Structural Basis for the Specific Neutralization of Stx2a with a Camelid Single Domain Antibody Fragment

Abstract

Background: Shiga toxin-producing Escherichia coli (STEC) are a subset of pathogens leading to illnesses such as diarrhea, hemolytic uremic syndrome and even death. The Shiga toxins are the main virulence factors and divided in two groups: Stx1 and Stx2, of which the latter is more frequently associated with severe pathologies in humans. Results: An immune library of nanobodies (Nbs) was constructed after immunizing an alpaca with recombinant Shiga toxin-2a B subunit (rStx2aB), to retrieve multiple rStx2aB-specific Nbs. The specificity of five Nbs towards rStx2aB was confirmed in ELISA and Western blot. Nb113 had the highest affinity (9.6 nM) and its bivalent construct exhibited a 100-fold higher functional affinity. The structure of the Nb113 in complex with rStx2aB was determined via X-ray crystallography. The crystal structure of the Nb113–rStx2aB complex revealed that five copies of Nb113 bind to the rStx2aB pentamer and that the Nb113 epitope overlaps with the Gb3 binding site, thereby providing a structural basis for the neutralization of Stx2a by Nb113 that was observed on Vero cells. Finally, the tandem-repeated, bivalent Nb1132 exhibits a higher toxin neutralization capacity compared to monovalent Nb113. Conclusions: The Nb of highest affinity for rStx2aB is also the best Stx2a and Stx2c toxin neutralizing Nb, especially in a bivalent format. This lead Nb neutralizes Stx2a by competing for the Gb3 receptor. The fusion of the bivalent Nb1132 with a serum albumin specific Nb is expected to combine high toxin neutralization potential with prolonged blood circulation.

Cryo-EM structure of P. falciparum circumsporozoite protein with a vaccine-elicited antibody is stabilized by somatically mutated inter-Fab contacts

“…This finding provides strong evidence for antigen-induced maturation of inter-Fab interactions for human antibodies, which may prove to be a common mechanism for increasing affinity against the PfCSP repeat region and for tandem repeat sequences in general. Recently, heavy-chain antibody fragments (nanobodies) derived from alpacas against a pentameric antigen were observed to have inter-nanobody contacts, suggesting that this mechanism may be present across certain antibodies in different species ( 31 ). A previous structure of antibody 2G12 to HIV Env revealed a novel domain swap within the Fabs of a single IgG molecule, where the heavy chain from one Fab paired with the light chain of the other Fab, such that a new V H -V H interface was formed that was also subject to somatic hypermutation ( 32 ).…”

Camelid VHHs Fused to Human Fc Fragments Provide Long Term Protection Against Botulinum Neurotoxin A in Mice

“…Camelid VHHs are a popular tool for constructing recombinant antibodies to detect and neutralize a range of targets [18,19,20,21,22,23,24]. In particular, it has been shown that HC-only antibodies or VHHs derived from HC-only antibodies, produced by camelids demonstrate strong anti-BoNT activities in animal models [6,43,44].…”

“…Clear advantages include the ability to recognize hidden antigenic sites that are inaccessible for conventional antibodies due to their structure; stability over a wide range of temperatures and pH; high solubility, as well as economical and facile expression and production in large quantities in microorganisms [15,17]. Several studies were conducted to test the potency of VHHs as inhibitors of viral infections [18] and different toxins, produced by such plants and microorganisms as Ricinus communis [19], Mycoplasma hominis [20], Clostridium tetani [21], Clostridium difficile [22], Crotalus durissus terrificus [23], and Shiga toxigenic Escherichia coli (STEC) [24]. It has been demonstrated that the antigen-binding region of VHHs produced by camelids showed strong anti-BoNT activities in animal models [25,26].…”

Cryo-EM structure of the circumsporozoite protein of Plasmodium falciparum with a vaccine-elicited antibody reveals maturation of inter-antibody contacts

“…This finding provides strong evidence for antigen-induced maturation of inter-Fab interactions for human antibodies, which may prove to be a common mechanism for increasing affinity against the PfCSP repeat region and for tandem repeat sequences in general. Recently, heavy chain antibody fragments (nanobodies) derived from Alpacas against a pentameric antigen were observed to have inter-nanobody contacts, suggesting that this mechanism may be present across certain antibodies in different animal kingdoms (31). A previous structure of antibody 2G12 to HIV Env revealed a novel domain swap within the Fabs of a single IgG molecule, where the heavy chain from one Fab paired with the light chain of the other Fab, such that a new VH-VH interface was formed that was also subject to affinity maturation (32).…”