Potent neutralizing nanobodies resist convergent circulating variants of SARS-CoV-2 by targeting novel and conserved epitopes

Abstract

There is an urgent need to develop effective interventions resistant to the evolving variants of SARS-CoV-2. Nanobodies (Nbs) are stable and cost-effective agents that can be delivered by novel aerosolization route to treat SARS-CoV-2 infections efficiently. However, it remains unknown if they possess broadly neutralizing activities against the prevalent circulating strains. We found that potent neutralizing Nbs are highly resistant to the convergent variants of concern that evade a large panel of neutralizing antibodies (Abs) and significantly reduce the activities of convalescent or vaccine-elicited sera. Subsequent determination of 9 high-resolution structures involving 6 potent neutralizing Nbs by cryoelectron microscopy reveals conserved and novel epitopes on virus spike inaccessible to Abs. Systematic structural comparison of neutralizing Abs and Nbs provides critical insights into how Nbs uniquely target the spike to achieve high-affinity and broadly neutralizing activity against the evolving virus. Our study will inform the rational design of novel pan-coronavirus vaccines and therapeutics.

Atomistic Simulations and Deep Mutational Scanning of Protein Stability and Binding Interactions in the SARS-CoV-2 Spike Protein Complexes with Nanobodies: Molecular Determinants of Mutational Escape Mechanisms

“…Consistent with the results of mutational scanning, the engineered charge reversal on R31 (R31D) is unable to recover the salt bridge and restore binding to the E484K mutant. 45, 46…”

“…46 The reported cryo-EM structures for different classes of nanobodies suggested mechanisms of high-affinity and broadly neutralizing activity by exploiting epitopes that are shared with antibodies as well as novel epitopes that are unique to the nanobodies. 46 Another diverse repertoire of high affinity nanobodies against SARS-CoV-2 S protein that are refractory to common escape mutants and showed synergistic neutralizing activity are characterized by proximal but non-overlapping epitopes suggesting that multimeric nanobody combinations can improve potency while minimizing susceptibility to escape mutations. 47 These studies identified a group of common resistant mutations in the dynamic RBM region (F490S, E484K, Q493K, F490L, F486S, F486L, and Y508H) that evade many individual nanobodies.…”

Keep out! SARS-CoV-2 entry inhibitors: their role and utility as COVID-19 therapeutics

“…Nanobodies are single domain antibodies that are generated from immunized llamas, camels and phage displays [85][86][87][88]. Recent published evidence shows that multivalent nanobodies are capable of both neutralizing circulating variants and preventing emergence of resistant escape mutants via binding to multiple, non-overlapping epitopes, avidity effects and binding to conserved epitopes largely inaccessible to normal antibodies [89][90][91][92][93][94][95][96]. Table 1 summarizes the main findings from these studies.…”

Deep Mutational Scanning of Dynamic Interaction Networks in the SARS-CoV-2 Spike Protein Complexes: Allosteric Hotspots Control Functional Mimicry and Resilience to Mutational Escape

“…25 Nonetheless, many potentially neutralizing nanobodies also recognize regions of RBD that are subject to escape variation, reducing their potential efficacy. 26-28 Potent neutralizing nanobodies that resist convergent circulating variants of SARS-CoV-2 by targeting novel and conserved epitopes were recently reported. 26 Cryo-EM structure determination of different classes of nanobodies revealed mechanisms of high-affinity and broadly neutralizing activity against the evolving virus by exploiting novel epitopes on the SARS-CoV-2 S protein that are not accessible to antibodies.…”

“…26 Cryo-EM structure determination of different classes of nanobodies revealed mechanisms of high-affinity and broadly neutralizing activity against the evolving virus by exploiting novel epitopes on the SARS-CoV-2 S protein that are not accessible to antibodies. 26…”