Nanobody cocktails potently neutralize SARS-CoV-2 D614G N501Y variant and protect mice

Abstract

Neutralizing antibodies are important for immunity against SARS-CoV-2 and as therapeutics for the prevention and treatment of COVID-19. Here, we identified high-affinity nanobodies from alpacas immunized with coronavirus spike and receptor-binding domains (RBD) that disrupted RBD engagement with the human receptor angiotensin-converting enzyme 2 (ACE2) and potently neutralized SARS-CoV-2. Epitope mapping, X-ray crystallography, and cryo-electron microscopy revealed two distinct antigenic sites and showed two neutralizing nanobodies from different epitope classes bound simultaneously to the spike trimer. Nanobody-Fc fusions of the four most potent nanobodies blocked ACE2 engagement with RBD variants present in human populations and potently neutralized both wild-type SARS-CoV-2 and the N501Y D614G variant at concentrations as low as 0.1 nM. Prophylactic administration of either single nanobody-Fc or as mixtures reduced viral loads by up to 104-fold in mice infected with the N501Y D614G SARS-CoV-2 virus. These results suggest a role for nanobody-Fc fusions as prophylactic agents against SARS-CoV-2.

A potent SARS-CoV-2 neutralising nanobody shows therapeutic efficacy in the Syrian golden hamster model of COVID-19

“…The site recognized by C5 and H3 overlapped with the ACE2 binding site on the top surface of the domain, whilst the second recognized by C1 and F2 corresponded to a location on the side of the RBD originally identi ed by the SARS-CoV antibody CR3022 24,26,47 and nanobody VHH72 48 . Consistent with other recent reports 10,17,39 nanobodies that bound to both sites showed very potent neutralization activity when con gured as multivalent trimers, with the C5 trimer demonstrating complete inhibition of infection of Vero cells at < 100 pM in a PRNT assay. This activity was translated into a marked disease-modifying effect in the Syrian golden hamster model of COVID-19 with treated animals showing minimal weight loss and very limited pulmonary infection and associated changes following a single dose of C5 trimer 24 h post virus challenge.…”

“…Nasal administration appeared to promote faster recovery than IP perhaps re ecting increased levels of the C5 trimer reaching the sites of infection in the lungs. Recently, mice challenged intranasally with SARS-CoV-2, and then treated prophylactically IP with a nanobody Fc fusion has also been shown to reduce viral load in the lungs 17 . More recently, Nebulli et al 18 , showed that nasal administration of a nanobody 6 h after viral challenge also reduced viral load and weight change in the Syrian hamster model.…”

“…Secondly, while conventional antibodies that comprise two disulphide-linked polypeptides, heavy and light chain, typically require mammalian cells for production, nanobodies can be manufactured using readily available microbial systems. The potency of nanobodies against SARS-CoV-2 7 infection has been demonstrated in cell-based assays [8][9][10][11][12][13][14][15][16] and most recently in animal studies 17,18 . Several strategies for engineering VHH into a multivalent species are known.…”

The development of Nanosota-1 as anti-SARS-CoV-2 nanobody drug candidates

“…Nanobody therapeutics derived from camelid antibodies has advantages relative to conventional antibodies. Although several studies have reported nanobody drug candidates that specifically target SARS-CoV-2 ( Huo et al, 2020 ; Hanke et al, 2020 ; Xiang et al, 2020 ; Schoof et al, 2020 ; Wrapp et al, 2020a ; Pymm et al, 2021 ; Nambulli et al, 2021 ), as of this writing, only two studies have evaluated their nanobody drug candidates in an animal model for anti-SARS-CoV-2 therapeutic efficacy ( Pymm et al, 2021 ; Nambulli et al, 2021 ). None of these studies have evaluated their nanobody drug candidates for in vitro thermostability, in vivo stability, or tissue biodistribution.…”

The success of SARS-CoV-2 vaccines and challenges ahead

“…A change in the vaccine composition is considered if an antigenic variant is responsible for disease outbreaks in disparate locations, especially in different countries. A similar approach could be used for SARS-CoV-2, with several labs sharing a panel of human monoclonal antibodies that have been mapped to different epitopes on the spike protein (Barnes et al, 2020;Dejnirattisai et al, 2021;Liu et al, 2021b;Piccoli et al, 2020;Pymm et al, 2021;Weisblum et al, 2020), as well as post-infection and post-vaccination human sera to support the antigenic characterization of circulating viruses, VOIs, and VOCs. Assays, shared reagents, and the criteria for considering whether a strain requires a COVID-19 vaccine to be updated all need to be defined.…”