Evaluation of the effects of Loxosceles intermedia's venom in zebrafish.

The zebrafish is an animal model of increasing use in many biomedical fields of study, including toxicology, inflammation, and tissue regeneration. In this paper, we have investigated the inflammatory effects of Loxosceles intermedia's venom (LIV) on zebrafish, as well as the effects of Maresin 2 (Mar2) and Resolvin D5 (RvD5), two specialized pro-resolving mediators (SPMs), in the context of tissue regeneration after fin fold amputation. Furthermore, increasing concentrations of LIV (250-2000 ng) were assayed for their haemolytic effects in vitro, and, afterwards, the same concentrations were evaluated in vivo, when injected intraperitoneally. LIV caused haemolysis in human red blood cells (RBCs), but not in zebrafish RBCs. The survival curve was also not altered by LIV injection, regardless of venom dosage. Histological analysis of renal and hepatic tissues, as well as the whole animal, revealed no pathological differences between LIV-injected and PBS-injected groups. Fin fold regeneration was not altered between LIV-injected and control groups, nor in the presence of MaR2 and RvD5. Results of swimming behavioral analysis also did not differ between groups. Moreover, in silico data indicated differences between human and zebrafish cell membrane lipid constitutions, such as in phospholipases D preferred substrates, that could lead to the protection of zebrafish against LIV. Although our data implies that zebrafish cannot be used as a toxicological model for LIV studies, the absence of observed toxicological effects paves the way for the comprehension of the venom's mechanism of action in mammals and the fundamental evolutionary processes involved.

An effective strategy for the humanization of antibody fragments under an accelerated timeline.

The use of monoclonal antibodies (mAbs) in therapy is gradually advancing and discussions entail its safety, rentability and effectiveness. To this date, around a hundred mAbs have been approved by the FDA for the treatment of various diseases. Aiming for their large-scale production, recombinant DNA technology is mainly employed, and antibodies can be expressed in various eukaryotic and prokaryotic systems. Moreover, considering their heterologous origin and potential immunogenicity, various strategies have been developed for mAb humanization, considering that around 50 % of commercial mAbs are humanized. Hence, we introduce LimAb7, a mouse mAb capable of binding and neutralizing brown spider's Loxosceles intermedia dermonecrotic toxins in vivo/in vitro. This antibody has been produced in mouse and humanized scFv and diabody formats, however results indicated losses in antigen-binding affinity, stability, and neutralizing ability. Intending to develop evolved, stable, and neutralizing antibody fragments, we report for the first time the design of humanized antibody V-domains produced as Fab fragments, against spider venom toxins. Improvements in constructs were observed regarding their physicochemical stability, target binding and binding pattern maintenance. As their neutralizing features remain to be characterized, we believe this data sheds new light on antibody humanization by producing a parental molecule in different recombinant formats.

Epitope mapping from Mycobacterium leprae proteins: Convergent data from in silico and in vitro approaches for serodiagnosis of leprosy.

Knowledge of immunodominant B-cell epitopes is essential to design powerful diagnostic strategies aiming for antibody detection. Outstanding progress in computational prediction has achieved a significant contribution to the biomedical fields, including immunodiagnosis. In silico analysis may have an even more important role when information concerning antigens from etiologic agents of neglected diseases, such as leprosy, is scarce. The aim of this study was to provide mapping of B-cell epitopes from two Mycobacterium leprae-derived antigens (Ag85B and ML2055), confirm their antigenicity, and to assess the ability of in silico immunoinformatics tools to accurately predict them. Linear B-cell epitopes predicted by ABCpred and SVMTrip servers were compared to antigenic regions of synthetic overlapping peptides that exhibited reactivity to antibodies from patients with leprosy. Our in vitro results identified several immunodominant regions that had also been indicated by in silico prediction, providing agreement between experimental and simulated data. After chemical synthesis, we used enzyme-linked immunosorbent assays to determine the effectiveness of the first identified sequence (GTNVPAEFLENFVHG) which had 72 % sensitivity and 78 % specificity (AUC = 0.79) while the second one (PVSSEAQPGDPNAPS) had 72 % sensitivity and 93.8 % specificity (AUC = 0.85). Using dot blotting, an easy-to-read visual test, both peptides could distinguish sera from patients with leprosy from those with tuberculosis and from sera of healthy volunteers. Our findings suggest that these synthetic peptides, with some refinement, may be useful as serological diagnostic antigens for leprosy. In addition, it was displayed that immunoinformatics provides reliable information for mapping potential B-cell epitopes for development of peptide-based diagnostic assays for neglected diseases.

