JPWO2019191257A5 - - Google Patents

Claims (28)

A method for producing recombinant influenza hemagglutinin (HA) polypeptides containing consensus amino acids:
a. Select more than one influenza HA polypeptide sequence and align the sequence;
b. Calculating a pairwise similarity / dissimilarity matrix;
c. Identifying and creating clusters of similar sequences from the pairwise similarity / dissimilarity matrix;
d. Within each cluster, the pairwise alignment method is used to determine if there is a consensus amino acid for each position in the sequence alignment, where the frequency of amino acids at a given position is 50% or greater. The amino acid is designated as a consensus amino acid, and if the frequency of the amino acid at a given position is less than 50%, the amino acid is designated as a variable amino acid;
e. Generate a first sequence containing consensus amino acids and variable amino acids for each cluster;
f. In some cases, when multiple clusters are analyzed:
i. Aligning the sequence generated in step (e) for each cluster;
ii. The pairwise alignment method is used to determine if there is a consensus amino acid for each position in the sequence alignment, and if the frequency of the amino acid at a given position is 50% or higher, the amino acid is consensus. If designated as an amino acid and the frequency of the amino acid at a given position is less than 50%, then the amino acid is designated as a variable amino acid; as well as iii. By generating a second sequence containing consensus amino acids and variable amino acids, the first sequence generated in the cluster step (e) and the first sequence generated in another cluster or a plurality of clusters can be obtained. To compare;
g. Within the first sequence generated in step (e) or the second sequence generated in step (f) (iii):
i. Generating a series of test sequences based on the first or second sequence, in which the test amino acid is located at a variable amino acid position;
ii. Performing molecular modeling for each of the test sequences;
iii. Determining the consensus amino acid for each variable amino acid position by determining the consensus amino acid for each variable amino acid position by selecting the amino acid (s) that result in the polypeptide having a negative total energy value, as well as h. The method comprising producing a recombinant influenza HA polypeptide comprising the consensus amino acid. The method of claim 1, wherein aligning the sequences comprises using MAFFT, MUSCLE, Clustal OMEGA, FASTA, combinations thereof or any other multi-sequence alignment software package. The method of claim 1 or 2, wherein calculating the pairwise similarity / dissimilarity matrix comprises using a BLOSUM, PAM, IDENTITY substitution matrix or a combination thereof. Identifying and creating clusters of similar sequences from a pairwise similarity / dissimilar matrix uses K-means clustering, minimax clustering, principal component analysis (PCA), multidimensional scaling (MDS), or a combination thereof. The method according to any one of claims 1 to 3, which comprises the above. The method of any one of claims 1-4, wherein molecular modeling comprises comparing to the crystal structure of an influenza HA polypeptide or protein. The method of any one of claims 1-5, wherein the molecular modeling comprises the use of Rosetta or any other molecular modeling software. The method according to any one of claims 1 to 6, wherein the test amino acid comprises any natural or unnatural amino acid found in a protein. A recombinant influenza HA polypeptide produced using the method according to any one of claims 1-7. Amino acid sequences selected from SEQ ID NOs: 1-16 or any one of these fragments, or at least 85%, 90%, 95%, 96%, 97 to any one of SEQ ID NOs: 1-16 or fragments thereof. The recombinant influenza HA polypeptide according to claim 8, comprising an amino acid sequence having%, 98% or 99% identity. A recombinant trimer HA protein comprising one or more of the recombinant HA polypeptides according to claim 8 or 9. An isolated nucleic acid encoding the recombinant HA polypeptide according to claim 8 or 9 or the recombinant trimer HA protein according to claim 10. The vector containing the nucleic acid according to claim 11. An isolated cell comprising the vector according to claim 12. 13. The isolated cell of claim 13, which is a mammalian cell. The isolated cell according to claim 14, which is a HEK293T cell or a CHO cell. 13. The isolated cell of claim 13, which is an insect cell. A fusion protein comprising the recombinant HA polypeptide according to claim 8 or 9 or the recombinant trimer HA protein according to claim 10. An influenza virus-like particle (VLP) comprising the recombinant HA polypeptide according to claim 8 or 9 or the recombinant trimer HA protein according to claim 10. The influenza VLP according to claim 18, further comprising one or more of an influenza neurominidase (NA) protein, an influenza matrix (M1) protein, a human immunodeficiency virus (HIV) gag protein or a combination thereof. The recombinant HA polypeptide according to claim 8 or 9, the recombinant trimer HA protein according to claim 10, the fusion protein according to claim 17, or the influenza VLP and pharmaceutical according to claim 18 or 19. A pharmaceutical composition comprising an acceptable carrier, excipient or adjuvant. 20. The pharmaceutical composition of claim 20, which elicits an immune response against one or more influenza strains, types and / or subtypes. 20. A pharmaceutical composition for use in a method of immunizing a subject against influenza virus, wherein the method comprises administering to the subject an effective amount of the pharmaceutical composition. The pharmaceutical composition. The pharmaceutical composition according to claim 20, which is used in a method for inducing an immune response to an influenza virus in a subject, wherein the method administers an effective amount of the pharmaceutical composition to the subject. The pharmaceutical composition comprising. The pharmaceutical composition according to claim 22 or 23, wherein the influenza virus is a seasonal or pandemic influenza virus. The pharmaceutical composition of claim 23 or 24, wherein the immune response comprises the production of antibodies against one or more influenza virus strains, types or subtypes. The pharmaceutical composition according to any one of claims 22 to 25, wherein the subject is a mammal. The pharmaceutical composition according to claim 26, wherein the subject is a human. The pharmaceutical composition according to any one of claims 22 to 27, wherein administration is performed via an intramuscular, intranasal, intradermal, subcutaneous, oral or intravenous route.