c) the bulk of the side chain. In contrast, non-conservative amino acid substitutions generally disrupt and/or alter (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge or hydrophobicity of the molecule at the site of the substitution, and/or (c) the bulk of the side chain. 
     A “deletion” refers to a change in a reference amino acid sequence (e.g., any of SEQ ID NOs:1-119) that results in the absence of one or more amino acid residues. A deletion removes at least 1, 2, 3, 4, 5, 10, 20, 50, 100, or 200 amino acids residues or a range of amino acid residues bounded by any of these values (e.g., a deletion of 5-10 amino acids). A deletion may include an internal deletion or a terminal deletion (e.g., an N-terminal truncation or a C-terminal truncation of a reference polypeptide). A “variant” of a reference polypeptide sequence may include a deletion relative to the reference polypeptide sequence. For example, SEQ ID NO:3 (amino acids 18-532) and SEQ ID NO:5 (amino acids 538-1091) include N-terminal deletions and C-terminal deletions relative to reference sequence SEQ ID NO:1 (amino acids 1-1162). 
     The disclosed recombinant IBV S protein may include an N-terminal methionine residue that does not occur naturally in the native amino acid of the protein. For example, the amino acid sequence of recombinant IBV S proteins contemplated herein may include an N-terminal deletion relative to the amino acid sequence of the full-length S protein, and further, may be modified to include an N-terminal methionine residue that is not present in the amino acid sequence of the full-length S protein. For example a recombinant IBV S protein may include a deletion of amino acids 1-17 which are replaced with an N-terminal methionine. 
     The words “insertion” and “addition” refer to changes in an amino acid sequence resulting in the addition of one or more amino acid residues. An insertion or addition may refer to 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, or 200 amino acid residues or a range of amino acid residues bounded by any of these values (e.g., an insertion or addition of 5-10 amino acids). A “variant” of a reference polypeptide sequence may include an insertion or addition relative to the reference polypeptide sequence. 
     A “fusion polypeptide” refers to a polypeptide comprising at the N-terminus, the C-terminus, or at both termini of its amino acid sequence a heterologous amino acid sequence, for example, a heterologous amino acid sequence that extends the half-life of the fusion polypeptide in serum. A “variant” of a reference polypeptide sequence may include a fusion polypeptide comprising the reference polypeptide. In some embodiments, the disclosed recombinant S IBV proteins may be defined as fusion polypeptides that include IBV amino acid sequences optionally fused to non-IBV amino acid sequences heterologous amino acid sequences (i.e., heterologous amino acid sequences). 
     A “fragment” is a portion of an amino acid sequence that is identical in sequence to but shorter in length than a reference sequence (e.g., any of SEQ ID NOs:1-191). A fragment may comprise up to the entire length of the reference sequence, minus at least one amino acid residue. For example, a fragment may comprise from 5 to 1000 contiguous amino acid residues of a reference polypeptide. In some embodiments, a fragment may comprise at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 contiguous amino acid residues of a reference polypeptide; or a fragment may comprise no more than 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 contiguous amino acid residues of a reference polypeptide; or a fragment may comprise a range of contiguous amino acid residues of a reference polypeptide bounded by any of these values (e.g., 400-600 contiguous amino acid residues). Fragments may be preferentially selected from certain regions of a molecule. The term “at least a fragment” encompasses the full-length polypeptide. A “variant” of a reference polypeptide sequence may include a fragment of the reference polypeptide sequence. For example, SEQ ID NO:3 (amino acids 18-532) and SEQ ID NO:5 (amino acids 538-1091) comprise fragments of reference sequence SEQ ID NO:1 (amino acids 1-1162). 
     “Homology” refers to sequence similarity or, interchangeably, sequence identity, between two or more polypeptide sequences. Homology, sequence similarity, and percentage sequence identity may be determined using methods in the art and described herein. 
     The phrases “percent identity” and “% identity,” as applied to polypeptide sequences, refer to the percentage of amino acid residue matches between at least two polypeptide sequences aligned using a standardized algorithm. Methods of polypeptide sequence alignment are well-known. Some alignment methods take into account conservative amino acid substitutions, non-conservative amino acid substitutions, deletions, and/or insertions. Percent identity for amino acid sequences may be determined as understood in the art. (See, e.g., U.S. Pat. No. 7,396,664, which is incorporated herein by reference in its entirety). A suite of commonly used and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) (Altschul, S. F. et al. (1990) J. Mol. Biol. 215:403 410), which is available from several sources, including the NCBI, Bethesda, Md., at its website. The BLAST software suite includes various sequence analysis programs including “blastp,” which is used to align a known amino acid sequence with other amino acids sequences from a variety of databases. 
     Percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence as defined by a particular SEQ ID number, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, at least 600, at least 650, at least 700, at least 750, at least 800, at least 850, at least 900, at least 950, or at least 1000 contiguous amino acid residues of any of SEQ ID NOs:1-119; or a fragment of no more than 15, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, at least 700, at least 750, at least 800, at least 850, at least 900, at least 950, or at least 1000 contiguous amino acid residues of any of SEQ ID NOs:1-119; or over a range bounded by any of these values (e.g., a range of 500-600 amino acid residues). Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured. 
     In some embodiments, a “variant” of a particular polypeptide sequence may be defined as a polypeptide sequence having at least 20% sequence identity to the particular polypeptide sequence over a certain length of one of the polypeptide sequences using blastp with the “BLAST 2 Sequences” tool available at the National Center for Biotechnology Information&#39;s website. (See Tatiana A. Tatusova, Thomas L. Madden (1999), “Blast 2 sequences—a new tool for comparing protein and nucleotide sequences”, FEMS Microbiol Lett. 174:247-250). In other embodiments, a pair of polypeptides may show, for example, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length of one of the polypeptides, or range of percentage identity bounded by any of these values (e.g., range of percentage identity of 80-99%). 
     The disclosed variants and mutants of a reference polypeptide may possess one or more biological activities associated with the reference polypeptide, or alternatively, the disclosed variants and mutants of a reference polypeptide may lack one or more biological activities associated with the reference polypeptide. For example, the disclosed recombinant IBV spike proteins may possess one or more biological activities associated with the wild-type IBV spike protein, or the disclosed recombinant IBV spike proteins may lack one or more biological activities associated with the wild-type IBV spike protein. 
     Polynucleotides, Nucleic Acid, and Nucleic Acid Sequences 
     The terms “polynucleotide,” “nucleic acid” and “nucleic acid sequence” refer to a polymer of DNA or RNA nucleotide of genomic or synthetic origin (which may be single-stranded or double-stranded and may represent the sense or the antisense strand). The polynucleotides contemplated herein may encode and may be utilized to express one or more IBV polypeptides such as the disclosed recombinant spike ectodomain proteins of IBV. 
     The terms “nucleic acid” and “oligonucleotide,” as used herein, refer to polydeoxyribonucleotides (containing 2-deoxy-ribose), polyribonucleotides (containing ribose), and to any other type of polynucleotide that is an N glycoside of a purine or pyrimidine base. As used herein, the terms “A,” “T,” “C”, “G” and “U” refer to adenine, thymine, cytosine, guanine, uracil as a nucleotide base, respectively. There is no intended distinction in length between the terms “nucleic acid,” “oligonucleotide,” and “polynucleotide,” and these terms will be used interchangeably. These terms refer only to the primary structure of the molecule. Thus, these terms include double- and single-stranded DNA, as well as double- and single-stranded RNA. For use in the present invention, an oligonucleotide also can comprise nucleotide analogs in which the base, sugar or phosphate backbone is modified as well as non-purine or non-pyrimidine nucleotide analogs. 