A new insight into the cellular mechanisms of envenomation: Elucidating the role of extracellular vesicles in Loxoscelism.

Envenomation by the Loxosceles genus spiders is a recurring health issue worldwide and specially in the Americas. The physiopathology of the envenomation is tightly associated to the venom's rich toxin composition, able to produce a local dermonecrotic lesion that can evolve systemically and if worsened, might result in multiple organ failure and lethality. The cellular and molecular mechanisms involved with the physiopathology of Loxoscelism are not completely understood, however, the venom's Phospholipases D (PLDs) are known to trigger membrane injury in various cell types. Here, we report for the first time the Loxosceles venom's ability to stimulate the production of extracellular vesicles (EVs) in various human cell lineages. Components of the Loxosceles venom were also detectable in the cargo of these vesicles, suggesting that they may be implicated in the process of extracellular venom release. EVs from venom treated cells exhibited phospholipase D activity and were able to induce in vitro hemolysis in human red blood cells and alter the HEK cell membranes' permeability. Nonetheless, the PLD activity was inhibited when an anti-venom PLDs monoclonal antibody was co-administered with the whole venom. In summary, our findings shed new light on the mechanisms underlying cellular events in the context of loxoscelism and suggest a crucial role of EVs in the process of envenomation.

Acanthamoeba spp. monoclonal antibody against a CPA2 transporter: a promising molecular tool for acanthamoebiasis diagnosis and encystment study.

Free-living amoeba of the genus Acanthamoeba are ubiquitous protozoa involved in opportunistic and non-opportunistic infection in humans, such as granulomatous amoebic encephalitis and amoebic keratitis. Both infections have challenging characteristics such as the formation of the resistant cysts in infected tissues, hampering the treatment and most usual diagnosis depending on time-consuming and/or low sensitivity techniques. The use of monoclonal antibodies presents itself as an opportunity for the development of more effective alternative diagnostic methods, as well as an important and useful tool in the search for new therapeutic targets. This study investigated the possibility of using a previously produced monoclonal antibody (mAb3), as a diagnostic tool for the detection of Acanthamoeba trophozoites by direct and indirect flow cytometry and immunofluorescence. Immunoprecipitation assay and mass spectrometry allowed the isolation of the antibody's target and suggested it is a transporter part of the CPA (cation: proton antiporter) superfamily. In vitro tests indicate an important role of this target in Acanthamoeba's encystment physiology. Our results support the importance of studying the role of CPA2 transporters in the context of acanthamoebiasis, as this may be a way to identify new therapeutic candidates.

Engineering a single-chain antibody against Trypanosoma cruzi metacyclic trypomastigotes to block cell invasion.

Trypanosoma cruzi is a flagellate protozoan pathogen that causes Chagas disease. Currently there is no preventive treatment and the efficiency of the two drugs available is limited to the acute phase. Therefore, there is an unmet need for innovative tools to block transmission in endemic areas. In this study, we engineered a novel recombinant molecule able to adhere to the T. cruzi surface, termed scFv-10D8, that consists of a single-chain variable fragment (scFv) derived from mAb-10D8 that targets gp35/50. The synthetic gene encoding scFv-10D8 was cloned and fused to a 6×His tag and expressed in a prokaryotic expression system. Total periplasmic or 6xHis tag affinity-purified fractions of scFv-10D8 retained the capacity to bind to gp35/50, as shown by Western blot analyses. Pre-incubation of metacyclic trypomastigotes with scFv-10D8 showed a remarkable reduction in cell invasion capacity. Our results suggest that scFv-10D8 can be used in a paratransgenic approach to target parasites in insect vectors, avoiding dissemination of infective forms. Such advances in the development of this functional molecule will surely prompt the improvement of alternative strategies to control Chagas disease by targeting mammalian host stages.

