Influenza hemagglutinin proteins and methods of use thereof
Inventors
Marshall, Christopher Patrick • Alff, Peter Joseph • Bertuccioli, Claudio • Yondola, Mark Andrew
Assignees
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Publication Number
US-11129887-B2
Publication Date
2021-09-28
Expiration Date
Abstract
In some embodiments the present invention provides influenza hemagglutinin (“HA”) polypeptides, proteins, and protein complexes that comprise a stalk domain that is engineered to facilitate maintenance of its native trimeric conformation, even if the head domain of the HA protein is removed or disrupted. In some embodiments, the present invention provides compositions comprising such polypeptides, proteins, and protein complexes, and methods of use of such proteins and compositions, for example as vaccine immunogens.
Core Innovation
The disclosure relates to engineered influenza HA immunogens that are folded in native conformation and maintain a trimeric stalk domain comprised of three protomers. The constructs are configured so that the head domain can be removed or disrupted while the stalk remains stabilized in its native trimeric stalk conformation. The disclosure further includes headless influenza HA and head-on influenza HA.
In one aspect, the stalk is stabilized by point mutations to tyrosine at amino acid positions 403, 406, 411, 422, 429, 432, 433, and 435, relative to SEQ ID NO: 1 numbering, and by incorporating one or more di-tyrosine cross-links in the stalk domain. In this configuration, the head domain comprises one or more artificially-introduced protease recognition motifs to enable proteolytically cleaving the head domain.
In another aspect, the disclosure includes head-on influenza HA in which the stalk domain comprises point mutations to tyrosine at specified positions and is subjected to a DT cross-linking reaction to cross-link tyrosine residues in the stalk domain. The disclosure also describes using to-tyrosine mutations for generating antibodies, and using HA analytes comprising the tyrosine point mutations for assaying anti-HA antibody binding or titer.
Claims Coverage
The independent claims cover four inventive areas: producing a headless influenza HA protein complex, producing a head-on engineered influenza HA protein, generating an antibody using an influenza HA immunogen, and assaying binding or titer of an anti-HA antibody using an HA analyte. Across these independent claims, the core inventive features are specified tyrosine-point mutations in the stalk domain and, depending on the method, DT cross-links and/or artificially introduced protease recognition motifs.
Headless influenza HA produced by tyrosine-stalk stabilization and protease motif cleavage
Obtaining an influenza HA protein folded into its native conformation having a head domain and a trimeric stalk domain comprised of three protomers, wherein the stalk domain comprises a point mutation to tyrosine at one or more of amino acid positions 403, 406, 411, 422, 429, 432, 433 and 435 (SEQ ID NO: 1 numbering) and comprises one or more di-tyrosine cross-links, wherein the head domain comprises one or more artificially-introduced protease recognition motifs, and subsequently proteolytically cleaving the head domain at the one or more artificially-introduced protease recognition motifs, thereby producing a headless influenza HA protein complex.
Head-on influenza HA with DT-cross-linked tyrosine-stabilized stalk
Obtaining an influenza HA protein folded into its native conformation having a trimeric stalk domain and a head domain, wherein the stalk domain comprises a point mutation to tyrosine at one or more of amino acid positions 403, 406, 422, 429, 432, 433 and 435 (SEQ ID NO: 1 numbering), and performing a DT cross-linking reaction to cross-link tyrosine residues in the stalk domain, thereby producing an engineered head-on influenza HA protein having a DT-cross-linked stalk domain.
Antibody generation using HA with to-tyrosine stalk mutations
Administering an influenza hemagglutinin (HA) polypeptide, protein or protein complex that comprises a to-tyrosine mutation at one or more of amino acid positions 403, 406, 422, 429, 432, 433, and 435 (SEQ ID NO: 1 numbering) to an animal, wherein the location of amino acid positions 403, 406, 422, 429, 432, 433 and 435 (or corresponding residues) is determined by alignment to SEQ ID NO: 1.
Anti-HA antibody binding or titer assay using HA with tyrosine stalk mutations
Using as an analyte an influenza hemagglutinin (HA) polypeptide, protein or protein complex comprising a point mutation to tyrosine at one or more of amino acid positions 403, 406, 422, 429, 432, 433, and 435 (SEQ ID NO: 1 numbering), wherein the location of amino acid positions 403, 406, 422, 429, 432, 433 and 435 (or corresponding residues) is determined by alignment to SEQ ID NO: 1, for assaying the binding or titer of an anti-HA antibody.
The core claim set centers on engineered influenza HA proteins using specified tyrosine point mutations in the stalk domain, with stabilization achieved either by DT cross-linking for head-on constructs or by combining stalk DT cross-links with artificially introduced protease recognition motifs for producing headless HA protein complexes. The engineered HA forms are then used to generate antibodies and as analytes to assay anti-HA antibody binding or titer.
Stated Advantages
Stabilization of the HA stalk via DT cross-links to maintain or support native-conformation features of the stalk domain.
Generation of conformationally locked headless and “head-on” HA constructs through proteolytic cleavage at artificially introduced protease recognition motifs.
DT stability under physiological conditions as non-hydrolyzable cross-links.
Documented Applications
Making headless influenza HA protein complexes by producing DT-cross-linked, protease-cleavable native-conformation HA followed by proteolytic head removal.
Making engineered “head-on” influenza HA proteins by obtaining native-conformation HA with tyrosine-point mutations and performing DT cross-linking in the stalk domain.
Generating an antibody against an influenza hemagglutinin (HA) polypeptide, protein or protein complex using an influenza HA immunogen comprising the specified to-tyrosine mutations.
Assaying the binding or titer of an anti-HA antibody using an influenza HA analyte comprising the specified point mutations to tyrosine.
Vaccine immunizations and antibody generation using engineered influenza HA immunogens.
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