     A “fragment” of a polynucleotide is a portion of a polynucleotide sequence which is identical in sequence to but shorter in length than a reference sequence. A fragment may comprise up to the entire length of the reference sequence, minus at least one nucleotide. For example, a fragment may comprise from 5 to 1000 contiguous nucleotides of a reference polynucleotide. In some embodiments, a fragment may comprise at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 250, or 500 contiguous nucleotides of a reference polynucleotide; in other embodiments a fragment may comprise no more than 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 250, or 500 contiguous nucleotides of a reference polynucleotide; in further embodiments a fragment may comprise a range of contiguous nucleotides of a reference polynucleotide bounded by any of the foregoing values (e.g. a fragment comprising 20-50 contiguous nucleotides of a reference polynucleotide). Fragments may be preferentially selected from certain regions of a molecule. The term “at least a fragment” encompasses the full-length polynucleotide. A “variant,” “mutant,” or “derivative” of a reference polynucleotide sequence may include a fragment of the reference polynucleotide sequence. 
     Regarding polynucleotide sequences, percent identity may be measured over the length of an entire defined polynucleotide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures, or Sequence Listing, may be used to describe a length over which percentage identity may be measured. 
     Regarding polynucleotide sequences, “variant,” “mutant,” or “derivative” may be defined as a nucleic acid sequence having at least 50% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the “BLAST 2 Sequences” tool available at the National Center for Biotechnology Information&#39;s website. (See Tatiana A. Tatusova, Thomas L. Madden (1999), “Blast 2 sequences—a new tool for comparing protein and nucleotide sequences”, FEMS Microbiol Lett. 174:247-250). Such a pair of nucleic acids may show, for example, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length. 
     A “recombinant nucleic acid” is a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques known in the art. The term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid. Frequently, a recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence. Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a cell. 
     The term “promoter” as used herein refers to a cis-acting DNA sequence that directs RNA polymerase and other trans-acting transcription factors to initiate RNA transcription from the DNA or RNA (in an RNA virus) template that includes the cis-acting DNA of RNA sequence. A promoter may be “operably linked” to a coding sequence meaning that the promoter promotes transcription of the coding sequence, for example, as part of a vector. 
     Spike Protein of Infectious Bronchitis Virus 
     Referring now to the spike protein (S) of Infectious Bronchitis Virus (IBV), the S protein is expressed as a polypeptide having a length which typically is ˜1160-1170 amino acids depending on the particular variant of IBV. The S protein is expressed as a type I membrane protein. (See, e.g., Shen et al., Virology, 326 (2004) 288-298; and Winter et al., J. Virol., March 2008, p. 2765-2771; the contents of which are incorporate herein by reference in their entireties). The N-terminal amino sequence of the S protein (i.e., about amino acids 1-17) functions as a leader sequence, which directs the nascent S protein into the lumen of the endoplasmic reticulum (ER) and as such functions as a signal peptide. The signal peptide of the S protein is subsequently cleaved from the S protein to provide the N-terminus of the S1 domain (i.e., between amino acids 17 and 18). The S protein is cleaved again by furin endoprotease at a recognition site (RFRR/S) at about amino acid positions 534-538 to provide the C-terminus for the S1 subunit and to provide the N-terminus of the S2 subunit (i.e., between amino acids 537 and 538 in this example). The S2 subunit of the S protein includes a membrane anchor sequence at about amino acid positions 1096-1115 and a cytosolic portion from about amino acid positions 1116 to the C-terminus (i.e., to about amino acid position 1160-1170). The S1 subunit and the S2 subunit in the lumen of the ER associate together non-covalently to form the mature S protein. The mature S protein comprising the non-covalently associated S1 subunit and S2 subunit self-associates to form a multimeric structure, typically a trimeric structure (i.e., 3×(S1/S2). Subsequently, the S protein is transported to the surface of the cell where the S1 subunits and the N-terminal portions of the S2 subunits are expressed extracellularly and otherwise are referred to as the “ectodomain.” The membrane anchor sequences of the S2 subunits anchor the S protein in the cell membrane whereas and the C-terminal portions of the S2 subunits are expressed intracellularly. 
     As discussed herein and as known in the art, most serotypes of IBV are related to antigenic determinants in the S1 subunit. However, because the S1 subunit and the S2 subunit associate naturally in IBV, it is thought that the S2 subunit may be important in order for the S1 subunit to be presented in its natural state where it can function as a surface protein for recognizing the cellular receptor for IBV. 
     The present inventors have hypothesized that the presence of the S2 domain and the association of the S1 domain with the S2 domain may be important for creating a recombinant antigenic S protein that best mimics the natural, mature S protein to generate a protective immune response against IBV. However, the S1 domain and the S2 domain are not covalently associated in the natural, mature S protein. Furthermore, ideally, an antigenic protein for use in vaccination method against IBV will be soluble in aqueous solution. However, the natural, mature S protein is a membrane-bound protein. Finally, because the natural, mature S protein is multimeric, the present inventors have hypothesized that it may be important that a recombinant antigenic S protein adopt a multimeric structure in order to generate neutralizing antibodies that are an important part of a protective immune response. The presently disclosed recombinant proteins were designed to address these issues. 
     Pharmaceutical Compositions and Vaccines 
     The compositions disclosed herein may include pharmaceutical compositions such as vaccine compositions comprising the presently disclosed recombinant proteins and/or vectors for expressing the presently disclosed recombinant protein, 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. 
     The disclosed compositions may include additional components such as carriers, diluents, excipients, and surfactants, as known in the art. Further, the compositions may include preservatives (e.g., anti-microbial or anti-bacterial agents such as benzalkonium chloride). The compositions also may include buffering agents (e.g., in order to maintain the pH of the composition between 6.5 and 7.5). 
     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), or reducing the effects of the pathogen. 
     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 disclosed herein may be co-administered or sequentially administered with other immunological, antigenic or vaccine or therapeutic compositions, including an adjuvant, or a chemical or biological agent given in combination with an antigen to enhance immunogenicity of the antigen. 
     Adjuvants. The compositions disclosed herein optionally include an adjuvant. The term “adjuvant” refers to a compound or mixture that enhances an immune response. An adjuvant can serve as a tissue depot that slowly releases the antigen and also as a lymphoid system activator that non-specifically enhances the immune response. Examples of adjuvants that may be utilized in the disclosed compositions include but are not limited to, co-polymer adjuvants (e.g., Pluronic L121® brand poloxamer 401, CRL1005, or a low molecular weight co-polymer adjuvant such as Polygen® adjuvant), poly (I:C), R-848 (a Th1-like adjuvant), resiquimod, imiquimod, PAM3CYS, aluminum phosphates (e.g., AlPO 4 ), loxoribine, potentially useful human adjuvants such as BCG (Bacille Calmette-Guerin) and  Corynebacterium parvum , CpG oligodeoxynucleotides (ODN), cholera toxin derived antigens (e.g., CTA1-DD), lipopolysaccharide adjuvants, complete Freund&#39;s adjuvant, incomplete Freund&#39;s adjuvant, saponin (e.g., Quil-A), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions in water (e.g., MF59 available from Novartis Vaccines, Montanide ISA 720, or Montanide ISA 71 VG), keyhole limpet hemocyanins, and dinitrophenol. 
     Prime-Boost Vaccination Regimen 
     As used herein, a “prime-boost vaccination regimen” refers to a regimen in which a subject is administered a first composition and then after a determined period of time (e.g., after about 2, 3, 4, 5, or 6 weeks), 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 disclosed methods may include priming a subject with a first composition by administering the first composition at least one time, allowing a predetermined length of time to pass (e.g., at least about 2, 3, 4, 5, or 6 weeks), and then boosting by administering the same composition or a second, different composition. 
