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 sequence operably linked to the promoter, wherein the nucleic acid encodes a fusion protein comprising:
(i) a modified ActA comprising more than the first 59 amino acids of ActA, and less than the first 380 amino acids of ActA; and
(ii) mesothelin, or an antigen derived from mesothelin.
2 . The polynucleotide of claim 1 , wherein the mesothelin is human mesothelin.
3 . The polynucleotide of claim 1 , wherein the promoter is actA promoter.
4 . The polynucleotide of claim 1 , wherein the modified ActA comprises at least the first 85 amino acids of ActA and less than the first 125 amino acids of ActA.
5 . The polynucleotide of claim 1 , wherein the modified ActA consists of amino acids 1-100 of ActA.
6 . The polynucleotide of claim 5 , wherein the promoter is the actA promoter.
7 . A plasmid comprising the polynucleotide of claim 1 .
8 . A Listeria bacterium comprising the polynucleotide of claim 1 .
9 . The Listeria bacterium of claim 8 , which is Listeria monocytogenes.
10 . The Listeria bacterium of claim 8 which is an actA deletion mutant or an actA insertion mutant.
11 . The Listeria bacterium of claim 8 , wherein the Listeria bacterium comprises the polynucleotide in its genome.
12 . The Listeria bacterium of claim 11 , wherein the polynucleotide has been integrated into a virulence gene in the genome, wherein the integration of the polynucleotide:
(a) disrupts expression of the virulence gene; or
(b) disrupts a coding sequence of the virulence gene.
13 . The Listeria bacterium of claim 12 , wherein the virulence gene is actA or inlB.
14 . The Listeria bacterium of claim 11 , wherein the nucleic acid encoding the fusion protein has been integrated into a virulence gene in the genome, wherein the integration of the nucleic acid:
(a) disrupts expression of the virulence gene; or
(b) disrupts a coding sequence of the virulence gene.
15 . A vaccine comprising the Listeria bacterium of claim 8 .
16 . A method for stimulating an immune response to mesothelin, or to an antigen derived from mesothelin, in a mammal comprising administering an effective amount of the Listeria bacterium of claim 8 , or an effective amount of a composition comprising the Listeria bacterium, to the mammal.
17 . A Listeria bacterium comprising a genome, wherein the genome comprises a polynucleotide comprising a nucleic acid encoding mesothelin, or an antigen derived from mesothelin, wherein the polynucleotide has been integrated into a virulence gene in the genome, wherein the integration of the polynucleotide:
(a) disrupts expression of the virulence gene; or
(b) disrupts a coding sequence of the virulence gene.
18 . The Listeria bacterium of claim 17 , wherein all or part of the virulence gene has been deleted.
19 . The Listeria bacterium of claim 17 , wherein none of the virulence gene has been deleted.
20 . The Listeria bacterium of claim 17 , wherein the virulence gene is actA or inlB.
21 . The Listeria bacterium of claim 17 , which is Listeria monocytogenes.
22 . The Listeria bacterium of claim 17 , wherein the mesothelin is human mesothelin.
23 . A vaccine comprising the Listeria bacterium of claim 17 .
24 . A method for stimulating an immune response to mesothelin, or to an antigen derived from mesothelin, in a mammal, comprising administering an effective amount of the Listeria bacterium of claim 17 , or an effective amount of a composition comprising the Listeria bacterium, to the mammal.
25 . A method of producing a Listeria bacterium for use in a vaccine, comprising integrating a polynucleotide into a virulence gene in the genome of the Listeria bacterium, wherein the polynucleotide comprises a nucleic acid encoding mesothelin, or an antigen derived from mesothelin, and wherein the integration of the polynucleotide:
(a) disrupts expression of the virulence gene; or
(b) disrupts a coding sequence of the virulence gene.
26 . A Listeria bacterium produced by the method of claim 25 .
27 . A polynucleotide comprising a first nucleic acid encoding actA-N100, operably linked and in frame with, a second nucleic acid encoding mesothelin, or an antigen derived from mesothelin.
28 . The polynucleotide of claim 27 , wherein the mesothelin is human mesothelin.
29 . A Listeria bacterium comprising the polynucleotide of claim 27 .
30 . The Listeria bacterium of claim 29 , wherein the polynucleotide is genomic.
31 . The Listeria bacterium of claim 30 , wherein the polynucleotide is integrated into the actA gene or inlB gene.
32 . The Listeria bacterium of claim 29 , wherein the polynucleotide is plasmid-based.
33 . The Listeria bacterium of claim 29 that is Listeria monocytogenes.
34 . The Listeria bacterium of claim 29 , wherein the polynucleotide is operably linked to an actA promoter.
35 . A vaccine comprising the Listeria bacterium of claim 29 .
36 . A method for stimulating immune response to mesothelin, or an antigen derived from mesothelin, in a mammal comprising administering the Listeria bacterium of claim 29 to the mammal.
37 . A polynucleotide comprising a first nucleic acid encoding a modified actA, where the modified actA comprises:
a. amino acids 1-59 of actA; and
b. an inactivating mutation in, deletion of, or truncation prior to, at least one domain for actA-mediated regulation of the host cell cytoskeleton,
wherein the first nucleic acid is operably linked and in frame with a second nucleic acid encoding mesothelin, or an antigen derived from mesothelin.
38 . A Listeria bacterium containing the polynucleotide of claim 37 .
39 . The Listeria bacterium of claim 37 , wherein the polynucleotide is operably linked with one or more of:
a. actA promoter; or
b. a bacterial promoter that is not actA promoter.
40 . The Listeria bacterium of claim 37 that is Listeria monocytogenes.
41 . A vaccine comprising the Listeria bacterium of claim 37 .
42 . A method for stimulating immune response to mesothelin, or an antigen derived from mesothelin, in a mammal comprising administering the Listeria bacterium of claim 37 to the mammal.
43 . The Listeria bacterium of claim 14 , wherein the virulence gene is a prfA-dependent gene.
44 . The Listeria bacterium of claim 14 , wherein the virulence gene is not a prfA-dependent gene.