title: Pathogenicity and immunogenicity of a vaccine strain of Listeria monocytogenes that relies on a suicide plasmid to supply an essential gene product.
abstract: Listeria monocytogenes is a bacterial pathogen that elicits a strong cellular immune response and thus has potential use as a vaccine vector. An attenuated strain, L. monocytogenes dal dat, produced by deletion of two genes (dal and dat) used for d-alanine synthesis, induces cytotoxic T lymphocytes and protective immunity in mice following infection in the presence of d-alanine. In order to obviate the dependence of L. monocytogenes dal dat on supplemental d-alanine yet retain its attenuation and immunogenicity, we explored mechanisms to allow transient endogenous synthesis of the amino acid. Here, we report on a derivative strain, L. monocytogenes dal dat/pRRR, that expresses a dal gene and synthesizes d-alanine under highly selective conditions. We constructed the suicide plasmid pRRR carrying a dal gene surrounded by two res1 sites and a resolvase gene, tnpR, which acts at the res1 sites. The resolvase gene is regulated by a promoter activated upon exposure to host cell cytosol. L. monocytogenes dal dat/pRRR was thus able to grow in liquid culture and to infect host cells without d-alanine supplementation. However, after infection of these cells, resolvase-mediated excision of the dal gene resulted in strong down-regulation of racemase expression. As a result, this system allowed only transient growth of L. monocytogenes dal dat/pRRR in infected cells and survival in animals for only 2 to 3 days. Nevertheless, mice immunized with L. monocytogenes dal dat/pRRR generated listeriolysin O-specific effector and memory CD8(+) T cells and were protected against lethal challenge by wild-type Listeria. This vector may be an attractive vaccine candidate for the induction of protective cellular immune responses.
authors.affiliation: 203C Johnson Pavilion, 3610 Hamilton Walk, Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.
authors.forename: Xinyan
authors.initials: X
authors.lastname: Zhao
authors.forename: Zhongxia
authors.initials: Z
authors.lastname: Li
authors.forename: Baiyan
authors.initials: B
authors.lastname: Gu
authors.forename: Fred R
authors.initials: FR
authors.lastname: Frankel
mesh_terms: D000411:Alanine Racemase / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001428:Bacterial Vaccines / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology*; D002478:Cells, Cultured; D041722:Genes, Transgenic, Suicide; D008089:Listeria monocytogenes / Q000235:genetics* / Q000276:immunology / Q000472:pathogenicity*; D008088:Listeriosis / Q000276:immunology / Q000382:microbiology / Q000517:prevention & control; D051379:Mice; D010957:Plasmids; D045522:Recombinases / Q000235:genetics
chemical_list: D001428:Bacterial Vaccines; D045522:Recombinases; D000411:Alanine Racemase