Expression of a leptospiral leucine-rich repeat protein using a food-grade vector in Lactobacillus plantarum, as a strategy for vaccine delivery

Abstract

In this study, a first food-grade mucosal vaccine against leptospirosis was developed without the use of antibiotic resistance gene. This expression system is based on a food-grade host/vector system of Lactobacillus plantarum and a new vaccine candidate antigen, a leucine-rich repeat (LRR) protein of Leptospira borgpetersenii. The LRR of interest from serovar Sejroe is encoded by two overlapping genes and these genes were fused together by site-directed mutagenesis. The mutant gene thus obtained could be successfully expressed in this system as was shown by western blot analysis and liquid chromatography-mass spectrometry (LC-MS/MS) analysis. In addition, this analysis showed that the mutant LRR protein fused to a homologous signal peptide of L. plantarum could be exported to the cell surface as a result of the native LPXAG motif of the heterologous LRR protein, which presumably is responsible for anchoring the protein to the cell wall of L. plantarum. This new strategy could be an essential tool for further studies of leptospirosis mucosal vaccine delivery.

Leptospira borgpetersenii Leucine-Rich Repeat Proteins and Derived Peptides in an Indirect ELISA Development for the Diagnosis of Canine Leptospiral Infections

“…The LipL32 recombinant protein of L. interrogans was purchased from Rekom Biotech (Spain). The rKU_Sej_LRR_2012M protein was produced by Escherichia coli BL21 Star TM (DE3) expression system, containing the pET161_hKU_R21M_2012 plasmid, as previously described [ 43 , 44 ]. Briefly, the protein expression was induced with 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG; Sigma–Aldrich, Burlington, MA, USA) and incubated at 30 °C, 200 rpm for 12 h. Cells were harvested by centrifugation at 4000× g for 15 min at 4 °C and washed with lysis-equilibration-wash buffer (LEW buffer; 50 mM NaH 2 PO 4 , 300 mM NaCl, pH 8.0).…”

“…The L. borgpetersenii serovar Sejroe clone R21-N2012 (Accession: JN627494.1) was mutated and fused with clone R21-C2012 (Accession: JN627492.1) to create the recombinant LRR protein, named rKU_Sej_LRR_2012M [ 42 , 43 ]. In this study, the LRR protein rKU_Sej_LRR_2012M of Leptospira was evaluated as an antigen for the development of an indirect ELISA and for the analysis of canine antisera against Leptospira.…”

Leptospiral Immunoglobulin-Like Domain Proteins: Roles in Virulence and Immunity

“…Triacylated lipopeptides stimulate signaling through TLR1/TLR2 heterodimers ( 140 ), and the lipid moiety influences the appearance of specific IgG subtypes in mice orally immunized with Lactobacillus plantarum expressing N-terminal triacylated OspA ( 141 ). L. plantarum is being explored as a vehicle to orally deliver leptospiral immunogens ( 142 ) in part because of its ability to stimulate innate immune memory via macrophages ( 143 ).…”

Living Therapeutics: The Next Frontier of Precision Medicine

“…This study demonstrates the potential of engineered commensals as delivery vehicles for exogenous antigens, though further optimization to improve immunogenicity and duration of immunity are required. More recent studies have used similar surface-tethered antigen strategies to generate potential vaccines against influenza and Leptospira infections …”

“…More recent studies have used similar surface-tethered antigen strategies to generate potential vaccines against influenza 72 and Leptospira infections. 73 Enhanced Antigen Presentation. Engineered commensal microbes have also been combined with clever antigen design to improve their efficacy as vaccine delivery vehicles (Figure 3B).…”