title: Efficacy of a Lactococcus lactis ΔpyrG vaccine delivery platform expressing chromosomally integrated hly from Listeria monocytogenes.
abstract: Listeria monocytogenes is a significant food-borne pathogen and the causative agent of listeriosis, a disease which manifests as meningitis in immunocompromised adults or infection of the fetus and miscarriage in pregnant women. We have previously used Lactococcus lactis, a GRAS (Generally Regarded As Safe) organism, as a vaccine vector against listeriosis by engineering plasmid-mediated expression of the immunodominant antigen from L. monocytogenes, listeriolysin O (LLO). However, the environmental release of an engineered vaccine vector carrying a replicating plasmid during clinical usage may raise safety concerns. Here we describe the integration of the LLO gene (hly) into the L. lactis chromosome through homologous double crossover to allow stable expression, in order to avoid the use of antibiotic selection markers and to eliminate the requirement for a plasmid-based system. The approach was designed to simultaneously eliminate the pyrG gene encoding the CTP synthase which is responsible for converting UTP to CTP in a unique step in the de novo pyrimidine synthesis in L. lactis. This gene was targeted in order to restrict bacterial replication outside of the host (biological containment). The resulting cytidine auxotroph was able to secrete LLO constitutively and could elicit LLO(91-99)-specific CD8(+) T lymphocytes in the murine infection model. Moreover, protection against lethal challenge with L. monocytogenes was accomplished after intraperitoneal (IP) vaccination with the constructed strain. The implications for the use of cytidine auxotropy in biological containment are discussed.
authors.affiliation: School of Pharmacy, University College Cork, Cork, Ireland.
authors.forename: Mohammed
authors.initials: M
authors.lastname: Bahey-El-Din
authors.forename: Pat G
authors.initials: PG
authors.lastname: Casey
authors.forename: Brendan T
authors.initials: BT
authors.lastname: Griffin
authors.forename: Cormac Gm
authors.initials: CG
authors.lastname: Gahan
mesh_terms: D000328:Adult; D000818:Animals; D001427:Bacterial Toxins / Q000235:genetics* / Q000276:immunology*; D001428:Bacterial Vaccines / Q000008:administration & dosage* / Q000235:genetics*; D001483:Base Sequence; D057005:Bioengineering; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D002876:Chromosomes, Bacterial / Q000235:genetics; D003264:Containment of Biohazards; D017931:DNA Primers / Q000235:genetics; D005260:Female; D005516:Food Microbiology; D018014:Gene Transfer Techniques; D005818:Genetic Engineering; D005822:Genetic Vectors; D006360:Heat-Shock Proteins / Q000235:genetics* / Q000276:immunology*; D006460:Hemolysin Proteins / Q000235:genetics* / Q000276:immunology*; D006801:Humans; D013294:Lactococcus lactis / Q000235:genetics* / Q000276:immunology*; D008089:Listeria monocytogenes / Q000235:genetics* / Q000276:immunology* / Q000472:pathogenicity; D008088:Listeriosis / Q000276:immunology / Q000517:prevention & control*; D051379:Mice; D008807:Mice, Inbred BALB C; D011247:Pregnancy
chemical_list: D001427:Bacterial Toxins; D001428:Bacterial Vaccines; D017931:DNA Primers; D006360:Heat-Shock Proteins; D006460:Hemolysin Proteins; C038705:hlyA protein, Listeria monocytogenes