     For example, the methods may include administering a first pharmaceutical composition and optionally may include administering a second pharmaceutical composition to augment or boost an immunogenic response induced by the first pharmaceutical composition. The first and second pharmaceutical compositions may be the same or different. The optionally administered second pharmaceutical composition may be administered prior to, concurrently with, or after administering the first pharmaceutical composition. In some embodiments, the first composition is administered and then the second composition is administered after waiting at least about 2, 3, 4, 5, or 6 weeks. The first composition (and the second composition) may be administered one or more times. 
     Characterization of the Immune Response in Vaccinated Subjects 
     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. Pat. 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, 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 or viremia. 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. 
     Antigens and Dose 
     The compositions disclosed herein optionally may include an antigen (e.g., a recombinant spike ectodomain of IBV) or a plurality of antigens (e.g., different recombinant spike ectodomains of different strains of IBV). A “plurality” or antigens as used herein means “more than one” and may mean more than 1, 2, 3, 4, 5, 10, 25, 50, or 100 antigens. 
     The composition, kits, and methods contain or utilize a protein, polypeptide, peptide, or plurality thereof as an antigen. The compositions, kits, and methods may be utilized to induce an antibody response and/or a cell-mediated response against infection by IBV. 
     Conventional vaccines and methods typically involve administering at least about 3 μg of an antigen per dose to a subject. (See, e.g., Scheifele et al. 2005, Hum. Vaccin. 1:180-186; Evans et al. 2001, Vaccine 19:2080-2091; and Kenney et al., N. Engl. J. Med. 351:2295-2301, the contents of which are incorporated herein by reference in their entireties). However, a dose as low as 1 μg of an antigen per dose to a subject also has been proposed. (See U.S. Pat. No. 6,372,223, the content of which is incorporated herein by reference in its entirety). In some embodiments, the vaccines and methods disclosed herein may include or utilize at least about 1, 2, 3, 4, 5, 10, 15, 20, 25, or 30 μg of an antigen per dose to a subject. 
     Suitable antigens may include polypeptides, peptides, or panels thereof that comprise one or more epitopes of a protein expressed by a pathogen associated with a disease. For example suitable polypeptides, peptides, or panels thereof may comprise one or more epitopes of a protein associated with a pathogen. Suitable polypeptides may comprise the full-length amino acid sequence of a corresponding protein of a pathogen or a fragment thereof. For example, suitable fragments may include 5-2000 amino acids (or from 5-1000, 5-100, 5-50, 5-25, 5-15, 10-2000, 10-1000, 10-50, 10-25, or 10-15 amino acids) and include at least one epitope of the protein from which the fragment is derived. Suitable antigens for the compositions, kits, and methods may include a plurality of peptides derived from a protein of a pathogen. For example, a suitable antigen may comprise a plurality of at least 2, 3, 4, 5, 10, 25, 50, 100, or more different peptides comprising at least about a 10-20 amino acid sequence from a protein of a pathogen. The different peptide antigens may overlap at the N-terminus, the C-terminus, or both termini with at least one other peptide antigen of the composition, for example, by at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. 
     Vectors 
     The term “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” refers to recombinant viral nucleic acid that has been engineered to express a heterologous polypeptide (e.g., a recombinant IBV S protein as disclosed herein). The recombinant viral nucleic acid typically includes cis-acting elements for expression of the heterologous polypeptide. The recombinant viral nucleic acid typically is capable of being packaged into a virus that is capable of infecting a host cell. For example, the recombinant viral nucleic acid may include cis-acting elements for packaging. Preferably, the viral vector is not replication competent, is attenuated, or at least does not cause disease. The viral vector may naturally be non-pathogenic to the host. Alternatively the viral vector may be genetically altered by modern molecular biological methods (e.g., restriction endonuclease and ligase treatment, and rendered less virulent than wild type), typically by deletion of specific genes. For example, the recombinant viral nucleic acid may lack a gene essential for production of infectious or virulent virus. 
     The recombinant viral nucleic acid may function as a vector for the immunogenic IBV S protein by virtue of the recombinant viral nucleic acid containing foreign DNA or RNA. The recombinant viral nucleic acid, packaged in a virus may be introduced into a vaccinee by standard methods for vaccination with live vaccines. A live vaccine of the invention can be administered at, for example, about 10 4  to 10 8  viruses/dose, or 10 6  to 10 9  pfu/dose. Actual dosages of such a vaccine can be readily determined by one of ordinary skill in the field of vaccine technology. 
     Numerous virus species can be used as the recombinant virus vectors for the composition disclosed herein. A preferred recombinant virus vector for a viral vaccine may include a recombinant paramyxovirus. (See, e.g., Bukreyev and Collins, 2008; Bukreyev et al., 2005; DiNapoli et al., 2007; DiNapoli et al., 2009; Ge et al., 2007; Ge et al., 2010; Huang et al., 2003a; Huang et al., 2004; Nakaya et al., 2001; Nayak et al., 2009; Park et al., 2006; Swayne et al., 2003). Other suitable viral vectors may include recombinant adenovirus, herpesvirus, retrovirus, or poxvirus vectors. Coronavirus and influenza virus transgenes have been expressed from replication-defective recombinant adenovirus, and the recombinant adenoviruses have proven to be stable and to induce strong immune responses (Toro et al., 2012a; Toro et al., 2012c; Toro et al., 2007; Toro et al., 2008). 
     Suitable virus species for vectors may include virus species or strains that naturally are not virulent for chickens. Preferred virus species for vectors include lentogenic Newcastle disease strains. Such strains are naturally not virulent, pathogenic, or exhibit only low pathogenicity for chickens. Other vectors used in the poultry industry to vaccinate chickens include herpesvirus of turkeys (HVT). These viruses also are not naturally virulent for chickens and do not need to be modified further in order to reduce their virulence. In particular, a HVT vector vaccine may be utilized for in ovo administration. (See, e.g., Roh et al., Poult. Sci. 2016 May; 95(5):1020-4; the content of which is incorporated herein by reference in its entirety). 
     Codon Optimization 
     The transgene expressed in the vectors disclosed herein may have the native polynucleotide sequence of an IBV gene or may have a polynucleotide sequence that has been modified. For example, the presently disclosed vectors may express polypeptides from polynucleotides that encode the polypeptides where the polynucleotides contain codons that are optimized for expression in a particular host. For example, presently disclosed vectors may include one or more polypeptides from IBV where the encoding polynucleotide sequence is optimized to include codons that are most prevalent in an avian such as a chicken. Codon usage for the chicken genome has been reported. (See Rao et al., DNA Res. 2011 December, 18(6):499-512, which is incorporated herein by reference). Accordingly, a polynucleotide encoding the amino acid sequence of a recombinant protein with the segments N ter -S1-Spacer-S2 ecto -MD-C ter  is contemplated herein wherein the polynucleotide&#39;s nucleic acid sequence has been codon-optimized for expressing these protein segments listed as SEQ ID NOs: 3, 5, 11, 13, 19, 21, 27, 29, 35, 37, 43, 45, 51, 53, 59, 61, 67, 69, 75, 77, 83, 85, 91, 93, 97, 99, 101, 106, 107, 110, 111, 114, 115, 118, 119, or variants thereof in chicken (i.e., codon-optimized based on codon usage for the chicken genome). 
