Nanobody repertoires generated against wild-type SARS-CoV-2 remains efficacious.

Nanobodies targeting the S1-RBD, S1 non-RBD, and S2 portions of spike effectively neutralize lentivirus pseudotyped with delta and omicron BA.1 SARS-CoV-2 spikes (PSV) from infecting ACE2 expressing HEK293T cells. (A) The half-maximal inhibitory concentration (IC50) is reported for the indicated nanobodies against wild-type (Mast, Fridy et al. 2021), delta, and omicron BA.1 PSV. Nanobodies are grouped by epitope and arranged within each epitope by neutralization efficacy against the wild-type PSV. n ≥ 4 (B) The structure of the RBD of spike delta (PDB ID: 7SBO) and omicron BA.1 (PDB ID: 7T9K) is shown, with heat-mapped epitopes of neutralization colored from pale white (epitopes with weak neutralization against SARS-CoV-2) to dark red (strong neutralization against SARS-CoV-2). The color bar scale for each epitope is the neutralizing strength of each nanobody epitope, which is the normalized -log ratio of nanobody binding (IC50) of variant to wild-type SARS-CoV-2 Spike S1. A higher value of -log ratio corresponds to potent neutralization of the variant by the nanobody. All structure representations were created on ChimeraX (Pettersen, Goddard et al. 2021).

Affinities of the nanobody repertoire against SARS-CoV-2 variants.

(A) Each nanobody is plotted against their affinity (KD) for SARS-CoV-2 Spike S1 from wild-type, delta and omicron BA.1 and BA.4 strains. Nanobodies are characterized into their respective epitope groups according to Mast et al (Mast, Fridy et al. 2021). (B) The structure of the RBD of spike delta (PDB ID: 7SBO), omicron BA.1 (PDB ID: 7T9K) and omicron BA.4 RBD (modeled) is shown, with heat-mapped epitopes of binding colored from pale white (epitopes with weak binding against SARS-CoV-2) to dark red (strong binding against SARS-CoV-2). The nanobodies that contributed to epitope mapping are in bold in panel A. The color bar scale for each epitope shows the binding affinity strength of each nanobody epitope, which is the normalized -log ratio of nanobody binding (KD) of variant to wild-type SARS-CoV-2 Spike S1. A higher value of -log ratio corresponds to strong binding of the nanobody to the variant. S1-RBD-16 showed binding to omicron BA.1 in ELISA. *S1-RBD-11 was not tested against omicron BA.4 and #S1-65 was not tested against BA.1. All structure representations were created on ChimeraX (Pettersen, Goddard et al. 2021).

Potent neutralization by broadly active nanobodies.

Nanobodies targeting the S1-RBD of spike and raised against the original wild-type sequence remain highly efficacious in neutralizing an evolved variant of SARS-CoV-2, omicron variant BA.5. Neutralization curves are plotted from the results of a plaque-forming reduction neutralization test with the indicated nanobodies. Serial dilutions of each nanobody were incubated with ∼200 SARS-CoV-2 virions for 60 min and then overlaid on a monolayer of TMPRSS2-expressing Vero E6 cells. After 72 h, cells were fixed and stained with crystal violet stain (1% w/vol in 20% ethanol) to visualize individual viral plaques. Plaques were enumerated for each nanobody dilution and used to fit the neutralization curves depicted in the figure. The colored shaded areas denote 90% confidence intervals for each fitted curve. n ≥ 3.

Refining epitopes of the nanobody repertoire.

All epitopes are mapped on the structure of the RBD (PDB ID: 6M0J). (A) The epitopes of six RBD nanobody groups (colored dark gray) able to bind/neutralize one or more variants. Four of these epitope groups: I, III, IV and V were further refined resulting in an additional six epitope groups – creating a total of 12 epitope groups able to bind one or more variants of concern. (B) Summary of variant specific and pan-variant nanobodies. (C) Nanobody groups predicted to bind/neutralize the circulating variants XBB and BQ.1 and BQ.1.1. All structure representations were created on ChimeraX (Pettersen, Goddard et al. 2021).

Persistence of synergistic neutralization with nanobody cocktails against SARS-CoV-2 Variants of Concern.

(A) S1-1 synergizes with S1-23 in neutralizing SARS-CoV-2 pseudovirus. The upper left panel shows two representations of spike with the accessible S1-1 (dark goldenrod) and S1-23 (sky blue) epitopes (PDB ID: 6VYB). The measured affinities for S1-1 and S1-23 (Table 1) for the RBD of wild-type, delta, and omicron BA.1 are displayed. Both S1-1 and S1-23 neutralize wild-type (i), whereas only S1-1 neutralizes delta at the concentrations shown (ii). In spite of a lack of neutralization at these concentrations, S1-23, synergizes with S1-1 and enhances its neutralization of delta SARS-CoV-2 pseudovirus (ii). As neither S1-1 or S1-23 are able to bind to the RBD of omicron BA.1, neither nanobody neutralizes omicron BA.1 SARS-CoV-2 pseudovirus (iii). (B) S1-36 synergizes with S1-RBD-22 in neutralizing SARS-CoV-2 pseudovirus. As in A, the upper left panel shows two representations of spike with the accessible S1-36 (cornflower blue) and S1-RBD-22 (sandy brown) epitopes. The measured affinities for S1-36 and S1-RBD-22 are displayed. Both S1-36 and S1-RBD-22 neutralize wild-type (i), whereas only S1-RBD-22 effectively neutralizes delta and omicron BA.1 SARS-CoV-2 pseudovirus at the concentrations shown (ii and iii, respectively). However, S1-36 synergizes with S1-RBD-22 and enhances its neutralization of the three depicted SARS-CoV-2 pseudoviruses (i, ii, and iii). (C) An example of no interactions (synergistic or antagonistic) between S1-1 and LaM2 (Fridy, Li et al. 2014), a non-specific nanobody that does not bind the RBD of delta. (D) An example of antagonism, where higher concentrations of S1-23 interferes with the ability of S1-RBD-16 to neutralize delta SARS-CoV-2 pseudovirus. These nanobodies have adjacent epitopes on the RBD of spike and were previously shown to interfere with each other’s binding to their respective epitope (Mast, Fridy et al. 2021). n = 4. Source data in Supplementary Table 1.

Nanobody binding and neutralization characterization; related to Figure 2.

Nanobodies effective against circulation variants of concern.

The nanobody epitopes (red) that retain effectiveness against wild-type (PDB ID: 7KNB), delta (PDB ID: 7V7O), omicron BA.1 and omicron BA.4 (the nanobody epitopes were mapped on PBD ID: 7XO5 for both BA.1 and BA.4 due to lack of suitable BA.4 structure). The spike trimer (silver where glycans are represented as light blue sticks) for each variant is displayed in three views: top = side-view of spike trimer; middle = birds-eye view looking down on the S1 domain and; bottom = birds-eye view (same as middle view) with the S1 domain made transparent to see the S2-domain. All structure representations were created on ChimeraX (Pettersen, Goddard et al. 2021).

(A) Nanobody epitope groups overlapping with the three mAb epitope classes (class 1, class 2 and class 3). Nanobody groups are colored in gold and mAb class footprints are outlined in black. mAb epitopes are taken from Cox et al (Cox, Peacock et al.). (B) A single RBD subunit with the ACE2 footprint/RBM mapped in green. All epitopes are mapped on the structure of the RBD (PDB ID: 6M0J). All structure representations were created on ChimeraX (Pettersen, Goddard et al.).