Identification of a common epitope in knottins and phospholipases D present in Loxosceles sp venom by a monoclonal antibody.

In the Americas and specially in Brazil, the Loxosceles intermedia, Loxosceles gaucho and Loxosceles laeta are the three most medically relevant brown spider species, and whose bites can lead to the condition known as loxoscelism. Here, we report the development of a tool capable of identifying a common epitope amongst Loxosceles sp. venom's toxins. A murine monoclonal antibody (LmAb12) and its recombinant fragments (scFv12P and diabody12P) have been produced and characterized. This antibody and its recombinant constructs were able to recognize proteins of Loxosceles spider venoms with specificity. The scFv12P variant was also able to detect low concentrations of Loxosceles venom in a competitive ELISA assay, displaying potential as a venom identification tool. The primary antigenic target of LmAb12 is a knottin, a venom neurotoxin, that has a shared identity of 100 % between the L. intermedia and L. gaucho species and high similarity to L. laeta. Furthermore, we observed LmAb12 was able to partially inhibit in vitro hemolysis, a cellular event typically induced by the Loxosceles sp. venoms. Such behavior might be due to LmAb12 cross-reactivity between the antigenic target of LmAb12 and the venom's dermonecrotic toxins, the PLDs, or even the existence of synergism between these two toxins.

Prevalence of immune-mediated inner ear disease in non-segmental vitiligo: A cross-sectional study.

BACKGROUND: Sensorineural hearing loss (SNHL) is associated with non-segmental vitiligo (NSV); however, the aetiology of SNHL has not been explored. The concomitance of autoimmune disease in vitiligo patients demands the investigation of immune-mediated inner ear disease (IMIED) as a cause of SNHL in NSV. The anti-Hsp70 antibody is a serological marker of IMIED, which may help in the early diagnosis of this disease. OBJECTIVE: To evaluate the prevalence of IMIED in NSV patients. METHODS: Cross-sectional study involving NSV adult patients and a control group, evaluated through audiometry and serological dosage of the anti-Hsp70 antibody. RESULTS: In total, 112 cases and 23 controls were evaluated. Bilateral SNHL was found in 28 (25.0%; 95%CI 17.9%-32.1%) patients and in 1 (4.3%) control (p = 0.019). Six cases (5.4%; 95%CI 2.7%-8.0%) presented bilateral SNHL of unexplained aetiology, and anti-Hsp70 antibody positivity, fulfilling the diagnostic criteria for IMIED. No controls met the diagnostic criteria for IMIED. Serum anti-Hsp70 antibodies were higher in cases with IMIED: median 220.9 vs. 85.1 ng/ml (p = 0.001). CONCLUSION: The prevalence of IMIED is remarkable in NSV adult patients.

Targeting platelet GPVI with glenzocimab: a novel mechanism for inhibition.

Platelet glycoprotein VI (GPVI) is attracting interest as a potential target for the development of new antiplatelet molecules with a low bleeding risk. GPVI binding to vascular collagen initiates thrombus formation and GPVI interactions with fibrin promote the growth and stability of the thrombus. In this study, we show that glenzocimab, a clinical stage humanized antibody fragment (Fab) with a high affinity for GPVI, blocks the binding of both ligands through a combination of steric hindrance and structural change. A cocrystal of glenzocimab with an extracellular domain of monomeric GPVI was obtained and its structure determined to a resolution of 1.9 Å. The data revealed that (1) glenzocimab binds to the D2 domain of GPVI, GPVI dimerization was not observed in the crystal structure because glenzocimab prevented D2 homotypic interactions and the formation of dimers that have a high affinity for collagen and fibrin; and (2) the light variable domain of the GPVI-bound Fab causes steric hindrance that is predicted to prevent the collagen-related peptide (CRP)/collagen fibers from extending out of their binding site and preclude GPVI clustering and downstream signaling. Glenzocimab did not bind to a truncated GPVI missing loop residues 129 to 136, thus validating the epitope identified in the crystal structure. Overall, these findings demonstrate that the binding of glenzocimab to the D2 domain of GPVI induces steric hindrance and structural modifications that drive the inhibition of GPVI interactions with its major ligands.

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.

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.

Loxoscelism: Advances and Challenges in the Design of Antibody Fragments with Therapeutic Potential.

Envenoming due to Loxosceles spider bites still remains a neglected disease of particular medical concern in the Americas. To date, there is no consensus for the treatment of envenomed patients, yet horse polyclonal antivenoms are usually infused to patients with identified severe medical conditions. It is widely known that venom proteins in the 30-35 kDa range with sphingomyelinase D (SMasesD) activity, reproduce most of the toxic effects observed in loxoscelism. Hence, we believe that monoclonal antibody fragments targeting such toxins might pose an alternative safe and effective treatment. In the present study, starting from the monoclonal antibody LimAb7, previously shown to target SMasesD from the venom of L. intermedia and neutralize its dermonecrotic activity, we designed humanized antibody V-domains, then produced and purified as recombinant single-chain antibody fragments (scFvs). These molecules were characterized in terms of humanness, structural stability, antigen-binding activity, and venom-neutralizing potential. Throughout this process, we identified some blocking points that can impact the Abs antigen-binding activity and neutralizing capacity. In silico analysis of the antigen/antibody amino acid interactions also contributed to a better understanding of the antibody's neutralization mechanism and led to reformatting the humanized antibody fragment which, ultimately, recovered the functional characteristics for efficient in vitro venom neutralization.

Exploration and Modulation of Antibody Fragment Biophysical Properties by Replacing the Framework Region Sequences.

In order to increase the successful development of recombinant antibodies and fragments, it seems fundamental to enhance their expression and/or biophysical properties, such as the thermal, chemical, and pH stabilities. In this study, we employed a method bases on replacing the antibody framework region sequences, in order to promote more particularly single-chain Fragment variable (scFv) product quality. We provide evidence that mutations of the VH- C-C' loop might significantly improve the prokaryote production of well-folded and functional fragments with a production yield multiplied by 27 times. Additional mutations are accountable for an increase in the thermal (+19.6 °C) and chemical (+1.9 M) stabilities have also been identified. Furthermore, the hereby-produced fragments have shown to remain stable at a pH of 2.0, which avoids molecule functional and structural impairments during the purification process. Lastly, this study provides relevant information to the understanding of the relationship between the antibodies amino acid sequences and their respective biophysical properties.

Immunodetection of the "brown" spider (Loxosceles intermedia) dermonecrotoxin with an scFv-alkaline phosphatase fusion protein.

Bites by spiders from Loxosceles genus often lead to a wide variance in envenomation profile of patients and diagnosis is difficult due to the number of diseases that mimic loxoscelism. In such a context, it is of interest to consider the design of standardized recombinant colorimetric antibodies for diagnosis and specific detection of individual circulating toxins in biological fluids of envenomed patients. We have previously prepared a monoclonal murine IgG (LiMab7) that reacts with Loxosceles intermedia venom components of 32-35kDa and neutralizes the dermonecrotic activity of the venom. Here, we re-engineered LiMab7 into a colorimetric bifunctional protein consisting in the corresponding single-chain antibody fragment (scFv) fused to alkaline phosphatase (AP) of Escherichia coli. The immune tracer was tested in two different types of immunoassays and it proved to be efficient in both. Thus, this recombinant fusion protein (scFv-LiMab7/AP) can be used for rapid and specific immunotitration of L. intermedia venom with a linear range of 39-20000ng/mL and a detection limit of 39ng/mL without any cross-reaction.