Identification and immunogenicity analysis of predominant T-B combined antigenic epitopes on the outer membrane protein Loa22 of Leptospira interrogans strains

Abstract

Objective To screen and identify the predominant T- and B-cell (T-B) combined antigenic epitopes on the outer membrane protein Loa22 of pathogenic Leptospira interrogans (L.interrogans) stains and to further analyze their immunogenicity. Methods PCR analysis was used to detect loa22 gene in L. interrogans strains belonging to eight different serogroups or serovars prevalent in China. The PCR products were sequenced after T-A cloning. A prokaryotic expression system for loa22 gene of L. interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai was constructed. The expressed recombinant protein rLoa22 was extracted by Ni-NTA affinity chromatography. The rabbit anti-rLoa22 serum samples and IgG were prepared. The T-B combined antigenic epitopes on Loa22 protein were predicted by using professional bioinformatic softwares. Phage display in combination with Western blot assay and ELISA were performed to identify the immunogenicity of the recombinant phage PⅢ protein-displayed and artificially-synthesized T-B combined antigenic epitopes, respectively. MTS assay and ELISA were performed to detect the activation of T cells and the expression of IL-2, IL-4 and IFN-γ induced by T-B combined antigenic epitopes, respectively. Results All of the tested pathogenic Leptospira strains were positive for loa22 gene, sharing 85.5%-99.8% homologies in nucleotide sequences and 93.9%-99.5% homologies in amino acid sequences. The constructed prokaryotic expression system for loa22 gene efficiently expressed the rLoa22 protein. Among four T-B combined antigenic epitopes (Loa22-77, Loa22-90, Loa22-125 and Loa22-157), only Loa22-90 epitope presented a strong positive band in Western blot analysis. The proliferation of CD4+ T cells and the expression of IL-2 (Th1) and IL-4 (Th2) were significantly enhanced by the stimulation with Loa22-90 epitope peptide (P<0.05). Conclusion Loa22 protein is a sequence-conserved genus-specific outer membrane protein of L. interrogans. The Loa22-90 epitope is the predominant T-B combined antigenic epitope of Loa22 protein, which might be used as a candidate antigenic epitope in the development of multiple antigenic peptide (MAP) vaccines against Leptospira infection. Key words: Leptospira; Outer membrane protein; Loa22; T-B combined antigenic epitope; Immunogenicity