Albert Einstein College of Medicine

University of Pittsburgh

Other

Stoyan S Bardarov

William R Jacobs, Jr

Graham F Hatfull

Tsungda Hsu

Ruth McAdam

Vasan Sambandamurthy

Svetoslav Bardarov, Legal Representative

Sheldon Morris

Methods of treating a mammal that is deficient in CD4and/or CD8lymphocytes are provided. The methods comprise inoculating the mammal with an attenuated mycobacterium in thecomplex. In these methods, the mycobacterium comprises two deletions, wherein a virulent mycobacterium in thecomplex having either deletion exhibits attenuated virulence. Use of these mycobacteria for the manufacture of a medicament for the treatment of mammals deficient in CD4and/or CD8lymphocytes is also provided.

[object Object]

Non-naturally occurring mycobacteria in thecomplex are provided. These mycobacteria have a deletion of an RD1 region or a region controlling production of a vitamin, and exhibit attenuated virulence in a mammal when compared to the mycobacteria without the deletion. Also provided are non-naturally occurring mycobacteria that have a deletion of a region controlling production of lysine, and mycobacteria comprising two attenuating deletions. Vaccines comprising these mycobacteria are also provided, as are methods of protecting mammals from virulent mycobacteria using the vaccines. Also provided are methods of preparing these vaccines which include the step of deleting an RD1 region or a region controlling production of a vitamin from a mycobacterium in thecomplex.

The present invention provides a method for high frequency of allelic exchange in the slow-growing mycobacteria using in vitro generated specialized transducing mycobacteriophages, as well as the recombinant slow-growing mycobacteria generated using the disclosed method. A transducing mycobacteriophage of the present invention comprises a conditional mycobacteriophage containing an,bacteriophage lambda cosmid inserted into a non-essential region of the mycobacteriophage, said cosmid containing a mutated DNA substrate which is homologous to a wildtype nucleic acid sequence of a slow-growing mycobacterium. When slow-growing mycobacteria infected with the conditional transducing phage are cultured under conditions wherein the conditional transducing phage does not replicate, the mutated DNA substrate is incorporated into the chromosomal DNA of the slow-growing mycobacteria by homologous recombination, thereby generating the recombinant slow-growing mycobacteria of the present invention. The disclosed method may be used to produce mycobacterial auxotrophs, including leucine and lysine auxotrophs.

One step allelic exchange in mycobacteria using in vitro generated conditional transducing phages

This invention is directed to L5 shuttle phasmids capable of delivering foreign DNA into mycobacteria and to methods of producing L5 shuttle phasmids. In addition, this invention is directed to a method of generating mycobacterial mutations and to a method of producing mycobacterial vaccines.

L5 shuttle phasmids

The present invention provides a conditional shuttle phasmid constructed by inserting a cosmid into a non-essential region of the TM4 mycobacteriophage that introduces DNA of interest into mycobacteria, especially M. tuberculosis complex organisms and other slow growing mycobacteria. The present invention provides a recombinant mycobacterium which expresses a DNA of interest incorporated into its chromosome by a TM4 conditional shuttle phasmid containing the DNA of interest. The present invention further provides a mycobacterial auxotrophic mutant and a method of generating auxotrophic mutants.

Growing community of inventors

Bronx, NY, United States of America

Stoyan S Bardarov