CHIMERIC VIRUS-LIKE PARTICLES
Chimeric virus-like particles including gag polypeptides are described. Virus-like particles are generated with a gag polypeptide and lipid raft-associated polypeptide linked to an antigen that is not naturally associated with a lipid raft. Preferred methods of generation include expression in insect cells.
1 . A chimeric virus-like particle comprising:
(a) a gag polypeptide; and
(b) a non-viral lipid raft-associated polypeptide.
2 . The virus-like particle of claim 1 , wherein the lipid raft-associated polypeptide is selected from the group consisting of a GPI anchor polypeptide, a myristoylation sequence polypeptide, a palmitoylation sequence polypeptide, a double acetylation sequence polypeptide, a signal transduction polypeptide, and a membrane trafficking polypeptide.
3 . The virus-like particle of claim 1 , wherein the lipid raft-associated polypeptide is selected from the group consisting of a GPI anchor polypeptide, a myristoylation sequence polypeptide, a palmitoylation sequence polypeptide, a double acetylation sequence polypeptide, a cavelin polypeptide, a flotillin polypeptide, a syntaxin-1 polypeptide, a syntaxin-4 polypeptide, a synapsin I polypeptide, an adducin polypeptide, a VAMP2 polypeptide, a VAMP/synaptobrevin polypeptide, a synaptobrevin II polypeptide, a SNARE polypeptide, a SNAP-25 polypeptide, a SNAP-23 polypeptide, a synaptotagmin I polypeptide, and a synaptotagmin II polypeptide.
4 . The virus-like particle of claim 1 further comprising a hemagglutinin polypeptide.
5 . The virus-like particle of claim 4 further comprising a neuraminidase polypeptide.
6 . A chimeric virus-like particle comprising:
(a) a gag polypeptide; and
(b) a lipid raft-associated polypeptide linked to an antigen to form a linkage, wherein said antigen is not naturally associated with a lipid raft.
7 . The virus-like particle of claim 6 , wherein said linkage is selected from the group consisting of: a covalent bond, an ionic interaction, a hydrogen bond, an ionic bond, a van der Waals force, a metal-ligand interaction, and an antibody-antigen interaction.
8 . The virus-like particle of claim 6 , wherein said linkage is a covalent bond.
9 . The virus-like particle of claim 8 , wherein said covalent bond is selected from the group consisting of: a peptide bond, a carbon-oxygen bond, a carbon-sulfur bond, a carbon-nitrogen bond, a carbon-carbon bond, and a disulfide bond.
10 . The virus-like particle of claim 6 , wherein said lipid raft-associated polypeptide is an integral membrane protein.
11 . The virus-like particle of claim 6 , wherein said lipid raft-associated polypeptide is selected from the group consisting of: a hemagglutinin polypeptide, a neuraminidase polypeptide, a fusion protein polypeptide, a glycoprotein polypeptide, and an envelope protein polypeptide.
12 . The virus-like particle of claim 11 , wherein said lipid raft-associated polypeptide is a hemagglutinin polypeptide.
13 . The virus-like particle of claim 12 further comprising a neuraminidase polypeptide.
14 . The virus-like particle of claim 11 , wherein said lipid raft-associated polypeptide is a neuraminidase polypeptide.
15 . The virus-like particle of claim 14 further comprising a hemagglutinin polypeptide.
16 . The virus-like particle of claim 6 , wherein said antigen is selected from the group consisting of: a protein, a polypeptide, a glycopolypeptide, a lipopolypeptide, a peptide, a polysaccharide, a polysaccharide conjugate, a peptide or non-peptide mimic of a polysaccharide, a small molecule, a lipid, a glycolipid, and a carbohydrate.
17 . The virus-like particle of claim 6 wherein the virus-like particle comprises insect cell glycosylation.
18 . The virus-like particle of claim 6 wherein the virus-like particle comprises mammalian cell glycosylation.
19 . The virus-like particle of claim 6 further comprising a second lipid-raft associated polypeptide.
20 . The virus-like particle of claim 6 , further comprising an adjuvant in admixture with said virus-like particle.
21 . The virus-like particle of claim 20 , wherein said adjuvant is located inside said virus-like particle.
22 . The virus-like particle of claim 21 , wherein said adjuvant is covalently linked to said gag polypeptide to form a covalent linkage.
23 . The virus-like particle of claim 20 , wherein said adjuvant is located outside said virus-like particle.
24 . The virus-like particle of claim 23 , wherein said adjuvant is covalently linked to said lipid raft-associated polypeptide to form a covalent linkage.
25 . The virus-like particle of claim 20 , wherein said adjuvant comprises an adjuvant-active fragment of flagellin.
