Virus-like particles with high-density coating for inducing the expression of antibodies
The invention relates to a fusion protein comprising a polypeptide of interest, a transmembrane domain and an HIV gag polypeptide, or their functionally equivalent variants. The invention also relates to the polynucleotides, vectors, host cells and virus-like particles expressing or presenting said fusion proteins and to the pharmaceutical, immunogenic or vaccines composition containing said fusion proteins, polynucleotides, vectors, host cells and virus-like particles and their use in human and veterinary medicine.
1. A virus-like particle comprising a fusion protein which comprises from the N- to the C-terminus:
(a) a polypeptide of interest, or a functionally equivalent variant thereof,
(b) a transmembrane domain, or a functionally equivalent variant thereof, and
(c) a HIV gag polypeptide, or a functionally equivalent variant thereof
wherein the HIV gag polypeptide or its functionally equivalent variant lacks myristoylation sequence; or
wherein the transmembrane domain comprises (a) the transmembrane domain of (i) the HIV gp41 polypeptide, (ii) the human CD22 molecule, (iii) the human CD36 molecule, or (iv) the human CD44 molecule; (b) the R696A mutant of HIV-1 Env; or (c) the functionally equivalent variants of (a) and (b).
2. A fusion protein according to claim 1 , which comprises from the N- to the C-terminus:
(a) a polypeptide of interest, or a functionally equivalent variant thereof,
(b) a transmembrane domain, or a functionally equivalent variant thereof, and
(c) an HIV gag polypeptide, or a functionally equivalent variant thereof.
3. A polynucleotide encoding a fusion protein according to claim 2 .
4. The polynucleotide according to claim 3 comprising sequence SEQ ID NO: 9 and/or comprising a sequence which sequence is codon-optimized for expression in human beings, companion or farm animals.
5. A vector comprising a polynucleotide according to claim 3 .
6. A host cell comprising a fusion protein according to claim 2 , a polynucleotide encoding the fusion protein or a vector comprising the polynucleotide.
7. A method for preparing a virus-like particle loaded with a polypeptide of interest comprising the steps of:
(a) expressing in a cell a fusion protein according to claim 2 and
(b) recovering the VLPs from the extracellular medium.
8. A pharmaceutical composition comprising a therapeutically effective amount of the virus-like particle according to claim 1 .
9. An immunogenic or vaccine composition comprising a therapeutically effective amount of the virus-like particle according to claim 1 .
10. The immunogenic or vaccine composition according to claim 9 wherein composition is essentially free from aluminum phosphate.
11. A method for the treatment or prevention of a disease caused by a pathogen or a tumor in a subject, wherein the pathogen or tumor contain the polypeptide of interest or a region thereof, preferably the polypeptide of interest is an HIV polypeptide and the infection is an HIV infection, and more preferably the HIV polypeptide comprises the membrane proximal external region of gp41 or functionally equivalent variant thereof, preferably wherein the subject is previously treated with a conjugate comprising the polypeptide of interest forming part of the fusion protein coupled to a carrier, preferably the carrier is KLH, the method comprising administering to the subject the fusion protein according to claim 2 , a polynucleotide encoding the fusion protein, a vector comprising the polynucleotide, a virus-like particle comprising the fusion protein, or a pharmaceutical, immunogenic or vaccine composition comprising the fusion protein or the virus-like particle.
12. The virus-like particle according to claim 1 , wherein the polypeptide of interest, transmembrane domain and HIV gag polypeptide, or their functionally equivalent variants, are joined by a linker, or a functionally equivalent variant thereof.
13. The virus-like particle according to claim 1 wherein the polypeptide of interest is immunogenic.
14. The virus-like particle according to claim claim 12 wherein the linker is selected from the group consisting of:
(a) a linker comprising glycine residues,
(b) a linker comprising glycine residues and serine residues,
(c) a linker comprising three glycine residues and one serine residue and
(d) a linker comprising sequences SEQ ID NO: 23 or SEQ ID NO: 24.
15. The virus-like particle according to claim 1 wherein the polypeptide of interest comprises at least one polypeptide from a virus, bacteria, fungi, protozoa, parasite, allergen or tumor marker or a functionally equivalent variant thereof.
16. The fusion protein according to claim 15 wherein the polypeptide of interest derives from an HIV env protein or a product resulting from the processing of an HIV env protein, preferably the HIV env protein is HIV gp41.
17. The virus-like particle according to claim 16 wherein the polypeptide of interest comprises from the N-to the C-terminus:
(a) the heptad repeat 2 of the HIV gp41 polypeptide or a functionally equivalent variant thereof, and
(b) the membrane proximal external region of the HIV gp41 polypeptide or functionally equivalent variant thereof.
18. The virus-like particle according to claim 16 comprising sequence SEQ ID NO: 28.