Physicochemical and immunological characterization of chitosan-coated bacteriophage nanoparticles for in vivo mycotoxin modeling.

To propose a novel modeling of aflatoxin immunization and surrogate toxin conjugate from AFB1 vaccines, an immunogen based on the mimotope, (i.e. a peptide-displayed phage that mimics aflatoxins epitope without toxin hazards) was designed. The recombinant phage 3P30 was identified by phage display technology and exhibited the ability to bind, dose dependent, specifically to its cognate target - anti-AFB1 antibody. In immunization assay, the phage-displayed mimotope and its peptide chemically synthesized were able to induce specific anti-AFB1 antibodies, indicating the proof of concept for aflatoxin mimicry. Furthermore, the phage 3P30 was homogeneously coated with chitosan, which also provided a tridimensional matrix network for mucosal delivery. After intranasal immunization, chitosan coated phages improved specific immunogenicity compared to the free antigen. It can be concluded that affinity-selected phage may contribute to the rational design of epitope-based vaccines in a prospectus for the control of aflatoxins and possibly other mycotoxins, and that chitosan coating improved the vectorization of the vaccine by the mucosal route.

Phage display and synthetic peptides as promising biotechnological tools for the serological diagnosis of leprosy.

BACKGROUND: The diagnosis of leprosy is primarily based on clinical manifestations, and there is no widely available laboratory test for the early detection of this disease, which is caused by Mycobacterium leprae. In fact, early detection and treatment are the key elements to the successful control of leprosy. METHODOLOGY/PRINCIPAL FINDINGS: Peptide ligands for antibodies from leprosy patients were selected from phage-displayed peptide libraries. Three peptide sequences expressed by reactive phage clones were chemically synthesized. Serological assays that used synthetic peptides were evaluated using serum samples from leprosy patients, household contacts (HC) of leprosy patients, tuberculosis patients and endemic controls (EC). A pool of three peptides identified 73.9% (17/23) of multibacillary (MB) leprosy patients using an enzyme-linked immunosorbent assay (ELISA). These peptides also showed some seroreactivities to the HC and EC individuals. The peptides were not reactive to rabbit polyclonal antisera against the different environmental mycobacteria. The same peptides that were conjugated to the carrier protein bovine serum albumin (BSA) induced the production of antibodies in the mice. The anti-peptide antibodies that were used in the Western blotting analysis of M. leprae crude extracts revealed a single band of approximately 30 kDa in one-dimensional electrophoresis and four 30 kDa isoforms in the two-dimensional gel. The Western blotting data indicated that the three peptides are derived from the same bacterial protein. CONCLUSIONS/SIGNIFICANCE: These new antigens may be useful in the diagnosis of MB leprosy patients. Their potentials as diagnostic reagents must be more extensively evaluated in future studies using a large panel of positive and negative sera. Furthermore, other test approaches using peptides should be assessed to increase their sensitivity and specificity in detecting leprosy patients. We have revealed evidence in support of phage-displayed peptides as promising biotechnological tools for the design of leprosy diagnostic serological assays.

Synthetic peptides for in vitro evaluation of the neutralizing potency of Loxosceles antivenoms.

