Recombinant spike ectodomain proteins, compositions, vectors, kits, and methods for immunizing against avian infectious bronchitis virus
Inventors
van Santen, Vicky L. • Toro, Haroldo E.
Assignees
Auburn University • US Department of Agriculture USDA
Publication Number
US-10772953-B2
Publication Date
2020-09-15
Expiration Date
2038-08-09
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Abstract
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 relate to, include, and/or utilize a soluble, multimerized form of the (IBV) spike (S) protein ectodomain. 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.
Core Innovation
The invention discloses recombinant spike ectodomain proteins, compositions, vectors, kits, and methods for inducing an immune response against avian infectious bronchitis virus (IBV). Specifically, it relates to a soluble, multimerized form of the IBV spike (S) protein ectodomain that includes the entire ectodomain portion comprising the S1 subunit and the ectodomain of the S2 subunit. These recombinant proteins and associated compositions and vectors are intended for immunizing poultry to prevent disease associated with IBV infection or to protect poultry from IBV infection.
The problem addressed arises from IBV being the most common cause of respiratory disease in chickens worldwide, despite extensive vaccination programs using type-specific live-attenuated vaccines. IBV exhibits high genetic diversity due to frequent mutations and recombination events, leading to the emergence of numerous antigenically distinct serotypes that do not confer cross-protection. Existing vaccination programs are challenged by IBV's rapid evolution and the possible contribution of attenuated vaccines to the emergence of vaccine-like viral subpopulations with altered phenotypes and increased virulence.
Current vaccines largely use sequences encoding only the S1 subunit of the spike glycoprotein because it mediates viral attachment and induces neutralizing antibodies. However, immunization with S1 alone has been shown to provide inadequate protection. The invention posits and demonstrates that inclusion of the S2 ectodomain with S1 in the recombinant antigen provides improved binding to chicken tissues and induces a more effective protective immune response than S1 alone. The recombinant proteins embody the ectodomain of the spike protein in a multimeric, soluble form, often containing a heterologous multimerization domain, thereby mimicking the native multimeric conformation important for immunogenicity.
Claims Coverage
The patent claims include two independent methods for vaccinating chickens against virulent IBV infection, involving compositions with vectors expressing multimeric recombinant proteins comprising a spike protein ectodomain sequence with specific structural features. The main inventive features revolve around the structure of the recombinant protein and its expression via vectors, particularly viral vectors.
Multimeric recombinant spike ectodomain protein expression by a vector
A method using a vector to express a multimeric recombinant protein comprising an amino acid sequence with the formula Nter-S1-Spacer-S2ecto-MD-Cter, where S1 is the S1 domain of IBV spike protein or variant, S2ecto is the ectodomain of the S2 domain or variant, MD is a heterologous multimerization domain, and Spacer is a sequence lacking the furin cleavage motif Arg-X-(Arg/Lys)-Arg.
Inclusion of a signal peptide facilitating protein translocation
An embodiment where the expressed multimeric recombinant protein includes an N-terminal signal peptide (SP), native or non-native, enabling translocation into the endoplasmic reticulum, represented by Nter-SP-S1-Spacer-S2ecto-MD-Cter.
Spacer sequence composition and cleavage site mutation
Use of a spacer sequence between S1 and S2ecto that is glycine rich with more than 50% glycine residues and that does not comprise the amino acid sequence Arg-X-(Arg/Lys)-Arg to prevent furin cleavage and maintain covalent linkage of S1 and S2ecto domains.
Multimerization domain conferring trimer formation
The multimerization domain (MD) is heterologous to the spike protein and includes a trimerization motif that results in soluble trimers of the recombinant protein in aqueous solution, mimicking the native multimeric structure of the spike.
Recombinant protein soluble and lacking transmembrane and cytoplasmic domains
The recombinant protein lacks the native transmembrane and cytoplasmic domains of the IBV spike protein, rendering it soluble in aqueous solutions and suitable for immunization purposes.
Using a viral vector for in vivo expression of the recombinant protein
Administration of a composition comprising a viral vector engineered to express the multimeric recombinant spike ectodomain protein in chickens, enabling in situ production of immunogenic antigen.
Use of specific sequences with defined identity to SEQ ID NOs
The recombinant protein's S1 domain comprises an amino acid sequence of SEQ ID NO:91 or a variant with at least about 95% sequence identity; the S2 ectodomain comprises SEQ ID NO:93 or a similar variant.
The claims cover methods of vaccinating chickens by administering compositions containing vectors that express a multimeric recombinant spike ectodomain protein with controlled structural features including a modified spacer preventing cleavage, a multimerization domain for trimer formation, optional signal peptides, and soluble forms lacking transmembrane and cytoplasmic domains, delivered by viral vectors to induce protective immunity against IBV infection.
Stated Advantages
The recombinant S-ectodomain protein provides improved binding affinity to chicken tissues compared to S1 protein alone.
Vaccination with the recombinant S-ectodomain protein yields significantly reduced viral loads and improved protection of tracheal integrity as compared to vaccination with S1 protein alone.
Including the S2 domain with S1 in the antigen induces a better protective immune response against IBV infection.
Documented Applications
Immunization of poultry, specifically chickens, against disease associated with infectious bronchitis virus infection.
Vaccination of chickens using compositions comprising recombinant spike ectodomain proteins or vectors expressing these proteins to protect from virulent IBV challenge.
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