Structure-guided multivalent nanobodies block SARS-CoV-2 infection and suppress mutational escape

Abstract

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic continues to spread with devastating consequences. For passive immunization efforts, nanobodies have size and cost advantages over conventional antibodies. Here, we generated four neutralizing nanobodies that target the receptor-binding domain of the SARS-CoV-2 spike protein. We defined two distinct binding epitopes using x-ray crystallography and cryo-electron microscopy. Based on the structures, we engineered multivalent nanobodies with more than 100-fold improved neutralizing activity than monovalent nanobodies. Biparatopic nanobody fusions suppressed the emergence of escape mutants. Several nanobody constructs neutralized through receptor-binding competition, while other monovalent and biparatopic nanobodies triggered aberrant activation of the spike fusion machinery. These premature conformational changes in the spike protein forestalled productive fusion, and rendered the virions non-infectious.

A combination of cross-neutralizing antibodies synergizes to prevent SARS-CoV-2 and SARS-CoV pseudovirus infection

“…Recent reports on SARS-CoV-2 mutations in both human and mink populations give rise to concerns about viral escape from current vaccines and therapeutics in development (Andreano et al, 2020; Greaney et al, 2021a; Kemp et al, 2021; Mallapaty, 2020; Oude Munnink et al, 2021; Tegally et al, 2020; Voloch et al, 2020). However, antibody cocktails that bind to distinct epitopes can increase neutralization breadth and may help prevent escape mutations (Baum et al, 2020; Du et al, 2020; Greaney et al, 2021b; Hansen et al, 2020; Koenig et al, 2021). We previously reported that CV38-142 does not compete for RBD binding with other potent antibodies in our sample set, which are encoded by diverse germline genes, such as CV07-200 (IGHV1-2), CV07-209 (IGHV3-11), CV07-222 (IGHV1-2), CV07-250 (IGHV1-18), CV07-262 (IGHV1-2), CV38-113 (IGHV3-53), and CV38-183 (IGHV3-53) (Kreye et al, 2020).…”

SARS-CoV-2 mutations in MHC-I-restricted epitopes evade CD8 + T cell responses

“…Viruses employ numerous strategies to evade CD8 + T cell immune responses (40)(41)(42). The SARS-CoV-2 encoded ORF8 protein is hypothesized to down-regulate the surface expression of MHC-I molecules (43), and several reports linked mutations within the viral spike protein to the evasion of neutralizing antibody responses (28)(29)(30)). Yet, we still lack a comprehensive understanding of the intrinsic capabilities of SARS-CoV-2 for immune evasion.…”

“…Compelling evolutionary evidence for CTL-mediated control of RNA viruses causing chronic infections like HIV and HCV is provided by mutations occurring in viral epitopes CORONAVIRUS SARS-CoV-2 mutations in MHC-I-restricted epitopes evade CD8 + T cell responses which directly interfere with MHC-I-restricted T cell antigen recognition and killing by CTLs (24)(25)(26)(27). While several mutations in SARS-CoV-2 have recently been associated with an escape from antibody responses (28)(29)(30), the extent to which SARS-CoV-2 mutations may upend the presentation of virusderived peptides via MHC-I remains to be determined. In this study, we used deep viral genome sequencing to identify nonsynonymous mutations in previously reported MHC-I epitopes.…”

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

“…Likewise, antibody fragments, especially camelid V H H single-domain antibodies, or nanobodies have also been successfully developed for virus neutralization [5][6][7][8][9][10][11][12] . Compared to mAbs, nanobodies (Nbs) are substantially smaller (~ 15 kDa) yet can still bind virus antigens with excellent specificity.…”