Salvatore Giovanni De-Simone

Immunome and venome of Bothrops jararacussu: A proteomic approach to study the molecular immunology of snake toxins

A combination of anti-bothropic and anti-crotalic sera has been reported to be more effective in neutralizing the effects of Bothrops jararacussu venom than anti-bothropic serum alone. The role of proteins from B. jararacussu venom in the horse immune response was evaluated via the analysis of cross-reactivity with homologous and heterologous sera. Many of the proteins in B. jararacussu venom were identified via 2D gel electrophoresis. Western blots revealed that anti-jararacussu showed higher reactivity to l-aminoxidase (LAOs) and snake venom metalloproteinase, (SVMPs) and weaker reactivity towards Snake venom serine proteases (SVSPs), PLA(2), C-type lectin and cysteine-rich proteins. Anti-jararaca preferentially recognized LAOs, SVMPs and SVSPs. Both of these sera failed to recognize low-molecular weight proteins. Anti-crotalic serum clearly recognized LAOs, C-type lectin, SVSP, cysteine-rich proteins, SVMP and Asp49-PLA(2). The cross-reactivity with anti-PLA(2) revealed the immunoreactivity of these antibodies to proteins with molecular masses in a range that is poorly recognized by other studied anti-sera. Our results suggest that the contribution of anti-crotalic serum to the neutralization of B. jararacussu by may be due to its cross-reactivity with proteins such as C-type lectins, SVSPs, Asp49-PLA(2). These results also reinforce the importance of neutralizing the highly toxic proteins inclusive those with low immunogenicity in commercial antivenom production to obtain a highly protective serum against snake venoms.

Trypanosoma cruzi: isolation and characterization of aspartyl proteases.

Two aspartyl proteases activities were identified and isolated from Trypanosoma cruzi epimastigotes: cruzipsin-I (CZP-I) and cruzipsin-II (CZP-II). One was isolated from a soluble fraction (CZP-II) and the other was solubilized with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CZP-I). The molecular mass of both proteases was estimated to be 120 kDa by HPLC gel filtration and the activity of the enzymes was detected in a doublet of bands (56 and 48 kDa) by substrate-sodium dodecyl sulphate-polyacrylamide-gelatin gel electrophoresis. Substrate specificity studies indicated that the enzymes consistently hydrolyze the cathepsin D substrate Phe-Ala-Ala-Phe (4-NO2)-Phe-Val-Leu-O4MP but failed to hydrolyze serine and other protease substrates. Both proteases activities were strongly inhibited by the classic inhibitor pepstatin-A (> or =68%) and the aspartic active site labeling agent, 1,2-epoxy-3-(phenyl-nitrophenoxy) propane (> or =80%). These findings show that both proteases are novel T. cruzi acidic proteases. The physiological function of these enzymes in T. cruzi has under investigation.

Chagas disease-specific antigens: characterization of epitopes in CRA/FRA by synthetic peptide mapping and evaluation by ELISA-peptide assay

BackgroundThe identification of epitopes in proteins recognized by medically relevant antibodies is useful for the development of peptide-based diagnostics and vaccines. In this study, epitopes in the cytoplasmic repetitive antigen (CRA) and flagellar repetitive antigen (FRA) proteins from Trypanosoma cruzi were identified using synthetic peptide techniques and pooled sera from Chagasic patients. The epitopes were further assayed with an ELISA assay based on synthetic peptides.MethodsTwenty-two overlapping synthetic peptides representing the coding sequence of the T. cruzi CRA and FRA proteins were assessed by a Spot-synthesis array analysis using sera donated by patients with Chagas disease. Shorter peptides were selected that represented the determined epitopes and synthesized by solid phase synthesis to evaluate the patterns of cross-reactivities and discrimination through an ELISA-diagnostic assay.ResultsThe peptide Spot-synthesis array successfully identified two IgG antigenic determinants in the CRA protein and four in FRA. Bioinformatics suggested that the CRA antigens were unique to T. cruzi while the FRA antigen showed similarity with sequences present within various proteins from Leishmania sp. Subsequently, shorter peptides representing the CRA-1, CRA-2 and FRA-1 epitopes were synthesized by solid phase synthesis and assayed by an ELISA-diagnostic assay. The CRA antigens gave a high discrimination between Chagasic, Leishmaniasis and T. cruzi-uninfected serum. A sensitivity and specificity of 100% was calculated for CRA. While the FRA antigen showed a slightly lower sensitivity (91.6%), its specificity was only 60%.ConclusionsThe epitopes recognized by human anti-T. cruzi antibodies have been precisely located in two biomarkers of T. cruzi, CRA and FRA. The results from screening a panel of patient sera through an ELISA assay based on peptides representing these epitopes strongly suggest that the sequences from CRA would be useful for the development of diagnostic reagents that could improve upon the sensitivity and specificity of currently available diagnostic tests. Overall, the results provide further evidence of the usefulness of identifying specific linear B-cell epitopes for improving diagnostic tools.

