Cryo-electron Microscopy Structure of S-Trimer, a Subunit Vaccine Candidate for COVID-19

Abstract

Within a year after its emergence, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected over 100 million people worldwide with a death toll over 2 million. Vaccination remains the best hope to ultimately put this pandemic to an end. Here, using Trimer-Tag technology, we produced both wild-type (WT) and furin site mutant (MT) S-Trimers for COVID-19 vaccine studies. Cryo-EM structures of the WT and MT S-Trimers, determined at 3.2 Å and 2.6 Å respectively, revealed that both antigens adopt a tightly closed conformation and their structures are essentially identical to that of the previously solved full-length WT S protein in detergent. The tightly closed conformation is stabilized by fatty acid and polysorbate 80 binding at the receptor binding domains (RBDs) and the N terminal domains (NTDs) respectively. Additionally, we identified an important pH switch in the WT S-Trimer that shows dramatic conformational change and accounts for its increased stability at lower pH. These results validate Trimer-Tag as a platform technology in production of metastable WT S-Trimer as a candidate for COVID-19 subunit vaccine. IMPORTANCE Effective vaccine against SARS-CoV-2 is critical to end the COVID-19 pandemic. Here, using Trimer-Tag technology, we are able to produce stable and large quantities of WT S-Trimer, a subunit vaccine candidate for COVID-19 with high safety and efficacy from animal and Phase 1 clinical trial studies. Cryo-EM structures of the S-Trimer subunit vaccine candidate show that it predominately adopts tightly closed pre-fusion state, and resembles that of the native and full-length spike in detergent, confirming its structural integrity. WT S-Trimer is currently being evaluated in global Phase 2/3 clinical trial. Combining with published structures of the S protein, we also propose a model to dissect the conformation change of the spike protein before receptor binding.

Efficacy of the adjuvanted subunit protein COVID-19 vaccine, SCB-2019: a phase 2 and 3 multicentre, double-blind, randomised, placebo-controlled trial

“…11 Clover Biopharmaceuticals has developed a vaccine candidate, SCB-2019, consisting of a recombinant SARS-CoV-2 S protein stabilised in the native prefusion trimeric conformation using its proprietary Trimer-Tag technology. 12 Preclinical studies have shown that adjuvanted SCB-2019 elicits protective neutralising antibody responses against SARS-CoV-2 challenge in non-human primates. 13 A phase 1 study in adults showed robust immune responses with SCB-2019 when adjuvanted with the toll-like receptor agonist CpG-1018 combined with alum, 14 with antibodies persisting at more than baseline for 6 months after vaccination.…”

Uncovering cryptic pockets in the SARS-CoV-2 spike glycoprotein

“…It appears that the LA pocket also has long-range effects on allosteric networks that include neighboring chains, NTD, and distant S1/S2 cleavage sites (Oliveira et al, 2022;Tan et al, 2022). The NTD contains a cryptic site that binds a polysorbate (PS) detergent molecule if it is present in vaccine formulation (Bangaru et al, 2020;Ma et al, 2021). The same pocket can accommodate heme metabolites biliverdin and bilirubin, the presence of which change the NTD epitope presentation and modulate the antibody response (Rosa et al, 2021).…”

Selective reversed-phase high-performance liquid chromatography method for the determination of intact SARS-CoV-2 spike protein

“…A potential quality issue for COVID-19 vaccines, such as SCB-2019, is uncontrolled cleavage during production [27] . Since both comS-protein A and comS-protein B retain the native S1/S2 protease cleavage site, the susceptibility to cleavage is increased [9] , which can be assessed by RP-HPLC analysis with Gradient I.…”