ABSTRACT
BACKGROUND/PURPOSE: Leptospirosis is a neglected zoonosis, imposing significant human and veterinary public health burdens. In this study, recombinant LipL3293-147 and LipL32148-184 middle domain of LipL3293-184, and LipL32171-214, and LipL32215-272 of c-terminal LipL32171-272 truncations were defined for immunodominance of the molecule during Leptospira infections revealed by leptospirosis sera. RESULTS: IgM-dominant was directed to highly surface accessible LipL32148-184 and Lipl32171-214. IgG dominance of LipL32148-184 revealed by rabbit anti-Leptospira sera and convalescent leptospirosis paired sera were mapped to highly accessible surface of middle LipL32148-184 truncation whereas two LipL32148-184 and LipL32215-272 truncations were IgG-dominant when revealed by single leptospirosis sera. The IgM-dominant of LipL32148-214 and IgG-dominant LipL32148-184 peptides have highly conserved amino acids of 70% identity among pathogenic and intermediate Leptospira species and were mapped to the highly surface accessible area of LipL32 molecule that mediated interaction of host components. IgG dominance of two therapeutic epitopes located at LipL32243-253 and LipL32122-130 of mAbLPF1 and mAbLPF2, respectively has been shown less IgG-dominant (<30%), located outside IgG-dominant regions characterized by leptospirosis paired sera. CONCLUSION: The IgM- and IgG-dominant LipL32 could be further perspectives for immunodominant LipL32-based serodiagnosis and LipL32 epitope-based vaccine.
Subject(s)
Antibodies, Monoclonal/immunology , Bacterial Outer Membrane Proteins/immunology , Immunodominant Epitopes/immunology , Leptospira/immunology , Leptospirosis/immunology , Adolescent , Adult , Aged , Animals , Antibodies, Bacterial/blood , Antibodies, Monoclonal/therapeutic use , Bacterial Outer Membrane Proteins/chemistry , Humans , Immunoglobulin G/blood , Immunoglobulin M/blood , Middle Aged , Peptides/chemistry , Peptides/immunology , Rabbits , Serologic Tests , Young AdultABSTRACT
The SAP genes of Fasciola encode proteins belonging to the saposin-like protein family. The saposin signature, a compact domain of mainly alpha-helical character, contains six conserved cysteine residues and has been implicated in membrane-binding, pore formation, and subsequent cell lysis in several family members. Recombinant SAP-2 of F. hepatica has been shown to induce lysis of human erythrocytes and peripheral blood mononuclear cells. This suggests that the SAPs are involved in the nutrition of Fasciola as the released content of lysed host cells is available for further enzymatic processing and uptake by the parasite. In the present study a new SAP-3 cDNA was obtained in an immunoscreen of an adult stage F. gigantica cDNA library with an antiserum against the parasite's excretion/secretion antigens. SAP-1 and SAP-2 cDNAs were isolated from F. gigantica cDNA libraries using oligonucleotide primers specific to the SAP-1 and SAP-2 DNA sequences from F. hepatica. Transcripts of the three SAPs are present from the metacercarial to the adult stage and are located to the gut epithelium. In immatures SAP-1 RNA is the predominant product whereas in adults SAP-2 and -3 are the more abundant products. Polyclonal anti-SAP-1 and SAP-2 antisera confirmed the tissue-specificity and revealed the subcellular localization of SAPs in large granules concentrated in the apical part of the gut epithelial cells of the parasite. Interestingly, evolutionary conservation of the Fasciola SAP sequences among other trematodes is low at 20-30% sequence identity comparable to the Entamoeba amoebapore sequences.