Oligopeptidase B-2 from Leishmania amazonensis with an unusual C-terminal extension

The oligopeptidase B serine protease is an important virulence factor and therapeutic target in Trypanosoma infections. Recently, the Leishmania major Genome Project identified a new oligopeptidase B that was denominated oligopeptidase B-like, herein named oligopeptidase B-2. In this study, a complete open reading frame of oligopeptidase B-2 from Leishmania amazonensis (PH8 strain) was amplified by PCR using primers designed for the oligopeptidase B-2 gene of L. major. The 2,715 bp fragment coded for a protein of 905 amino acids with a predicted molecular mass of 103,918.9 Da and theoretical pI of 5.82. The encoded protein displayed ∼96% identity with L. major and ∼75% identity with Trypanosoma cruzi and T. brucei oligopeptidases B-2, and ∼21% identity with Escherichia coli and L. amazonensis classical oligopeptidase B. An unusual C-terminal extension was found in relation to the classical trypanosomatid oligopeptidase B. By sequence alignment, we determined a catalytic triad (Ser 629, Asp 717 and His 758), S1 subsite (Glu 674 and Glu 676) and suggest a difference in the S2 subsite of L. amazonensis oligopeptidase B-2. We also found that the oligopeptidase B-2 gene is expressed in all cycle stages of L. amazonensis. A phylogenetic analysis indicated that oligopeptidase B-2 is a new member of oligopeptidase B.

On the quaternary structure of a C-type lectin from Bothrops jararacussu venom – BJ-32 (BjcuL)

BJ-32 (also known as BjcuL) is a C-type lectin from the venom of Bothrops jararacussu with specificity for beta-galactosides and a remarkable ability to agglutinate several species of trypanosomatids. Our objective was to study the oligomerization state of native BJ-32 by using different biophysical and computational methods. Small-angle X-ray light scattering (SAXS) experiments disclosed a compact, globular protein with a radius of gyration of 36.72+/-0.04A and molecular weight calculated as 147.5+/-2.0kDa. From analytical ultracentrifugation analysis, it was determined that the BJ-32 sedimentation profile fits nicely to a decamer model. The analysis of the intrinsic emitted fluorescence spectra for BJ-32 solutions indicated that association of subunits in the decamer is accompanied by changes in the environment of Tryptophan residues. Both ab initio and comparative models of BJ-32 supported the resemblance of the decamer in the crystallographic structure from a close homologue, the rattlesnake venom lectin (RSL) from Crotalus atrox.

Identification of linear B epitopes of pertactin of Bordetella pertussis induced by immunization with whole and acellular vaccine

