Engineered Listeria and methods of use thereof
The invention provides a bacterium containing a polynucleotide comprising a nucleic acid encoding a heterologous antigen, as well as fusion protein partners. Also provided are vectors for mediating site-specific recombination and vectors comprising removable antibiotic resistance genes.
1. A polynucleotide comprising:
(a) a promoter; and
(b) a nucleic acid operably linked to the promoter, wherein the nucleic acid encodes a fusion protein comprising:
(i) a truncated ActA polypeptide, the sequence of which consists of the first 59 to 379 contiguous amino acid residues of SEQ ID NO: 38 modified by a deletion of one or more of residues 31-60, wherein the first amino acid residue of SEQ ID NO: 38 is valine, or the first amino acid residue of SEQ ID NO: 38 is substituted with methionine; and
(ii) a heterologous antigen.
2. The polynucleotide of claim 1 , wherein the promoter is an actA promoter.
3. The polynucleotide of claim 1 , wherein the deletion comprises deleting residue 47 of SEQ ID NO: 38, and wherein the first amino acid residue of SEQ ID NO: 38 is optionally substituted with methionine.
4. The polynucleotide of claim 1 , wherein the deletion comprises deleting residue 60 of SEQ ID NO: 38, and wherein the first amino acid residue of SEQ ID NO: 38 is optionally substituted with methionine.
5. The polynucleotide of claim 4 , wherein the promoter is an actA promoter.
6. The polynucleotide of claim 1 , wherein the heterologous antigen is non-Listerial.
7. The polynucleotide of claim 6 , wherein the heterologous antigen is mesothelin or PSCA.
8. A plasmid comprising the polynucleotide of claim 1 .
9. A Listeria bacterium comprising the polynucleotide of claim 1 .
10. The Listeria bacterium of claim 9 , which is Listeria monocytogenes.
11. The Listeria bacterium of claim 10 which is attenuated for cell-to-cell spread and/or entry into nonphagocytic cells.
12. The Listeria bacterium of claim 11 which comprises an attenuating mutation in actA and/or inlB.
13. The Listeria bacterium of claim 9 , wherein the Listeria bacterium comprises the polynucleotide in its genome.
14. The Listeria bacterium of claim 13 , wherein the polynucleotide or the nucleic acid encoding the fusion protein has been integrated into a virulence gene in the genome.
15. The Listeria bacterium of claim 14 , wherein the virulence gene is a prfA-dependent gene.
16. The Listeria bacterium of claim 14 , wherein the virulence gene is not a prfA-dependent gene.
17. The Listeria bacterium of claim 14 , wherein the virulence gene is actA or inlB.
18. A vaccine comprising the Listeria bacterium of claim 9 .