RESUMEN

Cobra (Naja kaouthia) venom contains many toxins including α-neurotoxin (αNTX) and phospholipase A2 (PLA2), which can cause neurodegeneration, respiratory failure, and even death. The traditional antivenom derived from animal serum faces many challenges and limitations. Heavy-chain-only antibodies (HCAb), fusing VHH with human IgG Fc region, offer advantages in tissue penetration, antigen binding, and extended half-life. This research involved the construction and transient expression of two types of VHH-FC which are specific to α-Neurotoxin (VHH-αNTX-FC) and Phospholipase A2 (VHH-PLA2-FC) in Nicotiana benthamiana leaves. The recombinant HCAbs were incubated for up to six days to optimize expression levels followed by purification by affinity chromatography and characterization using LC/Q-TOF mass spectrometry (MS). Purified proteins demonstrated over 92 % sequence coverage and an average mass of around 82 kDa with a high-mannose N-glycan profile. An antigen binding assay showed that the VHH-αNTX-Fc has a greater ability to bind to crude venom than VHH-PLA2-Fc.

RESUMEN

Scorpion envenoming is a serious health problem which can cause a variety of clinical toxic effects. Of the many scorpion species native to Iran, Hottentotta saulcyi is important because its venom can produce toxic effects in man. Nowadays, antivenom derived from hyper immune horses is the only effective treatment for sever scorpion stings. Current limitations of immunotherapy urgently require an efficient alternative with high safety, target affinity and more promising venom neutralizing capability. Recently, heavy chain-only antibodies (HC-Abs) found naturally in camelid serum met the above mentioned advantages. In this study, immuno-reactivities of polyclonal antibodies were tested after successful immunization of camel using H. saulcyi scorpion crude venom. The lethal potency of scorpion venom in C57BL/6 mice injected intraperitoneally was determined to be 2.7 mg/kg. These results were followed by the efficient neutralization of lethal activity of H. saulcyi scorpion venom by injection of antivenom and purified IgG fractions into mice intraperitonelly or intravenously, respectively. HC-Ab camelid antivenom could be considered as a useful serotherapeutics instead of present treatment for scorpion envenomation.

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RESUMEN

Hottentotta saulcyi, medically important scorpion species, causes some of harmful toxic exposure in Iran. Administrated, conventional antivenom-based immunotherapy is still limited and hardly meet ideal characteristic of effective treatment for scorpion envenomation. In this study we aimed to develop a neutralizing agent directed against scorpion venom based on VHH, variable domain of the Camelidae heavy chain antibody or Nanobody. This promising biomolecule is well-established as an advantageous tool for therapeutic purposes due to its small size, stability, monomeric performance and less immunogenicity. In this study, a large Nb library was constructed and phage displayed after successful camel immunization using H. saulcyi scorpion crude venom. After a series of biopanning rounds on Sephadex G50 purified venom fraction and screening by monoclonal phage ELISA, the best reactive Nb was retrieved and designated Nb12. The selected Nb was then expressed as soluble protein in Escherichia coli, purified and confirmed by SDS-PAGE analysis and western blotting. The lead candidate Nb12 bound scorpion venom with Kaff value of 5×10(7)M(-1). Nb12 was shown to be capable of neutralizing 2 LD50 of whole venom of scorpion toxin when injected in the ratio of the Nb/toxin of 1.4:1 into C57BL/6 mice. In challenge experiment, Nb succeeded to rescue all i.p. lethal dose injected mice even when administrated i.v., 20min after envenoming. These results with ease of production and superior neutralizing activity make Nb a suitable anti-toxin candidate for treatment of scorpion envenoming.

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RESUMEN

Specific treatment is not available for human botulism. Current remedial mainstay is the passive administration of polyclonal antibody to botulinum neurotoxin (BoNT) derived from heterologous species (immunized animal or mouse hybridoma) together with supportive and symptomatic management. The antibody works extracellularly, probably by blocking the binding of receptor binding (R) domain to the neuronal receptors; thus inhibiting cellular entry of the holo-BoNT. The antibody cannot neutralize the intracellular toxin. Moreover, a conventional antibody with relatively large molecular size (150 kDa) is not accessible to the enzymatic groove and, thus, cannot directly inhibit the BoNT zinc metalloprotease activity. Recently, a 15-20 kDa single domain antibody (V(H)H) that binds specifically to light chain of BoNT serotype A was produced from a humanized-camel VH/V(H)H phage display library. The V(H)H has high sequence homology (>80%) to the human VH and could block the enzymatic activity of the BoNT. Molecular docking revealed not only the interface binding between the V(H)H and the toxin but also an insertion of the V(H)H CDR3 into the toxin enzymatic pocket. It is envisaged that, by molecular linking the V(H)H to a cell penetrating peptide (CPP), the CPP-V(H)H fusion protein would be able to traverse the hydrophobic cell membrane into the cytoplasm and inhibit the intracellular BoNT. This presents a novel and safe immunotherapeutic strategy for botulism by using a cell penetrating, humanized-single domain antibody that inhibits the BoNT by means of a direct blockade of the groove of the menace enzyme.

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