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A fundamental issue of the characterization of single-chain variable fragments (scFvs), capable of neutralizing scorpion toxins, is their cross-neutralizing ability. This aspect is very important in Mexico because all scorpions dangerous to humans belong to the Centruroides genus, where toxin sequences show high identity. Among toxin-neutralizing a...
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... 10FG2 showed similar affinities for both toxins with KDs of 1.1 nM and 1.8 nM for CsEM1a and CsEd, respectively. In the case of LR, greater differences were observed with KDs of 1.29 nM and 0.61 nM for CsEM1a and CsEd, respectively (Table 1) The kinetic constants of the molecular interactions obtained from the sensorgrams generated at different concentrations ( Figure 2a) were used to calculate the corresponding affinities. scFv 10FG2 showed similar affinities for both toxins with KDs of 1.1 nM and 1.8 nM for CsEM1a and CsEd, respectively. ...Context 2
... 10FG2 showed similar affinities for both toxins with KDs of 1.1 nM and 1.8 nM for CsEM1a and CsEd, respectively. In the case of LR, greater differences were observed with KDs of 1.29 nM and 0.61 nM for CsEM1a and CsEd, respectively (Table 1). Molecular interactions were performed at 25 • C with a flow rate of 50 µL min −1 . ...Context 3
... results of these analyses are shown in Tables S1 and S2. Figure 3a shows minimal differences in the superposition of the structural models of the toxins with the two scFvs, where some of the most important contacts at the interface scFv-toxin are highlighted (Figure 3b-e). The interactions at the interface of toxin Cn2 with both scFvs were used as a control (Tables S1 and S2) since this toxin is recognized with greater affinity by both scFvs [11]. During the MD of the different complexes, the structural similarities shared between CsEM1a and CsEd toxins with Cn2 toxin were reflected in the results, as they showed that the main contacts are kept (Tables S1 and S2). ...Context 4
... interactions at the interface of toxin Cn2 with both scFvs were used as a control (Tables S1 and S2) since this toxin is recognized with greater affinity by both scFvs [11]. During the MD of the different complexes, the structural similarities shared between CsEM1a and CsEd toxins with Cn2 toxin were reflected in the results, as they showed that the main contacts are kept (Tables S1 and S2). These observations explain the ability of scFvs LR and 10FG2 of recognizing this group of toxins. ...Context 5
... the case of dissociation, from the koff values, we were able to determine that the retention times (T R ) show greater differences in the average binding time of the toxin-antibody complex. For example, scFv LR interaction with CsEd toxin remained for almost 71 min (Table 1), while for CsEM1a toxin, it was only 21 min. We have reported that, for a good neutralization of this epitope present in Cn2 and Css2 toxins, retention times must be longer than 250 min [12]. ...Context 6
... results of the MD show that the wide cross-reactivity of scFvs 10FG2 and LR with the different toxins studied in this work is explained mainly through the conservation of interactions with the three toxins (Cn2, CsEM1a, and CsEd). The similarity of the sequences in the epitopes of the toxins favors the level of recognition shown by the scFvs 10FG2 and LR (Figures 1 and 3a and Supplementary Materials Tables S1 and S2). However, the differences in the toxin sequences influence the affinity levels of the scFvs (Table 1). ...Context 7
... similarity of the sequences in the epitopes of the toxins favors the level of recognition shown by the scFvs 10FG2 and LR (Figures 1 and 3a and Supplementary Materials Tables S1 and S2). However, the differences in the toxin sequences influence the affinity levels of the scFvs (Table 1). The affinity of the scFv LR for the CsEd toxin is greater than that of the CsEM1a toxin, while in the case of the scFv 10FG2, the affinity is greater for CsEM1a toxin. ...Context 8
... Figure 3b,c it can be seen that Y9 of CsEM1a establishes a variety of interactions, such as hydrogen bonds with Y60 backbone of scFv 10FG2, hydrophobic contacts with Y59 and Y60, an aromatic-aromatic interactions with Y59, as well as a cation-Pi interaction with K65. This is in contrast to S9 of the CsEd toxin (Table S1). When analyzing the area of the structure where Y9 is located, it is observed that the temperature B factors are lower for these three residues S8, Y9, and T10 (Table S3). ...Context 9
... these samples, one of every 5000 frames was taken and submitted to the PIC (Protein Interactions Calculator) software using default values [25] and to PISA software [26] for analyses of the interface between the scFvs and each of the toxins evaluated. Table S1: Comparison of interactions between scFv 10FG2 and the indicated toxins, Table S2: Comparison of interactions between scFv LR and the indicated toxins, Table S3: Temperature B factors of the first 20 residues of toxins CsEM1a and CsEd and their difference during interaction with scFv 10FG2. ...Similar publications
Among other scorpion species, Colombia has two genera of the Buthidae family Centruroides and Tityus, considered to be dangerous to humans. This research shares scientific knowledge aiming to a better understanding about the pathophysiological effects of such venoms. The venom of the three species: Centruroides margaritarus, Tityus pachyurus, and T...
