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

The Mode of Replication Is a Major Factor in Segregational Plasmid Instability in Lactococcus lactis

The effects of the rolling-circle and theta modes of replication on the maintenance of recombinant plasmids in Lactococcus lactis were studied. Heterologous Escherichia coli or bacteriophage λ DNA fragments of various sizes were inserted into vectors based on either the rolling-circle-type plasmid pWV01 or the theta-type plasmid pAMβ1. All pAMβ1 derivatives were stably maintained. pWV01 derivatives, however, showed size-dependent segregational instability, in particular when large DNA fragments were inserted. All recombinant pWV01 derivatives generated high-molecular-weight plasmid multimers (HMW) in amounts that were positively correlated with plasmid size and inversely correlated with the copy numbers of the monomeric plasmid forms. Formation of HMW or reductions in copy numbers were not observed with pAMβ1 derivatives. The results indicate that HMW formation and/or reduction in plasmid copy numbers is an important factor in the maintenance of pWV01 derivatives. It is concluded that theta-type plasmids are superior to rolling-circle-type plasmids for cloning in lactococci

Production of cytokines in human MDDCs co-cultured with purified recombinant _wtOspA or _mutOspA.

<p>Monocytes were isolated from human PBMCs by MACS using the Monocyte Isolation Kit II. Monocytes were derived to immature DCs (MDDCs) by cultivating the cells in the presence of 100 nM GM-CSF and 10 nM IL-4 for 5 days. MDDCs (2×10⁵ cells/well) were seed in 24 well plates and co-cultured with 2.5 µg/ml of _wtOspA or _mutOspA. After 48 h of stimulation, supernatants were collected and TNFα (A), IL-12 (B), IFNγ (C) and IL-10 (D) cytokine production was measured by sandwich ELISA (Quantikine). *<i>p</i><0.001,**<i>p</i><0.05. Results are representative of one of three independent experiments.</p

Localization of recombinant antigens in <i>L. plantarum</i>.

<p>Localization of the recombinant antigens was studied by (A) serial cell fractionation, (B) live-cell ELISA (lcELISA) and (C) Immunofluorescence Assay (IFA). (A) Immunoblot of cellular fractions of <i>L. plantarum</i> expressing OspA antigens: C, cytosol; M, membrane; CW, cell wall. <i>L. plantarum</i> was treated with 250 kU/ml Lysozyme (Lyz) for 45 min to digest the cell wall; protoplasts were disrupted and, membrane and cell wall fractions were separated by ultracentrifugation. 3 µg of each fraction was analyzed in a 12% SDS-PAGE, transferred to PVDF membrane, and tested by immunoblot with OspA-specific monoclonal antibody LA2.2 (mAb LA2.2). (B) Live recombinant <i>L. plantarum</i> were treated during 0, 5 or 45 min with Lyz and then subjected to lcELISA using mAb LA2.2 and anti-mouse IgG secondary antibody labeled with alkaline phosphatase. The Optical Density at 405 nm (OD₄₀₅) of the mean endpoint titer was determined. The average of triplicate samples per sample was determined and the error bar indicates standard deviation. (C) Live recombinant <i>L. plantarum</i> were treated with or without Lyz for 30 min. After cell wall removal, the cells were incubated with mAb LA2.2 followed by Alexa Fluor 488-labeled goat anti-mouse IgG (1∶250) antibodies. Immunofluorescence staining was visualized using a Zeiss inverted Axiovert 200 microscope, and the images were acquired using AxioVision software. *<i>p</i><0.001. Results are representative of one of three independent experiments.</p

Production of cytokines in human MDDC co-cultured with recombinant <i>Lactobacillus</i>.

<p>Monocytes were isolated from human PBMCs by MACS using the Monocyte Isolation Kit II. Monocytes were derived to immature DCs (MDDCs) by cultivating the cells in the presence of 100 nM GM-CSF and 10 nM IL-4 for 5 days. MDDCs (2×10⁵ cells/well) were co-cultured with UV-killed recombinant <i>Lactobacillus</i> expressing OspA (LpA) or the mutant OspA_D17 (LpA_D17) at MOI 10:1 colony-forming units per DC. 100 ng/ml <i>Escherichia coli</i> O111:B4 lipopolysaccharide (LPS) and <i>L. plantarum</i> (Lp) were used as positive and negative control, respectively. After 48 h of stimulation, supernatants were collected and TNF (A), IL-12 (B), IFNγ (C), IL-6 (D) and IL-10 (E) cytokine production was measured by sandwich ELISA (Quantikine). *<i>p</i><0.05,**<i>p</i><0.001. Results are representative of one of three independent experiments.</p

Antibody response to oral administration of recombinant <i>L. plantarum</i>: mucosal IgA.

<p>C3H-HeJ mice were inoculated intragastrically with <i>L. plantarum</i> expressing OspA (LpA) or the mutant OspA_D17 (LpA_D17). Control mice were inoculated with <i>L. plantarum</i> (Lp). Specific mucosal anti-OspA IgA antibodies were measured by indirect ELISA in broncheoalveolar lavage (BAL) (A) and stool (B) collected on day 68. The results corresponding to each mouse are expressed as Optical Density at 450 nm (OD₄₅₀) of the mean endpoint titer. Results are representative of one of three independent experiments. <i>n</i> = 4 mice per group. ns, not significant.</p