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

Hsieh, Ching Lin; Goldsmith, Jory A.; Schaub, Jeffrey M.; DiVenere, Andrea M.; Kuo, Hung Che; Javanmardi, Kamyab; Le, Kevin; Wrapp, Daniel; Lee, Alison G.; Liu, Yutong; Chou, Chih‐Ju; Byrne, Patrick O.; Hjorth, Christy K.; Johnson, Nicole V.; Ludes-Meyers, John; Nguyen, Annalee W.; Park, Juyeon; Wang, Nianshuang; Amengor, Dzifa; Lavinder, Jason J.; Ippolito, Gregory C.; Maynard, Jennifer A.; Finkelstein, Ilya J.; McLellan, Jason S.

doi:10.1126/science.abd0826

Cited by 1,091 publications

(1,360 citation statements)

References 35 publications

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“…NSEM has proved especially important because it reveals the threedimensional structural integrity of individual molecules, allowing us to discriminate between preparations that looked similar by bulk methods such as SDS-PAGE and SEC (Extended Data Figure 1). The observed variability in the spike fraction between preparations suggests a fragile S ectodomain, and measures to overcome the issue have been previously reported 11,12 . Here we link the apparent fragility of spike ectodomain 2P to its rapid denaturation upon storage at 4 °C (Figure 1 b-e).…”

mentioning

confidence: 65%

“…SARS-CoV-2 ectodomain constructs were produced and purified 1,11 as follows. A gene encoding residues 1-1208 of the SARS-CoV-2 S (GenBank: MN908947) with proline substitutions at residues 986 and 987, a “GSAS” substitution at the furin cleavage site (residues 682-685), a C-terminal T4 fibritin trimerization motif, an HRV3C protease cleavage site, a TwinStrepTag and an 8XHisTag was synthesized and cloned into the mammalian expression vector pαH.…”

Section: Methodsmentioning

confidence: 99%

“…These results suggested that cold-induced denaturation of spike was associated with increased RBD-exposure of the S ectodomain, and we thus asked whether a “down” state stabilized S ectodomain might be resistant to cold-induced denaturation (Figure 2). We included in this analysis a variant that combined the recently described rS2d 9 and HexaPro mutations 11 (Figure 2a,b). This new variant, named rS2d-HexaPro, showed the highest production yields of the all-RBD-down spike constructs (Figure 2c).…”

mentioning

confidence: 99%

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Cold sensitivity of the SARS-CoV-2 spike ectodomain

Edwards

Mansouri²,

Stalls³

et al. 2020

Preprint

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SummaryThe impact of COVID-19 and the urgency to develop a vaccine against the SARS-CoV-2 virus cannot be overstated. The viral fusion spike (S) protein ectodomain is the primary target for vaccine development. Here we report an unexpected cold sensitivity of a stabilized SARS-CoV-2 ectodomain construct currently being widely used for immunogen design. We found that when stored at 22 or 37 °C for 1 week, the S-protein displayed well-ordered trimeric spikes by negative stain electron microscopy. However, storage at 4 °C reduced the trimeric spikes to <10%, accompanied by decreased stability and enhanced exposure of the ACE-2 receptor binding site. Well-formed S particles could be recovered from cold-stored samples by a brief incubation at 37 °C. Our results will have broad impact on structural, functional and vaccine studies using the SARS-CoV-2 S ectodomain.HighlightsSARS-CoV-2 S ectodomain construct, widely used for vaccine studies, exhibits cold sensitivity.Negative stain electron microscopy shows disintegration of spike structure upon storage at 4 °C.Differential scanning calorimetry measurements confirm destabilization by cold.Cold storage alters antigenicity of SARS-CoV-2 spike.Brief incubation at 37 °C restored spike integrity after cold-storage.

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confidence: 65%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Cold sensitivity of the SARS-CoV-2 spike ectodomain

Edwards

Mansouri²,

Stalls³

et al. 2020

Preprint

View full text Add to dashboard Cite

show abstract

“…Depending on the expression system yields and posttranslational modifications vary. Also, specifically for recombinant spike, modifications like deletion of the polybasic cleavage site [40][41][42] , inclusion of two (or more) stabilizing mutations 13,40,43,44 , inclusion of trimerization domains as well as the mode of purification (soluble protein versus membrane extraction) might influence the generated immune response. The advantage of these vaccines is, that they can be produced without handling live virus.…”

Section: Recombinant Protein Vaccinesmentioning

confidence: 99%

SARS-CoV-2 vaccines in development

Krammer

2020

Nature

1,871

1,781

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This is a PDF file of a peer-reviewed paper that has been accepted for publication. Although unedited, the content has been subjected to preliminary formatting. Nature is providing this early version of the typeset paper as a service to our authors and readers. The text and figures will undergo copyediting and a proof review before the paper is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers apply.

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“…Most of the COVID-19 vaccine candidates reported are focused on a pre-fusion-stabilized S protein, either as recombinant protein with adjuvant or delivered from viral vectors or as DNA or mRNA vaccines 8–15 . The pre-fusion-stabilized version of SARS-CoV-2 S-protein contains two proline substitutions (2P), at amino acid positions 986 and 987, located near the apex of the central helix and heptad repeat 1 16 . Structural studies reveal that the pre-fusion stabilized S closely resembles native S protein on the virion surface; a structure targeted by many reported effective neutralizing antibodies 17–19 .…”

Section: Introductionmentioning

confidence: 99%

Immunogenicity of novel mRNA COVID-19 vaccine MRT5500 in mice and non-human primates

Kalnin

Plitnik²,

Kishko

et al. 2020

Preprint

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An effective vaccine to address the global pandemic of coronavirus disease 2019 (COVID-19) is an urgent public health priority. Novel synthetic mRNA and vector-based vaccine technologies offer an expeditious development path alternative to traditional vaccine approaches. Here we describe the efforts to utilize an mRNA platform for rational design and evaluations of mRNA vaccine candidates based on Spike (S) glycoprotein of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the virus causing COVID-19. Several mRNA constructs expressing various structural conformations of S-protein, including wild type (WT), a pre-fusion stabilized mutant (2P), a furin cleavage-site mutant (GSAS) and a double mutant form (2P/GSAS), were tested in a preclinical animal model for their capacity to elicit neutralizing antibodies (nAbs). The lead 2P/GSAS candidate was further assessed in dose-ranging studies in mice and Cynomolgus macaques. The selected 2P/GSAS vaccine formulation, now designated MRT5500, elicited potent nAbs as measured in two types of neutralization assays. In addition, MRT5500 elicited TH1-biased responses in both mouse and non-human primate species, a result that helps to address a hypothetical concern regarding potential vaccine-associated enhanced respiratory diseases associated with TH2-biased responses. These data position MRT5500 as a viable vaccine candidate for clinical development against COVID-19.

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

Cited by 1,091 publications

References 35 publications

Cold sensitivity of the SARS-CoV-2 spike ectodomain

Cold sensitivity of the SARS-CoV-2 spike ectodomain

SARS-CoV-2 vaccines in development

Immunogenicity of novel mRNA COVID-19 vaccine MRT5500 in mice and non-human primates

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