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Snakebite accident treatment requires the administration of antivenoms that provide efficacy and effectiveness against several snake venoms of the same genus or family. The low number of immunogenic components in venom mixtures that allow the production of antivenoms consequently gives them partial neutralization and a suboptimal pharmacological response. This study evaluates the immunorecognition and neutralizing efficacy of the polyvalent anticoral antivenom from the Instituto Nacional de Salud (INS) of Colombia against the heterologous endemic venoms of Micrurus medemi , and M. sangilensis , and M. helleri by assessing immunoreactivity through affinity chromatography, ELISA, Western blot, and neutralization capability. Immunorecognition towards the venoms of M. medemi and M. sangilensis showed values of 62% and 68% of the protein composition according to the immunoaffinity matrix, respectively. The analysis by Western blot depicted the highest recognition patterns for M. medemi , followed by M. sangilensis , and finally by M. helleri . These findings suggest that the venom compositions are closely related and exhibit similar recognition by the antivenom. According to enzyme immunoassays, M. helleri requires a higher amount of antivenom to achieve recognition than the others. Besides reinforcing the evaluation of INS antivenom capability, this work recommends the use of M. helleri in the production of Colombian antisera.

Nucleic acid nanoparticles (NANPs) are extensively investigated as diagnostic and therapeutic tools. These innovative particles can be composed of RNA, DNA, and/or modified nucleic acids. Due to the regulatory role of nucleic acids in the cellular system, NANPs have the ability to identify target molecules and regulate expression of genes in disease pathways. However, translation of NANPs in clinical settings is hindered due to inefficient intracellular delivery, chemical instability, and off-target immunostimulatory effects following immune recognition. The composition of nucleic acids forming NANPs has been demonstrated to influence immunorecognition, subcellular compartmentalization, and physicochemical properties of NANPs. This chapter first outlines the methods used to generate a panel of NANPs with a uniform shape, size, charge, sequence, and connectivity. This includes the procedures for replacing the RNA strands with DNA or chemical analogs in the designated NANPs. Second, this chapter will also describe experiments to assess the effect of the chemical modification on enzymatic and thermodynamic stability, delivery efficiency, and subcellular compartmentalization of NANPs., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Naja nivea (Cape Cobra) is endemic to southern Africa. Envenoming by N. nivea is neurotoxic, resulting in fatal paralysis. Its venom composition, however, has not been studied in depth, and specific antivenoms against it remain limited in supply. Applying a protein decomplexation approach, this study unveiled the venom proteome of N. nivea from South Africa. The major components in the venom are cytotoxins/cardiotoxins (~75.6% of total venom proteins) and alpha-neurotoxins (~7.4%), which belong to the three-finger toxin family. Intriguingly, phospholipase A 2 (PLA 2 ) was undetected-this is a unique venom phenotype increasingly recognized in the African cobras of the Uraeus subgenus. The work further showed that VINS African Polyvalent Antivenom (VAPAV) exhibited cross-reactivity toward the venom and immunorecognized its toxin fractions. In mice, VAPAV was moderately efficacious in cross-neutralizing the venom lethality with a potency of 0.51 mg/mL (amount of venom completely neutralized per milliliter of antivenom). In the challenge-rescue model, VAPAV prevented death in 75% of experimentally envenomed mice, with slow recovery from neurotoxicity up to 24 h. The finding suggests the potential para-specific utility of VAPAV for N. nivea envenoming, although a higher dose or repeated administration of the antivenom may be required to fully reverse the neurotoxic effect of the venom.

Nucleic acid nanoparticles, or NANPs, rationally designed to communicate with the human immune system, can offer innovative therapeutic strategies to overcome the limitations of traditional nucleic acid therapies. Each set of NANPs is unique in their architectural parameters and physicochemical properties, which together with the type of delivery vehicles determine the kind and the magnitude of their immune response. Currently, there are no predictive tools that would reliably guide the design of NANPs to the desired immunological outcome, a step crucial for the success of personalized therapies. Through a systematic approach investigating physicochemical and immunological profiles of a comprehensive panel of various NANPs, the research team developes and experimentally validates a computational model based on the transformer architecture able to predict the immune activities of NANPs. It is anticipated that the freely accessible computational tool that is called an "artificial immune cell," or AI-cell, will aid in addressing the current critical public health challenges related to safety criteria of nucleic acid therapies in a timely manner and promote the development of novel biomedical tools., (© 2022 The Authors. Small published by Wiley-VCH GmbH.)

