USE OF E. COLI SURFACE ANTIGEN 3 SEQUENCES FOR THE EXPORT OF HETEROLOGOUS ANTIGENS
The present invention provides an export signal system based on E. coli CS3 antigen for directing the secretion of foreign antigens from host cells. In particular, the invention describes genetic constructs encoding fusion proteins that contain a CS3 export signal fused to at least one heterologous amino acid sequence. Attenuated microorganisms expressing the fusion proteins and pharmaceutical compositions comprising such attenuated microorganisms are also disclosed.
1 . A genetic construct comprising a promoter operably linked to a nucleic acid encoding a fusion protein, wherein said fusion protein comprises an amino acid sequence from E. coli CS3 protein consisting essentially of an export signal fused to at least one heterologous antigen amino acid sequence.
2 . The genetic construct of claim 1 , wherein the promoter is an inducible promoter.
3 . The genetic construct of claim 2 , wherein the promoter is a Salmonella ssaG promoter.
4 . The genetic construct of claim 1 , wherein the amino acid sequence from E. coli CS3 protein is SEQ ID NO: 8.
5 . The genetic construct of claim 1 , wherein the heterologous antigen amino acid sequence is an amino acid sequence selected from the group consisting of enterotoxigenic E. coli heat stable toxin, enterotoxigenic E. coli heat labile toxin, Chlamydia pmpE, Chlamydia pmpiI, Chlamydia pmpG, Chlamydia htrA, a peptide comprising C. difficile Toxin A C-terminal repeat region, a peptide comprising C. difficile Toxin B C-terminal repeat region, a Hepatitis A antigen, Hepatitis B antigen , Hepatitis C antigen, a Helicobacter antigen, a Herpes Simplex virus antigen, and a human papilloma virus antigen.
6 . The genetic construct of claim 1 , wherein the fusion protein further comprises a linker amino acid sequence positioned between the CS3 export amino acid sequence and the heterologous antigen amino acid sequence.
7 . An attenuated microorganism comprising the genetic construct of claim 1 .
8 . (canceled)
9 . (canceled)
10 . The microorganism of claim 7 , wherein the microorganism is an attenuated Salmonella.
11 . The microorganism of claim 10 , wherein the attenuated Salmonella has a deletion or inactivation of a gene involved in the biosynthesis of aromatic compounds.
12 . (canceled)
13 . The microorganism of claim 10 , wherein the attenuated Salmonella has a deletion or inactivation of a gene encoded on the Salmonella pathogenicity island 2 (SPI-2).
14 . The microorganism of claim 13 , wherein the gene encoded on SPI-2 is ssaV.
15 - 17 . (canceled)
18 . A pharmaceutical composition comprising the microorganism of claim 7 and a pharmaceutically acceptable carrier.
19 - 25 . (canceled)
26 . A method for inducing an immune response in a subject comprising administering the composition of claim 18 to the subject.
27 - 32 . (canceled)
33 . A method for exporting a heterologous antigen from a cell comprising expressing in the cell a genetic construct encoding a fusion protein, wherein said fusion protein comprises an amino acid sequence from E. coli CS3 protein consisting essentially of an export signal fused to at least one heterologous antigen amino acid sequence.
34 . The method of claim 33 , wherein the fusion protein is expressed from an inducible promoter.
35 . The method of claim 34 , wherein said promoter is inducible in vivo.
36 . The method of claim 34 , wherein the inducible promoter is a Salmonella ssaG promoter.
37 - 45 . (canceled)
46 . The method of claim 33 , wherein the amino acid sequence from E. coli CS3 protein is SEQ ID NO: 8.