Ordered flagellin array as an immunostimulant
Bacterial flagellin protein is modified to improve adjuvant activity.
1. An adjuvant formulation comprising: an ordered array comprising: flagellin protein covalently linked through an unnatural amino acid to a virus-like particle (VLP) wherein the flagellin protein is modified to: (a) comprise the unnatural amino acid at a site corresponding to M1 of SEQ ID NO:1, wherein the unnatural amino acid is selected from homopropargylglycine (HPG), p-acetyl-L-phenylalanine, p-propargyloxyphenylalanine, and p-azido-L-phenylalanine; and (b) methionine residues corresponding to M310 and M466 of SEQ ID NO:1 are substituted with a non-polar amino acid other than methionine; and a pharmaceutically acceptable excipient.
2. The adjuvant formulation of claim 1 , wherein the flagellin protein comprises SEQ ID NO:7.
3. A flagellin protein modified to comprise (a) an unnatural amino acid selected from homopropargylglycine (HPG), p-acetyl-L-phenylalanine, p-propargyloxyphenylalanine, and p-azido-L-phenylalanine at a site corresponding to amino acid residue M1, of SEQ ID NO:1; and (b) methionine residues corresponding to M310 and M466 of SEQ ID NO:1 are substituted with a non-polar amino acid other than methionine.
4. The flagellin protein of claim 3 , wherein the protein comprises a single unnatural amino acid.
5. The flagellin protein of claim 3 , wherein the unnatural amino acid is homopropargylglycine (HPG).
6. A flagellin protein comprising a homopropargylglycine or methionine at the amino acid position corresponding to G239 of SEQ ID NO:1.
7. The flagellin protein of claim 6 , wherein the methionine residues corresponding to M310 and M466 of SEQ ID NO:1 are substituted with a non-polar amino acid other than methionine.
8. The flagellin protein of claim 6 , having the amino acid sequence of SEQ ID NO:3.
9. A flagellin protein modified to comprise
(a) homopropargylglycine (HPG) at the amino acid residue corresponding to M1 of SEQ ID NO:1
(b) an amino acid substitution selected from A2I or A2L; and
(c) methionine residues corresponding to M310 and M466 of SEQ ID NO:1 are substituted with a non-polar amino acid other than methionine.
10. A flagellin protein having the amino acid sequence of SEQ ID NO:7.