Toxins, Vol. 15, Pages 15: Monoclonal-Based Antivenomics Reveals Conserved Neutralizing Epitopes in Type I PLA2 Molecules from Coral Snakes

1. IntroductionCoral snakes are a large monophyletic group of over 80 small- to medium-sized colorful species of the Elapid family. Notable for their colorful red, yellow/white, and black colored banding, basal coral snake lineages originated in Asia [1,2] and are currently represented by two genera in the Old World (Calliophis and Sinomicrurus) and by three (Leptomicrurus, Micruroides, and Micrurus) in the New World [1,3,4]. However, separating Micrurus from Leptomicrurus is not a scientific consensus [3,5]. Extant New World coral snakes are widely distributed in the southern range of many temperate U.S. states, throughout Central America, and most of South America to central Argentina [6].Envenomation by New World coral snakes is characterized by local manifestations, including myonecrosis [7,8], cardiovascular effects [9], and predominantly systemic neurotoxicity leading to respiratory arrest and death in severe cases [10,11,12]. A number of micrurine species have their venoms analyzed by venomic and transcriptomic approaches, including those from M. surinamensis [13,14]; M. corallinus [14,15,16]; M. altirostris [16]; M. nigrocinctus [17]; M. mipartitus [18]; M. frontalis, M. ibiboboca, M. lemniscatus, and M. spixii, [14,19,20,21]; M. tener tener [22]; M. laticollaris [23]; M. fulvius [24,25]; M. mosquitensis and M. alleni [26]; M. dumerilii [27]; M. tschudii [28]; M. clarki [29]; M. pyrrhocryptus [30]; M. ruatanus [31]; M. browni browni [32]; and more recently M. yatesi [33]. These studies have revealed that post-synaptic α-neurotoxins of the three-finger toxins (3FTx) family [34] and pre-synaptic phospholipase A2 (PLA2) molecules [35,36,37] represent the main toxin classes of Micrurus venoms. However, the relative abundance of these toxins varies widely between species [38]. Venomics combined with protein biochemical characterizations has contributed to a deeper understanding of the molecular determinants of the clinical effects of coral snake envenomation [22,23,32,39,40].The Brazilian commercial Elapidic antivenom is produced at Butantan Institute (São Paulo) and Ezequiel Dias Foundation (Minas Gerais) by hyper-immunization of horses with equal amounts of venom from M. corallinus and M. frontalis [41], two snake species endemic to the south and southeast regions of Brazil. It has been reported that this antivenom is inefficient in fully neutralizing the neurotoxicity and lethality of some heterologous Micrurus species present in different geographic regions of the country [42,43,44,45]. An interesting aspect of some Micrurus venoms, especially those from M. lemniscatus and M. altirostris, is that despite having suitable antigens inducing a satisfactory immune response in horses, the generated antibodies have poor neutralizing capacity [44]. Additionally, the Brazilian commercial antivenom is ineffective in neutralizing lethality. In the case of M. altirostris, it has been demonstrated that its venom departs from others in immunochemical profile, biological activities, and toxin composition [16,42,44,45,46]. This species is distributed in southern Brazil, Uruguay, northeast Argentina, and Paraguay [47].New strategies and immunization approaches are thus needed to generate improved antivenoms with a broader clinical usefulness landscape. In this regard, lethality and neurotoxicity among Micrurus species can be ascribed to just a few toxins, mainly PLA2 and 3FTx families [13,25,39,40]. This offers the possibility of generating an antivenom comprising a restricted set of anti-PLA2 and anti-3FTx neutralizing antibodies, like those already developed against certain scorpion and snake venoms [48,49,50]. As a proof-of-concept and the first step towards this goal, we have produced and analyzed the immunoreactivity of a set of monoclonal antibodies against the most toxic components of M. altirostris venom. Two PLA2-specific monoclonal antibodies cross-reacted with all the PLA2 molecules from M. altirostris venom, inhibiting their catalytic activity and myotoxicity. They also exhibited paraspecificity against PLA2s from Naja naja venom, demonstrating the conservation of paraspecific neutralizing epitopes across the Elapid family. 3. DiscussionSince the first experimental snake antivenom was raised in pigeons against the venom from Sistrurus catenatus Sewall, 1887, antivenom technology (essentially using immunization of large mammals with a mixture of crude venoms as antigen) has remained relatively unchanged for most of the 20th century [52]. Only recently, new therapeutic monoclonal antibodies or fragments of antibodies for antagonizing the effects of envenomation caused by spiders, scorpions, and snakes have been reported [48,52,53,54,55,56,57,58,59]. Monoclonal and recombinant antibodies have demonstrated effective neutralization activity of low-complexity venoms [59]. In this respect, El Ayeb and Rochat (1988) first generated monoclonal antibodies specific for the Androctonus australis AaH2 toxin, which exhibited neutralizing activity [60]. Licea et al. [61] also characterized a monoclonal antibody that neutralized Centruroides noxius venom on the ratio of 6 ug F(ab’)2 per ug of dose of venom. These approaches have been improving, and new applications in the production of antivenoms are emerging, like the production of humanized-camel single-domain antibodies, scFvs, or