Envenomation by two medically important Sundaic pit vipers, Trimeresurus wiroti (Malaysia) and Trimeresurus puniceus (Indonesia), causes hemotoxic syndrome with a potentially fatal outcome. Research on the compositions and antigenicity of these pit viper venoms is however lacking, limiting our understanding of the pathophysiology and treatment of envenomation. This study investigated the venom proteomes of both species through a protein decomplexation strategy, applying C 18 reverse-phase high-performance liquid chromatography followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and protein identification through nano-electrospray ionization liquid chromatography-tandem mass spectrometry (nano-ESI-LCMS/MS) of trypsin-digested peptides. The venom antigenicity was profiled against the Thai Green Pit Viper Antivenom (GPVAV, a hetero-specific antivenom), using indirect enzyme-linked immunosorbent assay (ELISA). The venom proteomes of T. wiroti and T. puniceus consisted of 10 and 12 toxin families, respectively. The major proteins were of diverse snake venom serine proteases (19-30% of total venom proteins), snake venom metalloproteinases (17-26%), disintegrins (9-16%), phospholipases A 2 (8-28%) and C-type lectins (~8%). These were putative snake toxins implicated in hemorrhage and coagulopathy, consistent with clinical hemotoxicity. GPVAV showed strong immunorecognition toward high and medium molecular weight proteins (e.g., SVMP and PLA 2 ) in both venoms, while a lower binding activity was observed toward small proteins such as disintegrins. Conserved antigenicity in the major hemotoxins supported toxicity cross-neutralization by GPVAV and indicated that the immunorecognition of low molecular weight toxins may be optimized for improved binding efficacy. Taken together, the study provides insights into the pathophysiology and antivenom treatment of envenomation caused by T. wiroti and T. puniceus in the region., (Copyright © 2021 Elsevier Inc. All rights reserved.)

With recent advances in nanotechnology and therapeutic nucleic acids (TNAs), various nucleic acid nanoparticles (NANPs) have demonstrated great promise in diagnostics and therapeutics. However, the full realization of NANPs' potential necessitates the development of a safe, efficient, biocompatible, stable, tissue-specific, and non-immunogenic delivery system. Exosomes, the smallest extracellular vesicles and an endogenous source of nanocarriers, offer these advantages while avoiding complications associated with manufactured agents. The lipid membranes of exosomes surround a hydrophilic core, allowing for the simultaneous incorporation of hydrophobic and hydrophilic drugs, nucleic acids, and proteins. Additional capabilities for post-isolation exosome surface modifications with imaging agents, targeting ligands, and covalent linkages also pave the way for their diverse biomedical applications. This review focuses on exosomes: their biogenesis, intracellular trafficking, transportation capacities, and applications with emphasis on the delivery of TNAs and programmable NANPs. We also highlight some of the current challenges and discuss opportunities related to the development of therapeutic exosome-based formulations and their clinical translation., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The relatively straightforward methods of designing and assembling various functional nucleic acids into nanoparticles offer advantages for applications in diverse diagnostic and therapeutic approaches. However, due to the novelty of this approach, nucleic acid nanoparticles (NANPs) are not yet used in the clinic. The immune recognition of NANPs is among the areas of preclinical investigation aimed at enabling the translation of these novel materials into clinical settings. NANPs' interactions with the complement system, coagulation systems, and immune cells are essential components of their preclinical safety portfolio. It has been established that NANPs' physicochemical properties-composition, shape, and size-determine their interactions with immune cells (primarily blood plasmacytoid dendritic cells and monocytes), enable recognition by pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs) and RIG-I-like receptors (RLRs), and mediate the subsequent cytokine response. However, unlike traditional therapeutic nucleic acids (e.g., CpG oligonucleotides), NANPs do not trigger a cytokine response unless they are delivered into the cells using a carrier. Recently, it was discovered that the type of carrier provides an additional tool for regulating both the spectrum and the magnitude of the cytokine response to NANPs. Herein, we review the current knowledge of NANPs' interactions with various components of the immune system to emphasize the unique properties of these nanomaterials and highlight opportunities for their use in vaccines and immunotherapy.

