Engineered influenza antigenic polypeptides and immunogenic compositions thereof

A method of engineering a mosaic influenza neuraminidase (NA) polypeptide, comprising:

• aligning NA amino acid sequences from multiple circulating strains of a particular type and/or subtype of influenza virus to generate an alignment;
• identifying positions of amino acids corresponding to epitopes and antigenic regions or defined set of residue positions;
• compiling amino acid residues across the alignment at the identified positions for epitopes and antigenic regions, or defined set of residue positions thereby generating antigenic repertoires;
• defining a set of amino acid sequence patterns within the antigenic repertoires for each epitope and antigenic region, or defined set of residue positions, wherein each amino acid sequence pattern in the set is represented only once;
• selecting one or more sequences based on the defined amino acid sequence patterns in the set for each epitope or antigenic region, or defined set of residue positions, thereby resulting in combinations of selected sequences for epitopes and antigenic regions, or defined set of residue positions across the alignment according to pre-determined criteria;
• and
• inserting selected sequences into corresponding locations in a structural backbone of NA, thereby generating engineered mosaic influenza NA polypeptides, wherein the engineered mosaic influenza NA polypeptides are distinct from naturally circulating strains;
• and
• evaluating each of the engineered mosaic influenza NA polypeptides based on conformational stability and breadth of coverage across naturally occurring strains.