Structure-based design of a highly stable, covalently-linked SARS-CoV-2 spike trimer with improved structural properties and immunogenicity

Abstract

The continued threat of SARS-CoV-2 to global health necessitates development of improved research tools and vaccines. We present an improved SARS-CoV-2 spike ectodomain, 'VFLIP', bearing five proline substitutions, a flexible cleavage site linker, and an inter-protomer disulfide bond. VFLIP displays significantly improved stability, high-yield production and retains its trimeric state without exogenous trimerization motifs. High-resolution cryo-EM and glycan profiling reveal that the VFLIP quaternary structure and glycosylation mimic the native spike on the viral surface. Further, VFLIP has enhanced affinity and binding kinetics relative to other stabilized spike proteins for antibodies in the Coronavirus Immunotherapeutic Consortium (CoVIC), and mice immunized with VFLIP exhibit potent neutralizing antibody responses against wild-type and B.1.351 live SARS-CoV-2. Taken together, VFLIP represents an improved tool for diagnostics, structural biology, antibody discovery, and vaccine design.

Broad neutralization of SARS-CoV-2 variants by circular mRNA producing VFLIP-X spike in mice

“…Notably, neutralizing antibodies have been recently shown to mainly target prefusion conformation, suggesting that vaccines utilizing prefusion-stabilized spikes might elicit greater neutralizing titers 7 . In contrast to that notion, spike proteins kept in closed conformation, including VFLIP, have been demonstrated to elicit more potent neutralizing responses than the more opened conformation spikes S-2P and HexaPro 19,38 . In addition, the VFLIP spike displays more native-like glycosylation profiles than other prefusion-stabilized spikes, presumably better preserving the antigenicity of spike immunogen 19,39,40 .…”

“…In contrast to that notion, spike proteins kept in closed conformation, including VFLIP, have been demonstrated to elicit more potent neutralizing responses than the more opened conformation spikes S-2P and HexaPro 19,38 . In addition, the VFLIP spike displays more native-like glycosylation profiles than other prefusion-stabilized spikes, presumably better preserving the antigenicity of spike immunogen 19,39,40 . Therefore, a balance in prefusion-stabilized and metastable states, opened and closed structures, as well as glycosylation profiles might be required when revising next-generation vaccines against SARS-CoV-2.…”

“…(A) Schematic representation of HexaPro-X and VFLIP-X showing the S1 and S2 subunits. Amino acid positions described in the original HexaPro 49 and VFLIP 19 spikes are shown in blue and red colors, respectively. The positions of six rationally substituted amino acids (K417N, L452R, T478K, E484K, N501Y and D614G) are indicated by green color.…”

Structure-function relations of the SARS-CoV-2 spike protein and impact of mutations in the variants of concern

“…In the prefusion S trimer, the HR1 region is spring-loaded in the form a bundle of short helical segments under a "crown" formed by the S1 moiety (Figure 3), while HR2 is part of the flexible stalk (Figure 2B). To stabilize the prefusion spike trimer used in the current vaccines was engineered to introduce proline residues at the connections between the short helices of the spring-loaded HR1 [73][74][75]. Because prolines are helix-breaking residues, the mutated HR1 cannot spring out to forming a long helix as shown in Figure 3C, resulting in an overall stabilized from that is non-fusogenic.…”

“…Dans le trimère S de pré-fusion, la région HR1 est montée sur ressort sous la forme d'un faisceau de segments hélicoïdaux courts sous une « couronne » formée par la sous-unité S1 (Figure 3), tandis que HR2 fait partie de la tige flexible connectant à la membrane virale (Figure 2B). Pour stabiliser le spicule dans la forme pré-fusion utilisée dans les vaccins actuels, des mutations en proline ont été introduites aux connexions entre les hélices courtes de la HR1 chargée par ressort [73][74][75]. Les prolines étant des résidus de rupture d'hélice, le HR1 muté ne peut pas se déployer pour former une longue hélice, comme le montre la Figure 3C, ce qui donne un trimère globalement stabilisé qui n'est pas fusogène.…”