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.

FIGS. 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 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) 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 less than the first 265 amino acids of ActA.

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 4, wherein the modified ActA consists of amino acids 1-100 of ActA.

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. A plasmid comprising the polynucleotide of claim 1.

10. A Listeria bacterium comprising the polynucleotide of claim 1.

11. The Listeria bacterium of claim 10, which is Listeria monocytogenes.

12. The Listeria bacterium of claim 11 which is attenuated for cell-to-cell spread or entry into nonphagocytic cells.

13. The Listeria bacterium of claim 12 which is an actA deletion mutant or an actA insertion mutant.

14. The Listeria bacterium of claim 13, wherein the promoter is an actA promoter.

15. The Listeria bacterium of claim 10, wherein the Listeria bacterium comprises the polynucleotide in its genome.

16. The Listeria bacterium of claim 15, 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.

17. The Listeria bacterium of claim 16, wherein the virulence gene is actA or inlB.

18. The Listeria bacterium of claim 15, 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.

19. A vaccine comprising the Listeria bacterium of claim 10.

20. A method for stimulating an immune response to the heterologous antigen in a mammal comprising administering an effective amount of the Listeria bacterium of claim 10, or an effective amount of a composition comprising the Listeria bacterium, to the mammal.

21. A Listeria bacterium comprising a genome, wherein the genome comprises a polynucleotide comprising a nucleic acid encoding a heterologous antigen, wherein the nucleic acid has been integrated into a virulence gene in the genome, wherein integration of the polynucleotide (a) disrupts expression of the virulence gene; or(b) disrupts a coding sequence of the virulence gene.

22. The Listeria bacterium of claim 21, wherein all or part of the virulence gene has been deleted.

23. The Listeria bacterium of claim 21, wherein none of the virulence gene has been deleted.

24. The Listeria bacterium of claim 21, wherein the virulence gene is actA or inIB.

25. The Listeria bacterium of claim 21, which is Listeria monocytogenes.

26. The Listeria bacterium of claim 21, wherein the heterologous antigen is from, or is derived from, a cancer cell, tumor, or infectious agent.

27. The Listeria bacterium of claim 21, further comprising: a second nucleic acid encoding a second heterologous antigen that has been integrated into a second virulence gene.

28. The Listeria bacterium of claim 21, wherein the nucleic acid encodes a fusion protein comprising the heterologous antigen and a modified ActA.

29. A vaccine comprising the Listeria bacterium of claim 20.

30. A method for stimulating an immune response to the heterologous antigen in a mammal, comprising administering an effective amount of the Listeria bacterium of claim 21, or an effective amount of a composition comprising the Listeria bacterium, to the mammal.

31. 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 a heterologous antigen and wherein the integration of the polynucleotide(a) disrupts expression of the virulence gene; or(b) disrupts a coding sequence of the virulence gene.

32. The method of claim 31, wherein the polynucleotide is integrated into the virulence gene by homologous recombination.

33. The method of claim 31, wherein all or part of the virulence gene is deleted during integration of the polynucleotide into the virulence gene.

34. The method of claim 31, wherein the virulence gene is actA or inlB.

35. A Listeria bacterium produced by the method of claim 31.

36. A polynucleotide comprising a first nucleic acid encoding actA-N100, operably linked and in frame with, a second nucleic acid encoding a heterologous antigen.

37. A Listeria bacterium comprising the polynucleotide of claim 36.

38. The Listeria bacterium of claim 37, wherein the polynucleotide is genomic.

39. The Listeria bacterium of claim 37, wherein the polynucleotide is integrated into actA or inlB.

40. The Listeria bacterium of claim 37, wherein the polynucleotide is plasmid-based.

41. The Listeria bacterium of claim 37 which is Listeria monocytogenes.

42. The Listeria bacterium of claim 37, wherein the heterologous antigen is, or is derived from, a cancer cell, tumor, or infectious agent.

43. The Listeria bacterium of claim 37, wherein the heterologous antigen is immunologically cross-reactive with, or shares at least one epitope with, the cancer, tumor, or infectious agent.

44. The Listeria bacterium of claim 37, wherein the polynucleotide is operably linked to an actA promoter.

45. A vaccine comprising the Listeria bacterium of claim 37.

46. A method for stimulating immune response to an antigen from, or derived from, a cancer or infectious agent, comprising administering the Listeria bacterium of claim 37 to a mammal having the cancer or infectious agent, and wherein the heterologous antigen shares at least one epitope with or is immunologically cross-reactive with the antigen from, or derived from, the cancer or infectious agent.

