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

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Published inInfection and Immunity Vol. 70; no. 1; pp. 303 - 314
Main Authors MEDERLE, I, BOURGUIN, I, ENSERGUEIX, D, BADELL, E, MONIZ-PEIREIRA, J, GICQUEL, B, WINTER, N
Format Journal Article
LanguageEnglish
Published Washington, DC American Society for Microbiology 01.01.2002
Subjects
Animals
Antibodies, Viral - immunology
Antigens, Viral - genetics
Antigens, Viral - immunology
Bacteriology
Bacteriophages
Biological and medical sciences
Cells, Cultured
Chromosomes, Bacterial
Cloning, Molecular - methods
DNA, Viral
Female
Fundamental and applied biological sciences. Psychology
gag gene
Gene Expression
Gene Products, gag - genetics
Gene Products, gag - immunology
Gene Products, nef - genetics
Gene Products, nef - immunology
Genetic Vectors - genetics
Genetics
Life Sciences
Macrophages - cytology
Macrophages - immunology
Mice
Mice, Inbred BALB C
Microbial Immunity and Vaccines
Microbiology
Microbiology and Parasitology
Mutagenesis, Insertional - methods
Mutagenesis, Site-Directed
Mycobacterium bovis
Mycobacterium bovis - genetics
Mycobacterium bovis - virology
nef gene
Operon
Plasmids
Simian immunodeficiency virus
Simian Immunodeficiency Virus - genetics
Simian Immunodeficiency Virus - immunology
T-Lymphocytes - immunology
Vaccines, antisera, therapeutical immunoglobulins and monoclonal antibodies
Mycobacterial infection
Site specificity
Specificity
Gene
Bacteria
Vector
Immunopathology
Immune response
Mycobacterium bovis
Operon
Retroviridae
Chromosome
Gene expression
Lentivirus
Immune deficiency
Infection
Virus
Antigen
Plasmid
Tuberculosis
Mycobacteriales
Bacteriosis
Mycobacteriaceae
Replication
Actinomycetes
Simian immunodeficiency virus
PROTECTIVE IMMUNITY
RHESUS-MONKEYS
BACILLE CALMETTE-GUERIN
TUBERCULOSIS
OSPA LIPOPROTEIN
MICE
INFECTION
LYME-DISEASE VACCINE
SURFACE PROTEIN-A
NEF
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Abstract Classifications Services IAI Citing Articles Google Scholar PubMed Related Content Social Bookmarking CiteULike Delicious Digg Facebook Google+ Mendeley Reddit StumbleUpon Twitter current issue Spotlights in the Current Issue IAI About IAI Subscribers Authors Reviewers Advertisers Inquiries from the Press Permissions & Commercial Reprints ASM Journals Public Access Policy Connect to IAI IAI RSS Feeds 1752 N Street N.W. • Washington DC 20036 202.737.3600 • 202.942.9355 fax • journals@asmusa.org Print ISSN: 0019-9567 Online ISSN: 1098-5522 Copyright © 2014 by the American Society for Microbiology.   For an alternate route to IAI .asm.org, visit: IAI       
AbstractList 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.
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.
ABSTRACT 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.
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Author E. Badell
B. Gicquel
I. Méderlé
N. Winter
I. Bourguin
J. Moniz-Peireira
D. Ensergueix
AuthorAffiliation Unité de Génétique Mycobactérienne, Institut Pasteur, 75724 Paris Cedex 15, France, 1 Laboratorio de Microbiologia Molecular, Faculdade de Farmacia, Universidade de Lisboa, 1600 Lisbon, Portugal 2
AuthorAffiliation_xml – name: Unité de Génétique Mycobactérienne, Institut Pasteur, 75724 Paris Cedex 15, France, 1 Laboratorio de Microbiologia Molecular, Faculdade de Farmacia, Universidade de Lisboa, 1600 Lisbon, Portugal 2
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  fullname: MEDERLE, I
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  surname: ENSERGUEIX
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  fullname: MONIZ-PEIREIRA, J
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  givenname: N
  surname: WINTER
  fullname: WINTER, N
  organization: Unité de Génétique Mycobactérienne, Institut Pasteur, 75724 Paris, France
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Issue 1
Keywords Mycobacterial infection
Site specificity
Specificity
Gene
Bacteria
Vector
Immunopathology
Immune response
Mycobacterium bovis
Operon
Retroviridae
Chromosome
Gene expression
Lentivirus
Immune deficiency
Infection
Virus
Antigen
Plasmid
Tuberculosis
Mycobacteriales
Bacteriosis
Mycobacteriaceae
Replication
Actinomycetes
Simian immunodeficiency virus
PROTECTIVE IMMUNITY
RHESUS-MONKEYS
BACILLE CALMETTE-GUERIN
TUBERCULOSIS
OSPA LIPOPROTEIN
MICE
INFECTION
LYME-DISEASE VACCINE
SURFACE PROTEIN-A
NEF
Language English
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PMCID: PMC127622
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@pasteur.fr.
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Bivalent recombinant strains of Mycobacterium bovis BCG (rBCG) expressing the early regulatory nef and the structural gag(p26) genes from the simian...
ABSTRACT Bivalent recombinant strains of Mycobacterium bovis BCG (rBCG) expressing the early regulatory nef and the structural gag (p26) genes from the simian...
Bivalent recombinant strains of Mycobacterium bovis BCG (rBCG) expressing the early regulatory nef and the structural gag (p26) genes from the simian...
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StartPage 303
SubjectTerms Animals
Antibodies, Viral - immunology
Antigens, Viral - genetics
Antigens, Viral - immunology
Bacteriology
Bacteriophages
Biological and medical sciences
Cells, Cultured
Chromosomes, Bacterial
Cloning, Molecular - methods
DNA, Viral
Female
Fundamental and applied biological sciences. Psychology
gag gene
Gene Expression
Gene Products, gag - genetics
Gene Products, gag - immunology
Gene Products, nef - genetics
Gene Products, nef - immunology
Genetic Vectors - genetics
Genetics
Life Sciences
Macrophages - cytology
Macrophages - immunology
Mice
Mice, Inbred BALB C
Microbial Immunity and Vaccines
Microbiology
Microbiology and Parasitology
Mutagenesis, Insertional - methods
Mutagenesis, Site-Directed
Mycobacterium bovis
Mycobacterium bovis - genetics
Mycobacterium bovis - virology
nef gene
Operon
Plasmids
Simian immunodeficiency virus
Simian Immunodeficiency Virus - genetics
Simian Immunodeficiency Virus - immunology
T-Lymphocytes - immunology
Vaccines, antisera, therapeutical immunoglobulins and monoclonal antibodies
Title Plasmidic versus Insertional Cloning of Heterologous Genes in Mycobacterium bovis BCG: Impact on In Vivo Antigen Persistence and Immune Responses
URI http://iai.asm.org/content/70/1/303.abstract
https://www.ncbi.nlm.nih.gov/pubmed/11748196
https://search.proquest.com/docview/18219601
https://hal.inrae.fr/hal-02682618
https://pubmed.ncbi.nlm.nih.gov/PMC127622
Volume 70
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