     Recombinant Proteins, Compositions, Vectors, Kits, and Methods for Immunizing Against Avian Infectious Bronchitis Virus 
     Disclosed are recombinant IBV spike ectodomain proteins, compositions, vectors, kits, and methods for inducing an immune response against avian infectious bronchitis virus (IBV). In particular, the compositions, vectors, kits, and methods may be utilized to immunize poultry against disease associated with IBV infection or to protect poultry from IBV infection. 
     One aspect of the subject matter of this disclosure is a recombinant protein of IBV that may be referred to as a recombinant S protein of IBV that includes ectodomain portions of the native S protein from which the recombinant S protein is derived. The disclosed recombinant protein may include in succession from N-terminus to C-terminus an amino acid sequence represented by the formula N ter -S1-Spacer-S2 ecto -MD-C ter  or optionally represented by the formula N ter -S1-S2 ecto -MD-C ter . 
     In the disclosed formula, “N ter ” represents the N-terminus of the protein and “C ter ” represents the C-terminus of the protein. As discussed herein, the N-terminus of the protein may be modified to include a non-naturally occurring moiety, such as an N-terminal acetyl group, and the C-terminus of the protein may be modified to include a non-naturally occurring moiety, such as an amide group. 
     In the disclosed formula, “S1” represents the S1 domain of a spike protein (S) of IBV or a variant thereof. In some embodiments, S1 comprises an amino acid sequence of any of SEQ ID NOs:3, 11, 19, 27, 35, 43, 51, 59, 67, 75, 83, 91, 99, 106, 110, 114, and 118 or an amino acid sequence having at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs:3, 11, 19, 27, 35, 43, 51, 59, 67, 75, 83, 91, 99, 106, 110, 114, and 118. 
     In the disclosed formula, “S2 ecto ” represents the ecto-subdomain portion of the S2 domain of the spike protein (S) or a variant thereof (i.e., the extracellular portion of the S2 domain or a variant thereof). In some embodiments, S2 ecto  comprises an amino acid sequence of any of SEQ ID NOs:5, 13, 21, 29, 37, 45, 53, 61, 69, 77, 85, 93, 101, 107, 111, 115, and 119 or an amino acid sequence having at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs:5, 13, 21, 29, 37, 45, 53, 61, 69, 77, 85, 93, 101, 107, 111, 115, and 119. 
     In the disclosed formula, “Spacer” represents a spacer sequence of amino acids between S1 and S2 ecto . The Spacer typically does not comprise the amino acid sequence Arg-X-(Arg/Lys)-Arg. The amino acid sequence Arg-X-(Arg/Lys)-Arg-Ser is the recognition sequence for the furin endoprotease which cleaves between the arginine and serine residues where the serine residue provides the N-terminus for the S2 subunit. The amino acid sequence Arg-X-(Arg/Lys)-Arg-Ser is normally present in the native amino acid sequence of the S protein between the S1 domain and the S2 domain at about amino acid numbers 534-537 and is cleaved by the furin endoprotease during natural maturation of the S protein as discussed herein. Because the amino acid sequence Arg-X-(Arg/Lys)-Arg is not present in the Spacer of the recombinant protein, S1 and S2 remain covalently linked by the Spacer in the recombinant S protein. In the disclosed recombinant proteins, the native spacer between the S1 domain and the S2 domain may be replaced and/or mutated so as not to contain the amino acid sequence Arg-X-(Arg/Lys)-Arg. 
     In some IBV strains, the S2 ectodomain includes an additional recognition sequence for the furin endoprotease at about amino acid numbers 687-691 (e.g., Arg-Arg-Lys-Arg-Ser). (See, e.g., Yamada et al., J. Virol., September 2009, p. 8744-8758; the content of which is incorporated herein by reference in its entirety). Therefore, preferably in the disclosed recombinant proteins, the recombinant protein does not include the sequence Arg-X-(Arg/Lys)-Arg anywhere in the amino acid sequence of the recombinant protein. For recombinant S proteins derived from strains of IBV that include the additional recognition sequence for the furin endoprotease at about amino acid numbers 687-691, the amino acid sequence of the S2 ectodomain here may be replaced and/or mutated so as not to contain the amino acid sequence Arg-X-(Arg/Lys)-Arg. 
     In some embodiments of the disclosed recombinant proteins, the Spacer sequence may be relatively flexible, for example, so as to permit S1 and S2 ecto  to mimic their natural interaction in the S protein. In some embodiments, the Spacer sequence is of sufficient length to permit the domains to mimic their natural interaction in the S protein. Suitable spacer sequences may include, but are not limited to, amino acid sequences of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids or more, or a range bounded by any of these values (e.g., a spacer of 3-15 amino acids). In some embodiments, the Spacer sequence includes amino acids that provide flexibility to the Spacer and/or are small, neutral amino acids. In some embodiments, the spacer sequence comprises only glycine and/or serine residues or is rich in glycine and/or serine residues. For example, in some embodiments, the spacer sequence comprises at least about 50% glycine and/or serine residues, or at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% glycine and/or serine residues. 
     In the disclosed formula, “MD” represents a multimerization domain that is not present in the ectodomain of the S protein. As such, MD is a heterologous sequence with respect to the S protein. The multimerization domain is an amino acid sequence that self-associates. In some embodiments, the multimerization domain comprises a trimerization motif as known in the art and the recombinant protein forms trimers in aqueous solution. (See, e.g., Kammerer et al., PNAS USA, Sep. 27, 2005, vol. 102, no. 39, pages 13891-13896; Kammerer et al., PLoSONE, August 2012, Volume 7, Issue 8, e43603; Sliepen et al., J. Biol. Chem., Volume 290, Number 12, Mar. 20, 2015, 7436-7442; Alvarez-Cienfuegos et al., Scientific Reports, 6:28643, 1-14; the content of which is incorporated by reference in its entirety). 
     Optionally, the recombinant protein may include one or more additional spacer sequences. In some embodiments, the recombinant protein includes an additional Spacer between S2 ecto  and MD and the recombinant protein may be described as having a formula N ter -SP-S1-Spacer1-S2 ecto -Spacer2-MD-C ter , where Spacer1 and Spacer2 may be the same or different. 
     The disclosed recombinant proteins typically comprise the ectodomain portion of S or a variant thereof. The disclosed recombinant proteins typically do not comprise the transmembrane anchoring domain of the S protein. Similarly, the disclosed recombinant proteins typically do not comprise the cytoplasmic domain of the S protein. 
     The disclosed recombinant proteins typically are not membrane proteins. The disclosed recombinant proteins preferably are soluble in aqueous solution. 
     The disclosed recombinant proteins may include an N-terminal leader sequence that facilitates translocation of the protein into the endoplasmic reticulum and may otherwise be referred to as a “signal peptide.” For example, the recombinant proteins may be described as having a formula N ter -SP-S1-Spacer-S2 ecto -MD-C ter , where SP is a signal peptide. 
     In the disclosed formula, SP may comprise the native signal peptide of the S protein or a variant thereof. In some embodiments, SP comprises an amino acid sequence of any of SEQ ID NOs:2, 10, 18, 26, 34, 42, 50, 58, 66, 74, 82, 90, 105, 109, 113, or 117 or an amino acid sequence having at least about 50%, 60%, 70%, 80%, 90%, or 95% sequence identity to any of SEQ ID NOs: 2, 10, 18, 26, 34, 42, 50, 58, 66, 74, 82, 90, 105, 109, 113, or 117. 
