Surface expression of biologically active proteins in bacteria

• US 20050003510A1
• Filed: 01/28/2004
• Published: 01/06/2005
• Est. Priority Date: 01/29/2003
• Status: Active Grant

• Current Assignee: Osel, Inc.
• Original Assignee: Osel, Inc.
• Inventors: Lewicki, John A., Xu, Qiang, Chang, Chia-Hwa, Liu, Xiaowen
• Granted Patent: US 7,754,467 B2
• Time in Patent Office: Days
• US Class Current: 435/252.300
• CPC Class Codes: A61K 2300/00 Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
• CPC Class Codes: A61K 35/747 Lactobacilli, e.g. L. acidophilus or L. brevis
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 38/162 from virus
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 31/10 Antimycotics
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 31/18 for HIV
• CPC Class Codes: C07K 14/335 from Lactobacillus (G)
• CPC Class Codes: C07K 14/70514 CD4
• CPC Class Codes: C07K 2319/035 containing a signal for targeting to the external surface of a cell, e.g. to the outer membrane of Gram negative bacteria, GPI- anchored eukaryote proteins
• CPC Class Codes: C12N 15/62 DNA sequences coding for fusion proteins
• CPC Class Codes: C12N 15/746 for lactic acid bacteria (Streptococcus; Lactococcus; Lactobacillus; Pediococcus; Enterococcus; Leuconostoc; Propionibacterium; Bifidobacterium; Sporolactobacillus)

Abstract

Methods and compositions for targeting heterologous polypeptides to bacterial cell walls are provided.

Claims

1. A Lactobacillus bacterium comprising an expression cassette, the expression cassette comprising a promoter operably linked to polynucleotide encoding a signal sequence and a biologically-active polypeptide, wherein the biologically active polypeptide is linked to a heterologous carboxyl terminal cell wall targeting region and wherein the heterologous carboxyl terminal cell wall targeting region comprises in the following order:

a cell wall associated sequence;

LPQ(S/A/T)(G/A); and

a hydrophobic sequence.

2. The Lactobacillus bacterium of claim 1, wherein the cell wall associated sequence comprises at least 50 amino acids.

3. The Lactobacillus bacterium of claim 1, wherein the cell wall associated sequence comprises at least 200 amino acids.

4. The Lactobacillus bacterium of claim 1, wherein the heterologous carboxyl terminal cell wall targeting region further comprises a charged sequence at the carboxyl terminus of region.

5. The Lactobacillus bacterium of claim 1, wherein the Lactobacillus bacterium is a vagina-colonizing strain.

6. The Lactobacillus bacterium of claim 1, wherein the Lactobacillus bacterium is selected from the group consisting of L. jensenii, L. gasseri, and L. casei.

7. The Lactobacillus bacterium of claim 1, wherein the cell wall targeting region comprises the amino acid sequence LPQSG (SEQ ID NO:

11).

8. The Lactobacillus bacterium of claim 1, wherein the cell wall targeting region comprises the amino acid sequence LPQAG (SEQ ID NO:

12).

9. The Lactobacillus bacterium of claim 1, wherein the cell wall targeting region comprises the amino acid sequence LPQTG (SEQ ID NO:

13).

10. The Lactobacillus bacterium of claim 1, wherein the cell wall targeting region comprises the amino acid sequence LPQTA (SEQ ID NO:

14).

11. The Lactobacillus bacterium of claim 1, wherein the cell wall targeting region comprises SEQ ID NO:

7.

12. The Lactobacillus bacterium of claim 1, wherein the cell wall targeting region comprises SEQ ID NO:

8.

13. The Lactobacillus bacterium of claim 1, wherein the biologically active polypeptide is expressed in the cell wall of the bacterium.

14. The Lactobacillus bacterium of claim 1, wherein the biologically-active polypeptide is between 10 and 600 amino acids.

15. The Lactobacillus bacterium of claim 1, wherein the biologically active protein binds to a pathogen when the biologically active protein is contacted with the pathogen.

16. The Lactobacillus bacterium of claim 15, wherein the pathogen is a bacterial pathogen.

17. The Lactobacillus bacterium of claim 15, wherein the pathogen is a fungal pathogen.

18. The Lactobacillus bacterium of claim 15, wherein the pathogen is a viral pathogen.

19. The Lactobacillus bacterium of claim 18, wherein the viral pathogen is HIV.

20. The Lactobacillus bacterium of claim 19, wherein the biologically active protein is CD4 or an HIV-binding fragment of CD4.

21. The Lactobacillus bacterium of claim 19, wherein the biologically active protein is 2D-CD4.

22. The Lactobacillus bacterium of claim 18, wherein the biologically active protein is cyanovirin-N or a virus-binding fragment of cyanovirin-N.

23. The Lactobacillus bacterium of claim 18, wherein the viral pathogen is herpes simplex virus.

24. The Lactobacillus bacterium of claim 18, wherein the biologically active protein is herpes simplex virus entry mediator C (HveC) or a virus-binding fragment of HveC.

25. The Lactobacillus bacterium of claim 1, wherein the biologically active polypeptide is released from the Lactobacillus bacterium.

26. The Lactobacillus bacterium of claim 4, wherein the biologically active polypeptide is anchored to the cell wall of the Lactobacillus bacterium.

