Structural basis for the inhibition of HIV-1 Nef by a high-affinity binding single-domain antibody

Abstract

BackgroundThe HIV-1 Nef protein is essential for AIDS pathogenesis by its interaction with host cell surface receptors and signaling factors. Despite its critical role as a virulence factor Nef is not targeted by current antiviral strategies.ResultsWe have determined the crystal structure of the complex formed by a camelid single-domain antibody fragment, termed sdAb19, bound to HIV-1 Nef together with a stabilizing SH3 domain. sdAb19 forms a stoichiometric 1:1 complex with Nef and binds to a conformationally conserved surface at the C-terminus of Nef that overlaps with functionally important interaction sites involved in Nef-induced perturbations of signaling and trafficking pathways. The antibody fragment binds Nef with low nanomolar affinity, which could be attenuated to micromolar affinity range by site-directed mutagenesis of key interaction residues in sdAb19. Fusion of the SH3 domain to sdAb19, termed Neffin, leads to a significantly increased affinity for Nef and formation of a stoichiometric 2:2 Nef–Neffin complex. The 19 kDa Neffin protein inhibits all functions of Nef as CD4 and MHC-I downregulation, association with Pak2, and the increase in virus infectivity and replication.ConclusionsTogether, sdAb19 and Neffin thus represent efficient tools for the rational development of antiviral strategies against HIV-1 Nef.

Fully Human VH Single Domains That Rival the Stability and Cleft Recognition of Camelid Antibodies

“…The domains are biophysically and structurally well characterized and have been shown to possess high thermodynamic and colloidal stabilities, together with a preference for binding within pockets of protein structure (high cleft binding propensity) (5,6). These features are achieved by means of an extended CDR3 3 loop, which has been shown to be capable of protruding deeply into enzyme active sites and otherwise cryptic epitopes of viruses and G-protein-coupled receptors (7)(8)(9). Residues in the light chain interface differ between in human domains and V H H domains, resulting in a hydrophilic nature of the camelid surface (2).…”

Brief introduction of current technologies in isolation of broadly neutralizing HIV-1 antibodies

“…The DNA sequence will be optimized in order to improve the stability. In addition to heavy-chain variable domains, there are other types of single domain antibodies which have been studied, including CH2 antibodies, and light-chain variable domain antibodies, and some other forms of nanobodies (Duarte et al, 2016;Li et al, 2016;Gong et al, 2016;Louis et al, 2014;Lulf et al, 2014;Bouchet et al, 2012;Bouchet et al, 2011;Boudet et al, 1995).…”

Anti-Ebola therapies based on monoclonal antibodies: current state and challenges ahead

“…The use of properly engineered mAb fragments, instead of full-length mAbs, is a therapeutic alternative that has yet to be investigated for Ebola. The use of antibody fragments to neutralize viruses or prevent virus infection is not novel; proof of concept experiments have shown their potential applications in the context of different viral infections such us HIV (Lülf et al, 2014), Influenza A H1N5 (Bal et al, 2015), SARS (Sui et al, 2004), HPV (Culp et al, 2007), and West Nile virus (Gould et al, 2005). A recent contribution by Rodríguez-Martínez et al (2015) offers proof-of-principle of the application of three anti-EBOV mAb fragments, containing the variable regions the mAbs KZ52, 13C6, and 13F6, in immunological assays to specifically detect recombinant GP.…”