Heat shock fusion-based vaccine system
Disclosed are self-epitope-containing heat shock fusion proteins, DNA constructs encoding such fusion proteins, and methods of use. More specifically, disclosed are ubiquitin fusion proteins comprising ubiquitin fused to a plurality of identical or non-identical self-epitopes at specified locations. Immunization of an animal with these ubiquitin fusion proteins elicits an immune response to self-antigens present on endogenous proteins. Generation of an immune response to a specified self-antigen is a mechanism to decrease the levels of the endogenous protein below base-line.
1 . A fusion protein comprising a heat shock protein fused to a single epitope-containing segment, the epitope-containing segment comprising two or more identical self-epitopes.
2 . The fusion protein of claim 1 wherein the heat shock protein is ubiquitin and the fusion protein is a ubiquitin fusion protein.
3 . The ubiquitin fusion protein of claim 2 wherein the epitope-containing segment is fused to ubiquitin at a fusion site selected from the group consisting of the N-terminus, the C-terminus and an internal fusion site.
4 . The ubiquitin fusion protein of claim 2 wherein the N-terminal residue of ubiquitin is a residue other than methionine, and the N-terminal residue other than methionine is fused to the C-terminal residue of a second, unmodified ubiquitin protein.
5 . The ubiquitin fusion protein of claim 2 wherein the N-terminal residue of ubiquitin is a residue other than methionine, and the N-terminal residue other than methionine is fused to the C-terminal residue of a C-terminal ubiquitin subdomain competent to specify cleavage by a ubiquitin-specific protease between the C-terminal residue of the C-terminal ubiquitin subdomain and the N-terminal residue other than methionine.
6 . The ubiquitin fusion protein of claim 5 wherein at least one epitope-containing segment is positioned between the C-terminal residue of the C-terminal ubiquitin subdomain and the N-terminal residue other than methionine, and the C-terminus of the C-terminal subdomain is modified to inhibit cleavage by a ubiquitin-specific protease.
7 . The ubiquitin fusion protein of claim 2 which is post-translationally modified by the addition of fatty acids to enhance immunogenicity.
8 . The ubiquitin fusion protein of claim 2 wherein the epitope-containing segment contains from about 2 to about 30 self-epitopes.
9 . The ubiquitin fusion protein of claim 2 wherein the identical self-epitopes are B-cell epitopes.
10 . The ubiquitin fusion protein of claim 2 wherein the identical self-epitopes are T-cell epitopes.
11 . The ubiquitin fusion protein of claim 2 wherein the identical self-epitopes are structural mimics of biomolecules.
12 . The ubiquitin fusion protein of claim 2 wherein the identical self-epitopes represent epitopes from the proteins selected from the group consisting of gonadotropin releasing hormone, tumor necrosis factor, immunoglobulins, chorionic gonadotrophin, inhibin, growth hormones and sperm proteins.
13 . The ubiquitin fusion protein of claim 2 wherein the identical self-epitopes are gonadotropin releasing hormone epitopes.
14 . The ubiquitin fusion protein of claim 13 wherein the epitope-containing segment is comprised of amino acids QHWSYGLRPGQHWSYGLRPG (SEQ ID NO: 26), and is inserted between position 35 and 36 of ubiquitin.
15 . The ubiquitin fusion protein of claim 13 wherein the epitope-containing segment is comprised of amino acids QHWSYGLRPGQHWSYGLRPGQHWSYGLRPGQHWSYGLRPGC (SEQ ID NO: 34) and is fused via its N-terminal amino acid to the C-terminal residue of ubiquitin, the ubiquitin fusion protein being cleavable by a ubiquitin specific protease.
16 . The ubiquitin fusion protein of claim 15 which is further conjugated to an immunogenic carrier protein.
17 . The ubiquitin fusion protein of claim 2 wherein the internal fusion sites comprises a region of ubiquitin linking two domains of secondary structure, the two domains of secondary structure being selected from the group consisting of β-strand and α-helix.
18 . The ubiquitin fusion protein of claim 2 wherein the epitope-containing segment is fused to the C-terminus of ubiquitin and the C-terminus of ubiquitin is modified to inhibit cleavage of the ubiquitin fusion protein by a ubiquitin-specific protease.
19 . The ubiquitin fusion protein of claim 18 wherein the C-terminus of ubiquitin is modified at amino acid 76.
20 . The ubiquitin fusion protein of claim 19 wherein the modification at amino acid 76 of ubiquitin is a substitution of an amino acid selected from the group consisting of alanine, valine, and cysteine for the wild-type glycine amino acid residue.
21 . The ubiquitin fusion protein of claim 20 wherein the substituted amino acid is valine.
22 . The ubiquitin fusion protein of claim 21 wherein the epitope-containing segment comprises the amino acids sequence QHWSYGLRPGQHWSYGLRPGQHWSYGLRPGQHWSYGLRPG (SEQ ID NO: 35).
23 . The ubiquitin fusion protein of claim 22 which is further conjugated to an immunogenic carrier protein.
24 - 116 . (canceled)