Non-hemolytic LLO fusion proteins and methods of utilizing same
The present invention provides recombinant proteins or peptides comprising a mutated listeriolysin O (LLO) protein or fragment thereof, comprising a substitution or internal deletion of the cholesterol-binding domain or a portion thereof, fusion proteins or peptides comprising same, nucleotide molecules encoding same, and vaccine vectors comprising or encoding same. The present invention also provides methods of utilizing recombinant proteins, peptides, nucleotide molecules, and vaccine vectors of the present invention to induce an immune response to a peptide of interest.
1. A recombinant Listeria expressing a recombinant protein, said recombinant protein comprising a listeriolysin 0 (LLO) protein comprising a mutation of amino acid residues on positions 2, 9 and 10 in the cholesterol-binding domain (CBD) of said LLO protein, and wherein said CBD is set forth in SEQ ID NO: 18.
2. The recombinant Listeria of claim 1 , wherein said amino acid residues on positions 2, 9 and 10 of SEQ ID NO: 18 correspond to C484, W491 and W492 of an LLO protein, and wherein said LLO protein is set forth in SEQ ID NO: 37.
3. The recombinant Listeria of claim 1 , wherein said LLO protein comprises a deletion of the signal peptide sequence thereof.
4. The recombinant Listeria of claim 1 , wherein said LLO protein comprises the signal peptide sequence thereof.
5. The recombinant Listeria of claim 1 , wherein said recombinant protein further comprises a heterologous peptide of interest.
6. The recombinant Listeria of claim 5 , wherein said heterologous peptide of interest is an antigenic peptide.
7. The recombinant Listeria of claim 6 , wherein said antigenic peptide is a B-cell receptor (BCR) peptide.
8. The recombinant Listeria of claim 6 , wherein said antigenic peptide is a Human Papilloma Virus (HPV)-16-E6, HPV-16-E7, HPV-18-E6, HPV-18-E7, a Her/2-neu antigen, a Prostate Specific Antigen (PSA), Prostate Stem Cell Antigen (PSCA), a Stratum Corneum Chymotryptic Enzyme (SCCE) antigen, Wilms tumor antigen 1 (WT-1), human telomerase reverse transcriptase (hTERT), Proteinase 3, Tyrosinase Related Protein 2 (TRP2), High Molecular Weight Melanoma Associated Antigen (HMW-MAA), synovial sarcoma, X (SSX)-2, carcinoembryonic antigen (CEA), MAGE-A, interleukin-13 Receptor alpha (IL13-R alpha), Carbonic anhydrase IX (CAIX), survivin, GP100, or Testisin.
9. An immunogenic composition comprising the recombinant Listeria of claim 5 and an adjuvant.
10. The immunogenic composition of claim 9 , wherein said adjuvant comprises a granulocyte/macrophage colony-stimulating factor (GM-CSF) protein, a nucleotide molecule encoding a GM-CSF protein, saponin QS21, monophosphoryl lipid A, or an unmethylated CpG-containing oligonucleotide.
11. The recombinant Listeria of claim 1 , wherein said mutation is a deletion mutation, a point mutation or a substitution mutation.
12. The recombinant Listeria of claim 1 , wherein said recombinant protein exhibits a greater than 100-fold reduction in hemolytic activity relative to wild-type LLO.
13. A method for inducing an immune response in a subject, comprising administering to said subject the recombinant Listeria of claim 5 , thereby inducing an immune response against said antigenic peptide.
14. A method for inducing an immune response in a subject, comprising administering to said subject the immunogenic composition of claim 9 , thereby inducing an immune response against said heterologous peptide of interest.
15. A method for inducing an immune response in a subject against a B-cell receptor (BCR)-expressing lymphoma, the method comprising the step of administering to said subject the recombinant Listeria of claim 7 , thereby inducing an immune response against a BCR-expressing lymphoma.
16. A method for inducing an immune response in a subject against a HPV-E7-expressing tumor, the method comprising the step of administering to said subject the recombinant Listeria of claim 9 .
17. The method of claim 16 , wherein said HPV-E7-expressing tumor is cervical cancer or head-and-neck cancer.
18. A method for treating, inhibiting or suppressing a HPV-E7-expressing tumor, the method comprising the step of administering to said subject the recombinant Listeria of claim 9 .
19. The method of claim 18 , wherein said HPV-E7-expressing tumor is cervical cancer or head-and-neck cancer.