Insight into Vaccine Development for Alphacoronaviruses Based on Structural and Immunological Analyses of Spike Proteins

Outbreaks of coronaviruses, especially SARS-CoV-2, pose a serious threat to global public health. Development of vaccines to prevent the coronaviruses that can infect humans has always been a top priority. ABSTRACT Coronaviruses that infect humans belong to the Alphacoronavirus (including HCoV-229E) and Betacoronavirus (including SARS-CoV and SARS-CoV-2) genera. In particular, SARS-CoV-2 is currently a major threat to public health worldwide. The spike (S) homotrimers bind to their receptors via the receptor-binding domain (RBD), which is a major target to block viral entry. In this study, we selected Alphacoronavirus (HCoV-229E) and Betacoronavirus (SARS-CoV and SARS-CoV-2) as models. Their RBDs exist two different conformational states (“lying” or “standing”) in the prefusion S-trimer structure. Then, the differences in the immune responses to RBDs from these coronaviruses were analyzed structurally and immunologically. Our results showed that more RBD-specific antibodies (antibody titers: 1.28 × 105 and 2.75 × 105, respectively) were induced by the S-trimer with the RBD in the standing state (SARS-CoV and SARS-CoV-2) than the S-trimer with the RBD in the lying state (HCoV-229E; antibody titers: <500), and more S-trimer-specific antibodies were induced by the RBD in the SARS-CoV and SARS-CoV-2 (antibody titers: 6.72 × 105 and 5 × 105, respectively) than HCoV-229E (antibody titers: 1.125 × 103). Besides, we found that the ability of the HCoV-229E RBD to induce neutralizing antibodies was lower than S-trimer, and the intact and stable S1 subunit was essential for producing efficient neutralizing antibodies against HCoV-229E. Importantly, our results reveal different vaccine strategies for coronaviruses, and S-trimer is better than RBD as a target for vaccine development in Alphacoronavirus. Our findings will provide important implications for future development of coronavirus vaccines. IMPORTANCE Outbreaks of coronaviruses, especially SARS-CoV-2, pose a serious threat to global public health. Development of vaccines to prevent the coronaviruses that can infect humans has always been a top priority. Coronavirus spike (S) protein is considered a major target for vaccine development. Currently, structural studies have shown that Alphacoronavirus (HCoV-229E) and Betacoronavirus (SARS-CoV and SARS-CoV-2) RBDs are in “lying” and “standing” states in the prefusion S-trimer structure. Here, we evaluated the ability of S-trimer and RBD to induce neutralizing antibodies among these coronaviruses. Our results showed that the S-trimer and RBD are both candidates for subunit vaccines in Betacoronavirus (SARS-CoV and SARS-CoV-2) with an RBD standing state. However, for Alphacoronavirus (HCoV-229E) with an RBD lying state, the S-trimer may be more suitable for subunit vaccines than the RBD. Our results will provide novel ideas for the development of vaccines targeting S protein in the future.

[1]  Y. Hu,et al.  [Asymptomatic infection of COVID-19 and its challenge to epidemic prevention and control]. , 2020, Zhonghua liu xing bing xue za zhi = Zhonghua liuxingbingxue zazhi.

[2]  D. Dimitrov,et al.  Enhanced elicitation of potent neutralizing antibodies by the SARS-CoV-2 spike receptor binding domain Fc fusion protein in mice , 2020, Vaccine.

[3]  Yuquan Wei,et al.  A vaccine targeting the RBD of the S protein of SARS-CoV-2 induces protective immunity , 2020, Nature.

[4]  P. Hotez,et al.  Yeast-Expressed SARS-CoV Recombinant Receptor-Binding Domain (RBD219-N1) Formulated with Aluminum Hydroxide Induces Protective Immunity and Reduces Immune Enhancement , 2020, bioRxiv.

[5]  Linqi Zhang,et al.  Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor , 2020, Nature.

[6]  R. Scheuermann,et al.  A Sequence Homology and Bioinformatic Approach Can Predict Candidate Targets for Immune Responses to SARS-CoV-2 , 2020, Cell Host & Microbe.