The wide distribution of king cobra (Ophiophagus hannah), a medically important venomous snake in Asia could be associated with geographical variation in the toxicity and antigenicity of the venom. This study investigated the lethality of king cobra venoms (KCV) from four geographical locales (Malaysia, Thailand, Indonesia, China), and the immunological binding as well as in vivo neutralization activities of three antivenom products (Thai Ophiophagus hannah monovalent antivenom, OHMAV; Indonesian Serum Anti Bisa Ular, SABU; Chinese Naja atra monovalent antivenom, NAMAV) toward the venoms. The Indonesian and Chinese KCV were more lethal (median lethal dose, LD 50 ~0.5 μg/g) than those from Malaysia and Thailand (LD 50 ~1.0 μg/g). The antivenoms, composed of F(ab)' 2 , were variably immunoreactive toward the KCV from all locales, with OHMAV exhibited the highest immunological binding activity. In mice, OHMAV neutralized the neurotoxic lethality of Thai KCV most effectively (normalized potency = 118 mg venom neutralized per g antivenom) followed by Malaysian, Indonesian and Chinese KCV. In comparison, the hetero-specific SABU was remarkably less potent by at least 6 to10 folds, whereas NAMAV appeared to be non-effective. The finding supports that a specific king cobra antivenom is needed for the effective treatment of king cobra envenomation in each region., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Nucleic acid nanoparticles (NANPs) have evolved as a new class of therapeutics with the potential to detect and treat diseases. Despite tremendous advancements in NANP development, their immunotoxicity, one of the major impediments in clinical translation of traditional therapeutic nucleic acids (TNAs), has never been fully characterized. Here, we describe the first systematically studied immunological recognition of 25 representative RNA and DNA NANPs selected to have different design principles and physicochemical properties. We discover that, unlike traditional TNAs, NANPs used without a delivery carrier are immunoquiescent. We show that interferons (IFNs) are the key cytokines triggered by NANPs after their internalization by phagocytic cells, which agrees with predictions based on the experiences with TNAs. However, in addition to type I IFNs, type III IFNs also serve as reliable biomarkers of NANPs, which is usually not characteristic of TNAs. We show that overall immunostimulation relies on NANP shapes, connectivities, and compositions. We demonstrate that, like with traditional TNAs, plasmacytoid dendritic cells serve as the primary interferon producers among all peripheral blood mononuclear cells treated with NANPs, and scavenger receptor-mediated uptake and endosomal Toll-like receptor signaling are essential for NANP immunorecognition. The TLR involvement, however, is different from that expected for traditional TNA recognition. Based on these results, we suggest that NANP technology may serve as a prototype of auxiliary molecular language for communication with the immune system and the modulation of immune responses.

Bradykinin-potentiating peptides (BPPs) from the South American pit viper snake venom were the first natural inhibitors of the human angiotensin I-converting enzyme (ACE) described. The pioneer characterization of the BPPs precursor from the snake venom glands by our group showed for the first time the presence of the C-type natriuretic peptide (CNP) in this same viper precursor protein. The confirmation of the BPP/CNP expression in snake brain regions correlated with neuroendocrine functions stimulated us to pursue the physiological correlates of these vasoactive peptides in mammals. Notably, several snake toxins were shown to have endogenous physiological correlates in mammals. In the present work, we expressed in bacteria the BPPs domain of the snake venom gland precursor protein, and this purified recombinant protein was used to raise specific polyclonal anti-BPPs antibodies. The correspondent single protein band immune-recognized in adult rat brain cytosol was isolated by 2D-SDS/PAGE and/or HPLC, before characterization by MS fingerprint analysis, which identified this protein as superoxide dismutase (SOD, EC 1.15.1.1), a classically known enzyme with antioxidant activity and important roles in the blood pressure modulation. In silico analysis showed the exposition of the BPP-like peptide sequences on the surface of the 3D structure of rat SOD. These peptides were chemically synthesized to show the BPP-like biological activities in ex vivo and in vivo pharmacological bioassays. Taken together, our data suggest that SOD protein have the potential to be a source for putative BPP-like bioactive peptides, which once released may contribute to the blood pressure control in mammals., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Biomimetic nanoparticles are promising materials for biomedical and biotechnological applications. Cationic poly(N-isopropylacrylamide) (PNIPAM) nanogels containing charged amine groups brought by addition of 2-aminoethylmethacrylate hydrochloride (AEMH) or N-(3-aminopropyl) methacrylamide hydrochloride (APMH) as co-monomers were prepared by surfactant-free precipitation polymerization. The influence of the relative amount and mode of addition of the co-monomer on both the size and the amine group density of the nanogel particles was studied. Two nanogels, one prepared using APMH (1%mol/mol NIPAM, in batch) and another with AEMH (2%mol/mol NIPAM, by shot addition) as co-monomers, were selected for the covalent coupling of a Protein L-mimic ligand to free amine groups on the particles. The ability of the synthesized biomimetic nanoparticles for recognizing and binding human IgG (hIgG) molecules was assessed and the selectivity toward immunoglobulin molecules evaluated., (Copyright © 2013 Elsevier B.V. All rights reserved.)