Methods of generating broadly protective vaccine compositions comprising neuraminidase
The present disclosure relates to a cluster-based consensus approach for generating recombinant neuraminidase (NA) polypeptides. The disclosure further relates to influenza vaccine compositions comprising the recombinant NA polypeptides.
1 . 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.
2 . The method of claim 1 , wherein aligning the sequences comprises using MAFFT, MUSCLE, CLUSTAL OMEGA, FASTA, a combination thereof, or any other multiple sequence alignment software packages.
3 . The method of claim 1 , wherein calculating the pairwise similarity/dissimilarity matrices comprises using BLOSUM, PAM, IDENTITY substitution matrices, or a combination thereof.
4 . The method of claim 1 , wherein identifying and creating clusters of similar sequences from the pairwise similarity/dissimilarity matrices comprise using K-means clustering, minimax clustering, principle component analysis (PCA), multidimensional scaling (MDS), or a combination thereof.
5 . The method of claim 1 , wherein molecular modeling comprises comparing to a crystal structure of an influenza NA polypeptide or protein.
6 . The method of claim 1 , wherein molecular modeling comprises use of Rosetta or any other molecular modeling software.
7 . The method of claim 1 , wherein the test amino acids comprise any natural or non-natural amino acid found in proteins.
8 . The method of claim 1 , further comprising after step (e) if a plurality of clusters is analyzed:
i. aligning the sequences generated in step (e) for each cluster;
ii. 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; and
iii. generating a second sequence comprising consensus amino acids and variable amino acids,
wherein the consensus amino acid for each variable amino acid position is determined in step (f) within said second sequence generated.
9 . The method of claim 8 , wherein in step (f) (i) the set of test sequences is generated based on the second sequence.