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Genetic Alteration of Mycobacterium smegmatis To Improve Mycobacterium-Mediated Transfer of Plasmid DNA into Mammalian Cells and DNA Immunization

Yongkai Mo,^1, Natalie M. Quanquin,^1, William H. Vecino,^1, Uma Devi Ranganathan,¹ Lydia Tesfa,¹ William Bourn,² Keith M. Derbyshire,^3,4 Norman L. Letvin,⁵ William R. Jacobs Jr.,^1,6 and Glenn J. Fennelly^7,8*

Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York 10461,¹ Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Observatory, 7925, Cape Town, South Africa,² Division of Infectious Disease, Wadsworth Center, New York State Department of Health,³ Department of Biomedical Sciences, University at Albany, Albany, New York 12201,⁴ Division of Viral Pathogenesis, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02115,⁵ Howard Hughes Medical Institute, Albert Einstein College of Medicine, Bronx, New York 10461,⁶ Department of Pediatrics, Albert Einstein College of Medicine, Bronx, New York 10461,⁷ The Lewis M. Fraad Department of Pediatrics, Jacobi Medical Center, Bronx, New York 10461⁸

Received 28 November 2006/ Returned for modification 9 January 2007/ Accepted 15 July 2007

Mycobacteria target and persist within phagocytic monocytes and are strong adjuvants, making them attractive candidate vectors for DNA vaccines. We characterized the ability of mycobacteria to deliver transgenes to mammalian cells and the effects of various bacterial chromosomal mutations on the efficiency of transfer in vivo and in vitro. First, we observed green fluorescent protein expression via microscopy and fluorescence-activated cell sorting analysis after infection of phagocytic and nonphagocytic cell lines by Mycobacterium smegmatis or M. bovis BCG harboring a plasmid encoding the fluorescence gene under the control of a eukaryotic promoter. Next, we compared the efficiencies of gene transfer using M. smegmatis or BCG containing chromosomal insertions or deletions that cause early lysis, hyperconjugation, or an increased plasmid copy number. We observed a significant—albeit only 1.7-fold—increase in the level of plasmid transfer to eukaryotic cells infected with M. smegmatis hyperconjugation mutants. M. smegmatis strains that overexpressed replication proteins (Rep) of pAL5000, a plasmid whose replicon is incorporated in many mycobacterial constructs, generated a 10-fold increase in plasmid copy number and 3.5-fold and 3-fold increases in gene transfer efficiency to HeLa cells and J774 cells, respectively. Although BCG strains overexpressing Rep could not be recovered, BCG harboring a plasmid with a copy-up mutation in oriM resulted in a threefold increase in gene transfer to J774 cells. Moreover, M. smegmatis strains overexpressing Rep enhanced gene transfer in vivo compared with a wild-type control. Immunization of mice with mycobacteria harboring a plasmid (pgp120_h^E) encoding human immunodeficiency virus gp120 elicited gp120-specific CD8 T-cell responses among splenocytes and peripheral blood mononuclear cells that were up to twofold (P < 0.05) and threefold (P < 0.001) higher, respectively, in strains supporting higher copy numbers. The magnitude of these responses was approximately one-half of that observed after intramuscular immunization with pgp120_h^E. M. smegmatis and other nonpathogenic mycobacteria are promising candidate vectors for DNA vaccine delivery.

Published ahead of print on 30 July 2007.

Editor: J. B. Bliska

Y.M. and N.M.Q. contributed equally to this work.

Present address: PeerView Institute for Medical Education, 315 Bleecker Street, Suite 182, New York, NY 10014.