Efficient Targeting of Conserved Cryptic Epitopes of Infectious Agents by Single Domain Antibodies

Abstract

Antigen variation is a successful defense system adopted by several infectious agents to evade the host immune response. The principle of this defense strategy in the African trypanosome paradigm involves a dense packing of variant surface glycoproteins (VSG) exposing only highly variable and immuno-dominant epitopes to the immune system, whereas conserved epitopes become inaccessible for large molecules. Reducing the size of binders that target the conserved, less-immunogenic, cryptic VSG epitopes forms an obvious solution to combat these parasites. This goal was achieved by introducing dromedary Heavy-chain antibodies. We found that only these unique antibodies recognize epitopes common to multiple VSG classes. After phage display of their antigen-binding repertoire, we isolated a single domain antibody fragment with high specificity for the conserved Asn-linked carbohydrate of VSG. In sharp contrast to labeled concanavalin-A that stains only the flagellar pocket where carbohydrates are accessible because of less dense VSG packing, the single domain binder stains the entire surface of viable parasites, irrespective of the VSG type expressed. This corroborates the idea that small antibody fragments, but not larger lectins or conventional antibody fragments, are able to penetrate the dense VSG coat to target their epitope. The diagnostic potential of this fluorescently labeled binder was proven by the direct, selective, and sensitive detection of parasites in blood smears. The employment of this binder as a molecular recognition unit in immuno-toxins designed for trypanosomosis therapy becomes feasible as well. This was illustrated by the specific trypanolysis induced by an antibody::␤-lactamase fusion activating a prodrug.

Antigen recognition by single-domain antibodies: structural latitudes and constraints

“…Stijlemans et al . 68 hypothesized that the ability of a dromedary V H H, cAb-An33, to target a cryptic glycopeptide epitope conserved across all variant surface glycoprotein classes of Trypanosoma brucei was due to the V H H’s small size as well as, potentially, the nature of this epitope. This hypothesis was supported by the inability of rabbit and dromedary polyclonal conventional antibodies as well as a ~90-kDa lectin to access this site.…”

“…No studies have reported hapten-specific V NAR s, and only one study has described a carbohydrate-specific V H H directed against Neisseira meningitidis lipopolysaccharide; 120 at least two camelid V H Hs have been described that bind to glycopeptide epitopes. 68,76 No sdAbs of any type have been described that convincingly bind lipids or nucleic acids. Together, the consensus of the data is that it is probably difficult, but not impossible, for sdAb paratopes to accommodate haptens and that three CDRs are sufficient to form the binding pockets and grooves required for such interactions, although potential involvement of solubility-enhancing FR2 residues at the former V L interface in pocket formation may impose restrictions on hapten-binding specificities.…”

Similar inflammatory DC maturation signatures induced by TNF or Trypanosoma brucei antigens instruct default Th2‐cell responses

“…sVSG was prepared by osmotic shock as described previously [78]. Briefly, the DE52-purified parasites were resuspended in Balts-buffer (50 mM sodium phosphate buffer, pH 5.5) and incubation on ice for 30 min followed by a 5-min incubation at 371C.…”

Fragments d’anticorps à domaine unique

“…Ces anticorps dénommés HcAb pour heavy chain only antibodies lient leurs cibles par un seul domaine variable nommé VHH pour le différencier des VH conventionnels (Figure 1 [12] (➜). Les dAb peuvent aussi cibler des épitopes conservés et peu accessibles sur certaines protéines d'agents infectieux qui ont évolué pour présenter aux anticorps conventionnels des épitopes dominants hypervariables [13]. Par leur taille et leur structure, les dAb sont donc une source potentielle d'anticorps antiviraux neutralisants à spectre large [14].…”