recombinant human monoclonals obtained by phage display, among others [53,62,63]. Our present work aimed to generate monoclonal antibodies with broad specificity toward lethal toxins from Micrurus altirostris venom.Micrurus venoms are not readily available; since these snakes have an ophiophagus diet, they are hardly maintained in captivity. In addition, due to their small size and small gland, they produce a low amount of venom in each extraction, thus hindering the production of anti-elapidic serum [15]. In that sense, obtaining mABs is an important strategy to increase antibody titles. Micrurus venoms are relatively simple regarding the class of lethal toxin families, which comprise mainly 3FTXs and PLA2 molecules [25,29,39]. Likewise, the lethality of M. altirostris venom is triggered by the same protein classes. High toxicity (LD50 of 3.3 and 2.9 µg/mouse) was found on the venom fractions enriched with PLA2s (P4) and 3FTXs (P8), respectively (Table 1). Regarding the PLA2, the venomics analysis identified eight proteoforms (13.7% of total venom protein), and among them five were assigned for the transcripts F5CPF1, F5CPF0, and AED89576, pointing to the presence of isoforms. The transcript F5CPF0 was identified in RP-HPLC peak 20, which was specifically recognized by mAb 3B2. F5CPF1 and AED89576 corresponding to PLA2s, which share a conserved structural epitope recognized by mAb 1E8 (RP-HPLC peaks 21–25).It is worth mentioning that F5CPF1 and AED89576 showed close phylogenetic relationships with MmipPLA2, the most abundant PLA2 of M. mipartitus venom (accounting for nearly 10% of the total venom protein) [41]. Functional characterization of MmipPLA2 revealed a toxin with low enzymatic activity and myotoxicity but high lethality in mice [40]. A similar functional pattern was found for fraction P4 isolated from M. altirostris venom (Table 1, Figure 3D), highlighting the importance of developing inhibitors for each type of PLA2. The monoclonal antibodies produced here could inhibit the catalytic activity and in vivo myotoxicity of M. altirostris, N. naja venoms, and pooled PLA2. Furthermore, these antibodies increased the survival time of all envenomed animals by 50% by protecting more than 60% from death (Figure 5). It is worth emphasizing that the protocol used for the evaluation of survival uses premixed antibodies/venom proportions, which implies a limitation for a clinical application that must be observed in further experiments.The results indicate the involvement of two epitopes in this process, both antibodies inhibiting enzymatic and toxic activities. Moreover, these anti-PLA2 mAbs exhibited paraspecificity against PLA2 molecules from N. naja venom, suggesting the conservation of neutralizing paraspecific structural epitopes across the Elapidae family. Both mABs 1E8 and 3B2 recognized structural PLA2 epitopes, demonstrating that despite differences in primary structure, these toxin classes present high immunological similarity, corroborating the existence of antibodies sharing broad neutralization of family-specific snake venom toxic proteins. Chavanayarn et al., 2012 [62], produced Humanized Single Domain Antibodies against the PLA2s from Naja kaouthia, inhibiting enzymatic activity. By modeling and docking analysis, they suggested that these antibodies’ CDR loops are inserted into PLA2 catalytic grooves. We aligned the sequence from Naja naja, Naja atra, and M. altirostris PLA2s and verified manually that the residues from the calcium binding site and lipid binding site are shared between these molecules (Figure S3). Our data indicate that a similar mechanism may be involved. Future structural analyses are necessary to confirm this hypothesis.Our results suggest the applicability of mAb 1E8 and 3B2 to supplement current conventional antivenoms to improve the neutralization capacity of the antisera. For example, the Brazilian coral snake antivenom lacks neutralization activity towards the venom of M. altirostris [43,44,45]. The antivenomic analysis revealed low immunoreactivity for some toxins from M. altirostris venom, particularly PLA2 F5CPF0, which entirely escaped immunodepletion by coral snake antivenom [16]. However, this toxin was neutralized by monoclonal antibodies described in this work. Our results demonstrate potential applicability, especially to neutralize the lethal activity of coral snakes from venoms enriched with PLA2, including M. fulvius, M. moscatensis, and M. dumerilli, which have 58%, 55%, and 52% of PLA2, respectively [29]. Furthermore, other preparations may improve antivenoms’ effectiveness, such as combining polyclonal and monoclonal antibodies with neostigmine, a cholinesterase inhibitor that has been applied to overcome the effects of α-neurotoxins [64,65]. Future studies must confirm if this supplementation to antielapidic sera will produce more protection. Additionally, new immunization strategies were recently driven by the vaccine market and consequently brought a lot of innovation to this area; these include DNA or mRNA vaccines, among others [66,67], and they can be used to develop new antisera.Two divergent patterns of coral snakes’ venom composition, 3FTx-rich and PLA2-rich, have been revealed by proteomics studies [20,28,29]. Our work indicates the potential use of monoclonal antibodies 1E8 and 3B2 for neutralizing the PLA2 molecules of PLA2-rich venoms of coral snakes from Central and North America.

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