Therapeutic epitopes of Leptospira LipL32 protein and their characteristics.

Two LipL32-specific mouse monoclonal antibodies (mAbLPF1 and mAbLPF2) which neutralized Leptospira-mediated hemolysis in vitro and rescued hamsters from lethal Leptospira infection were produced. In this communication, locations and characteristics of the protective epitopes of the mAbs were studied by using a truncated LipL32 recombinant protein based-immunoassay and phage consensus mimotope identification and multiple alignments. The mAbLPF1 epitope consisted of P243, L244, I245, H246, L252 and Q253 on the LipL32 protein; it is mapped on the surface-exposed region of non-continuous ß13-turn and C-terminal amphipathic α6 helix with hydrophobic patch, contributing to phospholipid/host cell adhesion and membrane insertion on one side, and hydrophilic, acidic and basic amino acid residues on another side. The epitope peptide of the mAbLPF2 is linear 122PEEKSMPHW130 and located on surface-exposed α1 and α2 between ß7 and ß8 that bound to several host constituents. Both epitopes are highly conserved among the pathogenic and intermediately pathogenic Leptospira spp. and are absent from the LipL32 superfamily proteins of other microorganisms. This study not only enlightens the molecular mechanisms of the therapeutic mAbLPF1 and mAbLPF2, but also elaborates the potential of the two LipL32 regions as diagnostic and vaccine targets for leptospirosis.

Humanized-monoclonal antibody against heterologous Leptospira infection.

Patients with leptospirosis are commonly treated with antibiotics. Jarisch-Herxheimer reaction caused by toxic bacterial substances massively released as a result of the antibiotic mediated-bacterial lysis occurs in some patients which may aggravate the existing severe clinical manifestations. In this study, a humanized-murine single-chain monoclonal antibody (HuScFv) was produced and tested as an alternative of antibiotics for treatment of leptospirosis. Complementary DNA was prepared from total RNA of a murine hybridoma clone secreting monoclonal antibody (MAb) specific to LipL32 of pathogenic Leptospira spp. The MAb had therapeutic efficacy in Leptospira challenged hamsters. The VH and VL coding sequences were amplified using the cDNA as a template. The sequences were linked to form a single-chain variable murine DNA fragment (muscFv). CDR sequences of the muscFv were grafted onto the best matching human VH and VL immunoglobulin frameworks. After cloning of the humanized murine DNA sequences (huscFv) into a phagemid vector and the vector was introduced into competent Escherichia coli, the HuScFv was produced. On the same weight basis, the HuScFv possessed equal neutralizing activities to the murine ScFv counterpart against heterologous Leptospira-mediated hemolysis in vitro and rescued hamsters from a heterologous Leptospira lethal challenge. The HuScFv antibody has high therapeutic potential as an alternative to antibiotics for human leptospirosis, especially for drug hypersensitive patients.

Monoclonal antibodies to LipL32 protect against heterologous Leptospira spp. challenge.

A non-culture-based leptospirosis vaccine that cross-protects against infection caused by heterologous Leptospira spp. should replace the currently available products, which are qualitatively and quantitatively inadequate. With that in mind, two murine hybridomas secreting monoclonal antibodies (MAb) binding only to homogenates of pathogenic Leptospira spp., and not of the saprophytic L. biflexa, serogroup Patoc, serovar Patoc, were produced. The MAbs of both clones neutralized Leptospira-mediated human red blood cell lysis in vitro and rescued hamsters from lethal infection with heterologous Leptospira spp. The orthologous Leptospira spp. protein carrying the MAb epitope(s) was identified by two-dimensional gel electrophoresis (2DE)-based proteomics and database search. The epitopes of the MAbs were located on the major outer membrane protein LipL32 of the pathogenic Leptospira spp. The MAbs in their humanized version are potential leptospirosis immunotherapeutics. They are also suitable as detection reagents in antigen-based assays for the rapid diagnosis of leptospirosis. Recombinant LipL32 is a good candidate for a broad spectrum, non-culture-based leptospirosis vaccine.

Proteome and immunome of pathogenic Leptospira spp. revealed by 2DE and 2DE-immunoblotting with immune serum.