26 . A chimeric virus-like particle comprising:
(a) a gag polypeptide; and
(b) an RSV lipid raft-associated polypeptide.
27 . The virus-like particle of claim 26 wherein the virus-like particle comprises insect cell glycosylation.
28 . The virus-like particle of claim 26 wherein the virus-like particle comprises mammalian cell glycosylation.
29 . The virus-like particle of claim 26 further comprising a second lipid-raft associated polypeptide.
30 . The virus-like particle of claim 26 further comprising an antigen linked to the RSV lipid raft-associated polypeptide to form a linkage.
31 . The virus-like particle of claim 29 wherein the second lipid-raft associated polypeptide is or is linked to a second RSV antigen.
32 . The virus-like particle of claim 29 wherein the second lipid-raft associated polypeptide is linked to an RSV antigen.
33 . The virus-like particle of claim 26 , further comprising an adjuvant in admixture with said virus-like particle.
34 . A chimeric virus-like particle comprising:
(a) a gag polypeptide; and
(b) an enveloped virus lipid raft-associated polypeptide;
wherein the virus-like particle comprises insect cell glycosylation.
35 . The virus-like particle of claim 34 wherein the enveloped virus lipid raft-associated polypeptide is selected from the group consisting of a Paramyxoviridae and Herpesviridae lipid raft-associated polypeptide.
36 . The virus-like particle of claim 35 wherein the lipid raft-associated polypeptide is selected from the group consisting of parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus, Avian pneumovirus, Human metapneumovirus, herpes simplex virus 1 (HHV-1), herpes simplex virus 2(HHV-2), varicella zoster virus (HHV-3), Epstein Barr virus (HHV-4), and cytomegalovirus (CMV) (HHV-5) lipid raft-associated polypeptides.
37 . The virus-like particle of claim 34 further comprising a second lipid-raft associated polypeptide.
38 . The virus-like particle of claim 34 , further comprising an adjuvant in admixture with said virus-like particle.
39 . A chimeric virus-like particle expression vector system, which comprises:
(a) a first nucleotide sequence encoding a gag polypeptide; and
(b) a second nucleotide sequence encoding a lipid raft-associated polypeptide linked to an antigen, wherein said antigen is not naturally associated with a lipid raft,
wherein upon expression in a cellular host, said polypeptides form a virus-like particle.
40 . The virus-like particle expression vector system of claim 39 , wherein said first and second nucleotide sequences are in a single expression vector.
41 . The virus-like particle expression vector system of claim 40 , wherein said first and second nucleotide sequences are operably linked to a single promoter.
42 . The virus-like particle expression vector system of claim 39 , wherein said first and second nucleotide sequences are in multiple expression vectors.
43 . A method for producing a chimeric virus-like particle, comprising:
(a) providing one or more expression vectors, together which express a gag polypeptide and a lipid raft-associated polypeptide linked to an antigen, wherein said antigen is not naturally associated with a lipid raft;
(b) introducing said one or more expression vectors into a cell; and
(c) expressing said gag polypeptide and said lipid raft-associated polypeptide linked to an antigen to produce said virus-like particle.
44 . The method of claim 43 , further comprising the step of recovering said virus-like particle from the media in which said cell is cultured.
45 . The method of claim 43 , wherein said one or more expression vectors is a viral vector.
46 . The method of claim 45 , wherein said viral vector is selected from the group consisting of: a baculovirus, an adenovirus, a herpesvirus, a poxvirus and a retrovirus.
47 . The method of claim 46 , wherein said viral vector is a baculovirus.
48 . The method of claim 43 , wherein said cell is selected from the group consisting of: an insect cell and a mammalian cell.
49 . The method of claim 48 , wherein said cell is an insect cell.
50 . The method of claim 43 , wherein said one or more expression vectors is a baculovirus and said cell is an insect cell.
51 . The method of claim 43 , further comprising the step of recovering said virus-like particle from the media in which said cell is cultured.
52 . A method for treating or preventing a disease or symptom of the immune system, comprising administering an immunogenic amount of a chimeric virus-like particle, wherein said particle comprises a gag polypeptide and a lipid raft-associated polypeptide linked to an antigen, wherein said antigen is not naturally associated with a lipid raft.
53 . The method of claim 52 , wherein the administering induces a protective immunization response in the subject.
54 . The method of claim 52 , wherein the administering is selected from the group consisting of subcutaneous delivery, transcutaneous delivery, intradermal delivery, subdermal delivery, intramuscularly delivery, peroral delivery, oral delivery, intranasal delivery, buccal delivery, sublinqual delivery, intraperitoneal delivery, intravaginal delivery, anal delivery and intracranial delivery.