Figure 6. 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 PSV. 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 PSV (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 PSV (iii). (B) S1–36 synergizes with S1-RBD-22 in neutralizing SARS-CoV-2 PSV. 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 PSV 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 PSV. 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 Figure 6–Source data 2.