Surface display of antigens on gram-negative outer membrane vesicles
The present invention relates to vaccine compositions based on Gram-negative outer membrane vesicles displaying antigens of pathogens expressed as part of a fusion protein comprising N-terminal parts of surface expressed lipoproteins of Gram-negative bacteria, and use of such compositions in vaccination. The invention further relates to the fusion lipoproteins comprising N-terminal parts of surface expressed lipoproteins of Gram-negative bacteria and antigens of pathogens fused thereto, DNA constructs and bacterial host cells for expressing these fusion lipoproteins and to methods for producing outer membrane vesicles displaying the fusion lipoproteins.
1. A fusion lipoprotein comprising an N-terminal and a C-terminal fusion partner, wherein:
(a) the N-terminal fusion partner comprises in N- to C-terminal order:
i) a lipidated N-terminal cysteine;
ii) a tether of a surface exposed lipoprotein of a Gram-negative bacterium; and, optionally,
iii) a stretch of at least 5 contiguous amino acids that are located C-terminally of the tether of a surface exposed lipoprotein of a Gram-negative bacterium;
wherein the N-terminal fusion partner causes presentation of the fusion lipoprotein on the extracellular outer membrane surface of a Gram-negative bacterium upon expression therein; and,
(b) the C-terminal fusion partner comprises at least one epitope of an antigen associated with an infectious disease and/or a tumour,
wherein the C-terminal fusion partner does not comprise an amino acid sequence of at least 10 contiguous amino acids from the surface exposed lipoprotein from which the sequence of the N-terminal fusion partner originates; and
wherein the amino acid sequence of the fusion lipoprotein does not occur in nature.
2. The fusion lipoprotein according to claim 1 , wherein the tether is located adjacent to the lipidated N-terminal cysteine.
3. The fusion lipoprotein according to claim 1 , wherein the N-terminal fusion partner comprises an N-terminal fragment from a surface exposed lipoprotein of a Gram-negative bacterium and wherein the fragment causes surface expression of the fusion lipoprotein when expressed in the Gram-negative bacterium.
4. The fusion lipoprotein according to claim 1 , wherein the Gram-negative bacterium is of the genus Neisseria.
5. The fusion lipoprotein according to claim 3 , wherein the surface exposed lipoprotein is selected from the group consisting of fHbp, LpbB, TbpB, NHBA and Ag473.
6. The fusion lipoprotein according to claim 1 , wherein the N-terminal fusion partner comprises at least one of:
a) the amino acid sequence in positions 20-38 of SEQ ID NO: 1;
b) the amino acid sequence in positions 21-61 or positions 21-63 of SEQ ID NO: 2; or,
c) the amino acid sequence in positions 23-51 of SEQ ID NO: 3.
7. The fusion lipoprotein according to claim 6 , wherein the N-terminal fusion partner comprises at least one of:
a) the amino acid sequence in positions 20-50 of SEQ ID NO: 1;
b) the amino acid sequence in positions 21-73 or positions 21-75 of SEQ ID NO: 2; or,
c) the amino acid sequence in positions 23-63 of SEQ ID NO: 3.
8. The fusion lipoprotein according to claim 1 , wherein the C-terminal fusion partner does not comprise a contiguous amino acid sequence of at least 5 amino acids from the surface exposed lipoprotein from which the sequence of the N-terminal fusion partner originates.
9. The fusion lipoprotein according to claim 8 , wherein the C-terminal fusion partner comprises surface exposed epitopes from a proteinaceous antigen of an infectious agent or tumour.
10. The fusion lipoprotein according to claim 9 , wherein the C-terminal fusion partner comprises a surface exposed domain of a surface exposed bacterial protein or lipoprotein.
11. The fusion lipoprotein according to claim 10 , wherein bacterial protein comprises a Borrelia surface lipoprotein selected from the group consisting of OspA, OspB, OspC, OspF, VlsE, BbCRASP1, Vsp1, P35 (BBK32), P37 (BBK50), P39, P66, DpbA and BB017.
12. The fusion lipoprotein according to claim 6 , wherein the Borrelia surface lipoprotein comprises amino acids 29-273 of SEQ ID NO: 4, amino acids 29-273 of SEQ ID NO: 58 or amino acids 136-210 of SEQ ID NO: 59.
13. The fusion lipoprotein according to claim 1 , wherein the N-terminal fusion partner comprises a stretch of at least 10 contiguous amino acids that are located C-terminally of the tether of a surface exposed lipoprotein of a Gram-negative bacterium.