     In the disclosed formula, SP may comprise a non-native signal peptide of the S protein (i.e., a heterologous signal peptide relative to the S protein). Signal peptides are known in the art. The core of the signal peptide contains a long stretch of hydrophobic amino acids (about 5-16 residues long) that has a tendency to form a single alpha-helix and is also referred to as the “h-region.” Signal peptides may begin with a short positively charged stretch of amino acids, which may help to enforce proper topology of the polypeptide during translocation by what is known as the positive-inside rule. Because of its close location to the N-terminus this short positively charged stretch of amino acid is called the “n-region.” At the end of the signal peptide there is typically a stretch of amino acids that is recognized and cleaved by signal peptidase and therefore named the “cleavage site.” A database of signal peptides is provided at Signal Peptide Website. 
     The disclosed recombinant proteins further may comprise a tag for identifying and/or purifying the recombinant proteins. In some embodiments, the tag is present at the C-terminus and the recombinant protein may be described as having a formula N ter -SP-S1-Spacer-S2 ecto -MD-Tag-C ter . Suitable tags may include but are not limited to Streptag (i.e., a sequence comprising SEQ ID NO:102), for example where the recombinant protein may be described as having a formula N ter -SP-S1-Spacer-S2 ecto -MD-Streptag-C ter . Other suitable amino acid tag sequences may include, but are not limited to, a strep tag, poly(His) tag comprising 5-10 histidine residues, chitin binding protein (CBP), maltose binding protein (MBP), and glutathione-S-transferase (GST). 
     Also disclosed herein are polynucleotides that encode the disclosed recombinant proteins. The polynucleotides may include a coding sequence for the recombinant proteins that has been optimized for expression in an avian such as a chicken. 
     Also disclosed herein are vectors that express the disclosed recombinant proteins. For example, the disclosed vectors may comprise a polynucleotide encoding the amino acid sequence of the disclosed recombinant proteins operably linked to a promoter for expressing the polynucleotide and recombinant protein in a suitable cell line. As discussed herein, suitable vectors may include, but are not limited to, viral vectors. 
     Also disclosed herein are methods for making the disclosed recombinant proteins. The disclosed methods may include expressing the recombinant proteins in a cell line, for example, via a vector which optionally is a viral vector, or in a unicellular eukaryotic host. The recombinant proteins thus made optionally may be purified, for example, via a protein tag, and further processed or formulated for use (e.g., as part of a vaccine composition). 
     Also disclosed herein are methods for vaccinating a subject against infection by IBV. In some embodiments, the methods include administering to the subject (e.g., a chicken) a composition comprising the recombinant proteins as disclosed herein and a suitable carrier. In other embodiment, the methods include administering to the subject (e.g., a chicken) a composition comprising a vector that expresses the recombinant proteins as disclosed herein and a suitable carrier. The disclosed methods for vaccinating a subject may include a prime-boost vaccination regimen as known in the art. 
     EXAMPLES 
     The following examples are illustrative and are not intended to limit the disclosed subject matter. 
     Example 1 
     Title—Protection Against Infectious Bronchitis Virus by Spike Ectodomain Subunit Vaccine 
     Reference is made to the manuscript: Fatma Eldemery, Kellye S. Joiner, Haroldo Toro, and Vicky L. van Santen, “Protection against infectious bronchitis virus by spike ectodomain subunit vaccine,” Vaccine 35 (2017), 5864-5871; the content of which is incorporated herein by reference in its entirety. 
     Abstract 
     The avian coronavirus infectious bronchitis virus (IBV) S1 subunit of the spike (S) glycoprotein mediates viral attachment to host cells and the S2 subunit is responsible for membrane fusion. Using IBV Arkansas-type (Ark) S protein histochemistry, we show that extension of S1 with the S2 ectodomain improves binding to chicken tissues. Although the S1 subunit is the major inducer of neutralizing antibodies, vaccination with S1 protein has been shown to confer inadequate protection against challenge. The demonstrated contribution of S2 ectodomain to binding to chicken tissues suggests that vaccination with the ectodomain might improve protection compared to vaccination with S1 alone. Therefore, we immunized chickens with recombinant trimeric soluble IBV Ark-type S1 or S-ectodomain protein produced from codon-optimized constructs in mammalian cells. Chickens were primed at 12 days of age with water-in-oil emulsified S1 or S-ectodomain proteins, and then boosted 21 days later. Challenge was performed with virulent Ark IBV 21 days after boost. Chickens immunized with recombinant S-ectodomain protein showed statistically significantly (P&lt;0.05) reduced viral loads 5 days post-challenge in both tears and tracheas compared to chickens immunized with recombinant S1 protein. Consistent with viral loads, significantly reduced (P&lt;0.05) tracheal mucosal thickness and tracheal lesion scores revealed that recombinant S-ectodomain protein provided improved protection of tracheal integrity compared to S1 protein. These results indicate that the S2 domain has an important role in inducing protective immunity. Thus, including the S2 domain with S1 might be promising for better viral vectored and/or subunit vaccine strategies. 
     1. Introduction 
     Infectious bronchitis virus (IBV) is a highly prevalent coronavirus of chickens that causes economic losses worldwide despite extensive vaccination. Continuous emergence of new virus serotypes results from mutation and recombination followed by selection [1]. Routinely used live-attenuated IBV vaccines, which are affected by the same evolutionary processes, not only result in vaccine-like viruses with increased virulence and persistence [2, 3], but may also contribute genetic material for recombination with other vaccine or wild virus populations. We previously identified five minor vaccine virus subpopulations selected in chickens from Arkansas-Delmarva Poultry Industry (ArkDPI)-derived IBV vaccines, designated components (C) 1-5 [3, 4]. The selection of these viral subpopulations within 3 days post-vaccination suggests they replicate better in chickens than the predominant virus population in the vaccine prior to inoculation [3, 4]. 
     The spike (S) protein of IBV mediates viral entry into host cells [5, 6]. Its S1 subunit mediates viral attachment to host cells and induces virus-neutralizing antibodies that are important for host protective immune responses [7-9]. However, the S1 subunit shows extensive amino acid sequence variability among IBV strains, which leads to the virus&#39;s immunological escape [1, 10, 11]. The S2 subunit of S, responsible for membrane fusion, is more conserved among IBV strains [12]. The N-terminal portion of S2 contains immunodominant regions and a neutralizing epitope and therefore the S2 protein has been suggested for vaccine development [12, 13]. 
     Previous studies indicated that the S1 protein alone does not induce effective protection against IBV challenge. For instance, at least four immunizations with purified S1 glycoprotein were required to induce protection against nephropathogenic N1/62 strain challenge [14]. Similarly, three immunizations with KM91 S1 protein expressed by a recombinant baculovirus produced only 50% protection against virulent nephropathogenic KM91 strain challenge [15]. 
     The S1 subunit of IBV is sufficient for attachment [5, 16-19] and the S2 portion of coronavirus spike proteins has traditionally been considered to play a role only in subsequent entry [20, 21]. However, a role for the S2 ectodomain in binding to cells has been demonstrated for spike proteins of Massachusetts serotype IBVs, i.e. the highly-attenuated Beaudette strain and the virulent M41 strain [22, 23]. In the current study, we evaluated binding of trimeric Ark S-ectodomain compared to trimeric S1 subunit alone to multiple relevant chicken tissues. After confirming improved binding of Ark S-ectodomain, which might be explained by the presence of the S2 ectodomain altering the conformation of S1 and thus increasing its affinity for receptors, or by S2 directly contributing to interaction with receptors or co-receptors, we tested the hypothesis that immunization with recombinant soluble trimeric S-ectodomain provides more effective protection than immunization with trimeric S1 subunit alone. 
