Identification and mechanistic basis of non-ACE2 blocking neutralizing antibodies from COVID-19 patients with deep RNA sequencing and molecular dynamics simulations

Abstract

Variants of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) continue to cause disease and impair the effectiveness of treatments. The therapeutic potential of convergent neutralizing antibodies (NAbs) from fully recovered patients has been explored in several early stages of novel drugs. Here, we identified initially elicited NAbs (Ig Heavy, Ig lambda, Ig kappa) in response to COVID-19 infection in patients admitted to the intensive care unit at a single center with deep RNA sequencing (>100 million reads) of peripheral blood as a diagnostic tool for predicting the severity of the disease and as a means to pinpoint specific compensatory NAb treatments. Clinical data were prospectively collected at multiple time points during ICU admission, and amino acid sequences for the NAb CDR3 segments were identified. Patients who survived severe COVID-19 had significantly more of a Class 3 antibody (C135) to SARS-CoV-2 compared to non-survivors (15059.4 vs. 1412.7, p = 0.016). In addition to highlighting the utility of RNA sequencing in revealing unique NAb profiles in COVID-19 patients with different outcomes, we provided a physical basis for our findings via atomistic modeling combined with molecular dynamics simulations. We established the interactions of the Class 3 NAb C135 with the SARS-CoV-2 spike protein, proposing a mechanistic basis for inhibition via multiple conformations that can effectively prevent ACE2 from binding to the spike protein, despite C135 not directly blocking the ACE2 binding motif. Overall, we demonstrate that deep RNA sequencing combined with structural modeling offers the new potential to identify and understand novel therapeutic(s) NAbs in individuals lacking certain immune responses due to their poor endogenous production. Our results suggest a possible window of opportunity for administration of such NAbs when their full sequence becomes available. A method involving rapid deep RNA sequencing of patients infected with SARS-CoV-2 or its variants at the earliest infection time could help to develop personalized treatments using the identified specific NAbs.

Keywords: COVID-19; RNA sequencing; antibodies; intensive care; sepsis.

FIGURE 1

MD-derived electrostatic potential (ESP) maps and the equilibrated structure for NAb conformation. (A) Frames corresponding to conformation A (green arrow) and conformation B (red arrow). (B) Overall structure of conformation A between the spike RBD (rainbow colors with glycosylated N343 in ball-and-stick) and heavy (slate)/light (light blue) chains of the C135 Fab. (C) MD-derived ESP maps contoured at +5σ. Contact interface maps are colored: light chain in blue, heavy chain in green, and RBD in pink. (D) Closeup view of the RBD with the map from (C). (E) Interactions between the RBD and the NAb light chain. (F) Interactions among RBD, heavy/light chains with maps contoured +5σ. (G). Reduced contouring level to +2.5σ for the heavy chain to show the features for N343 glycan.

FIGURE 2

Comparison between conformations A and B of the NAb-spike complex. (A) Two orthogonal views of comparison with rotational axis and angle indicated upon alignment of the RBD (rainbow color) between conformation A (grey) and B (multicolor). (B) Corresponding MD-ESP maps contoured at +5σ. (C) Interactions of the RBD and heavy/light chains near the rotational axis in stereodiagram of conformation (A). (D) Conformation B in stereodiagram. (E) Conserved interaction surrounding T345 in the two conformations. (F) Alignment of heavy chain between the two conformations. (G) Alignment of light chain.

FIGURE 3

Comparison of MD-derived conformation A (blue) and B (magenta) with the starting conformation S (multicolor). (A) Starting conformation S. (B) Superposition between conformations S and (A). (C) Superposition between conformations S and (B). (D) Two orthogonal views of conformations S, A and (B). (E) Conformation S in context of the spike trimer with NTD and RBD of neighboring subunits included. (F) Superposition of conformations S and A in the context of the spike protein. (G) Superposition of conformations S and B in the trimer. (H) Compiled view of conformation S and A/B in the trimer.

FIGURE 4

Binding of the full-length NAb C135 to the spike trimer in two conformations. (A) Using the full-length anti-PD1 IgG4 to represent the full-length C135, constant domains are in blue, heavy chain in green, and light chain in cyan. (B) Two orthogonal views of the closed spike trimer (from PDB: 6vxx coordinates). (C) Two orthogonal views of the fully open (open-1) spike trimer (using symmetrized PDB: 6vyb coordinates). (D) Binding of one NAb C135 to the closed spike trimer in two orthogonal views. (E) Binding of three NAb C135 to the closed spike trimer in two orthogonal views. (F) Binding of three NAb C135 to the open spike trimer.

FIGURE 5

Modeling of the full-length NAb C135 with the full-length N⁰AT1/ACE2/spiker supercomplexes. (A) Two orthogonal views of three NAb C135 (blue, forest, and cyan) and three B⁰AT1/ACE2 (magenta, pink, and grey) dimers bound to one open spike trimer (black for the extended S2 central stalk, and multicolor for NTD and rainbow colors for RBDs). (B) Two NAb C135 bound to the B⁰AT1/ACE2 dimer in the opened ACE2 cleft in four orthogonal views. (C) B⁰AT1/ACE2 dimer with the spike RBD (rainbow color), but without the modeled NAb C135. (D) Two simultaneously modeled NAb C135 on one ACE2 dimer showing steric clashes between the constant domains and heavy/light chains of NAb C135.