In vitro and in vivo evaluation of listeria monocytogenes based protein and DNA delivery Sinha, Shubhra 10.4225/03/58b4bbc86a7e7 https://bridges.monash.edu/articles/thesis/In_vitro_and_in_vivo_evaluation_of_listeria_monocytogenes_based_protein_and_DNA_delivery/4700566 Listeria monocytogenes was chosen as a carrier for vaccine delivery to take advantage of several unique features. It is capable of invading, surviving and replicating in the majority of mammalian host cells. Due to its abilities to infect phagocytic and non-phagocytic cells and to survive in the cytoplasm of mammalian cells, it is a promising vector for oral delivery of vaccines, providing that safety measures can be built into the system to reduce its pathogenicity. Since the organism is capable of infecting the gastrointestinal epithelium, it is particularly attractive as a basis for designing a generic delivery system for oral vaccination. In this study, a stable recombinant suicidal strain of L. monocytogenes (rsΔ2), developed previously in our laboratory, was used for protein and DNA delivery. The L. monocytogenes strain (rsΔ2) contains the cell wall hydrolysis gene “ply118” and its associated holin gene from a Listeria specific phage A118. These genes are integrated into the L. monocytogenes genome and promote lysis of the rsΔ2 strain when the bacterial cells reach the cytoplasm of mammalian cells. The engineered plasmid pDuLX is a dual expression vector that can drive gene expression from each of two promoters, the Listeria promoter (phly) and the commonly used eukaryotic viral promoter (PCMV). An early objective of this study was to use the luciferase reporter gene to demonstrate that luciferase protein can be detected after uptake of the rsΔ2 strain containing pDuLX-Luc by Caco-2 cells. The luciferase reporter demonstrated that pDuLX can deliver protein and DNA to dividing Caco-2 human epithelial monolayers. Non-dividing (fully differentiated) cells were resistant to lipofectamine transfection, a finding that can be explained by lack of DNA delivery to the nucleus. It was demonstrated that when differentiated Caco-2 monolayers were treated with rsΔ2, the bacteria were able to deliver a significant quantity of luciferase protein produced by the Listeria cells. By implication the bacteria were also able to deliver DNA, but expression driven by the eukaryotic promoter in differentiated Caco-2 cells was not observed. The presence of luciferase in non-dividing Caco-2 cells was explained by activation of the promoter Phly in pDuLX which drove expression of luciferase when the bacteria were grown in charcoal-treated BHI medium. This results in presence of reporter protein in the Listeria cells prior to delivery into the host Caco-2 cells. Eukaryotic expression of luciferase in non-dividing cells was absent because the pDuLX plasmid lacked a mechanism to enter the nucleus. When the rsΔ2 strain was taken up by Caco-2 cells, there was little or no bacterial growth, whereas when the control Δ2 strain was taken up it was shown to be viable and multiplied by approximately three log cycles over a 2-day period within the Caco-2 cells. A small mass of protein or DNA also appeared to be delivered to host cells by the Δ2 strain, perhaps because some bacteria died but, despite the extent of bacterial growth, the mass of protein delivered to dividing Caco-2 cells by the Δ2 strain was considerably less than that delivered by the rsΔ2 strain. In vivo experiments to test the rsΔ2 strain as a potential delivery system for vaccines were carried out in a mouse model using ovalbumin as the test vaccine via either intramuscular or oral administration. After intramuscular injection, the bacterial strain, rs∆2(pDuLX-OVA) produced significantly higher titres of antibody and was also more effective at inducing targeted T cell lysis, when compared to negative controls and other strains. This could be explained by delivery of OVA originally expressed from the prokaryotic promoter present in pDuLX-OVA. Some activity was evident using rs∆2(p3L-OVA) which does not contain the prokaryotic promoter, suggesting that some transfection of mouse myocytes, other cells present in muscle, or infiltrating cells of the immune system, occurred after intramuscular injection. Delivery of the negative control rs∆2(pDuLX-Luc) confirmed that the observed activity was induced specifically by the ovalbumin vaccination. Tests of oral ovalbumin vaccination of mice were performed by gavage using rs∆2(pDuLX-OVA) and the negative control rs∆2(pDuLX-Luc). After a single dose there was evidence that the system was also effective as an oral vaccine. Further work will be required to investigate biological activity after multiple oral doses to optimise a dosage regimen for oral vaccination. 2017-02-27 23:52:38 In vivo DNA thesis(doctorate) Restricted access ethesis-20130430-12414 1959.1/860185 monash:118451 Protein Listeria Oral vaccine 2013