This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Méderlé, I.
Right arrow Articles by Winter, N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Méderlé, I.
Right arrow Articles by Winter, N.

 Previous Article  |  Next Article 

Infection and Immunity, January 2002, p. 303-314, Vol. 70, No. 1
0019-9567/01/$04.00+0     DOI: 10.1128/IAI.70.1.303-314.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Plasmidic versus Insertional Cloning of Heterologous Genes in Mycobacterium bovis BCG: Impact on In Vivo Antigen Persistence and Immune Responses

I. Méderlé,1 I. Bourguin,1 D. Ensergueix,1 E. Badell,1 J. Moniz-Peireira,2 B. Gicquel,1 and N. Winter1*

Unité de Génétique Mycobactérienne, Institut Pasteur, 75724 Paris Cedex 15, France,1 Laboratorio de Microbiologia Molecular, Faculdade de Farmacia, Universidade de Lisboa, 1600 Lisbon, Portugal2

Received 25 June 2001/ Returned for modification 15 August 2001/ Accepted 8 October 2001

Bivalent recombinant strains of Mycobacterium bovis BCG (rBCG) expressing the early regulatory nef and the structural gag(p26) genes from the simian immunodeficiency virus (SIV) SIVmac251 were engineered so that both genes were cotranscribed from a synthetic operon. The expression cassette was cloned into a multicopy-replicating vector, and the expression levels of both nef and gag in the bivalent rBCG(nef-gag) strain were found to be comparable to those of monovalent rBCG(nef) or rBCG(gag) strains. However, extrachromosomal cloning of the nef-gag operon into a replicative plasmid resulted in strains of low genetic stability that rapidly lost the plasmid in vivo. Thus, the nef-gag operon was inserted site specifically into the BCG chromosome by means of mycobacteriophage Ms6-derived vectors. The resulting integrative rBCG(nef-gag) strains showed very high genetic stability both in vitro and in vivo. The in vivo expression of the heterologous genes was much longer lived when the expression cassette was inserted into the BCG chromosome. In one of the strains obtained, integrative cloning did not reduce the expression levels of the genes even though a single copy was present. Accordingly, this strain induced cellular immune responses of the same magnitude as that of the replicative rBCG strain containing several copies of the genes.

* Corresponding author. Mailing address: Unité de Génétique Mycobactérienne, Institut Pasteur, 25-28 rue du Docteur Roux, 75724, Paris Cedex 15, France. Phone: (33)140613599. Fax: (33)145688843. E-mail: nwinter{at}pasteur.fr.

Editor: S. H. E. Kaufmann

Infection and Immunity, January 2002, p. 303-314, Vol. 70, No. 1
0019-9567/01/$04.00+0     DOI: 10.1128/IAI.70.1.303-314.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Campoy, S., Aranda, J., Alvarez, G., Barbe, J., Llagostera, M. (2006). Isolation and Sequencing of a Temperate Transducing Phage for Pasteurella multocida.. Appl. Environ. Microbiol. 72: 3154-3160 [Abstract] [Full Text]  
  • Cayabyab, M. J., Hovav, A.-H., Hsu, T., Krivulka, G. R., Lifton, M. A., Gorgone, D. A., Fennelly, G. J., Haynes, B. F., Jacobs, W. R. Jr., Letvin, N. L. (2006). Generation of CD8+ T-Cell Responses by a Recombinant Nonpathogenic Mycobacterium smegmatis Vaccine Vector Expressing Human Immunodeficiency Virus Type 1 Env. J. Virol. 80: 1645-1652 [Abstract] [Full Text]  
  • Abadie, V., Badell, E., Douillard, P., Ensergueix, D., Leenen, P. J. M., Tanguy, M., Fiette, L., Saeland, S., Gicquel, B., Winter, N. (2005). Neutrophils rapidly migrate via lymphatics after Mycobacterium bovis BCG intradermal vaccination and shuttle live bacilli to the draining lymph nodes. Blood 106: 1843-1850 [Abstract] [Full Text]  
  • Kanekiyo, M., Matsuo, K., Hamatake, M., Hamano, T., Ohsu, T., Matsumoto, S., Yamada, T., Yamazaki, S., Hasegawa, A., Yamamoto, N., Honda, M. (2005). Mycobacterial Codon Optimization Enhances Antigen Expression and Virus-Specific Immune Responses in Recombinant Mycobacterium bovis Bacille Calmette-Guerin Expressing Human Immunodeficiency Virus Type 1 Gag. J. Virol. 79: 8716-8723 [Abstract] [Full Text]  
  • Kocincova, D., Sonden, B., Bordat, Y., Pivert, E., de Mendonca-Lima, L., Gicquel, B., Reyrat, J.-M. (2004). The Hydrophobic Domain of the Mycobacterial Erp Protein Is Not Essential for the Virulence of Mycobacterium tuberculosis. Infect. Immun. 72: 2379-2382 [Abstract] [Full Text]  
  • Prina, E., Abdi, S. Z., Lebastard, M., Perret, E., Winter, N., Antoine, J.-C. (2004). Dendritic cells as host cells for the promastigote and amastigote stages of Leishmania amazonensis: the role of opsonins in parasite uptake and dendritic cell maturation. J. Cell Sci. 117: 315-325 [Abstract] [Full Text]  
  • Recchi, C., Sclavi, B., Rauzier, J., Gicquel, B., Reyrat, J.-M. (2003). Mycobacterium tuberculosis Rv1395 Is a Class III Transcriptional Regulator of the AraC Family Involved in Cytochrome P450 Regulation. J. Biol. Chem. 278: 33763-33773 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»