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

Abstract

BackgroundOver 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.Methodology/Principal FindingsWe 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.Conclusions/SignificanceLipid modification of OspA expressed in L. plantarum modulates the immune response to this antigen through a Th1/Th2 immune response.

Oral Immunization with Escherichia coli Expressing a Lipidated Form of LigA Protects Hamsters against Challenge with Leptospira interrogans Serovar Copenhageni

“…1B and C). Lipidation of OspA has been shown to be important for overcoming oral tolerance by inducing a Th1/Th2 immune response (19). Hamsters immunized by oral gavage with E. coli expressing the lipidated LigA7-13 antigen developed a protective immune response to lethal challenge by L. interrogans serovar Copenhageni.…”

“…Inclusion of the 16-amino-acid lipoprotein signal peptide of OspA tags the protein for translocation across the cytoplasmic membrane and subsequent lipidation. Lipidation is known to function as an adjuvant (16)(17)(18), and the immune response to oral immunization with OspA has been shown to be modulated by its lipidation (19).…”

Development of Oral Vaccines Against Lyme Disease

“…Analysis of localization and lipidation of the five vaccine constructs used in this study revealed that the OspA signal peptide tagged OspC K, OspB, BBK32, Salp15 and Salp25 for translocation across the cytoplasmic membrane, given that these proteins were primarily found in the envelope fraction (>60%) as opposed to the cytosolic fraction of the cell. Furthermore, OspC K, BBK32 and Salp15 constructs partitioned mostly into the detergent phase (Figure 3-1), the hydrophobic fraction of the E. coli cell envelope, consistent with protein lipidation and with our previous findings [110,114,115].…”

“…Our vaccine technology consisted of recombinant live E. coli expressing the leader sequence of B. burgdorferi OspA lipoprotein fused to the N-terminus of each candidate immunogen (OspC K, OspB, BBK32, Salp15 or Salp25), aiming to target them across the cytoplasmic membrane to the cell envelope as previously shown [110,114,115]. Analysis of localization and lipidation of the five vaccine constructs used in this study revealed that the OspA signal peptide tagged OspC K, OspB, BBK32, Salp15 and Salp25 for translocation across the cytoplasmic membrane, given that these proteins were primarily found in the envelope fraction (>60%) as opposed to the cytosolic fraction of the cell.…”

“…In a previous study, where we investigated the effect of the lipid modification of the leader peptide OspA in the localization of the antigen in live oral delivery vehicles, we showed that the lipidation of the leader peptide of OspA targeted the protein through the membrane and it accumulated in the cell wall. In contrast, mutation of the lipidation site generated a nonlipidated version of OspA that was localized mostly in the cell membrane [114]. We next applied this platform technology and developed Lactobacillus-based oral vaccines for plague [115] and E. colibased oral vaccines for Leptospirosis [110] where we were able to target the immunogens for translocation across the bacterial cytoplasmic membrane to the cell envelope as a consequence of lipid modification of the leader peptide OspA.…”