     2. Materials and Methods 
     2.1. Genes and Expression Vectors 
     The amino acid sequence of S proteins representing an IBV ArkDPI vaccine subpopulation previously designated C2 (GenBank accession ABY66333) was chosen to produce recombinant proteins. C2 was strongly selected in chickens after vaccination with an ArkDPI-derived attenuated vaccine [3, 4]. Its S1 is almost identical to that of the unattenuated parent ArkDPI isolate [24] and represents the consensus sequence of vaccine subpopulations rapidly positively selected in chickens after vaccination with ArkDPI-derived attenuated vaccines [2-4, 25, 26]. To generate recombinant S1 protein, a human codon-optimized sequence encoding C2 S1 [amino acids (AA) 19-538] was synthesized (GeneArt, Regensburg, Germany) and cloned into the pCD5 vector. To generate recombinant S-ectodomain, a human-codon optimized sequence encoding the C2 S2 ectodomain (S AA 544-1097) was cloned into the pCD5 vector already containing the S1 domain as described [22]. At the S1/S2 border, the furin cleavage site sequence RRSRR was replaced by GGGVP to avoid cleavage of the full length S-ectodomain [22]. These S1 and S-ectodomain-coding sequences were flanked by sequences encoding an N-terminal CD5 signal sequence and sequences encoding C-terminal artificial GCN4 trimerization motif and Strep-tag II for purification and detection of proteins, as described [16]. 
     2.2. Recombinant S Protein Production and Purification 
     Soluble trimeric recombinant S1 and S-ectodomain proteins were produced in human embryonic kidney (HEK) 293T cells as described [16, 22, 27]. In brief, the expression vectors encoding S1 or S-ectodomain were transfected into HEK293T cells and recombinant proteins purified from tissue culture supernatants 6 days post-transfection using Strep-Tactin® Sepharose columns according to the manufacturer&#39;s instructions (IBA GmbH, Göttingen, Germany). The concentration of purified proteins was determined by Qubit® 2.0 fluorometer (Invitrogen, Carlsbad, Calif.). The purified proteins were confirmed and concentrations normalized by electrophoresis in Mini-PROTEAN®TGX Stain-Free™ Precast Gels (Bio-Rad, Hercules, Calif.). 
     2.3. Binding to Tissues by Protein Histochemistry 
     The binding efficiency of S1 and S-ectodomain proteins to tissue sections prepared from healthy specific pathogen free (SPF) 40-day old white leghorn chickens was assessed by protein histochemistry as described [22, 27] with minor modifications: antigen retrieval was conducted at 80° C. for 30 min, Tris buffers were substituted for phosphate buffers, slides were blocked with universal negative serum (Biocare, Pacheco, Calif.) instead of 10% goat serum, and the addition of most reagents and washing steps were performed by an intelliPATH FLX automated slide stainer (Biocare, Pacheco, Calif.). S proteins and 3-amino-9-ethyl-carbazole (AEC+; Dako, Carpinteria, Calif.) were added manually. Briefly, S proteins (100 μg/ml for S1 and 50 μg/ml for S-ectodomain) pre-complexed with Strep-Tactin-HRP (IBA GmbH, Göttingen, Germany) were incubated with deparaffinized and rehydrated tissue sections overnight at 4° C. Bound S protein was visualized with AEC+chromogenic substrate. The tissues were counterstained with hematoxylin and mounted with Lerner AquaMount (Covance, Princeton, N.J.). Images were captured from an Olympus BX41 microscope with an Olympus DP71 12 mp camera. 
     2.4. Protection Trial 
     2.4.1. Chickens 
     White leghorn chickens hatched from SPF eggs (Charles River, North Franklin, Conn.) were maintained in Horsfall-type isolators in biosafety level 2 facilities. Experimental procedures and animal care were performed in compliance with all applicable federal and institutional animal guidelines. Auburn University College of Veterinary Medicine is an Association for Assessment and Accreditation of Laboratory Animal Care-accredited institution. 
     2.4.2. Experimental Design 
     Four groups of chickens (each n=16-17) were used. Chickens were primed at 12 days of age (DOA) by subcutaneous injection in the neck region of 0.2 ml containing 10 μg of S1 (group A) or 20 μg of S-ectodomain protein (group B) emulsified in Montanide™ ISA 71 VG adjuvant (Seppic, Paris, France). Twice the amount of S-ectodomain protein was used because recombinant S-ectodomain is 1.96-times the molecular weight of recombinant S1. Thus, approximately equimolar amounts of protein were administered. Chickens in groups A and B were subsequently boosted with the same adjuvanted protein 21 days later. Control group C (non-vaccinated) was primed and boosted with PBS and the adjuvant, and group D was the unvaccinated/unchallenged control group. Chickens in groups A, B and C were challenged 21 days after boost by ocular and nasal instillation of 10 5  50% embryo infective doses (EID 50 ) of a virulent IBV Ark-type strain (GenBank accession JN861120) previously characterized [28]. Protection was evaluated 5 days post-challenge (DPC) by viral load in tears and tracheas, tracheal histomorphometry, and tracheal histopathology lesion scoring. In addition, antibodies in sera specific for IBV or S protein were determined by ELISA before prime (11 DOA), three weeks after prime (32 DOA), two weeks after boost (45 DOA) and 5 days post-challenge. 
     2.4.3. Viral Load by qRT-PCR 
     Relative IBV RNA levels in tears and tracheas were determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Viral RNA was extracted from individual tear samples using the QIAmp viral RNA mini kit (Qiagen, Valencia, Calif.), and from homogenized tracheas with TriReagent® RNA/DNA/protein isolation reagent (Molecular Research Center, Cincinnati, Ohio) following the manufacturers&#39; protocols. Relative viral RNA concentrations in tear and tracheal samples were determined by TaqMan® qRT-PCR as described [29]. Data were analyzed by one-way analysis of variance (ANOVA) followed by Tukey&#39;s multiple comparisons post-test. 
     2.4.4. Tracheal Histomorphometry and Histopathology 
     Histomorphometry of the tracheal mucosa was evaluated blindly as described [30]. Briefly, formalin-fixed sections of trachea collected from challenged and control birds at 5 days post-challenge 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. The severity of lesions (tracheal deciliation and epithelial necrosis) was scored (1=normal, 2=mild, 3=moderate, 4=marked, 5=severe) and the average of the two scores determined as a lesion score for each chicken. Histomorphometric data were analyzed by one-way ANOVA followed by Tukey&#39;s multiple comparisons post-test. Lesion scores were analyzed by Kruskal-Wallis test followed by Dunn&#39;s multiple comparisons post-test. 
     2.4.5. Antibodies Measured by ELISA 
     IBV-Specific ELISA. 
     IBV-specific ELISA was performed as previously described [31]. Briefly, ELISA plates (Nunc MaxiSorp Immuno Plates; Thermo Scientific) were coated with heat-inactivated IBV (ArkDPI vaccine strain; S AA sequence GenBank # ABY66334) purified as described [31]. Individual chicken sera diluted 1:100 were loaded and plates incubated at 4° C. overnight. IBV-specific IgG was detected using biotinylated monoclonal mouse-anti chicken IgG [(clone G-1) Southern Biotechnology Associates, Inc., Birmingham, Ala.], streptavidin-conjugated HRP (Southern Biotechnology Associates, Inc.) and tetramethylbenzidine (TMB; Invitrogen Corp., Frederick, Md.) HRP substrate. Absorbance at 450 nm was measured with a Powerwave XS (BioTek Instruments, Inc., Winooski, Vt.). 
     S1 and S-Ectodomain Protein-Specific ELISA. 
