Antigen delivery platforms
The invention relates to platforms for delivery of herpes virus proteins to cells, particularly proteins that form complexes in vivo. In some embodiments these proteins and the complexes they form elicit potent neutralizing antibodies. Thus, presentation of herpes virus proteins using such platforms permits the generation of broad and potent immune responses useful for vaccine development.
1. A method of forming a protein complex comprising:
delivering to a cell, a self-replicating RNA molecule comprising a polynucleotide that comprises:
a) a first nucleotide sequence encoding a first protein or fragment thereof from cytomegalovirus (CMV), wherein the first nucleotide sequence is operably linked to a first subgenomic promoter and followed by b);
b) a second nucleotide sequence encoding a second protein or fragment thereof from said CMV, wherein the second nucleotide sequence is operably linked to a second subgenomic promoter and followed by c);
c) a third nucleotide sequence encoding a third protein or fragment thereof from said CMV, wherein the third nucleotide sequence is operably linked to a third subgenomic promoter and followed by d);
d) a fourth nucleotide sequence encoding a fourth protein or fragment thereof from said CMV, wherein the fourth nucleotide sequence is operably linked to an IRES or a viral 2A site and followed by e); and
e) a fifth nucleotide sequence encoding a fifth protein or fragment thereof from said CMV, wherein the fifth nucleotide sequence is operably linked to an IRES or a viral 2A site,
wherein the first protein is gH, the second protein is gL, the third protein is UL128, the fourth protein is UL130, and the fifth protein is UL131; and
maintaining the cell under conditions suitable for expression of the self-replicating RNA molecule, wherein the first, second, third, fourth and fifth CMV proteins or fragments thereof are expressed in an amount sufficient for the formation of a gH/gL/UL128/UL130/UL131 pentameric complex.
2. The method of claim 1 , wherein the first protein consists of SEQ ID NO: 32 or a fragment thereof.
3. The method of claim 1 , wherein the second protein consists of SEQ ID NO: 36 or a fragment thereof.
4. The method of claim 1 , wherein the third protein consists of SEQ ID NO: 44 or a fragment thereof.
5. The method of claim 1 , wherein the fourth protein consists of SEQ ID NO: 46 or a fragment thereof.
6. The method of claim 1 , wherein the fifth protein consists of SEQ ID NO: 48 or a fragment thereof.
7. The method of claim 1 , wherein the first protein consists of SEQ ID NO: 32 or a fragment thereof; the second protein consists of SEQ ID NO: 36 or a fragment thereof; the third protein consists of SEQ ID NO: 44 or a fragment thereof; the fourth protein consists of SEQ ID NO: 46 or a fragment thereof; and the fifth protein consists of SEQ ID NO: 48 or a fragment thereof.
8. The method of claim 7 , wherein the self-replicating RNA molecule is encoded by a DNA sequence selected from the group consisting of SEQ ID NO: 56 (vector A526) and SEQ ID NO: 57 (vector A527).
9. The method of claim 1 , wherein the self-replicating RNA molecule is an alphavirus replicon.
10. The method of claim 1 , wherein the first, second and/or third subgenomic promoter comprises SEQ ID NO:51.
11. The method of claim 1 , wherein the IRES, when present, comprises SEQ ID NO:49 or SEQ ID NO:50.
12. The method of claim 1 , wherein the viral 2A site, when present, comprises SEQ ID NO:2.
13. The method of claim 12 , wherein the viral 2A site, when present, comprises SEQ ID NO:3.
14. The method of claim 1 , comprising delivering the self-replicating RNA molecule and an RNA delivery system to the cell.
15. The method of claim 14 , wherein the RNA delivery system is a liposome, a polymeric nanoparticle, a lipid nanoparticle (LNP), an oil-in-water cationic nanoemulsion or combinations thereof.
16. The method of claim 1 , wherein a recombinant DNA molecule encodes the self-replicating RNA molecule.
17. The method of claim 16 , wherein the recombinant DNA molecule is a plasmid.
18. The method of claim 17 , wherein the recombinant DNA molecule comprises a DNA sequence selected from the group consisting of SEQ ID NO: 56 (vector A526) and SEQ ID NO: 57 (vector A527).
19. The method of claim 1 , wherein the cell is in vivo.