Entero toxigenic Escherichia coli (ETEC) is a pathogenic strain of E.coli can cause diarrhea and mortality in neonatal piglets (70). ETEC strains have two virulence factors: 1- F4 and F18 fimbriae mediate bacterial attachment to enterocytes brush borders.
2- Enterotoxins categorized as heat-labile and heat stable (71, 72). Piglets that express F4 receptor are sensitive to ETEC infections. Although, lactogenic uptake provide immunity for weaned piglet using vaccination of the sow, loss of maternal immunity cause post-weaning diarrhea (70). Currently, there are some polyclonal antibodies for weaned piglets but they aren’t cost-effective for field preparation (73). Harmsen et al (74) obtained K609 VHH capable of inhibiting ETEC F4 fimbriae adhesion to enterocyte receptors with an IC90 of 3.8 mg/L. However, K609 was not protective against fluid loss in jejunal segments up to 30%.
This specific VHH didn’t show sufficient immune- protective activity against ETEC in pigletsIt could be degraded by proteolytic digestion in oral immunotherapy (75). Currently, recombinant DNA technology provides the possibility of antibody engineering as a stable agent against the proteolytic enzymes. In order to improve the physicochemical characteristic of K906, another light chain-devoid Ab against ETEC infection was developed. Four VHH clones with 1.
5- to 3-fold increase in vitro stability were obtained using DNA shuffling with distanced predicted protease cleavage sites. Among them, the most stable clone, K922, retained 41% activity after incubation in gastric fluid and 90% in the jejunal fluid. Oral application of K922 pointed to improved proteolytic stability in piglets. Furthermore, K922 inhibits binding of F4 fimbriae at a high affinity. It could be as a useful agent against piglet diarrhea.As ETEC causes diarrhea in piglets, in-seed oral passive immunization is another important strategy to prevent the F4+ETEC gastric infections and prolong maternal lactogenic immunity.
With the aim to develop such a protective agent, VHHs against ETEC was grafted to the Fc fragment of a porcine immunoglobulin (IgG3 or IgA) and expressed in Arabidopsis thaliana seeds. Divalent antibodies expressed in seed successfully inhibit bacterial binding activity to porcine gut enterocytes. Meanwhile piglet feed-challenge experiment showed only the VHH-IgA (dose 20 mg/d per pig) was protective and declined bacteria shedding, compared with piglets receiving VHH-IgG3 seeds (76)Moonens developed four Nbs directed against the lectin domain of the F18 fimbria adhesion, FedF.
It is suggested that two mechanisms mediate the inhibitory function of Nbs. Selected Nbs interfere with F18 fimbriated E. coli bacteria attachment to the blood group antigen receptor on the FedF surface or conformational change induction in CDR3 region of the Nb displacing D0-E loop near the binding site, an essential loop for the interaction of F18 fimbriated bacteria and blood group antigen receptors (77).
Due to the importance of pig farming, selected Nb can be as a useful agent for expression in plants as a prophylactic agent to reduce ETEC and STEC infections. Escherichia coli strain O157: H7 is a major food-borne pathogen causative of human morbidity or mortality(70). Lee et al used RNA aptamers to detect this pathogen from other strains of E.
coli, so they used the subtractive cell-SELEX to detect O157: H7 strain. At the first, they produced an RNA library in E. coli K12 and extracted these RNAs to achieve these Aptamers.
RNAs were used after 6 rounds of the subtractive cell-SELEX process to identify the E. coli O157: H7 strain. Subsequent results were evaluated by using ELISA, Real Time-PCR and aptamer-bound precipitation experiments. The results of this study indicated that aptamers detect strain O157: H7 of E. coli by binding to the expressed O antigen on the bacterial wall of the lipopolysaccharide layer(78).The results of a similar study by Marton et al illustrated that the designed DNA aptamers could detect E. coli strains.
In this study, the SELEX method was used to isolate four single-stranded DNA (ssDNA) that have high affinity for E.coli cells. Identified aptamers were detected by fluorescence attached to them and detected by fluorescence microscopy. (79).
Taken together, Nbs are not only successful in neutralization of bacterial toxins, but they are potent inactivation of some lethal animal toxins as well. Scorpion envenoming is a frequently life-treating problem leading to morbidity and mortality in some tropic area in the world. Currently, immunotherapy based on hyper immune horse sera is widely used for envenomed patients. Nevertheless, antivenom therapy with adverse effects suffers from total inactivation of scorpion venom effects. Most of the animal toxins structurally composed of complicated low molecular peptide and protein. This make them as a suitable target for rapid access and appropriate interaction of VHH to peptide target and improve venom neutralization compared to conventional antibody.
In this regard several study used VHH fragments derived from immunized camelid for toxin neutralization in an in vitro or in vivo studies. Proof of concept, neutralization activity of VHH monomer, Nb12, against Hottentotta saulcyi venom was assessed in pre-incubating and challenge rescue experiments. The lethality of venom in C57 was 40 and 30 µg/mouse(18-20gr) in I.P and I.V rout (80). All treated mice administered in I.v survived the lethal dose of venom 1.
4-fold excess of Nb12 to toxin. Nb12 also was protective up to 30 min after i.p. venom injection and could only protect 20% of animals.
(81). Of the unique physicochemical property of VHH, they can successfully neutralize hazardous toxin effects and could be applied instead of traditional sera-therapy.