Design of a highly thermotolerant, immunogenic SARS-CoV-2 spike fragment

J Biol Chem. Jan-Jun 2021;296:100025. doi: 10.1074/jbc.RA120.016284. Epub 2020 Nov 23.


Virtually all SARS-CoV-2 vaccines currently in clinical testing are stored in a refrigerated or frozen state prior to use. This is a major impediment to deployment in resource-poor settings. Furthermore, several of them use viral vectors or mRNA. In contrast to protein subunit vaccines, there is limited manufacturing expertise for these nucleic-acid-based modalities, especially in the developing world. Neutralizing antibodies, the clearest known correlate of protection against SARS-CoV-2, are primarily directed against the receptor-binding domain (RBD) of the viral spike protein, suggesting that a suitable RBD construct might serve as a more accessible vaccine ingredient. We describe a monomeric, glycan-engineered RBD protein fragment that is expressed at a purified yield of 214 mg/l in unoptimized, mammalian cell culture and, in contrast to a stabilized spike ectodomain, is tolerant of exposure to temperatures as high as 100 °C when lyophilized, up to 70 °C in solution and stable for over 4 weeks at 37 °C. In prime:boost guinea pig immunizations, when formulated with the MF59-like adjuvant AddaVax, the RBD derivative elicited neutralizing antibodies with an endpoint geometric mean titer of ∼415 against replicative virus, comparing favorably with several vaccine formulations currently in the clinic. These features of high yield, extreme thermotolerance, and satisfactory immunogenicity suggest that such RBD subunit vaccine formulations hold great promise to combat COVID-19.

Keywords: ACE2; Pichia; glycosylation; microbial; thermostable.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Angiotensin-Converting Enzyme 2 / chemistry
  • Angiotensin-Converting Enzyme 2 / genetics
  • Angiotensin-Converting Enzyme 2 / immunology*
  • Animals
  • Antibodies, Neutralizing / biosynthesis
  • Antibodies, Viral / biosynthesis*
  • Binding Sites
  • COVID-19 / immunology
  • COVID-19 / prevention & control*
  • COVID-19 / virology
  • COVID-19 Vaccines / administration & dosage
  • COVID-19 Vaccines / biosynthesis*
  • COVID-19 Vaccines / genetics
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Female
  • Guinea Pigs
  • HEK293 Cells
  • Hot Temperature
  • Humans
  • Immunogenicity, Vaccine
  • Models, Molecular
  • Protein Binding
  • Protein Conformation, alpha-Helical
  • Protein Conformation, beta-Strand
  • Protein Domains
  • Protein Interaction Domains and Motifs
  • Protein Stability
  • Receptors, Virus / chemistry
  • Receptors, Virus / genetics
  • Receptors, Virus / immunology*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / immunology
  • SARS-CoV-2 / chemistry
  • SARS-CoV-2 / immunology*
  • Spike Glycoprotein, Coronavirus / chemistry
  • Spike Glycoprotein, Coronavirus / genetics
  • Spike Glycoprotein, Coronavirus / immunology*
  • Vaccination
  • Vaccine Potency


  • Antibodies, Neutralizing
  • Antibodies, Viral
  • COVID-19 Vaccines
  • Receptors, Virus
  • Recombinant Proteins
  • Spike Glycoprotein, Coronavirus
  • spike protein, SARS-CoV-2
  • ACE2 protein, human
  • Angiotensin-Converting Enzyme 2