In this study, proteomes of two pathogenic Leptospira spp., namely L. interrogans, serogroup Icterohaemorrhagiae, serovar Copenhageni and L. borgpetersenii, serogroup Tarassovi, serovar Tarassovi, were revealed by using two dimensional gel electrophoresis (2DE)-based-proteomics. Bacterial cells were disrupted in a lysis buffer containing 30 mM Tris, 2 M thiourea, 7 M urea, 4% CHAPS, 2% IPG buffer pH 3-10 and protease inhibitors and then subjected to sonication in order to solubilize as much as possible the bacterial proteins. The 2DE-separated components of both Leptospira homogenates were blotted individually onto membranes and antigenic components (immunomes) were revealed by probing the blots with immune serum of a mouse readily immunized with the homogenate of L. interrogans, serogroup Icterohaemorrhagiae, serovar Copenhageni. The immunogenic proteins of the two pathogenic Leptospira spp. could be grouped into 10 groups. These are: 1) proteins involved in the bacterial transcription and translation including beta subunit transcription anti-termination protein of DNA polymerase III, elongation factors Tu and Ts, and tRNA (guanine-N1)-methyltransferase; 2) proteins functioning as enzymes for metabolisms and nutrient acquisition including acetyl-Co-A acetyltransferase, putative glutamine synthetase, glyceraldehyde-3-phospahte dehydrogenase, NifU-like protein, 3-oxoacyl-(acyl-carrier-protein) reductase, oxidoreductase, sphingomyelinase C precursor, spermidine synthase, beta subunit of succinyl-CoA synthetase, and succinate dehydrogenase iron-sulfur subunit; 3) proteins/enzymes necessary for energy and electron transfer, i.e. electron transfer flavoprotein, and proton-translocating transhydrogenase; 4) enzymes for degradation of misfolded proteins, i.e. ATP-dependent Clp protease; 5) molecular chaperone, i.e. 60 kDa chaperonin; 6) signal transduction system, i.e. response regulator; 7) protein involved in immune evasion in host, i.e. peroxiredoxin; 8) cell structure proteins including MreB (cytoskeletal) and flagellin/ periplasmic flagellin; 9) lipoproteins/outer membrane proteins: LipL32, LipL41, LipL45 and OmpL1; and 10) various hypothetical proteins. Many immunogenic proteins are common to both Leptospira spp. These proteins not only are the diagnostic targets but also have potential as candidates of a broad spectrum leptospirosis vaccine especially the surface exposed components which should be vulnerable to the host immune effector factors.

Components of pathogenic Leptospira spp. with potentials for diagnosis of human leptospirosis.

Existing serological methods for diagnosis of leptospirosis are still unsatisfactorily due mainly to their low accuracy. In this study, serum samples of 18 clinically diagnosed-, IgM dipstick positive-, MAT positive-leptospirosis patients (group 1) were analyzed by IgG Western blotting against SDS-PAGE separated-whole cell homogenates of pathogenic and non-pathogenic Leptospira spp. belonging to 20 serovars of 15 serogroups. The samples of group 1 were collected from the patients at days 3 to 10 after the fever onset (fist samples). Second and third samples could be obtained from 4 patients. Sera of the 22 patients with other febrile illnesses (group 2) and 22 healthy counterparts (group 3) were used as patient- and normal- controls, respectively. Irrespective of the serovar or serogroup of the pathogenic Leptospira spp. used as antigen in the Western blotting, all of the 18 sera of patients with leptospirosis (group 1) gave characteristic diffuse antigen-antibody reactive bands located at approximately 35-38 and 22-26 kDa; and thus 100% diagnostic sensitivity of the Western blot assay. Some serum samples of the leptospirosis patients also reacted to components located at 80-100, approximately 70, 60, 54, and 48 kDa. More bands or the early recognized bands with increased intensity were observed when tested the second and third samples. The characteristic bands were not seen when homogenates of L. biflexa, serogroup Semaranga, serovar Patoc (saprophytic) and L. biflexa, serogroup Andamana, serovar Andamana (non-pathogenic but can infect host) were used in the assay. Sera of groups 2 and 3 did not react to the components at the seven locations implying 100% diagnostic specificity of the IgG Western blot assay. While awaiting validation with more patients' samples, the IgG Western Blot analysis aiming at the detection of the characteristic antigen-antibody reactive bands described in this study has high potential for early, rapid, simple and accurate diagnosis of human leptospirosis.

Mimotope of Leptospira from phage-displayed random peptide library is reactive with both monoclonal antibodies and patients' sera.

The study aim was to use random heptapeptide library displayed by bacteriophage T7 for identifying mimotopes from 15 monoclonal antibodies (MAbs) specific to Leptospira spp., and from four leptospirosis patient sera, respectively. The bound phages, selected from fourth round of bio-panning with each antibody, were cloned by plaque isolation and the binding specificity of individual clones were confirmed by enzyme-linked immunosorbent assay, before being further amplified and checked for phage peptide sequence using PCR and DNA sequencing. All together 150 phages were selected, mimotope from 86 phages (56.6%) were found to match with protein sequences of Leptospira from GenBank database. The predominant mimotopes were mimotope with sequence LTPCD that found in 27.3%, followed by TPCSK (16%), KSKKSS (4%), KTKRXAS (4%), SSKSYR (3.3%), DPNXNSF (3.3%), KSGRC (2.6%), TLINIF (2%), TPCI (2%), 1.33% each with mimotopes PKKS, PCNTKXTA, and CTKKK, and one phage each (0.66%) with mimotopes PTFGS, TNSKRK, SKSSRC, RSKRIR, VTNNTP, and CSNXSKR. Interestingly, mimotopes LTPCD, TPCSK, and TPCI were found to react with both MAb and patient's sera. The matched proteins from GenBank namely, leptospiral putative outer membrane protein (matched with mimotope PTFGS), thermolysin precursor protein (matched with mimotope TPCIXXGSAS), and hypothetical protein LIC12228 (matched with mimotope CSNXSKR), were found to locate at outer membrane of Leptospira. These phage mimotopes and matched proteins may have potential for further use as diagnostic reagent and immunogen against leptospirosis in the future. The results demonstrate that phage display technique has potential for rapidly identifying phage mimotopes that interact with leptospiral MAbs and patient's sera.

