Structure-based design of prefusion-stabilized SARS-CoV-2 spikes

By C. Hsieh, J. Goldsmith, Jeffrey M. Schaub, Andrea M. DiVenere, Hung-Che Kuo, Kamyab Javanmardi, Kevin C. Le, D. Wrapp, Alison G. Lee, Yutong Liu and 14 more. 2020

831 citations recorded by the source corpus.

Stabilizing the prefusion SARS-CoV-2 spike The development of therapeutic antibodies and vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is focused on the spike (S) protein that decorates the viral surface. A version of the spike ectodomain that includes two proline substitutions (S-2P) and stabilizes the prefusion conformation has been used to determine high-resolution structures. However, even S-2P is unstable and difficult to produce in mammalian cells. Hsieh et al. characterized many individual and combined structure-guided substitutions and identified a variant, named HexaPro, that retains the prefusion conformation but shows higher expression than S-2P and can also withstand heating and freezing. This version of the protein is likely to be useful in the development of vaccines and diagnostics. Science, this issue p. 1501 The design of stabilizing mutations in the SARS-CoV-2 spike protein allows for high-yield production of a critical vaccine antigen. The coronavirus disease 2019 (COVID-19) pandemic has led to accelerated efforts to develop therapeutics and vaccines. A key target of these efforts is the spike (S) protein, which is metastable and difficult to produce recombinantly. We characterized 100 structure-guided spike designs and identified 26 individual substitutions that increased protein yields and stability. Testing combinations of beneficial substitutions resulted in the identification of HexaPro, a variant with six beneficial proline substitutions exhibiting higher expression than its parental construct (by a factor of 10) as well as the ability to withstand heat stress, storage at room temperature, and three freeze-thaw cycles. A cryo–electron microscopy structure of HexaPro at a resolution of 3.2 angstroms confirmed that it retains the prefusion spike conformation. High-yield production of a stabilized prefusion spike protein will accelerate the development of vaccines and serological diagnostics for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Subfields: Ecology, Genetics, Infectious Diseases

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In Ecology

1. Jesse D. Bloom

In Genetics

1. Tyler N. Starr
2. K. H. Crawford
3. Allison J. Greaney
4. Jesse D. Bloom

In Infectious Diseases

1. Jesse D. Bloom
2. C. Hillyer

Citations in the corpus

• Escape from neutralizing antibodies by SARS-CoV-2 spike protein variants (2020, methodology): “To test whether neutralization escape mutations conferred loss of binding to the monoclonal antibodies, we expressed conformationally prefusion-stabilized S trimers (25), appended at their C-termini with NanoLuc luciferase (Figure 5A).”
• Complete mapping of mutations to the SARS-CoV-2 spike receptor-binding domain that escape antibody recognition (2020, positive): “We also expressed a recently reported spike protein construct with 4 additional proline substitutions that enhance thermostability, yield, and structural homogeneity, here referred to as S6P ecto (Hsieh et al., 2020) .”
• COVID-19 vaccines (2021, background): “Protein subunit vaccines consist of viral antigenic fragments produced by recombinant protein techniques [15].”
• Nanobody cocktails potently neutralize SARS-CoV-2 D614G N501Y variant and protect mice (2021, positive): “To visualize how the nanobodies bound to SARS-CoV-2 spike protein, we incubated WNb 2 and WNb 10 with the purified ectodomain of SARS-CoV-2 spike containing the six stabilizing proline mutations at a stochiometric ratio of 6:1 for 30 min at room temperature and applied to size-exclusion chromatograp”
• Efficient evolution of human antibodies from general protein language models (2023, neutral): “MEDI8852 variants were screened against HA H4 Hubei; MEDI8852 UCA variants against HA H1 Solomon; mAb114 and mAb114 UCA variants against ebolavirus GP; S309 variants against Wuhan-Hu-1 S-6P; and REGN10987 and C143 variants against Beta S-6P. b , Phylogenetic trees illustrating the evolutionary traje”