Computationally optimized broadly reactive antigens for H3N2, H2N2, and B influenza viruses

1. A recombinant influenza hemagglutinin (HA) polypeptide, comprising:

(i) an amino acid sequence at least 97.7% identical to residues 2-566 of SEQ ID NO: 8;

(ii) an amino acid sequence at least 99.6% identical to residues 2-562 of SEQ ID NO: 1;

(iii) an amino acid sequence at least 99.4% identical to residues 2-562 of SEQ ID NO: 2;

(iv) an amino acid sequence comprising residues 2-562 of SEQ ID NO: 3;

(v) an amino acid sequence at least 99.7% identical to residues 2-562 of SEQ ID NO: 4;

(vi) an amino acid sequence at least 99.6% identical to residues 2-584 of SEQ ID NO: 5;

(vii) an amino acid sequence at least 98.8% identical to residues 2-585 of SEQ ID NO: 6;

(viii) an amino acid sequence comprising residues 2-585 of SEQ ID NO: 7;

(ix) an amino acid sequence at least 98.4% identical to residues 2-566 of SEQ ID NO: 9;

(x) an amino acid sequence at least 97.8% identical to residues 2-566 of SEQ ID NO: 10; or

(xi) an amino acid sequence at least 98.9% identical to residues 2-566 of SEQ ID NO: 11.

2. The influenza HA polypeptide of claim 1 , comprising:

(i) an amino acid sequence at least 97.7% identical to SEQ ID NO: 8;

(ii) an amino acid sequence at least 99.6% identical to SEQ ID NO: 1;

(iii) an amino acid sequence at least 99.4% identical to SEQ ID NO: 2;

(iv) the amino acid sequence of SEQ ID NO: 3;

(v) an amino acid sequence at least 99.7% identical to SEQ ID NO: 4;

(vi) an amino acid sequence at least 99.6% identical to SEQ ID NO: 5;

(vii) an amino acid sequence at least 98.8% identical to SEQ ID NO: 6;

(viii) the amino acid sequence of SEQ ID NO: 7;

(ix) an amino acid sequence at least 98.4% identical to SEQ ID NO: 9;

(x) an amino acid sequence at least 97.8% identical to SEQ ID NO: 10; or

(xi) an amino acid sequence at least 98.9% identical to SEQ ID NO: 11.

3. The influenza HA polypeptide of claim 1 , wherein the amino acid sequence of the polypeptide comprises:

(i) no more than 10 amino acid substitutions relative to SEQ ID NO: 8;

(ii) no more than 2 amino acid substitutions relative to SEQ ID NO: 1;

(iii) no more than 3 amino acid substitutions relative to SEQ ID NO: 2;

(iv) no more than 1 amino acid substitution relative to SEQ ID NO: 4;

(v) no more than 2 amino acid substitutions relative to SEQ ID NO: 5;

(vi) no more than 7 amino acid substitutions relative to SEQ ID NO: 6;

(vii) no more than 9 amino acid substitutions relative to SEQ ID NO: 9;

(viii) no more than 10 amino acid substitutions relative to SEQ ID NO: 10; or

(ix) no more than 6 amino acid substitutions relative to SEQ ID NO: 11.

4. The influenza HA polypeptide of claim 1 , comprising the amino acid sequence of residues 2-566 of SEQ ID NO: 8, residues 2-562 of SEQ ID NO: 1, residues 2-562 of SEQ ID NO: 2, residues 2-562 of SEQ ID NO: 4, residues 2-584 of SEQ ID NO: 5, residues 2-585 of SEQ ID NO: 6, residues 2-566 of SEQ ID NO: 9, residues 2-566 of SEQ ID NO: 10 or residues 2-566 of SEQ ID NO: 11.

5. The influenza HA polypeptide of claim 1 , consisting of the amino acid sequence of residues 2-566 of SEQ ID NO: 8, residues 2-562 of SEQ ID NO: 1, residues 2-562 of SEQ ID NO: 2, residues 2-562 of SEQ ID NO: 3, residues 2-562 of SEQ ID NO: 4, residues 2-584 of SEQ ID NO: 5, residues 2-585 of SEQ ID NO: 6, residues 2-585 of SEQ ID NO: 7, residues 2-566 of SEQ ID NO: 9, residues 2-566 of SEQ ID NO: 10 or residues 2-566 of SEQ ID NO: 11.

6. The influenza HA polypeptide of claim 2 , comprising the amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11.

7. The influenza HA polypeptide of claim 2 , consisting of the amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11.

8. An isolated nucleic acid molecule encoding the influenza HA polypeptide of claim 1 .

9. The isolated nucleic acid molecule of claim 8 , wherein the nucleic acid molecule is codon-optimized for expression in mammalian cells.

10. A vector comprising the nucleic acid molecule of claim 8 .

11. The vector of claim 10 , further comprising a promoter operably linked to the nucleic acid sequence encoding the influenza HA polypeptide.

12. An isolated cell comprising the vector of claim 10 .

13. An influenza virus-like particle (VLP) comprising the influenza HA polypeptide of claim 1 .

14. The influenza VLP of claim 13 , further comprising an influenza neuraminidase (NA) protein, an influenza matrix (M1) protein, or both.

15. An influenza VLP comprising the influenza HA polypeptide of claim 1 , produced by transfecting a host cell with a vector encoding the HA polypeptide, a vector encoding an influenza NA protein and a vector encoding an influenza M1 protein under conditions sufficient to allow for expression of the HA, M1 and NA proteins.

16. A fusion protein comprising the influenza HA polypeptide of claim 1 .

17. A composition comprising the VLP of claim 13 and a pharmaceutically acceptable carrier.

18. A method of eliciting an immune response to influenza virus in a subject, comprising administering the VLP of claim 13 .

19. A method of eliciting an immune response against influenza virus in a subject, comprising administering to the subject a composition comprising the VLP of claim 13 and pharmaceutically acceptable carrier.

20. The method of claim 19 , wherein the composition further comprises an adjuvant.

21. The method of claim 19 , wherein the composition is administered intramuscularly.

22. The method of claim 19 , wherein the composition comprises about 1 to about 25 μg of the VLP.

23. The method of claim 22 , wherein the composition comprises about 15 μg of the VLP.