An important step in the development of therapeutic antivenoms is the pre-clinical testing using in vivo methods to assess their neutralizing potency. For spider antivenoms (Loxosceles species), horse serum potency against the necrotizing activities of Loxosceles intermedia crude venom is currently tested in rabbits. These procedures are time consuming and involve a large number of animals. The aim of this study was to develop an in vitro method to assess the neutralizing potency of anti-Loxosceles sera. We first demonstrated that it was not possible to establish a correlation between the ELISA antibody reactivity of horse anti-Loxosceles serum and their neutralizing potency. We then showed that the antivenoms recognized several peptide epitopes from different regions of SMase-D proteins, which are toxic antigens from Loxosceles venoms. The recognition of some peptides was observed only when high neutralizing potency sera was used. Based on these results, three peptides (peptide 1, DNRRPIWNLAHMVNA and peptide 3, DFSGPYLPSLPTLDA corresponding to residues 2-16 and 164-178, respectively, of SMase-1 protein from Loxosceles laeta, and peptide 2, EFVNLGANSIETDVS corresponding to residues 22-36 of A1H - LoxGa protein from Loxosceles gaucho and LiD1 protein from L. intermedia) were selected. The peptides were synthesized, coupled to bovine serum albumin (BSA), and used as antigens in indirect ELISA to test their reactivity with horse anti-Loxosceles serum of varying neutralizing potencies. We found certain assay conditions that discriminated between the high and low neutralizing potency sera. This study introduced an in vitro and peptide-based neutralization assay for anti-Loxosceles antivenoms.

Evaluation of the protective potential of a Taenia solium cysticercus mimotope on murine cysticercosis.

An NC-1 mimotope from Taenia solium cysticerci can help identify patients with neurocysticercosis through immunoassay. After chemical synthesis, an NC-1 peptide was coupled to bovine serum albumin (NC-1/BSA) for used as an immunogen in murine Taenia crassiceps cysticercosis, which is an experimental model of cysticercosis caused by T. solium. NC-1/BSA immunisation decreased parasitaemia by inducing 74% protection compared to the 77% protection obtained with T. crassiceps crude antigen. The influence of immunisation was also observed on the size and stage of development of the parasite. Antibodies from NC-1/BSA-immunised mice recognised proteins from the tegument and from the buddings, and intense immunostaining was observed in the final stage of the metacestode. The capacity of NC-1/BSA to induce protective antibodies which are reactive to proteins from the tegument of the metacestode suggests that this mimotope is a potential candidate for a vaccine against human and animal cysticercosis.

In vivo protection against Tityus serrulatus scorpion venom by antibodies raised against a discontinuous synthetic epitope.

Scorpion stings cause human fatalities in numerous countries. Serotherapy is the only specific means to try to circumvent the noxious effects of venom toxins. TsNTxP is a natural anatoxin from the venom of the scorpion Tityus serrulatus that may be useful to raise therapeutic anti-venom sera. Linear epitopes recognized by anti-TsNTxP antibodies have previously been mapped. Here, we attempted to identify discontinuous epitopes in TsNTxP since neutralizing epitopes are often associated with such complex entities. One hundred and fifty-three octadecapeptides with the general formula (P1)-(Gly-Gly)-(P2) were synthesized by the Spot method on cellulose membranes. P1 and P2 were octapeptides from the TsNTxP N-terminal and C-terminal sections, respectively. Each sequence of eight amino acids was frameshifted in turn by three residues, in order to cover TsNTxP entire sequence. Binding of neutralizing anti-TsNTxP rabbit antibodies to spotted peptides revealed GREGYPADGGGLPDSVKI as the more reactive peptide sequence. This epitope was made from the first eight residues of the protein (GREGYPAD) and from residues 47 to 54 (GLPDSVKI) of the C-terminal part of TsNTxP. BALB/c mice were immunized with synthetic GREGYPADGGGLPDSVKI peptide conjugated to ovalbumin. One week after the last immunization, in vivo protection assays showed that immunized mice could resist a challenge by an amount of T.serrulatus whole venom equivalent to 1.75 LD(100), a dose that killed all control non-immune mice. Based on molecular models of TsNTxP and related Tityus toxins, we found that the above peptide matches with a discontinuous epitope, well exposed at the toxin molecular surface which contains residues known to be important for the bioactivity of toxins.