     ELISA plates (Nunc MaxiSorp Immuno Plates; Thermo Scientific) were coated with 100 μl of 0.25 μg/ml of either recombinant S1 protein or S-ectodomain protein at 4° C. overnight. Plates were drained and blocked with 200 μl of 1% bovine serum albumin and 0.05% Tween 20 in PBS for 1 h at room temperature. Plates were drained and individual chicken sera (diluted 1:100) were loaded and incubated 30 min at room temperature. Plates were washed and antibodies detected using reagents in a commercial IBV ELISA kit (Idexx Laboratories, Inc., Westbrook, Me.) following instructions in the kit. Absorbance at 650 nm was measured with a Powerwave XS. Statistical analyses were performed using one-way ANOVA followed by Tukey&#39;s multiple comparisons test. 
     3. Results 
     3.1. S Binding to Tissues 
     The binding affinity of recombinant S-ectodomain to relevant chicken tissues was compared to that of recombinant S1 protein using protein histochemistry. As seen in  FIG. 1 , the S1 protein bound weakly to the epithelium of trachea, nasal mucosa, choana (not shown), cecal tonsils, and cloaca, and to secretory cells of trachea, nasal mucosa, and choana, while binding was not detected in the lung and kidney. Extension of S1 with S2 subunit ectodomain (S-ectodomain) increased binding affinity to trachea, choana, nasal mucosa, cloaca, and cecal tonsils and enabled binding to lung and kidney. It should be noted that the molar concentration of S-ectodomain used for spike histochemistry was approximately one-fourth that of S1, indicating that the binding affinity of S-ectodomain is much greater than that of S1. 
     3.2. Viral Load 
     Chickens immunized with recombinant S-ectodomain protein showed statistically significant (P&lt;0.05) reductions of viral RNA both in tears and tracheas 5 days post-challenge compared to chickens immunized with recombinant S1 protein or adjuvant alone ( FIG. 2 ). A significant (P&lt;0.05) reduction of the viral RNA in the S1-immunized group compared to mock-vaccinated chickens was detected only in tears. S1-protein immunization did not significantly reduce viral RNA levels in trachea. 
     3.3. Tracheal Histomorphometry and Histopathology 
     Consistent with the viral load results, the S-ectodomain-immunized chickens showed a significant reduction (P&lt;0.05) of tracheal mucosal thickness, lymphocyte infiltration, and lesion severity (tracheal deciliation and epithelial necrosis) 5 days post-challenge compared to recombinant S1 protein alone-immunized and adjuvant-only chickens ( FIG. 3 ). In contrast, no significant differences (P&lt;0.05) were detected between recombinant S1 protein-immunized and adjuvant-only groups. Remarkably, no significant differences in any of the tested tracheal histopathology parameters were detected between chickens immunized with S-ectodomain protein and unvaccinated/unchallenged controls, indicating that immunization with recombinant S-ectodomain protein provided complete protection of tracheal integrity. 
     3.4. Antibodies 
     Chickens immunized with S-ectodomain protein showed significant (P&lt;0.05) increases in IBV-specific antibodies in sera compared to those immunized with S1 protein alone and the non-vaccinated controls before challenge at 32 and 45 DOA, as well as 5 DPC ( FIG. 4A ). However, no significant differences were detected between S1 protein-immunized chickens and non-vaccinated controls. Consistent with IBV-specific antibodies, S-ectodomain protein-specific ELISA also revealed significant differences between the S-ectodomain protein-immunized group and the S1 protein-immunized group at all times post-immunization ( FIG. 4B ). S1 protein-specific ELISA did not indicate any significant differences between the chickens immunized with S-ectodomain protein compared to chickens immunized with S1 protein alone (not shown). Collectively, these results indicate the presence of antibodies directed against S2 and/or 5-ectodomain-specific conformational epitope(s) in chickens immunized with S-ectodomain protein. 
     4. Discussion 
     The evolutionary success of IBV and the problems associated with use of live-attenuated vaccines indicate an urgent need to develop novel vaccines. Alternative approaches such as subunit vaccines or viral-vectored vaccines expressing specific proteins would eliminate emergence of vaccine subpopulations and facilitate the rapid development of effective vaccines against new serotypes. We have demonstrated that trimeric S-ectodomain provides more effective protection than trimeric S1 protein. 
     Comparing the binding of recombinant S1 and S-ectodomain proteins of IBV Ark-type strain revealed that S-ectodomain shows increased binding affinity to chicken tissues including trachea, choana, nasal mucosa, cecal tonsils and cloaca. Interestingly, S1 protein was unable to bind to lung and kidney tissues, which are also target organs for IBV, and required the S2 ectodomain to bind. These results are consistent with reports by others, showing that while the S1 subunit of the embryo- and cell-culture-adapted Beaudette strain is unable to bind to chorioallantoic membrane, the Beaudette S-ectodomain binds efficiently [22]. Furthermore, the extension of the M41 S1 with the M41 S2 ectodomain domain increased binding to chicken trachea [23]. The M41 S1 shows only 77% amino acid sequence identity with the ArkDPI S1 used herein. Thus, the current results confirm these findings for another IBV serotype and additional tissues. Using chimeric S-ectodomain proteins, Promunktod et al. concluded that S2 does not contain an additional independent receptor binding site that would explain its contribution to the affinity of S for receptors [22]. Another possible explanation for improved tissue binding of S-ectodomain is that the S2 subunit is necessary for the S1 protein to adopt a conformation optimal for binding. Structures of trimeric S-ectodomains of other coronaviruses determined by cryo-electron microscopy, e.g. [32, 33], suggest that the trimeric structure is important for the conformation of S1, because the S1 domains of the monomers are interwoven in the trimer. In the recombinant S1 protein used in this study, the artificial trimerization domain immediately follows the S1 domain and could thus artificially constrain the trimeric S1 in a suboptimal conformation. When the S2 ectodomain is included between S1 and the trimerization domain, the trimers might be closer to their normal conformation. However, our unpublished results indicate that a single amino acid change in the S2 domain can reduce the binding of the S-ectodomain (S. Farjana et al., unpublished results). Thus, S2 may influence the conformation of S1 in a more specific way. 
     Most IBV neutralizing antibodies recognize conformational epitopes in S1 [8, 34-36]. Thus, if the S2 ectodomain allows S1 to adopt a conformation optimal for attachment, antibodies generated against this conformation might more effectively neutralize virus than antibodies generated against the suboptimal conformation of S1 adopted in the absence of S2. Therefore, we considered the possibility that extension of recombinant S1 protein with the S2 ectodomain would improve the protection afforded by a subunit vaccine. Indeed, our protection trial results indicated that immunization with trimeric S-ectodomain protein significantly reduces viral loads in tears and trachea, as well as tracheal damage, compared to immunization with trimeric S1 protein. Moreover, there were no significant differences in tracheal damage between chickens immunized with S-ectodomain protein and unvaccinated/unchallenged control chickens, indicating complete protection. Conversely, no significant differences were observed between chickens immunized with S1 protein and the mock-immunized group except for the viral load in tears. This limited protection conferred by S1 protein is in agreement with results of others [14] who found that at least four immunizations with the purified S1 glycoprotein of nephropathogenic N1/62 strain of IBV were necessary to induce protection, even though they used a considerably larger amount of purified S1 antigen (50 μg) for immunization. 
