Engineered listeria and methods of use thereof

Abstract

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.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 discloses pINT, a 6055 bp plasmid. Once pINT is integrated in a listerial genome, the Listeria can be isolated by erythromycin resistance (ErmC), followed by treatment with Cre recombinase to remove a region of the plasmid encoding the antibiotic resistance genes (CAT and ErmC).

FIG. 2 shows pKSV7, a 7096 plasmid that mediates homologous recombination.

FIG. 3 shows steps, or intermediates, occurring with pKSV7-mediated homologous recombination into a bacterial genome.

FIG. 4 discloses a method for preparing an insert bearing homologous arms, where the insert bearing the homologous arms is placed into pKSV7. The loxP-flanked region is bracketed by the homologous arms. After integration into a bacterial genome, transient exposure to Cre recombinase catalyzes removal of the antibiotic resistance gene. Integration occurs with deletion of part of the genome, corresponding to the region between areas matching the homologous arms.

FIG. 5 shows an alternate method for preparing an insert bearing homologous arms, where the insert bearing homologous arms is placed into pKSV7. The loxP-flanked region resides outside the homologous arms. After integration into a bacterial genome, transient exposure to Cre recombinase catalyzes removal of the antibiotic resistance gene (or other selection marker). Integration occurs with deletion of part of the genome, corresponding to the region between areas matching the homologous arms.

FIG. 6 discloses the preparation of an insert bearing homologous arms, where the insert bearing homologous arms is placed into pKSV7. The loxP-flanked region resides in between the homologous arms. In vectors prepared according to this figure, integration is not followed by deletion of any corresponding region of the genome.

FIG. 7 is a schematic disclosing some of the mesothelin constructs of the present invention, including, e.g., any promoters, secretory sequences, fusion protein partners, and so on.

FIG. 8 is a gel showing expression of mesothelin from various listerial constructs.

FIG. 9 is a gel showing expression of mesothelin from a number of listerial constructs.

FIGS. 10-12 show expression of interferon-gamma (IFNgamma) from spot forming cell (SFC) assays, and compare immune responses where mice had been vaccinated with various numbers (colony forming units; c.f.u.) of engineered L. monocytogenes.

FIG. 13 disclose numbers of tumor metastases on the surfaces of livers, after treating tumor-bearing mice with various preparations of recombinant L. monocytogenes. FIG. 13 reveals the raw data (photographs of fixed livers).

FIG. 14 also disclose numbers of tumor metastases on the surfaces of livers, after treatment of tumor-bearing mice with various preparations of recombinant L. monocytogenes.

FIG. 15 further disclose numbers of tumor metastases on the surfaces of livers, after treating tumor-bearing mice with recombinant L. monocytogenes.

FIG. 16 demonstrates increased survival to tumors by tumor-bearing mice with treatment with various preparations of recombinant L. monocytogenes.

FIG. 17 illustrates mesothelin constructs and secretion of mesothelin by various preparations of recombinant L. monocytogenes.

FIG. 18 discloses secretion of mesothelin and immune responses stimulated by various preparations of recombinant L. monocytogenes.

FIG. 19 shows secretion of mesothelin and immune responses stimulated by various preparations of recombinant L. monocytogenes.

FIG. 20 further reveals mesothelin expression and immune responses stimulated by various preparations of recombinant L. monocytogenes.

FIG. 21 additionally illustrates secretion of mesothelin and immune responses stimulated by various preparations of recombinant L. monocytogenes.

FIG. 22 demonstrates mesothelin expression and immune responses stimulated by various preparations of recombinant L. monocytogenes.

FIG. 23 discloses immune responses stimulated by vaccination with various preparations of recombinant Listeria.

FIG. 24 further discloses secretion of mesothelin and immune responses stimulated by various preparations of recombinant L. monocytogenes.

FIG. 25 reveals immune responses stimulated after vaccination with a number of preparations of recombinant Listeria.

FIG. 26 additionally discloses secretion of mesothelin and immune responses stimulated by various preparations of recombinant L. monocytogenes. hMeso6: L. monocytogenes ΔactAΔinlB encoding actA promoter; actA-N100-hMeso ΔSSΔGPI; integrated at actA locus. hMeso25: L. monocytogenes ΔactAΔinlB encoding actA promoter; actA-N100-hMeso ΔSSΔGPI; integrated at inlB locus.

FIG. 27 further demonstrates secretion of mesothelin and immune responses stimulated by various preparations of recombinant L. monocytogenes.

FIG. 28 shows photographs of fixed lungs.

FIG. 29 shows a histogram of data from the photographs of fixed lung.

FIG. 30 reveals the effectiveness of various preparations of recombinant Listeria in improving survival of tumor-bearing mice.

FIG. 31 discloses secretion of mesothelin and immune responses stimulated by various preparations of recombinant L. monocytogenes.

FIG. 32 compares mesothelin expression from various preparations of recombinant Listeria.

FIG. 33 depicts mesothelin secretion and immune responses stimulated after vaccination with recombinant L. monocytogenes.

FIG. 34 demonstrates immune response stimulated after vaccination with the preparations and doses of recombinant Listeria.

FIGS. 35A and 35B disclose numbers of tumor metastases on livers, after treatment of tumor-bearing mice with various preparations of recombinant L. monocytogenes. FIG. 35A illustrates raw data (photographs of fixed livers).

FIG. 36 demonstrates the effectiveness of various preparations of recombinant Listeria in improving survival of tumor-bearing mice.

FIG. 37 discloses immune response after vaccination with various preparations of recombinant Listeria, and compares CD4⁺ T cell and CD8⁺ T cell responses.

FIG. 38 reveals survival of tumor-bearing mice to the tumors after vaccination with various preparations of recombinant Listeria.

FIG. 39 further illustrates survival of tumor-bearing mice to the tumors after vaccination with various preparations of recombinant Listeria.

FIG. 40 discloses alignment of a phage integrase of the present invention with a another phage integrase (U153 int: SEQ ID NO: 1; lin 1231: SEQ ID NO:2).

FIG. 41 discloses alignment of yet another phage integrase of the present invention another phage integrase (PSA int: SEQ ID NO:3; lin 0071: SEQ ID NO:4).

FIG. 42 shows alignment of still another phage integrase of the present invention with a different phage integrase (PSA int: SEQ ID NO:5; lin 1765: SEQ ID NO:6).

FIG. 43 discloses alignment of a further phage integrase of the present invention with another phage integrase (PSA int: SEQ ID NO:7; lin 2601: SEQ ID NO:8).

FIG. 44 provides an alignment of an additional phage integrase of the present invention with a nucleic acid encoding another phage integrase (PSA int: SEQ ID NO:119; lmof6854_—2703: SEQ ID NO:120).

Claims

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; andb. 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; orb. 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.