[7]  A. M. Leontovich,et al.  The species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2 , 2020, Nature Microbiology.

[8]  B. Graham,et al.  Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation , 2020, Science.

[9]  K. Khoo,et al.  Cryo-EM analysis of a feline coronavirus spike protein reveals a unique structure and camouflaging glycans , 2020, Proceedings of the National Academy of Sciences.

[10]  John L Rubinstein,et al.  The human coronavirus HCoV-229E S-protein structure and receptor binding , 2019, eLife.

[11]  J. McLellan,et al.  The 3.1-Angstrom Cryo-electron Microscopy Structure of the Porcine Epidemic Diarrhea Virus Spike Protein in the Prefusion Conformation , 2019, Journal of Virology.

[12]  H. Kimura,et al.  A genetically encoded probe for imaging nascent and mature HA-tagged proteins in vivo , 2019, Nature Communications.

[13]  Alexandra C Walls,et al.  Structural basis for human coronavirus attachment to sialic acid receptors , 2019, Nature Structural & Molecular Biology.

[14]  Jian Peng,et al.  Zfp217 mediates m6A mRNA methylation to orchestrate transcriptional and post-transcriptional regulation to promote adipogenic differentiation , 2019, Nucleic acids research.

[15]  Robert,et al.  Stability and sub-cellular localization of DNA polymerase β is regulated by interactions with NQO1 and XRCC1 in response to oxidative stress , 2019, Nucleic acids research.

[16]  Zhènglì Shí,et al.  Origin and evolution of pathogenic coronaviruses , 2018, Nature Reviews Microbiology.

[17]  Daniel Wrapp,et al.  Stabilized coronavirus spikes are resistant to conformational changes induced by receptor recognition or proteolysis , 2018, Scientific Reports.

[18]  Xinquan Wang,et al.  Cryo-EM structure of the SARS coronavirus spike glycoprotein in complex with its host cell receptor ACE2 , 2018, PLoS pathogens.

[19]  J. Shang,et al.  Cryo-electron microscopy structure of infectious bronchitis coronavirus spike protein , 2018 .

[20]  Fang Li,et al.  Cryo-EM structure of infectious bronchitis coronavirus spike protein reveals structural and functional evolution of coronavirus spike proteins , 2018, PLoS pathogens.

[21]  J. Rini,et al.  Receptor-binding loops in alphacoronavirus adaptation and evolution , 2017, Nature Communications.

[22]  A. Walls,et al.  Glycan Shield and Fusion Activation of a Deltacoronavirus Spike Glycoprotein Fine-Tuned for Enteric Infections , 2017, Journal of Virology.

[23]  Fang Li,et al.  Cryo-Electron Microscopy Structure of Porcine Deltacoronavirus Spike Protein in the Prefusion State , 2017, Journal of Virology.

[24]  Barney S. Graham,et al.  Immunogenicity and structures of a rationally designed prefusion MERS-CoV spike antigen , 2017, Proceedings of the National Academy of Sciences.

[25]  Yi Shi,et al.  Cryo-EM structures of MERS-CoV and SARS-CoV spike glycoproteins reveal the dynamic receptor binding domains , 2017, Nature Communications.

[26]  G. Wong,et al.  Novel chimeric virus-like particles vaccine displaying MERS-CoV receptor-binding domain induce specific humoral and cellular immune response in mice , 2016, Antiviral Research.

[27]  Haixia Zhou,et al.  Cryo-electron microscopy structures of the SARS-CoV spike glycoprotein reveal a prerequisite conformational state for receptor binding , 2016, Cell Research.

[28]  S. Perlman,et al.  Introduction of neutralizing immunogenicity index to the rational design of MERS coronavirus subunit vaccines , 2016, Nature Communications.

[29]  Fang Li,et al.  Structure, Function, and Evolution of Coronavirus Spike Proteins. , 2016, Annual review of virology.

[30]  Frank DiMaio,et al.  Glycan shield and epitope masking of a coronavirus spike protein observed by cryo-electron microscopy , 2016, Nature Structural &Molecular Biology.