     One possible explanation for improved protection following immunization with S-ectodomain, as already mentioned, is that antibodies produced to S1 in the ectodomain conformation neutralize the challenge virus more effectively than antibodies produced to S1 protein alone. Alternatively, the conserved immunodominant linear neutralizing epitope within S2 [13] might also contribute to improved protection. Although we did not attempt to demonstrate neutralizing antibodies, our ELISA results using both purified IBV and S-ectodomain protein showed a significant increase of antibody level in chickens immunized with S-ectodomain protein compared to those immunized with S1 protein alone, indicating that antibodies to S2 epitopes were generated. Furthermore, a peptide near the amino terminal end of S2 has been shown to induce a protective cell-mediated response [37]. The adjuvant used has been reported to stimulate both antibody and cell-mediated immune responses [38-40]. The addition of the HA2 domain of the influenza hemagglutinin has also been demonstrated to increase the immunogenicity and protective capacity of IBV S1, possibly by increasing thermo stability [41]. 
     The findings that recombinant S-ectodomain protein shows improved binding to cell receptors and elicits improved protection against challenge suggests that the S2 domain has an important role in inducing protective immunity. Thus, including the S2 ectodomain with S1 provides a promising option for a subunit vaccine and expands options for better viral vectored vaccines. 
     Example 2 
     Title—Protection Against Infectious Bronchitis Virus by Spike Ectodomain Protein Expressed from Recombinant Newcastle Disease Virus Vector 
     Abstract 
     We previously demonstrated protection against infectious bronchitis virus (IBV) infection in chickens following subcutaneous vaccination with recombinant soluble trimeric recombinant spike (S)-ectodomain (e) protein. We now demonstrate proof-of-principle that vaccination with this recombinant protein delivered by a recombinant viral vector can also protect chickens against IBV infection. A recombinant LaSota strain Newcastle disease virus (NDV.Se) encoding Ark-type IBV S-ectodomain protein was generated and used to vaccinate chickens at one and again at 14 days of age. Vaccinated chickens challenged with virulent Ark-type IBV18 days after the second vaccination exhibited a lower incidence of respiratory signs and lower viral loads in tears five days post challenge than chickens unvaccinated chickens mock-vaccinated with LaSota NDV not expressing the recombinant protein. 
     Materials and Methods 
     Construction and Characterization of Recombinant LaSota NDV Expressing Soluble Trimeric IBV Ark-Type S-Ectodomain Protein. 
     A chicken-codon-optimized synthetic gene encoding the same recombinant S-ectodomain protein used for previous vaccination experiments described above 1731 was inserted into a full-length LaSota cDNA clone between the phosphoprotein (P) and matrix (M) genes as an additional transcription unit as previously described [12]. The recombinant protein encoded includes an N-terminal CD5 signal sequence, a C-terminal artificial GCN4 trimerization motif, and a C-terminal Strep-tag II for detection of the expressed protein. At the S1/S2 border, the furin cleavage site sequence RRSRR was replaced by GGGVP to avoid cleavage of the full length S-ectodomain [22]. Recombinant virus NDV.Se was rescued, propagated, and characterized as previously described [12]. 
     Vaccination and Challenge. 
     White leghorn chickens hatched from specific-pathogen-free (SPF) eggs were divided into three groups of twelve chickens each and maintained in Horsfall-type isolators in biosafety level 2 facilities. Experimental procedures and animal care were performed in compliance with all applicable federal and institutional animal guidelines. Auburn University College of Veterinary Medicine is an Association for Assessment and Accreditation of Laboratory Animal Care-accredited institution. At one day of age one group of chickens (vaccinated group) was vaccinated ocularly with 10 5  50% embryo infective doses (EID 50 ) recombinant NDV.Se. The other two groups, unvaccinated/challenged and unvaccinated/non-challenged control groups, were mock-vaccinated with LaSota NDV lacking the recombinant IBV S-ectodomain gene. At 14 days of age, vaccinated chickens were boosted ocularly with the same dose of NDV.Se and the control groups were mock-boosted with LaSota NDV. Nineteen days after boosting, chickens in the vaccinated and unvaccinated/challenged group were challenged by ocular and nasal instillation of 10 6  50% embryo infective doses (EID 50 ) of a virulent IBV Ark-type strain (GenBank accession JN861120) previously characterized [28]. 
     Evaluation of Protection. 
     Protection was assessed five days after challenge by respiratory signs and viral load in tears. The presence or absence of tracheal and/or nasal rales was determined for each individual bird without knowledge of treatment group by bringing the head of the bird near the examiner&#39;s ear and listening. RNA was isolated from tears collected five days post challenge from each individual bird and relative number of IBV RNA copy numbers determined as described above 
     Statistical Analysis. 
     The significance of differences in numbers of chickens in each group exhibiting respiratory signs was evaluated using Fisher&#39;s exact test. The significance of differences in relative viral RNA copy numbers in tears among groups was evaluated using log 10  relative copy numbers by one-way ANOVA followed by Tukey&#39;s multiple comparison test. Differences with P&lt;0.05 were considered statistically significant. 
     Results 
     Properties of Recombinant LaSota NDV Expressing Recombinant Soluble Trimeric IBV S-Ectodomain Protein. 
     Recombinant virus NDV.S had an intracerebral pathogenicity index (ICPI) in day-old chickens of 0.0 and exhibited mean death time in embryonating eggs &gt;150 hours, indicating even lower pathogenicity than the already low pathogenicity of the parental LaSota virus. Expression of the recombinant S protein in NDV.S-infected cells in vitro was confirmed by indirect immunofluorescence using sera both from chickens vaccinated with Ark-type live vaccine and chickens vaccinated with recombinant Ark-type S-ectodomain protein (results not shown). Chickens immunized with the recombinant NDV.Se or LaSota NDV did not exhibit any clinical signs. 
     Clinical Signs. 
     Only two of twelve chickens vaccinated with NDV.Se exhibited respiratory and/or nasal rales five days post-challenge with virulent IBV, compared to eight of twelve chickens mock-vaccinated with LaSota NDV ( FIG. 5 ). Fisher&#39;s exact test indicated that this difference in incidence of respiratory signs was statistically significant (P&lt;0.05). No chickens in the unvaccinated/non-challenged control group exhibited respiratory signs. 
     Viral Load in Tears. 
     The relative viral loads in tears five days post challenge, as reflected in relative IBV RNA copy numbers, were significantly lower (P&lt;0.05) in chickens vaccinated with NDV.Se compared to control chickens that had been mock-vaccinated with LaSota NDV and challenged ( FIG. 6 ). IBV RNA was not detected in unvaccinated/non-challenged control chickens. 
     Discussion 
     Our results indicate that it is possible to protect chickens from IBV infection by vaccination with soluble trimeric S-ectodomain protein delivered by a recombinant virus. In this initial experiment, the protection parameters assessed show statistically significant, but modest, protection. It should be noted that this experiment used a ten-fold lower dose of recombinant NDV vaccine virus than used in other vaccination-challenge studies in chickens using LaSota NDV-vectored recombinant vaccines (e.g. [12, 74, 75]). Furthermore, our experiment used a ten-fold higher dose of virulent IBV challenge virus than was used in our study demonstrating remarkable protection following immunization with recombinant S-ectodomain protein administered subcutaneously [73]. Thus there is potential for substantial improvement in degree of protection demonstrated by increasing the dose of recombinant vaccine virus and/or decreasing the challenge virus dose. 
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     In the foregoing description, it will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention. Thus, it should be understood that although the present invention has been illustrated by specific embodiments and optional features, modification and/or variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention. 
     Citations to a number of references are made herein. The cited references are incorporated by reference herein in their entireties. In the event that there is an inconsistency between a definition of a term in the specification as compared to a definition of the term in a cited reference, the term should be interpreted based on the definition in the specification.