Immune Response to Lactobacillus plantarum Expressing Borrelia burgdorferi OspA Is Modulated by the Lipid Modification of the Antigen

Over the past decade there has been increasing interest in the use of lactic acid bacteria as mucosal delivery vehicles for vaccine antigens, microbicides and therapeutics. We investigated the mechanism by which a mucosal vaccine based in recombinant lactic acid bacteria breaks the immunological tolerance of the gut in order to elicit a protective immune response.We analyzed how the lipid modification of OspA affects the localization of the antigen in our delivery vehicle using a number of biochemistry techniques. Furthermore, we examined how OspA-expressing L. plantarum breaks the oral tolerance of the gut by stimulating human intestinal epithelial cells, peripheral blood mononuclear cells and monocyte derived dendritic cells and measuring cytokine production. We show that the leader peptide of OspA targets the protein to the cell envelope of L. plantarum, and it is responsible for protein export across the membrane. Mutation of the lipidation site in OspA redirects protein localization within the cell envelope. Further, we show that lipidated-OspA-expressing L. plantarum does not induce secretion of the pro-inflammatory cytokine IL-8 by intestinal epithelial cells. In addition, it breaks oral tolerance of the gut via Th1/Th2 cell mediated immunity, as shown by the production of pro- and anti-inflammatory cytokines by human dendritic cells, and by the production of IgG2a and IgG1 antibodies, respectively.Lipid modification of OspA expressed in L. plantarum modulates the immune response to this antigen through a Th1/Th2 immune response

Distinct Peripheral Blood RNA Responses to Salmonella in Pigs Differing in Salmonella Shedding Levels: Intersection of IFNG, TLR and miRNA Pathways

Transcriptomic analysis of the response to bacterial pathogens has been reported for several species, yet few studies have investigated the transcriptional differences in whole blood in subjects that differ in their disease response phenotypes. Salmonella species infect many vertebrate species, and pigs colonized with Salmonella enterica serovar Typhimurium (ST) are usually asymptomatic, making detection of these Salmonella-carrier pigs difficult. The variable fecal shedding of Salmonella is an important cause of foodborne illness and zoonotic disease. To investigate gene pathways and biomarkers associated with the variance in Salmonella shedding following experimental inoculation, we initiated the first analysis of the whole blood transcriptional response induced by Salmonella. A population of pigs (n = 40) was inoculated with ST and peripheral blood and fecal Salmonella counts were collected between 2 and 20 days post-inoculation (dpi). Two groups of pigs with either low shedding (LS) or persistent shedding (PS) phenotypes were identified. Global transcriptional changes in response to ST inoculation were identified by Affymetrix Genechip® analysis of peripheral blood RNA at day 0 and 2 dpi. ST inoculation triggered substantial gene expression changes in the pigs and there was differential expression of many genes between LS and PS pigs. Analysis of the differential profiles of gene expression within and between PS and LS phenotypic classes identified distinct regulatory pathways mediated by IFN-γ, TNF, NF-κB, or one of several miRNAs. We confirmed the activation of two regulatory factors, SPI1 and CEBPB, and demonstrated that expression of miR-155 was decreased specifically in the PS animals. These data provide insight into specific pathways associated with extremes in Salmonella fecal shedding that can be targeted for further exploration on why some animals develop a carrier state. This knowledge can also be used to develop rational manipulations of genetics, pharmaceuticals, nutrition or husbandry methods to decrease Salmonella colonization, shedding and spread