Recombinant polypeptide construct comprising multiple enterotoxigenic Escherichia coli fimbrial subunits
Inventors
Assignees
Publication Number
US-10689422-B2
Publication Date
2020-06-23
Expiration Date
2026-01-10
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Abstract
The inventive subject matter relates to a recombinant polypeptide constructs comprising enterotoxigenic Escherichia coli fimbrial subunits. The recombinant polypeptide constructs comprise multiple subunits to the same or different ETEC fimbrial types. The constructs are useful for inclusion in immunogenic formulations for the induction of immunity against entertoxigenic Escherichia coli. The inventive subject matter also relates to the use of the recombinant polypeptide constructs in induce anti-enterotoxigenic Escherichia coli immunity.
Core Innovation
The invention relates to recombinant polypeptide constructs comprising enterotoxigenic Escherichia coli (ETEC) fimbrial subunits. These constructs include multiple subunits from the same or different ETEC fimbrial types and are designed to be used in immunogenic formulations to induce immunity against ETEC strains. The constructs utilize donor strand complementation to stabilize the fimbrial subunits, thereby enhancing their immunogenicity and stability.
The background describes the problem of ETEC as a principal cause of diarrhea, particularly in young children and travelers, with bacterial adherence mediated by fimbriae playing a key role in pathogenesis. Existing fimbriae are composed of major and minor subunits that are variably immunogenic and susceptible to proteolytic degradation. Natural fimbriae have limited quantities of minor (adhesin) subunits, making immune targeting of the adhesin subunits challenging. There is a need for stable immunogenic constructs that incorporate multiple fimbrial subunits to provide broad protection against diverse ETEC strains.
The invention addresses this problem by providing recombinant constructs that covalently connect major and minor fimbrial subunits derived from one or more fimbrial types via polypeptide linkers. The constructs are stabilized by donor strand complementation where a donor β strand from one subunit binds and stabilizes an adjacent subunit, either derived from the same (homologous) or different (heterologous) subunit of the same fimbrial type. Additionally, N-terminal deletions in some subunits prevent undesirable aggregation and proteolytic cleavage, improving manufacturability and immunogenicity. Multipartite fusion constructs comprising subunits from multiple fimbrial types are also disclosed to provide broad-spectrum protection.
Claims Coverage
The patent includes multiple independent claims covering methods for inducing immune responses using recombinant polypeptide constructs comprising stabilized ETEC fimbrial subunits.
Method of inducing immunity using stabilized recombinant fimbrial subunit constructs
A method involving constructing a recombinant polypeptide subunit vaccine or expression vector encoding ETEC fimbrial minor or major subunits connected via polypeptide linkers, stabilized by donor strand complementation. The construct includes a C-terminal donor β strand derived from a homologous or heterologous major fimbrial subunit, optionally with a histidine tag.
Multipartite fusion of recombinant polypeptide constructs from different fimbrial types
Connecting one or more recombinant polypeptide constructs derived from different ETEC fimbrial types to form multipartite fusion constructs with donor strand complementation stabilization and an optional C-terminal histidine tag.
Use of different expression vectors
The method wherein the recombinant polypeptide construct is expressed using DNA plasmid, viral vector, or bacterial vector expression systems.
Selection of fimbrial subunits from specific ETEC strains
Use of minor or major ETEC fimbrial subunits derived from class 5, CS3, and CS6 strains to compose the recombinant polypeptide construct.
Selection of specific minor and major fimbrial subunits
Use of minor subunits selected from CfaE, CsfD, CsuD, CooD, CsbD, CosD, CsdD, CotD and major subunits from CfaB, CsfA, CsuA1, CsuA2, CooA, CsdA, CosA, CsbA, CotA, CstG, CstH, CssA, and CssB or their immunogenic fragments or derivatives.
Specific polypeptide linkers
Using polypeptide linkers comprising SEQ ID No. 5 or a triglycine sequence between subunits.
Donor β strand length specification
The donor β strand used for stabilization contains 12 to 16 amino acids.
Inclusion of signal peptides
The minor or major fimbrial subunits optionally contain an 18-22 amino acid N-terminal signal peptide.
N-terminal deletion in major subunits
One or more major subunits may contain a deletion of 14 to 18 N-terminal amino acids to prevent undesirable associations.
Specific amino acid and nucleic acid sequences
The minor and major subunits can be selected from specific amino acid (e.g., SEQ ID Nos. 2, 4, 45, 46, 48, 52, 57, 60, 61, 69, 89) and nucleic acid sequences (e.g., SEQ ID Nos. 1, 44, 47, 50, 56, 68, 72) or derivatives thereof.
Routes of administration and mammalian subjects
Immunogenic compositions can be administered via subcutaneous, intradermal, sublingual, intrarectal, transdermal, intramuscular, oral, transcutaneous, or nasal routes to mammals, including humans.
The claims cover methods for inducing immune responses using recombinant polypeptide constructs composed of donor strand complemented ETEC fimbrial minor and major subunits connected via polypeptide linkers. The invention includes multipartite fusion constructs from different fimbrial types, various expression vectors, specific subunit selections, linker sequences, and modifications such as N-terminal deletions and signal peptides, with administration methods for mammals including humans.
Stated Advantages
The recombinant constructs enhance immune recognition of minor fimbrial subunits compared to native fimbriae.
Donor strand complementation stabilizes constructs against proteolytic degradation and misfolding.
Multipartite fusion constructs provide broad-spectrum immunity against multiple ETEC strains.
Deleting N-terminal regions reduces undesirable association and protease cleavage, improving stability and manufacturability.
Fusion constructs elicited higher and broader antibody responses than monomeric constructs or prototypical adhesins, as shown in immunogenicity studies.
Documented Applications
Use of recombinant polypeptide constructs in immunogenic compositions to induce immunity against enterotoxigenic Escherichia coli strains.
Formulations include subunit vaccines and DNA, viral, or bacterial vector-based live vaccines expressing the recombinant polypeptides.
Administered to mammals, including humans, via various routes such as oral, nasal, subcutaneous, intradermal, transdermal, sublingual, intramuscular, or rectal.
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