47. The method of claim 46, wherein the cancer comprises a tumor or pre-cancerous cell.

48. The method of claim 46, wherein the infectious agent comprises a virus, pathogenic bacterium, or parasitic organism.

49. A polynucleotide comprising a first nucleic acid encoding a modified actA, wherein 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 a heterologous antigen.

50. The polynucleotide of claim 49, wherein the domain is the cofilin homology region (KKRR) (SEQ ID NO:23).

51. The polynucleotide of claim 49, wherein the domain is the phospholipid core binding domain (KVFKKIKDAGKWVRDKI) (SEQ ID NO:20).

52. The polynucleotide of claim 49, wherein the at least one domain comprises all four proline-rich domains (FPPPP, FPPPP, FPPPP, FPPIP) (SEQ ID NO:21, SEQ ID NO:22) of ActA.

53. A Listeria bacterium containing the polynucleotide of claim 49.

54. The Listeria bacterium of claim 53, wherein the polynucleotide is genomic.

55. The Listeria bacterium of claim 53, wherein the polynucleotide is not genomic.

56. The Listeria bacterium of claim 53, wherein the polynucleotide is operably linked with one or more of: a. actA promoter; orb. a bacterial promoter that is not actA promoter.

57. The Listeria bacterium of claim 53 that is Listeria monocytogenes.

58. A vaccine comprising the Listeria bacterium of claim 57.

59. A method for stimulating immune response to an antigen from, or derived from, a cancer or infectious agent, comprising administering the Listeria bacterium of claim 53 to a mammal having the cancer or infectious agent, and wherein the heterologous antigen shares at least one epitope with or is immunologically cross-reactive with the antigen from, or derived from, the cancer or infectious agent.

60. The method of claim 59, wherein the cancer comprises a tumor or pre-cancerous cell.

61. The method of claim 59, wherein the infectious agent comprises a virus, pathogenic bacterium, or parasitic organism.

62. The method of claim 59, wherein the heterologous antigen is, or is derived from, a cancer cell, tumor, or infectious agent.

63. A plasmid comprising a first nucleic acid encoding a phage integrase, a second nucleic acid encoding a phage attachment site (attPP′ site), and a third nucleic acid encoding a heterologous antigen or regulatory nucleic acid, wherein: a. each of the nucleic acids is derivable from L. innocua 0071;b. each of the nucleic acids is derivable from L. innocua 1765;c. each of the nucleic acids is derivable from L. innocua 2601;d. each of the nucleic acids is derivable from L. monocytogenes f6854—2703; ore. the first nucleic acid encodes a phiC31 integrase,wherein the plasmid is useful for mediating site-specific integration of the nucleic acid encoding the heterologous antigen at a bacterial attachment site (attBB′ site) in a bacterial genome that is compatible with the attPP′ site of the plasmid.

64. A method of modifying a bacterial genome, comprising transfecting the bacterium with the plasmid of claim 63, and allowing integrase-catalyzed integration of the third nucleic acid into the bacterial genome under conditions suitable for integration.

65. A plasmid comprising: a. a first nucleic acid encoding a first region of homology to a bacterial genome,b. a second nucleic acid encoding a second region of homology to the bacterial genome, andc. a third nucleic acid comprising a bacterial attachment site (attBB′), wherein the third nucleic acid is flanked by the first and second nucleic acids, wherein the first nucleic acid and second nucleic acid are operably linked with each other and able to mediate homologous integration of the third nucleic acid into the bacterial genome.

66. A bacterium modified by integration of the plasmid of claim 65.

67. A bacterium wherein the genome of the bacterium comprises a polynucleotide containing two operably linked heterologous recombinase binding sites flanking a first nucleic acid, wherein the two sites are: a. two lox sites; orb. two Frt sites,

68. A method of excising the first nucleic acid of claim 67 from the bacterial genome, comprising contacting the genome with Cre recombinase or FLP recombinase, and allowing the recombinase to catalyze excision of the first nucleic acid, under conditions allowing or facilitating excision: a. wherein the first nucleic acid is flanked by lox sites and the recombinase is Cre recombinase; orb. wherein the first nucleic acid is flanked by Frt sites and the recombinase is FLP recombinase.

69. The Listeria bacterium of claim 18, wherein the virulence gene is a prfA-dependent gene.

70. The Listeria bacterium of claim 18, wherein the virulence gene is not a prfA-dependent gene.

71. The plasmid of claim 63, further comprising a first promoter operably linked with the first nucleic acid, and a second promoter operably linked with the third nucleic acid.

72. The Listeria bacterium of claim 66, wherein the integration is in a region of the genome that is necessary for mediating growth or spread.

73. The Listeria bacterium of claim 66, wherein the integration is in a region of the genome that is not necessary for mediating growth or spread.

74. The Listeria bacterium of claim 67, wherein each lox site is a loxP site.