Identification of immunodominant B- and T-cell combined epitopes in outer membrane lipoproteins LipL32 and LipL21 of Leptospira interrogans

Abstract

Leptospirosis is a serious infectious disease caused by pathogenic Leptospira. B- and T-cell-mediated immune responses contribute to the mechanisms of Leptospira interrogans infection and immune intervention. LipL32 and LipL21 are the conserved outer membrane lipoproteins of L. interrogans and are considered vaccine candidates. In this study, we identified B- and T-cell combined epitopes within LipL32 and LipL21 to further develop a novel vaccine. By using a computer prediction algorithm, two B- and T-cell combined epitopes of LipL21 and four of LipL32 were predicted. All of the predicted epitopes were expressed in a phage display system. Four epitopes, LipL21 residues 97 to 112 and 176 to 184 (LipL21(97-112) and LipL21(176-184), respectively) and LipL32(133-160) and LipL32(221-247) of LipL32 were selected as antigens by Western blotting and enzyme-linked immunosorbent assay. These selected epitopes were also recognized by CD4(+) T lymphocytes derived from LipL21- or LipL32-immunized BALB/c (H-2(d)) mice and mainly polarized the immune response toward a Th1 phenotype. The identification of epitopes that have both B- and T-cell immune reactivities is of value for studying the immune mechanisms in response to leptospiral infection and for designing an effective vaccine for leptospirosis.

FIG. 1.

SDS-PAGE and Western blot spectrum of each expressed epitope. (A) SDS-PAGE analysis of purified recombinant phage particles. Lane M, protein ladder; lane 1, wild-type M13KE particles; lanes 2 to 5, particles containing epitope fragments of LipL32_46-66, LipL32_133-160, LipL32_201-218, and LipL32_221-247; lanes 6 and 7, particles containing epitope fragments of LipL21_97-122 and LipL21_176-184. Panels B and C show Western blot analysis of the expressed epitope peptides using rabbit serum from L. interrogans serovar Lai-infected or recombinant protein-stimulated rabbits.

FIG. 2.

Direct binding assay of epitopes with antibodies in human serum. Histograms show the binding of the indicated epitopes in sera from patients with (+) or without (−) leptospiral infection. Compared with the control (M13KE), each epitope of LipL32 (residues 46 to 66, 133 to 160, 201 to 218, 221 to 247) and LipL21 (97 to 112 and 176 to 184) recognized the antibody in the patient sera. The data represent the average of three separate experiments.

FIG. 3.

Cell proliferation assay of splenocytes from mice immunized with LipL32 or LipL21 and stimulated with the different epitopes. Lymphocytes (5 × 10⁴) and mitomycin-inactivated allogeneic splenocytes (10⁵) were mixed and stimulated with phage particles containing epitopes from LipL32 (A) or LipL21 (B) for a proliferation assay. The response to each antigen is presented as the mean of three independent experiments. Lymphocytes isolated from mice treated with PBS served as controls to determine if the responses were LipL32 or LipL21 specific. Cells stimulated with ConA or wild-type phage were used as controls.

FIG. 4.

Cytokine profiles of LipL32- and LipL21-specific T cells. Splenocytes from mice immunized with recombinant LipL32 (A) or LipL21 (B) were isolated 10 days after the last immunization and restimulated with epitopes from corresponding proteins in vitro for 72 h together with antigen-presenting cells (APCs). Each value represents three tested mice and in triplicate.