title: Stable integration vector for nutrient broth-based selection of attenuated Listeria monocytogenes strains with recombinant antigen expression.
abstract: Recombinant Listeria monocytogenes strains induce strong cellular immune responses and may prove useful for antigen delivery for the vaccination of humans. However, the genetic systems currently available for the stable expression of recombinant antigens by L. monocytogenes rely on the use of antibiotic resistance genes. We report on a derivative, pPL2dalGlnA, of the Listeria monocytogenes pPL2 integration vector that completely lacks drug resistance genes. The selectable markers in pPL2dalGlnA are glutamine synthetase (GlnA) and alanine racemase (Dal). This novel vector was stably maintained in auxotropic L. monocytogenes strains that normally require d-alanine. The pPL2dalGlnA vector also partially restored the ability of an L. monocytogenes Deltadal Deltadat strain to colonize the spleens and livers of infected mice. A novel, highly attenuated strain of L. monocytogenes with quadruple deletions was also engineered by deleting the L. monocytogenes actA and plcB virulence genes from a Deltadal Deltadat strain. Infection of mice with recombinants of this mutant strain that express the antigen from pPL2dalGlnA were shown to elicit CD8(+) T-cell responses to human immunodeficiency virus Tat. This vector system is thus useful for stable antigen expression and vaccination studies.
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California 94720, USA. lenzl@njc.org
authors.forename: Laurel L
authors.initials: LL
authors.lastname: Lenz
authors.forename: William A
authors.initials: WA
authors.lastname: Huang
authors.forename: Chenghui
authors.initials: C
authors.lastname: Zhou
authors.forename: Zhongxia
authors.initials: Z
authors.lastname: Li
authors.forename: Richard
authors.initials: R
authors.lastname: Calendar
mesh_terms: D016915:AIDS Vaccines / Q000235:genetics / Q000276:immunology; D000411:Alanine Racemase / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001428:Bacterial Vaccines / Q000235:genetics* / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D005260:Female; D017353:Gene Deletion; D015870:Gene Expression; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D005822:Genetic Vectors; D005974:Glutamate-Ammonia Ligase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008089:Listeria monocytogenes / Q000235:genetics*; D008099:Liver / Q000382:microbiology; D051379:Mice; D008807:Mice, Inbred BALB C; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D011995:Recombination, Genetic; D012641:Selection, Genetic; D013154:Spleen / Q000382:microbiology; D014613:Vaccines, Attenuated / Q000235:genetics / Q000276:immunology; D014774:Virulence
chemical_list: D016915:AIDS Vaccines; D001428:Bacterial Vaccines; D011994:Recombinant Proteins; D014613:Vaccines, Attenuated; D000411:Alanine Racemase; C081173:glutamine synthetase I; D005974:Glutamate-Ammonia Ligase