Citations
... Based on these observations, mix-type assays were carried out [16], where 1 LD 50 of fresh C. huichol venom was mixed with scFvs LR and 10FG2 at a molar ratio of 1:10:10 (venom: scFv LR: scFv 10FG2). To estimate the amount of scFv to be used, in this experiment it was assumed that the proportion of toxins corresponds to 10% of the venom (average value with respect to the venoms studied so far [16,18,20]). ...
... In the case of Chui5 toxin, it also modified the activity of hNav1.5 channel. The presence of four components with a total abundance of 14% is significant since, typically, the abundances of the toxins correspond to less than 10% in the venom of Mexican scorpions [16,18,20]. ...
Centruroides huichol scorpion venom is lethal to mammals. Analysis of the venom allowed the characterization of four lethal toxins named Chui2, Chui3, Chui4, and Chui5. scFv 10FG2 recognized well all toxins except Chui5 toxin, therefore a partial neutralization of the venom was observed. Thus, scFv 10FG2 was subjected to three processes of directed evolution and phage display against Chui5 toxin until obtaining scFv HV. Interaction kinetic constants of these scFvs with the toxins were determined by surface plasmon resonance (SPR) as well as thermodynamic parameters of scFv variants bound to Chui5. In silico models allowed to analyze the molecular interactions that favor the increase in affinity. In a rescue trial, scFv HV protected 100% of the mice injected with three lethal doses 50 (LD50) of venom. Moreover, in mix-type neutralization assays, a combination of scFvs HV and 10FG2 protected 100% of mice injected with 5 LD50 of venom with moderate signs of intoxication. The ability of scFv HV to neutralize different toxins is a significant achievement, considering the diversity of the species of Mexican venomous scorpions, so this scFv is a candidate to be part of a recombinant anti-venom against scorpion stings in Mexico.
A key aspect during the development of antivenoms is the evaluation of the efficiency and security of the therapeutic molecules. In this work, we report the pharmacokinetic analysis of a neutralizing single chain antibody fragment named LR (scFv LR) where three sheep were used as a large animal model. The animals were injected through i.v. route with 2 mg of scFv LR. Blood samples were drawn every minute within the first 15 min, the sampling continues at 20, 25, 30, 45, 60, 90, 120 min, subsequently at 1-h intervals, 3, 4, 5, 6 h, two more samples at 9 and 12 h and, two more samples at 24 and 48 h and finally at one-day intervals during 4 days. scFv LR levels were measured from blood serum and urine samples by an ELISA. The pharmacokinetics of the experimental data was analyzed using the three-exponential kinetics. The value of the fast initial component (τ 1 = 0.409 ± 0.258 min) indicated that the scFv is distributed rapidly into the tissues. The mean residence time, MRT, was 45 ± 0.51 min and the clearance (CL), 114.3 ± 14.3 mL/min. From urine samples it was possible to detect significant amounts of scFv LR, which is evidence of renal elimination.
