Recombinant BCG strains with enhanced ability to escape the endosome
Mycobacterium strains that have an enhanced ability to elicit a MHC-Class I-restricted CD8 + T cell immune response are provided. The Mycobacterium strains are genetically engineered to express: a endosomalytic protein that is active at neutral pH (e.g. Perfringolysin O), permitting escape of the Mycobacterium from endosomes into the cytoplasm of the cell; and antigens of interest, such as tuberculosis antigens. The invention also provides vaccine preparations containing such Mycobacterium strains.
1. A method of enabling a recombinant Mycobacterium to
escape from endosomes, comprising, the
step of genetically engineering said Mycobacterium to: express a nucleic acid sequence that encodes an expressable and secretable Prefringolysin O (PFO) protein that is active at a pH of 6-8.
2. The method of claim 1 , wherein said genetically engineering
step further comprises engineering said Mycobacterium to include additional nucleotide sequences that code for one or more proteins of interest.
3. The method of claim 2 , wherein either
or both of said nucleic acid sequence encoding an expressable and secretable PFO protein or said one or more additional nucleotide sequences encoding one or more proteins of interest are present on a plasmid in the recombinant Mycobacterium.
4. The method of claim 2 wherein said one or more proteins of interest include one or more plasmodium antigens.
5. The method of claim 2 wherein said one or more proteins of interest include one or more Mycobacterium tuberculosis antigens.
6. The method of claim 5 wherein said one or more Mycobacterium tuberculosis antigens are selected from Ag85A, Ag85B, TB 10.4, Rv0125, Rv0203, Rv0287, Rv0288, Rv0603, Rv1196, Rv1223, Rv1271c, Rv1733c, Rv1738 Rv1804c, Rv1886, Rv2031c, Rv2032, Rv2253, Rv2290, Rv2389c, Rv2626c, Rv2627c, Rv2779c, Rv2873, Rv2875, Rv3017c, Rv3407, Rv3804c, Rv3810, and Rv3841.
7. The method of claim 1 wherein said recombinant Mycobacterium is Bacille Calmette Guerin (BCG).
8. The method of claim 7 wherein said BCG is BCG Danish 1331.
9. The method of claim 1 wherein said nucleic acid sequence that encodes an expressable and secretable PFO protein is present in the chromosome of said recombinant Mycobacterium.
10. The method of claim 1 wherein said genetically engineering step is performed by replacing a urease C gene in the chromosome of a
Mycobacterium with said nucleic acid sequence that encodes an expressable and
secretable PFO protein.