Leveraging coronavirus binding to gangliosides for innovative vaccine and therapeutic strategies against COVID-19

Abstract

Covid-19 is an infectious respiratory disease due to a coronavirus named SARS-CoV-2. A critical step of the infection cycle is the binding of the virus spike S protein to the cellular ACE-2 receptor. This interaction involves a receptor binding domain (RBD) located at the center of the S trimer, whereas the lateral N-terminal domain (NTD) displays a flat ganglioside binding site that enables the virus to bind to lipid rafts of the plasma membrane, where the ACE-2 receptor resides. S protein binding to lipid rafts can be blocked by hydroxychloroquine, which binds to gangliosides, and by azithromycin, which binds to the NTD. Based on these data, we identified the NTD of SARS-CoV-2 as a promising target for both therapeutic and vaccine strategies, a notion later supported by the discovery, in convalescent Covid-19 patients, of a neutralizing antibody (4A8) that selectively binds to the NTD. The 4A8 epitope overlaps the ganglioside binding domain, denying any access of the virus to lipid rafts when the antibody is bound to the S protein. Thus, our data explain why antibody binding to the tip of the NTD results in SARS-CoV-2 neutralization. The high level of conservation of the ganglioside binding domain of SARS-CoV-2 (100% identity in 584 of 600 isolates analyzed worldwide) offers unique opportunities for innovative vaccine/therapeutic strategies.

Keywords: Azithromycin; Coronavirus; Ganglioside; Hydroxychloroquine; Lipid raft; SARS-CoV-2; Vaccine.

Fig. 1

The 4A8 epitope overlaps the ganglioside and azithromycin binding domains of the NTD in the SARS-CoV-2 spike protein. A part of the 4A8 epitope lies within the 144–158 amino acid sequence. This region covers most of the azithromycin binding domain (amino acids 134–161) and a significant part of the ganglioside binding domain (amino acids 111–162). The ganglioside binding domain has a flat surface that can simultaneously accommodate two gangliosides [16]. The SARS-CoV-2 spike protein bound to the 4A8 neutralizing antibody (nAb) was obtained from pdb file # 7c2l [23].

Fig. 2

Amino acid sequence alignments of the ganglioside binding domain of the SARS-CoV-2 spike protein. Among 600 sequences of the ganglioside binding domain (fragment 111–162) retrieved from the NCBI server [25], 584 were identical to the reference sequence (6VSB_A). The 16 remaining sequences (each with a single mutation) are listed below the reference with amino acid changes highlighted. The degree of conservation observed in each column is symbolized by an asterisk (∗) when the residue is fully conserved, a colon () for distinct residues with strongly similar properties (scoring > 0.5 in the Gonnet PAM 250 matrix), and a period (.) for distinct residues with weakly similar properties (scoring ≤ 0.5 in the Gonnet PAM 250 matrix [16,17].

Fig. 3

Why antibody binding to the tip of the NTD blocks SARS-Cov-2 infection. (a) In the trimeric spike of SARS-Cov-2 envelope the RBD is located at the center of the spike, whereas the three NTD (one in each protein subunit) are positioned laterally [16]. The ganglioside binding domain is a large flat surface located at the tip of each NTD, allowing a multiple engagement of the whole spike to several lipid rafts. (b) the NTD binding to gangliosides induces the coalescence of lipid rafts together with a local modulation of membrane curvature that may facilitate the recruitment of the ACE-2 receptor. (c) Anti-NTD antibodies neutralize SARS-Cov-2 by blocking the initial step of the infection cycle, i.e. virus-ganglioside interactions in lipid rafts. Preventing virus binding to lipid rafts is thus likely to render ACE-2 inaccessible. Note that the complementary-determining region (CDR) of the 4A8 antibody has a negative surface potential (colored in red). Since the tip of the NTD has a positive surface potential, it fits with both the CDR of the 4A8 antibody and the negative charges of lipid raft gangliosides. In all panels, the trimeric structure of the SARS-CoV-2 spike is represented in surface rendition with subunits A, B and C colored in cyan, purple, and yellow. The surface potential of the A48 antibody is indicated in conventional colors (positive areas in blue, negative in red, apolar in white). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)