Evaluation of a monoclonal antibody-based dot-blot ELISA for detection of Leptospira spp in bovine urine samples.

OBJECTIVE: To evaluate the efficacy of a novel monoclonal antibody (MAb)-based dot-blot ELISA for detection of Leptospira antigens in urine samples of cattle. SAMPLE POPULATION: Blood and urine samples of 45 test cattle from 5 farms in Chonburi province and 20 control cattle from 2 farms in Khon Kaen province in Thailand. PROCEDURE: Blood and urine samples were assayed (microscopic agglutination test and urine antigen test) for Leptospira infection by use of an MAb-based dot-blot ELISA, and results for the ELISA were compared with those for dark-field microscopy (DFM), microbial culture, and a polymerase chain reaction (PCR) assay. RESULTS: All urine samples with positive results for DFM, microbial culture, PCR assay, or > 1 of these tests also had positive results when tested by use of the MAb-based dot-blot ELISA, except for 1 sample that had positive results only for the PCR assay. Detection limits of the dot-blot ELISA were 10(3) leptospires/mL of urine and 9.3 ng of Leptospira homogenate. Comparison revealed that the diagnostic sensitivity, specificity, efficacy (accuracy), positive predictive value, and negative predictive value for the ELISA were in agreement with results for DFM (100%, 72.72%, 80%, 57.14%, and 100%, respectively), microbial culture (100%, 61.54%, 66.62%, 28.57%, and 100%, respectively), and PCR assay (95.45%, 100%, 91.77%, 100%, and 95.83%, respectively). CONCLUSIONS AND CLINICAL RELEVANCE: The MAb-based dot-blot ELISA is suitable as a tool for detecting leptospires in urine samples of cattle.

Diagnosis of human leptospirosis by monoclonal antibody-based antigen detection in urine.

Hybridomas secreting specific monoclonal antibodies (MAb) to all members of the genus Leptospira (clone LF9) and those that are specific only to the pathogenic species (clones LD5 and LE1) were produced. MAb LF9, which was immunoglobulin G1 (IgG1), reacted to a 38-kDa component of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated whole-cell lysates of all Leptospira spp., while MAb LD5 and MAb LE1, which were IgG1 and IgG2a, respectively, reacted to the 35- to 36-kDa components of all serogroups of the pathogenic species of LEPTOSPIRA: The MAb LD5 was used in a dot blot-enzyme-linked immunosorbent assay (dot-ELISA) for detecting Leptospira antigen in urine samples serially collected from two groups of patients diagnosed with leptospirosis, i.e., 36 clinically diagnosed patients and 25 Leptospira culture confirmed patients. Their serum samples were tested serologically by IgM Dipstick assay, indirect immunofluorescence assay (IFA), and/or microscopic agglutination test (MAT). Urine samples of 26 patients diagnosed with other illnesses and 120 healthy individuals served as controls. For the first group of patients, who had been ill for an average of 3.4 days before hospitalization, the IgM Dipstick test, IFA, and MAT were positive for 69.4, 70.0, and 85.7% of patients, while the Leptospira antigenuria tested by the MAb-based dot-ELISA was positive for 75.0, 88.9, 97.2, 97.2, and 100% of patients on days 1, 2, 3, 7, and 14 of hospitalization, respectively. All but 1 of 11 patients whose serum samples collected on the first day of hospitalization were IgM seronegative, were positive by urine antigen test on day 1. This is strong evidence that detection of antigen in urine can provide diagnostic information that could be useful in directing early therapeutic intervention. The MAT was positive in 10 of 12 patients (83.3%) of the 25 culture-positive Leptospira patients who had been ill for an average of 5.04 days before hospitalization, and the Leptospira antigen was found in 64.0, 84.0, 96.0, 100, 100, 100, and 100% of the patients' urine samples collected on days 1, 2, 3, 4, 5, 6, and 7 of hospitalization, respectively. Leptospira antigenuria was found in 3 of the 26 patients diagnosed with other illnesses and 1 of the 120 healthy controls. The reasons for this positivity are discussed. The detection of antigen in urine by the monoclonal antibody-based dot-ELISA has high potential for rapid, sensitive, and specific diagnosis of leptospirosis at a low cost.