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Clostridium botulinum is considered as the most naturally lethal agent with extreme toxicity LD50 1?gr/kg in the oral rout. It possesses potent neurotoxins, botulinum neurotoxins (BoNT), responsible for botulinum syndrome in humans characterized by flaccid paralysis and protease activity. Proteolytic activity target is SNARE proteins that modulate neuro-transmitter release mediated by the metalloproteinase domain of the toxin.(45) So, SNARE protein is one of the important candidates for Aptamers or Nbs targeting. The neurotoxin is classified in seven types (A-G) which, A, B, E and F toxins lead to human diseases while BoNT/A and BoNT/ B are designed as two stable protease (46)
The second messenger signaling molecule bis (3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) connected to riboswitches of class I and II regulates some virulence action in bacteria, such as polysaccharide synthesis, cellular mobility, biofilm formation and pathogenicity mechanisms (47, 48). Luo et al(2013) developed an in silico study for the detection of clostridium tetani and botulinum using RNA Aptamers and found 4 classes of C-di-GMP (Ct-E88, Cb-17B, Cb-E43 and Cd-630 RNAs). They showed that nanoscale amount of aptamers were able to bind to the Ct-E88 and Cb-E43 classes of C-di-GMP of Clostridium tetani and Clostridium botulinum respectively, while Cb-17B and Cd-630 are needed to attach higher amount of Aptamers (100 times Weaker) (49).
Current effective immunotherapy consists of polyclonal antibodies from heterologous animals antibodies, if administrate prior the appearance of major clinical symptoms in carriers. However conventional antitoxin suffers from totally reversing of sever enzymatically effects highlights effective therapeutic for improving treatment strategy exploration
In one study, two high affinity VHHs were selected from alpacas immunized with BoNT/A and BoNT/B capable of inhibiting of protease activity in Ki and KD value of 1 nM in mammalian cell intoxication(50) . Another study is based on VHH development for enzymatic BoNT serotype A light chain (BoNT/A Lc) inhibition as substrate mimic around the high affinity VHH displayed on the surface of the yeast Saccharomyces cerevisiae. (51) Anti-toxin therapy based on Algal choloroplast provide a cost-effective and massive production of therapeutic proteins could be worth exploring as a vehicle for vaccine development and immunotoxin as well (52). It also enables recombinant proteins VHH expression in folded format as well as production in yeast Saccharomyces cerevisiae. Additionally, it provides a kind of recombinant expression system without any solubilizing tag and purification (53). High productive folded Pichia pastoris displaying anti-BoTN VHH fragments showed in vivo neutralization activity up to 4LD50 of the BoNT/E in 25% of mice population administered I.P (54). Taken together rapid appearance of the protective VNA in serum make it suitable tool for acute treatments for many toxin-mediated diseases.(55)