Structural analysis of full-length SARS-CoV-2 spike protein from an advanced vaccine candidate

Abstract

Vaccine efforts against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) responsible for the current COVID-19 pandemic are focused on SARS-CoV-2 spike glycoprotein, the primary target for neutralizing antibodies. Here, we performed cryo-EM and site-specific glycan analysis of one of the leading subunit vaccine candidates from Novavax based on a full-length spike protein formulated in polysorbate 80 (PS 80) detergent. Our studies reveal a stable prefusion conformation of the spike immunogen with slight differences in the S1 subunit compared to published spike ectodomain structures. Interestingly, we also observed novel interactions between the spike trimers allowing formation of higher order spike complexes. This study confirms the structural integrity of the full-length spike protein immunogen and provides a basis for interpreting immune responses to this multivalent nanoparticle immunogen.

Figure 1.. Evaluation of SARS-CoV-2 3Q-2P-FL spike glycoprotein.

(A) Linear diagram of the sequence/structure elements of the full-length SARS-CoV-2 spike (S) protein showing the S1 and S2 ectodomain. Structural elements include a cleavable signal sequence (SS, white), N-terminal domain (NTD, blue), receptor binding domain (RBD, green), subdomains 1 and 2 (SD1/SD2, light blue), protease cleavage site 2’ (S2’, arrow), fusion peptide (FP, red), heptad repeat 1 (HR1, yellow), central helix (CH, brown), heptad repeat 2 (HR2, purple), transmembrane domain (TM, black) and cytoplasmic tail (CT, white). The native furin cleavage site was mutated (RRAR →QQAQ) to be protease resistant and stabilized by introducing two proline (2P) substitutions at positions K986P and V987P to produce SARS-CoV-2 3Q-2P-FL spike. (B) Representative negative stain EM images and 2D classes of SARS-CoV-2 3Q-2P-FL, formulated in polysorbate 80 detergent in the presence of Matrix-M adjuvant. In the raw micrograph, spike rosettes are circled in yellow and Matrix-M adjuvant cages are circled in white. 2D classes showing individual spikes, higher order spike nanoparticles and Matrix-M cages of different sizes.

Figure 2.. Cryo-EM analysis of SARS-CoV-2 3Q-2P-FL spikes.

(A) Representative electron micrograph and 2D class averages of 3Q-2P-FL spikes showing free trimers and complexes of trimers (B) Side and top views of the B-factor-sharpened cryo-EM map of 3Q-2P-FL free trimers showing the spike in prefusion state, with the RBDs in closed conformation. The protomers are colored in blue, green and coral for clarity (C) Side and top view of the atomic model of free trimer represented as a ribbon diagram fit into the map density. The protomers are colored in blue, green and coral and the map is shown as a transparent gray density. (D) Comparison of 3Q-2P-FL spike with published structures (PDB IDs 6VXX and 6VSB) on a subunit level. PDB 6vXx is shown in cyan, PDB 6VSB shown in blue and 3Q-2P-FL spike in coral.

Figure 3.. Structural features of the SARS-CoV-2 3Q-2P-FL spike trimer.

(A) Comparison of the 615–635 loop between 3Q-2P-FL spike shown in coral and PDB 6X6P shown in blue. The residues that were built in 6X6P model but not in our model are shown in dark blue. Threonines at positions 618 and 632 flanking the gap in the 3Q-2P-FL trimer model are shown on both models to highlight their relative positions. (B) Interprotomeric salt-bridge interaction between D614 and K854 in 3Q-2P-FL spike trimer. (C) Linoleic acid (Dark blue) binding within a hydrophobic pocket of one RBD where the fatty acid head group reaches out to interact with the closed RBD of the adjacent protomer. The interacting residues are shown in pink (D) Polysorbate 80 detergent (blue) binding within the NTD with potential hydrogen bonding with R190 and H207. The interacting residues are shown in orange. Adjacent protomers are shown in yellow and gray in panels (B), (C) and (D).

Figure 4.. Trimer-trimer interactions and glycan analysis.

(A) Side and top views of the sharpened cryo-EM map of 3Q-2P-FL dimers of spike trimers. Individual spike trimmers are shown in blue and coral along a two-fold axis of symmetry (dotted line). (B) Top view of the B-factor-sharpened cryo-EM map of trimer-of-trimers complex with individual trimers colored in blue, coral and green. (C) Ribbon representation of a protomer from one trimer (blue) interacting with the protomer from the adjacent trimer (coral) docked into the dimers-of-trimers density (D) A close-up view of the interaction between the protomers of adjacent trimers. One protomer is shown as a ribbon diagram in blue while its binding partner is shown as surface in gray. Residues 621-PVAIHADQ-628 in the loop with potential interactions to the neighboring NTD are colored yellow and the residues in the NTD binding pocket are highlighted in coral. Residue D614 at the start of the loop is highlighted in dark blue. Glycosylation at residue 616 is not shown for clarity. (E) Changes occurring in the binding pocket in the bound state (gray) versus the free trimer (pink). Y145 and H146 switch positions to accommodate the loop better, also resulting in salt bridge formation between H146 and D627. It also results in stacking between W152 and H146 (F) Pseudoviruses expressing SARS-CoV-2 WT or mutant spikes (Mutant 1 has loop residues 621 -PVAIHADQ-628 replaced with a glycine-serine linker and mutant 2 has residues 619-EVPV-622 mutated to 619-DVST-622) were used to infect HeLa or HeLa-ACE2 cells for 42 to 48 hours. Infection was measured by luciferase intensity (Relative Light Unit) in the lysed cells following infection. (G) Site-specific glycan analysis of 3Q-2P-FL spike protein expressed in SF9 insect cell line. Proportions shown for no occupancy, oligomannose and complex/paucimannose PNGS are the average and SEM of 3–32 unique peptides for each glycosite except for sites 17, 709 and 717 where only a single peptide was observed.