Venomous snakebites are an important health problem in tropical and subtropical countries. King cobra (Ophiophagus hannah) is the largest venomous snake found in South and Southeast Asia. In this study, the O. hannah venom proteome and the venom components cross-reactive to N. kaouthia monospecific antivenin were studied. O. hannah venom consisted of 14 different protein families, including three finger toxins, phospholipases, cysteine-rich secretory proteins, cobra venom factor, muscarinic toxin, L-amino acid oxidase, hypothetical proteins, low cysteine protein, phosphodiesterase, proteases, vespryn toxin, Kunitz, growth factor activators and others (coagulation factor, endonuclease, 5’-nucleotidase). N. kaouthia antivenin recognized several functionally different O. hannah venom proteins and mediated paratherapeutic efficacy by rescuing the O. hannah envenomed mice from lethality. An engineered human ScFv specific to N. kaouthia long neurotoxin (NkLN-HuScFv) cross-neutralized the O. hannah venom and extricated the O. hannah envenomed mice from death in a dose escalation manner. Homology modeling and molecular docking revealed that NkLN-HuScFv interacted with residues in loops 2 and 3 of the neurotoxins of both snake species, which are important for neuronal acetylcholine receptor binding. The data of this study are useful for snakebite treatment when and where the polyspecific antivenin is not available. Because the supply of horse-derived antivenin is limited and the preparation may cause some adverse effects in recipients, a cocktail of recombinant human ScFvs for various toxic venom components shared by different venomous snakes, exemplified by the in vitro produced NkLN-HuScFv in this study, should contribute to a possible future route for an improved alternative to the antivenins.
There is a need for safe and broadly effective anti-HCV agents that can cope with genetic multiplicity and mutations of the virus. In this study, humanized-camel VHHs to genotype 3a HCV serine protease were produced and were linked molecularly to a cell penetrating peptide, penetratin (PEN). Human hepatic (Huh7) cells transfected with the JFH-1 RNA of HCV genotype 2a and treated with the cell penetrable nanobodies (transbodies) had a marked reduction of the HCV RNA intracellularly and in their culture fluids, less HCV foci inside the cells and less amounts of HCV core antigen in culture supernatants compared with the infected cells cultured in the medium alone. The PEN-VHH-treated-transfected cells also had up-regulation of the genes coding for the host innate immune response (TRIF, TRAF3, IRF3, IL-28B and IFN-β), indicating that the cell penetrable nanobodies rescued the host innate immune response from the HCV mediated-suppression. Computerized intermolecular docking revealed that the VHHs bound to residues of the protease catalytic triad, oxyanion loop and/or the NS3 N-terminal portion important for non-covalent binding of the NS4A protease cofactor protein. The so-produced transbodies have high potential for testing further as a candidate for safe, broadly effective and virus mutation tolerable anti-HCV agents.
Small molecular inhibitors and passive immunization against Ebola virus disease (EVD) have been tested in animal models, including rodents and non-human primates, as well as in clinical trials. Nevertheless, there is currently no Food and Drug Administration (FDA)-approved therapy, and alternative strategies must be pursued. The aim of this study was to produce cell-penetrable human single-chain antibodies (transbodies) that are able to interfere with the activities of interferon inhibitory domain (IID) of the VP35 protein, a multifunctional virulence factor of Ebola virus (EBOV). We speculated that effective VP35-IID-specific transbodies could inspire further studies to identify an alternative to conventional antibody therapies. Phage display technology was used to generate Escherichia coli-derived human single-chain antibodies (HuscFvs) that bind to IID. HuscFvs were linked to nona-arginine (R9) to make them cell penetrable. Transbodies of transformed E. coli clones 13 and 3, which were predicted to interact with first basic patch residues (R9-HuscFv13), central basic patch, and end-cap residues (R9-HuscFv3), effectively inhibited EBOV minigenome activity. Transbodies of E. coli clones 3 and 8 antagonized VP35-mediated interferon suppression in VP35-transduced cells. We postulate that these transbodies formed an interface contact with the IID central basic patch, end-cap, and/or residues that are important for IID multimeric formation for dsRNA binding. These transbodies should be evaluated further in vitro using authentic EBOV and in vivo in animal models of EVD before their therapeutic/prophylactic effectiveness is clinically evaluated.
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