Methods for constructing antibiotic resistance free vaccines

Patent metadata

Field	Value
Publication number	US-9700608-B2
Application number	US-201615296973-A
Country	US
Kind code	B2
Filing date	Oct 18, 2016
Priority date	Aug 13, 2004
Publication date	Jul 11, 2017
Grant date	Jul 11, 2017

Abstract

The present invention provides Listeria vaccine strains that express a heterologous antigen and a metabolic enzyme, and methods of generating same.

First claim

What is claimed is:

1. A method of inducing an immune response against an antigen-expressing tumor or a n antigen-expressing cancer in a subject, said method comprising the step of administering to the subject a recombinant Listeria strain auxotrophic for D-alanine synthesis, comprising a mutation in a D-alanine racemase gene and in a D-amino acid transferase gene in the chromosome of said recombinant Listeria , wherein said Listeria further comprises a plasmid, wherein the plasmid comprises:
a first nucleic acid sequence encoding a polypeptide, wherein said polypeptide comprises a heterologous antigen, and
a second nucleic acid sequence encoding a D-alanine racemase enzyme, wherein said plasmid does not confer antibiotic resistance upon said recombinant Listeria strain,
whereby said D-alanine racemase gene complements said mutation in said chromosome of said recombinant Listeria , wherein said plasmid is stably maintained in said recombinant Listeria strain in the absence of an antibiotic selection, wherein said first nucleic acid sequence is operably linked to a prokaryotic promoter/regulatory sequence, and wherein said administering induces an immune response against said antigen-expressing tumor or said antigen-expressing cancer in said subject.

2. The method of claim 1 , wherein said polypeptide is a fusion protein comprising said heterologous antigen and an additional polypeptide, wherein said additional polypeptide is a non-hemolytic fragment of a listeriolysin O (LLO) protein, a Proline, Glutamic acid, Serine and Threonine (PEST) amino acid sequence, or an Actin assembly-inducing protein (ActA) protein or a fragment thereof.

3. The method of claim 1 , wherein said second nucleic acid sequence is operably linked to said promoter/regulatory sequence.

4. The method of claim 1 , wherein said heterologous antigen is an HPV E6 or an HPV E7 antigen.

5. The method of claim 1 , wherein said cancer is a cervical cancer.

6. The method of claim 1 , wherein said immune response comprises reducing the size of a HPV antigen-expressing tumor or a HPV antigen-expressing cancer, protecting against recurrence of a HPV antigen-expressing tumor or a HPV antigen-expressing cancer, reducing the growth rate of a HPV antigen-expressing tumor or a HPV antigen-expressing cancer, or inhibiting the growth rate of a HPV antigen-expressing tumor or a HPV antigen-expressing cancer, or any combination thereof.

7. The method of claim 6 , wherein said immune response comprises treating said HPV antigen-expressing tumor or said HPV antigen-expressing cancer.

8. The method of claim 7 , wherein treating said HPV antigen-expressing tumor or said HPV antigen-expressing cancer comprises regression of an established HPV antigen-expressing tumor or an established HPV-expressing cancer.

9. The method of claim 1 , wherein said inducing an immune response comprises inducing a HPV antigen-specific tumor-infiltrating cytotoxic T-cells (CTL).

10. A method of regressing an antigen-expressing tumor or an antigen-expressing cancer in a subject, said method comprising the step of administering to the subject a recombinant Listeria strain auxotrophic for D-alanine synthesis, comprising a mutation in a D-alanine racemase gene and in a D-amino acid transferase gene in the chromosome of said recombinant Listeria , wherein said Listeria further comprises a plasmid, wherein the plasmid comprises:
a first nucleic acid sequence encoding a polypeptide, wherein said polypeptide comprises a heterologous antigen, and
a second nucleic acid sequence encoding a D-alanine racemase enzyme, wherein said plasmid does not confer antibiotic resistance upon said recombinant Listeria strain,
whereby said D-alanine racemase gene complements said mutation in said chromosome of said recombinant Listeria , wherein said plasmid is stably maintained in said recombinant Listeria strain in the absence of an antibiotic selection, and wherein said first nucleic acid sequence is operably linked to a prokaryotic promoter/regulatory sequence, wherein said administering regresses said antigen-expressing tumor or said antigen-expressing cancer in said subject.

11. The method of claim 10 , wherein said polypeptide is a fusion protein comprising said heterologous antigen and an additional polypeptide, wherein said additional polypeptide is a non-hemolytic fragment of a listeriolysin O (LLO) protein, a Proline, Glutamic acid, Serine and Threonine (PEST) amino acid sequence, or an Actin assembly inducing protein (ActA) protein or a fragment thereof.

12. The method of claim 10 , wherein said second nucleic acid sequence is operably linked to said promoter/regulatory sequence.

13. The method of claim 10 , wherein said heterologous antigen is an HPV E6 or an HPV E7 antigen.

14. The method of claim 10 , wherein said cancer is a cervical cancer.

15. The method of claim 10 , wherein said regressing said antigen-expressing tumor or said antigen-expressing cancer comprises reducing the size of a HPV antigen-expressing tumor or a HPV antigen-expressing cancer, reducing the growth rate of a HPV antigen-expressing tumor or a HPV antigen-expressing cancer, or inhibiting the growth rate of a HPV antigen-expressing tumor or a HPV antigen-expressing cancer, or any combination thereof.

16. The method of claim 15 , wherein said regressing said HPV antigen-expressing tumor or said HPV antigen-expressing cancer comprises treating said tumor or said cancer.

17. The method of claim 15 , wherein said regressing said HPV antigen-expressing tumor or said HPV antigen-expressing cancer comprises inducing an immune response against said HPV antigen-expressing tumor or said HPV antigen-expressing cancer.

18. The method of claim 17 , wherein said inducing an immune response comprises inducing HPV antigen-specific tumor-infiltrating cytotoxic T-cells (CTL).

Assignees

• Univ Pennsylvania

Inventors

• Paterson Yvonne
• Verch Thorsten

Classifications

• C12Y206/01021

  D-Amino-acid transaminase (2.6.1.21), i.e. D-alanine aminotransferase/transaminase or D-aspartic aminotransferase/transaminase · CPC title

• C12N9/16

  acting on ester bonds (3.1) · CPC title

• C12Y501/01001

  Alanine racemase (5.1.1.1) · CPC title

• A61K2039/523

  expressing foreign proteins · CPC title

• C12Y206/01

  Transaminases (2.6.1) · CPC title