Pertussis is a serious infectious disease of the respiratory tract caused by the gram-negative bacteria Bordetella pertussis. There has been a reemergence of this disease within the population of several countries that have well established vaccination programs. Analyzes of clinical isolates suggest an antigenic divergence between the vaccine-based strains to the circulating strains. Although antibodies against P.69 are involved in the observed protective immunity, the sequences recognized as antigenic determinants in P.133, the precursor for P.69, P.3.4 and P.30, have not be determined. Here, the precise mapping of linear B-cell epitopes within the predicted P.133 pertactin sequences was accomplished using the SPOT-synthesis of peptide arrays onto cellulose membranes and screening with murine sera generated by vaccination with either the Pertussis cellular (miPc) or Pertussis acellular (miPa) vaccine. A total of 23 major epitopes were identified by sera from miPc vaccinated mice, while thirteen were identified by sera from miPa vaccinated mice. Of these epitopes, 12 epitopes were specifically identified by antibodies produced in response to the miPc vaccine and two were specific to the miPa vaccine. These epitopes were distributed throughout the pertactin sequence but a significant number were concentrated to the P.30 Prn segment. An analysis of the epitope correlation homologies indicated that the variations from the observed mutations in pertactin would not constitute a problem using these vaccines. In addition, the mapping of epitopes demonstrated a higher number of linear B-cell epitopes immunized with the Pc vaccine than the Pa vaccine.

Serine Proteases and Vaccines against Leishmaniasis: A Dual Role

Serine proteases are involved in several biochemical processes that are essential for the biology of pathogens, including Leishmania sp. Considering their importance, an interest in serine proteases for vaccine development against leishmaniasis has been raised. As targets, these enzymes have demonstrated a dual role in a vaccine against leishmaniasis, both protective and a counter-protective, depending on the conditions that they are evaluated. In this work, serine proteases or inhibitors of them that have been used as components of vaccines to Leishmania sp. are presented, aiming to disseminate the knowledge gained about these proteases and their potential in potential vaccine against leishmaniasis.

Purification of equine IgG3 by lectin affinity and an interaction analysis via microscale thermophoresis

The availability of purified antibodies is a prerequisite for many applications and the appropriate choice(s) for antibody-purification is crucial. Numerous methods have been developed for the purification of antibodies from different sources with affinity chromatography-based methods being the most extensively utilized. These methods are based on high specificity, easy reversibility and biological interactions between two molecules (e.g., between receptor and ligand or antibody and antigen). However, no simple techniques have yet been described to characterize and purify subclasses of immunoglobulins (Ig) from some animals of biotechnology importance such as equines, which are frequently used to produce biotherapeutic antibodies. The sera of these animals present a large number of Ig classes that have a greater complexity than other animals. The implementation of an effective protocol to purify the desired antibody class/subclasses requires meticulous planning to achieve yields at a high purity. The IgG3 subclass of equine-Ig has recently been used as antigen in a new diagnostic test for allergic responses to horse sera-based therapies. Here, we defined a simple method using Jacalin lectin immobilized on Sepharose beads to prepare highly pure equine IgG3 antibodies with a determination of the affinity constants for Jacalin lectin and horse IgG3.

Spot Synthesis: An Optimized Microarray to Detect IgE Epitopes.

Peptide microarrays have become increasingly more affordable in recent years with the SPOT technique being one of the most frequently used methods for synthesis and screening of peptides in arrays. Here, a protocol is presented for the identification of the amino acid sites involved in the conversion of human IgG to IgE response during the passive administration of therapeutic, anti-snake venom sera. Similarly, the minimal region of both the IgG and IgE binding epitopes, important for its interaction with ligand, were identified. As the ratio of concentrations for IgG to IgE in human serum is 1:10,000, also presented is a reproductive protocol of chemiluminescence-scanning for the detection of both immunoglobulins.

Eletrochemical immunosensor development for detection of dengue

Background The development of biosensors for the diagnosis and monitoring of diseases, drug discovery, proteomics, and the environmental detection of pollutants and/or biological agents is an extremely significant problem. Fundamentally, a biosensor is derived from the coupling of a ligand-receptor binding reaction to a signal transducer. In this study we used as a model a peptide marker of DENV (dengue virus) type 1. The electrochemical technique of cyclic voltammetry was performed to detect the signal generated by the interaction between the peptide and antibody from patients’ blood samples. Graphite and gold electrodes modified with chitosan film were employed to evaluate the interaction antigen-antibodies. The construction of this immunosensor, able to identify in real time circulating antibodies or antigens can be applied in the diagnosis of dengue and other infectious and parasitic diseases.