Methods of generating broadly protective vaccine compositions comprising neuraminidase

A method for producing a recombinant influenza neuraminidase (NA) polypeptide comprising consensus amino acids, wherein the method comprises:

• a. selecting more than one influenza NA polypeptide sequence and aligning the sequences;
• b. calculating pairwise similarity/dissimilarity matrices;
• c. identifying and creating clusters of similar sequences from the pairwise similarity/dissimilarity matrices;
• d. within each cluster, determining whether there is a consensus amino acid for each position in the sequence alignment using a pairwise alignment method, wherein if the frequency of the amino acid at a given position is 50% or greater, that amino acid is designated a consensus amino acid, and if the frequency of the amino acid at a given position is less than 50%, that amino acid is designated as a variable amino acid;
• e. generating a first sequence comprising consensus amino acids and variable amino acids for each cluster;
• f. within the first sequence generated in step (e), determining a consensus amino acid for each variable amino acid position, by:
• i. generating a set of test sequences based on the first sequence, wherein test amino acids are placed at the variable amino acid positions;
• ii. performing molecular modeling for each of the test sequences;
• and
• iii. determining a consensus amino acid for each variable amino acid position by selecting amino acid(s) that result in a polypeptide having a negative total energy value and generating a candidate sequence comprising the selected consensus amino acid(s);
• g. producing the recombinant influenza NA polypeptide comprising the candidate sequence, wherein the candidate sequence differs from the more than one influenza NA polypeptide sequences of step (a);
• and
• h. isolating the produced recombinant influenza NA polypeptide.