27. A method of expressing a biologically active polypeptide in the cell wall of a Lactobacillus bacterium, the method comprising

providing a Lactobacillus bacterium comprising an expression cassette, the expression cassette comprising a promoter operably linked to a polynucleotide encoding a signal sequence and a biologically-active polypeptide, wherein the biologically active polypeptide is linked to a heterologous carboxyl terminal cell wall targeting region and wherein the heterologous carboxyl terminal cell wall targeting region comprises in the following order:

a cell wall associated sequence;

LPQ(S/A/T)(G/A);

and

a hydrophobic sequence; and

culturing the bacterium under conditions to induce expression of the polypeptide, thereby expressing a biologically active polypeptide in the cell wall of a Lactobacillus bacterium.

28. The method of claim 27, wherein the cell wall associated sequence comprises at least 50 amino acids.

29. The method of claim 27, wherein the cell wall associated sequence comprises at least 200 amino acids.

30. The method of claim 27, wherein the heterologous carboxyl terminal cell wall targeting region further comprises a charged sequence at the carboxyl terminus of region.

31. The method of claim 27, wherein the providing step comprises transferring the expression cassette into the bacterium.

32. The method of claim 27, wherein the cell wall targeting region comprises the amino acid sequence LPQSG (SEO ID NO:

11).

33. The method of claim 27, wherein the cell wall targeting region comprises the amino acid sequence LPQAG (SEQ ID NO:

12).

34. The method of claim 27, wherein the cell wall targeting region comprises the amino acid sequence LPQTG (SEQ ID NO:

13).

35. The method of claim 27, wherein the cell wall targeting region comprises the amino acid sequence LPQTA (SEQ ID NO:

14).

36. The method of claim 27, wherein the cell wall targeting region comprises SEQ ID NO:

7.

37. The method of claim 27, wherein the cell wall targeting region comprises SEQ ID NO:

8.

38. The method of claim 27, wherein the cell wall targeting region comprises at least 200 amino acids.

39. The method of claim 27, wherein the Lactobacillus bacterium is a vagina-colonizing strain.

40. The method of claim 27, wherein the Lactobacillus bacterium is selected from the group consisting of L. jensenii, L. gasseri, and L. casei.

41. The method of claim 27, wherein the biologically-active polypeptide is between 10 and 600 amino acids.

42. The method of claim 27, wherein the biologically active protein binds to a pathogen when the biologically active protein is contacted with the pathogen.

43. The method of claim 42, wherein the pathogen is a bacterial pathogen.

44. The method of claim 42, wherein the pathogen is a fungal pathogen.

45. The method of claim 42, wherein the pathogen is a viral pathogen.

46. The method of claim 45, wherein the viral pathogen is HIV.

47. The method of claim 46, wherein the biologically active protein is CD4 or an HIV-binding fragment of CD4.

48. The method of claim 46, wherein the biologically active protein is 2D-CD4.

49. The method of claim 45, wherein the biologically active protein is cyanovirin-N or a virus-binding fragment of cyanovirin-N.

50. The method of claim 45, wherein the viral pathogen is herpes simplex virus.

51. The method of claim 45, wherein the biologically active protein is herpes simplex virus entry mediator C (HveC) or a virus-binding fragment of HveC.

52. The method of claim 27, wherein the biologically active polypeptide is released from the Lactobacillus bacterium.

53. The method of claim 30, wherein the biologically active polypeptide is anchored in the cell wall of the Lactobacillus bacterium.

54. A method of providing a biologically active protein to a mammalian mucosal surface, the method comprising,

contacting a mucosal surface with a Lactobacillus bacterium recombinantly altered to express a signal sequence linked to a biologically-active polypeptide linked to a heterologous carboxyl terminal cell wall targeting region, the heterologous carboxyl terminal cell wall targeting region comprising in the following order:

a cell wall associated sequence;

LPQ(S/A/T)(G/A); and

a hydrophobic sequence,

wherein the biologically active polypeptide is expressed in an amount able to be detected in a sample collected from the mucosal surface.

55. The method of claim 54, wherein the cell wall associated sequence comprises at least 50 amino acids.

56. The method of claim 54, wherein the cell wall associated sequence comprises at least 200 amino acids.

57. The method of claim 54, wherein the heterologous carboxyl terminal cell wall targeting region further comprises a charged sequence at the carboxyl terminus of region.

58. The method of claim 54, wherein the Lactobacillus bacterium is selected from the group consisting of L. jensenii, L. gasseri, and L. casei.

59. The method of claim 54, wherein the mucosal surface resides within the vagina.

60. The method of claim 54, wherein the mucosal surface resides within the gastrointestinal tract.

61. The method of claim 54, wherein the contacting step comprises orally administering the Lactobacillus bacterium.

62. The method of claim 54, wherein the contacting step comprises vaginally administering the Lactobacillus bacterium.

63. The method of claim 54, wherein the contacting step comprises rectally administering the Lactobacillus bacterium.

64. An expression cassette comprising a promoter operably linked to a polynucleotide encoding a signal sequence and a biologically-active polypeptide, wherein the biologically active polypeptide is linked to SEQ ID NO:

7 or SEQ ID NO;

8.

65. A vector comprising the expression cassette of claim 64.