Received Sep 22; Accepted Feb Abstract Background Snakes of the genus Bothrops, popularly known as pit vipers, are responsible for most cases of snakebite in Brazil. Within this genus, Bothrops jararacussu and B. Regarding the treatment of snakebites by Bothrops jararacussu, questions have been raised about the effectiveness of the specific bothropic antivenom in neutralizing myotoxic effects; however, there are no accurate data for humans.
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Received Sep 18; Accepted Feb This article has been cited by other articles in PMC. Associated Data All relevant data are within the paper. Abstract Antivenoms, produced using animal hyperimmune plasma, remains the standard therapy for snakebites.
Although effective against systemic damages, conventional antivenoms have limited efficacy against local tissue damage. Additionally, the hypersensitivity reactions, often elicited by antivenoms, the high costs for animal maintenance, the difficulty of producing homogeneous lots, and the instability of biological products instigate the search for innovative products for antivenom therapy.
Complementarity determining regions CDRs and immunoglobulin frameworks FRs of 13 VHH-deduced amino acid sequences were identified, as well as the camelid hallmark amino acid substitutions in FR2. Identified VHHs could be a biotechnological tool to improve the treatment for snake envenomation, an important and neglected world public health problem. Introduction Snakebites represent a relevant public health problem, especially in subtropical and tropical countries.
Affecting mainly the rural population, about 5 million snakebites occur each year worldwide, causing approximately , deaths [ 1 — 3 ]. A large number of the victims experience permanent physical sequelaes due to inflammatory, hemorrhagic, coagulant, neurotoxic and myotoxic effects occasioned by the venom components. These signs are often aggravated by the difficulty of accessing health services in less developed regions [ 2 , 4 ].
It has been described that, worldwide, there are four families of venomous snakes, i. Viperidae, Elapidae, Atractaspididae, and Colubridae [ 5 ]. Viperidae and Elapidae families cause the most venomous bites. Beloging to the Viperidae family, the Bothrops genus is responsible for the majority of snake envenoming in Central and South America, occasioning both high morbidity and mortality [ 6 ]. Diagnosis of snakebite is based on clinic-epidemiological evalution and the current treatment consists, besides supportive care, of the intravenous administration of antivenoms [ 7 ].
Although they are effective against systemic toxic effects, conventional antivenoms poorly neutralize venom toxins in deep tissues, due to the discrepancies between the pharmacokinetic profiles of the low molecular weight toxins and antivenoms [ 9 ]. Additionally, the hypersensitivity reactions, often elicited by heterologous serum, the high costs for animal maintenance, the difficulty of producing homogeneous lots, the instability of biological products, together with animal rights and welfare instigate the search for innovative products for snakebite therapy [ 8 ].
Among proposed innovations the use of monoclonal antibodies and recombinant antibody fragments, like single chain variable fragment scFv , stand out [ 10 — 12 ].
While the high immunogenicity, as well as the high cost for monoclonal antibodies production limit the use of that technology, scFvs usually show lower affinity when compared with the antigen recognition region of conventional antibodies [ 13 ].
To overcome these issues, studies have proposed the use of recombinant antigen binding domains derived from camelid heavy chain antibodies, called VHH, for antivenom production [ 14 ]. With about 15 kDa, one-tenth the size of whole antibodies, VHH possess attractive physico-chemistry, pharmacokinetic and pharmacodynamic properties, and shows low immunogenicity.
VHHs present higher tissue penetration ability, and can act as potent enzyme inhibitors by penetrating in the toxin enzymatic clefts, normally inaccessible to conventional VHs [ 16 , 17 ]. Thus, VHHs have emerged as versatile biotechnological tools for antivenom development.
However, antivenoms composed entirely of small antibody fragments, including VHHs, would have limited therapeutic efficacy because of their short serum half-life profiles. Therefore, different strategies have been explored in order to extend the serum half-life of VHHs [ 18 ]. Besides allowing for the neutralization of toxins by small fragments in tissue compartments, the formed toxin-VHH complex can be eliminated rapidly through renal excretion.
In addition, these preparations ensure that a significant concentration of high molecular mass antibodies remains in circulation to neutralize toxins later in the course of envenomation [ 14 ]. Evaluating the features of VHH, the characteristics of bothropstoxins, and the necessity for developing new approaches to support the treatment or even to diagnose snakebites, this study aimed to select VHHs capable of recognizing and neutralizing myotoxic effects triggered by BthTX-I, BthTX-II, and B.
Animal health status was checked routinely by a veterinary. In order to verify the study viability and to determine a protocol for experimental humane endpoints, a pilot study was conducted previously, following the CONCEA Normative Resolution No. This protocol was conducted when severe neurotoxic and myotoxic signs were observed.
All mice groups were monitored regularly 5 min intervals until the end of experimental period and there were no unintended death of animals. Lama glama immunization and immune response monitoring One young adult male L.
Animal immune response was monitored by enzyme immunoassay. The assay was performed in triplicate. Excess antibody was removed by washing, and peroxidase conjugated mouse anti-rabbit IgG Sigma Aldrich was incubated at a dilution for 2 h in BS. TMB-Ultra Millipore was used to reveal the reaction. The negative control was performed using the llama pre-immune serum. Subsequently, PCR, performed using the cDNA as template and one pair of gene-specific primers, resulted in an amplification of the conventional and heavy-chain IgG repertoire gene fragments.
Amplified gene fragments were electrophoresed and the purified heavy chain amplicon was used as template in a nested PCR, resulting in fragments consisting of about bp [ 21 ]. Ligation products were transformed into electrocompetent E. The eluants were transferred, separately, to E. The negative control was carried out using the llama pre-immune serum. Positive clones were sequenced, analyzed and submitted to GenBank. The sonication process was carried out for 3. VHH protein concentration was determined by the Bradford method.
Llama postimmunization serum was used as positive control. The negative control was performed with the llama pre-immune serum After that, a solution of 1 M ethanolamine hydrochloride was injected in order to block remaining reactive groups in flow cell.
The control flow cell was prepared only with ethanolamine. The binding responses were calculated by subtracting the RUs obtained from both blank control cell and running buffer injection. Kinetic analyses were performed by fitting the obtained sensograms with the Langmuir model using the BIA-evaluation software GE Healthcare.
The fluorescent phospholipid was reconstituted in chloroform and dried over a low flow of N2. Reactions were revealed with TMB, and measured at nm. Neutralization of myotoxicity by in vitro pre-incubation To evaluate the VHH neutralization effect on myotoxicity induced by B. Then, each group received an i. To analyze the cross neutralization, 3 other groups of mice were treated with a myotoxic PLA2 of B. Table 1 Groups of mice subjected to in vivo neutralization assays.