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

Bangaru, Sandhya; Ozorowski, Gabriel; Turner, Hannah L.; Antanasijevic, Aleksandar; Huang, Deli; Wang, Xiaoning; Torres, Jonathan L.; Diedrich, Jolene K.; Tian, Jing-Hui; Portnoff, Alyse D.; Patel, Nita; Massare, Michael J.; Yates, John R.; Nemazee, David; Paulson, James C.; Glenn, Greg; Smith, Gale; Ward, Andrew B.

doi:10.1101/2020.08.06.234674

Cited by 50 publications

(82 citation statements)

References 61 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…In contrast to the structural differences described above for S-2P protein, both of our WT and MT S-Trimer were nearly identical to the recently published structures of full-length wild-type S (PDB ID: 6XR8 ) (11) and 3Q-2P-FL with two proline mutation (PDB ID: 7JJI) (7,14) purified in detergent from HEK293 and sf9 insect cell membranes, respectively. When (Fig.…”

Section: Conformational Change Of the S Protein Before Receptor Bindingsupporting

confidence: 81%

“…Although both oleic acid and linoleic acid can be ambiguously fitted into the density of the RBD region owing to the excellent quality of EM map, mass spectrometry analysis indicated both MT and WT samples contained a higher level of oleic acid than linoleic acid (Fig 4B and 4C). Recent studies revealed the presence of linoleic acid at the same binding site but with slightly different position (7,12). The oleic acid in our structure located in the hydrophobic pocket of the RBD domain engaged a salt bridge interaction with R408 and a hydrogen bond interaction with Q409 at the adjacent protomer through its carboxylic acid group, bringing the RBD domain in close proximity and resulting in the tightly closed conformation (Fig.…”

Section: Downloaded Frommentioning

confidence: 73%

“…Using Trimer-Tag technology (8), we produced both soluble wild-type (WT) and a furin site mutant (MT) (R685A) forms of S-Trimer from CHO cells in serum-free fed-batch processes in bioreactors with protein yield ranging from 0.5 -1 g/L. These antigen titers are three orders of magnitude higher than that reported previously for foldon derived S proteins with two Pro mutations (S-2P) (6,7), laying a solid foundation for scalability requirement of a successful COVID-19 vaccine. Both S-Trimers consist of the ectodomain (amino acid residues 1-1211) of SARS-CoV-2 Spike protein fused in-frame to the C-terminal region of human type I (α) collagen, which spontaneously form a disulfide-bond linked homo-trimer, thereby stabilizing the antigens in trimeric forms ( Fig.1 A and B).…”

Section: Constructs Design and Negative Stainingmentioning

confidence: 99%

“…In the case of SARS-CoV-2, this occurs via binding to ACE2 receptor expressed in target cells (5). Approaches for using a non-covalent trimer-foldon from T4 bacteriophage fibritin to stabilize the trimer conformation while simultaneously introducing mutations in viral antigens to abolish furin cleavage and stabilize the antigen in pre-fusion forms (6,7) are common strategies inherited from decades of HIV and RSV vaccine studies, but these have yet to yield a successful vaccine.…”

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

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

Jian

Sun

et al. 2021

J Virol

View full text Add to dashboard Cite

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.

show abstract

Section: Conformational Change Of the S Protein Before Receptor Bindingsupporting

confidence: 81%

Section: Downloaded Frommentioning

confidence: 73%

Section: Constructs Design and Negative Stainingmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

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

Jian

Sun

et al. 2021

J Virol

View full text Add to dashboard Cite

show abstract

“…Only the V367F mutation was found to be exposed to solvent and was therefore not considered for inclusion in stabilized RBD designs to avoid the risk of unfavorably altering antigenicity. The other four mutations were observed to be near or within a recently identified linoleic acid-binding pocket formed between adjacent RBDs in the closed Spike trimer (71,72), with Y365 identified as a key gating residue for this interaction (Figure 1A-B). The improved expression and stability observed by DMS for several mutations in the linoleic acid-binding pocket suggested that this region is structurally suboptimal in the isolated RBD.…”

Section: Resultsmentioning

confidence: 91%

Stabilization of the SARS-CoV-2 Spike receptor-binding domain using deep mutational scanning and structure-based design

Ellis

Brunette

Crawford

et al. 2021

Preprint

View full text Add to dashboard Cite

The unprecedented global demand for SARS-CoV-2 vaccines has demonstrated the need for highly effective vaccine candidates that are thermostable and amenable to large-scale manufacturing. Nanoparticle immunogens presenting the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein (S) in repetitive arrays are being advanced as second-generation vaccine candidates, as they feature robust manufacturing characteristics and have shown promising immunogenicity in preclinical models. Here, we used previously reported deep mutational scanning (DMS) data to guide the design of stabilized variants of the RBD. The selected mutations fill a cavity in the RBD that has been identified as a linoleic acid binding pocket. Screening of several designs led to the selection of two lead candidates that expressed at higher yields than the wild-type RBD. These stabilized RBDs possess enhanced thermal stability and resistance to aggregation, particularly when incorporated into an icosahedral nanoparticle immunogen that maintained its integrity and antigenicity for 28 days at 35-40°C, while corresponding immunogens displaying the wild-type RBD experienced aggregation and loss of antigenicity. The stabilized immunogens preserved the potent immunogenicity of the original nanoparticle immunogen, which is currently being evaluated in a Phase I/II clinical trial. Our findings may improve the scalability and stability of RBD-based coronavirus vaccines in any format and more generally highlight the utility of comprehensive DMS data in guiding vaccine design.

show abstract

SARS‐CoV‐2 spike protein aggregation is triggered by bacterial lipopolysaccharide

et al. 2022

View full text Add to dashboard Cite

SARS-CoV-2 spike (S) protein is crucial for virus invasion in COVID-19. Here, we showed that lipopolysaccharide (LPS) can trigger S protein aggregation at high doses of LPS and S protein. We demonstrated the formation of S protein aggregates by microscopy analyses, aggregation and gel shift assays. LPS at high levels boosts the formation of S protein aggregates as detected by amytracker and thioflavin T dyes that specifically bind to aggregating proteins. We validated the role of LPS by blocking the formation of aggregates by the endotoxin-scavenging thrombin-derived peptide TCP-25. Aggregation-prone sequences in S protein are predicted to be nearby LPS binding sites, while molecular simulations showed stable formation of S protein-LPS higher-order oligomers. Collectively, our results provide evidence of LPS-induced S protein aggregation.

show abstract

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

Cited by 50 publications

References 61 publications

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

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

Stabilization of the SARS-CoV-2 Spike receptor-binding domain using deep mutational scanning and structure-based design

SARS‐CoV‐2 spike protein aggregation is triggered by bacterial lipopolysaccharide

Contact Info

Product

Resources

About