Scorpion envenomation is a serious health problem in tropical and subtropical zones. The access to scorpion antivenom is sometimes limited in availability and specificity. The classical production process is cumbersome, from the hyper-immunization of the horses to the IgG digestion and purification of the F(ab)′2 antibody fragments. The production of recombinant antibody fragments in Escherichia coli is a popular trend due to the ability of this microbial host to produce correctly folded proteins. Small recombinant antibody fragments, such as single-chain variable fragments (scFv) and nanobodies (VHH), have been constructed to recognize and neutralize the neurotoxins responsible for the envenomation symptoms in humans. They are the focus of interest of the most recent studies and are proposed as potentially new generation of pharmaceuticals for their use in immunotherapy against scorpion stings of the Buthidae family. This literature review comprises the current status on the scorpion antivenom market and the analyses of cross-reactivity of commercial scorpion anti-serum against non-specific scorpion venoms. Recent studies on the production of new recombinant scFv and nanobodies will be presented, with a focus on the Androctonus and Centruroides scorpion species. Protein engineering-based technology could be the key to obtaining the next generation of therapeutics capable of neutralizing and cross-reacting against several types of scorpion venoms.
Key points
• Commercial antivenoms consist of predominantly purified equine F(ab)′2fragments.
• Nanobody-based antivenom can neutralize Androctonus venoms and have a low immunogenicity.
• Affinity maturation and directed evolution are used to obtain potent scFv families against Centruroides scorpions.
Strychnine poisoning induces seizures that result in loss of control of airway muscles, leading to asphyxiation and subsequent death. Current treatment options are limited, requiring hands-on medical care and isolation to low-stimulus environments. Anticonvulsants and muscle relaxants have shown limited success in cases of severe toxicity. Furthermore, nonfatal strychnine poisoning is likely to result in long-term muscular and cognitive damage. Due to its potency, accessibility, and lack of effective antidotes, strychnine poses a unique threat for mass casualty incidents. As a first step toward developing an anti-strychnine immunotherapy to reduce or prevent strychnine-induced seizures, a strychnine vaccine was synthesized using subunit keyhole limpet hemocyanin. Mice were vaccinated with the strychnine immunoconjugate and then given a 0.75 mg/kg IP challenge of strychnine and observed for seizures for 30 min. Vaccination reduced strychnine-induced events, and serum strychnine levels were increased while brain strychnine levels were decreased in vaccinated animals compared to the control. These data demonstrate that strychnine-specific antibodies can block the seizure-inducing effects of strychnine and could be used to develop a therapeutic for strychnine poisoning.
Previously, it was demonstrated that from the single chain fragment variable (scFv) 3F it is possible to generate variants capable of neutralizing the Cn2 and Css2 toxins, as well as their respective venoms (Centruroides noxius and Centruroides suffusus). Despite this success, it has not been easy to modify the recognition of this family of scFvs toward other dangerous scorpion toxins. The analysis of toxin-scFv interactions and in vitro maturation strategies allowed us to propose a new maturation pathway for scFv 3F to broaden recognition toward other Mexican scorpion toxins. From maturation processes against toxins CeII9 from C. elegans and Ct1a from C. tecomanus, the scFv RAS27 was developed. This scFv showed an increased affinity and cross-reactivity for at least 9 different toxins while maintaining recognition for its original target, the Cn2 toxin. In addition, it was confirmed that it can neutralize at least three different toxins. These results constitute an important advance since it was possible to improve the cross-reactivity and neutralizing capacity of the scFv 3F family of antibodies.
The methylotrophic yeast Pichia pastoris has been one of the most widely used organisms in recent years as an expression system for a wide variety of recombinant proteins with therapeutic potential. Its popularity as an alternative system to Escherichia coli is mainly due to the easy genetic manipulation and the ability to produce high levels of heterologous proteins, either intracellularly or extracellularly. Being a eukaryotic organism, P. pastoris carries out post-translational modifications that allow it to produce soluble and correctly folded recombinant proteins. This work, evaluated the expression capacity in P. pastoris of two single-chain variable fragments (scFvs) of human origin, 10FG2 and LR. These scFvs were previously obtained by directed evolution against scorpion venom toxins and are able to neutralize different toxins and venoms of Mexican species. The yield obtained in P. pastoris was higher than that obtained in bacterial periplasm (E. coli), and most importantly, biochemical and functional properties were not modified. These results confirm that P. pastoris yeast can be a good expression system for the production of antibody fragments of a new recombinant antivenom.
