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 modified ActA comprising a deletion of one or more amino acids of SEQ ID NO: 38 in a segment comprising amino acids 31-60, and which is truncated at about amino acid 100 of SEQ ID NO: 38 wherein the first amino acid residue of SEQ ID NO: 38 is optionally 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 modified ActA comprises from 1 to 10 conservative amino acid substitutions, relative to SEQ ID NO: 38.
4 . 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.
5 . 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.
6 . The polynucleotide of claim 5 , wherein the promoter is an actA promoter.
7 . The polynucleotide of claim 1 , wherein the heterologous antigen is non-Listerial.
8 . The polynucleotide of claim 1 , wherein the heterologous antigen is from, or is derived from, a cancer cell, tumor, or infectious agent.
9 . The polynucleotide of claim 8 , wherein the heterologous antigen is mesothelin, an antigen derived from mesothelin, PSCA, or an antigen derived from PSCA.
10 . A plasmid comprising the polynucleotide of claim 1 .
11 . A Listeria bacterium comprising the polynucleotide of claim 1 .
12 . The Listeria bacterium of claim 11 , which is Listeria monocytogenes.
13 . The Listeria bacterium of claim 12 which is attenuated for cell-to-cell spread and/or entry into nonphagocytic cells.
14 . The Listeria bacterium of claim 13 which comprises an attenuating mutation in actA and/or inlB.
15 . The Listeria bacterium of claim 11 , wherein the Listeria bacterium comprises the polynucleotide in its genome.
16 . The Listeria bacterium of claim 15 , wherein the polynucleotide or the nucleic acid encoding the fusion protein has been integrated into a virulence gene in the genome.
17 . The Listeria bacterium of claim 16 , wherein the virulence gene is a prfA-dependent gene.
18 . The Listeria bacterium of claim 16 , wherein the virulence gene is not a prfA-dependent gene.
19 . The Listeria bacterium of claim 16 , wherein the virulence gene is actA or inlB.
20 . A vaccine comprising the Listeria bacterium of claim 11 .
21 - 84 . (canceled)