Characterization of the Immunogenic and Antigenic Potential of Putative Lipoproteins from Leptospira interrogans

Abstract

The search for a vaccine capable of conferring heterologous protection, through the identification of conserved and cross-protective antigens, remains an ongoing priority in leptospirosis research. In the present study, an in silico analysis was used to identify potentially protective lipoproteins from Leptospira interrogans serovar Copenhageni. Eight putative lipoproteins were selected (LIC10009, LIC10054, LIC10091, LIC11058, LIC11567, LIC13059, LIC13305, and LIC20172), cloned and expressed in Escherichia coli and purified by affinity chromatography. The recombinant proteins were used to inoculate mice and the subsequent humoral immune response was evaluated by ELISA. Seven of the potential lipoproteins induced a significant IgG response. Furthermore, all of the recombinant proteins were recognized by antibodies present in the sera of severe leptospirosis patients. These putative lipoproteins exhibited potential for further evaluation as prospective vaccine candidates.

A Prime-Boost Strategy Using the Novel Vaccine Candidate, LemA, Protects Hamsters against Leptospirosis

“…The lemA coding sequence was amplified using oligonucleotides based on the lemA gene sequence from the L. interrogans serovar Copenhageni strain Fiocruz L1-130 genome. Cloning into the vector pQE30 and the expression and purification of recombinant LemA (rLemA) protein were performed as previously described (27). The protein concentration was determined by using a bicinchoninic acid (BCA) protein assay kit (Pierce; Thermo Scientific, Rockford, IL, USA).…”

“…The transfected cells were suspended in lysis buffer (PBS containing 4% [vol/vol] NP-40) and incubated on ice for 30 min. The expression of rLemA by cell lysates was analyzed by Western blot (WB) assay using mouse anti-LemA serum (1:100 in PBS) (27). The reactions were developed with ECL WB detection reagents (GE Healthcare, São Paulo, Brazil).…”

“…The antibody responses were monitored by indirect enzyme-linked immunosorbent assay (ELISA) using rLemA as previously described (27). Briefly, each well was coated with 50 ng of rLemA diluted in carbonate-bicarbonate buffer, pH 9.6.…”

“…was confirmed by PCR amplification using genomic DNA from 17 serovars, including serovars of L. borgpetersenii, L. interrogans, L. kirschneri, and L. santarosai. The PCR product was amplified with the primers used to clone the LemA coding region, as described previously (27). The 16S gene was amplified using the primer pair fD1_F and rP2_R (29) to confirm the identity of the DNA template.…”

“…DNA-type vaccine candidates have been evaluated in several leptospirosis vaccine studies (23)(24)(25), including prime-boost strategies (26). In a previous study, we reported that the LemA subunit vaccine preparation induced a strong humoral immune response in immunized mice and that it was recognized by antibodies present in sera of patients with severe leptospirosis (27). The aim of the present study was to determine, using prime-boost, DNA, and protein-based immunizations, whether LemA could induce a protective immune response in hamsters against challenge with a virulent strain of Leptospira interrogans.…”