[31]  Andrew J. Davison,et al.  Ratification vote on taxonomic proposals to the International Committee on Taxonomy of Viruses (2016) , 2016, Archives of Virology.

[32]  G. Gao,et al.  Epidemiology, Genetic Recombination, and Pathogenesis of Coronaviruses , 2016, Trends in Microbiology.

[33]  Barney S. Graham,et al.  Pre-fusion structure of a human coronavirus spike protein , 2016, Nature.

[34]  F. Dimaio,et al.  Cryo-electron microscopy structure of a coronavirus spike glycoprotein trimer , 2016, Nature.

[35]  G. Ye,et al.  Comparison of lentiviruses pseudotyped with S proteins from coronaviruses and cell tropisms of porcine coronaviruses , 2016, Virologica Sinica.

[36]  G. Gao,et al.  Recombinant Receptor Binding Domain Protein Induces Partial Protective Immunity in Rhesus Macaques Against Middle East Respiratory Syndrome Coronavirus Challenge , 2015, EBioMedicine.

[37]  Ulas Bagci,et al.  Evaluation of candidate vaccine approaches for MERS-CoV , 2015, Nature Communications.

[38]  M. Ar Gouilh,et al.  Genomic Analysis of 15 Human Coronaviruses OC43 (HCoV-OC43s) Circulating in France from 2001 to 2013 Reveals a High Intra-Specific Diversity with New Recombinant Genotypes , 2015, Viruses.

[39]  Lu Lu,et al.  Receptor-binding domain-based subunit vaccines against MERS-CoV , 2014, Virus Research.

[40]  M. Frieman,et al.  Purified coronavirus spike protein nanoparticles induce coronavirus neutralizing antibodies in mice , 2014, Vaccine.

[41]  R. Fouchier,et al.  MERS: emergence of a novel human coronavirus , 2014, Current Opinion in Virology.

[42]  Linqi Zhang,et al.  Structure of MERS-CoV spike receptor-binding domain complexed with human receptor DPP4 , 2013, Cell Research.

[43]  M. J. Adams,et al.  Ratification vote on taxonomic proposals to the International Committee on Taxonomy of Viruses (2013) , 2013, Archives of Virology.

[44]  A. Osterhaus,et al.  Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia. , 2012, The New England journal of medicine.

[45]  J. Rini,et al.  The X-ray Crystal Structure of Human Aminopeptidase N Reveals a Novel Dimer and the Basis for Peptide Processing* , 2012, The Journal of Biological Chemistry.

[46]  L. Enjuanes,et al.  Structural Bases of Coronavirus Attachment to Host Aminopeptidase N and Its Inhibition by Neutralizing Antibodies , 2012, PLoS pathogens.

[47]  Jincun Zhao,et al.  Immunization with an attenuated severe acute respiratory syndrome coronavirus deleted in E protein protects against lethal respiratory disease , 2010, Virology.

[48]  Fang Li,et al.  Crystal structure of NL63 respiratory coronavirus receptor-binding domain complexed with its human receptor , 2009, Proceedings of the National Academy of Sciences.

[49]  Shibo Jiang,et al.  The spike protein of SARS-CoV — a target for vaccine and therapeutic development , 2009, Nature Reviews Microbiology.

[50]  Rodrigo Lopez,et al.  Clustal W and Clustal X version 2.0 , 2007, Bioinform..

[51]  Shibo Jiang,et al.  Antigenic and Immunogenic Characterization of Recombinant Baculovirus-Expressed Severe Acute Respiratory Syndrome Coronavirus Spike Protein: Implication for Vaccine Design , 2006, Journal of Virology.

[52]  S. Harrison,et al.  Structure of SARS Coronavirus Spike Receptor-Binding Domain Complexed with Receptor , 2005, Science.

[53]  S. Perlman,et al.  A Severe Acute Respiratory Syndrome-Associated Coronavirus-Specific Protein Enhances Virulence of an Attenuated Murine Coronavirus , 2005, Journal of Virology.

[54]  K. Subbarao,et al.  Long-term protection from SARS coronavirus infection conferred by a single immunization with an attenuated VSV-based vaccine , 2005, Virology.

[55]  P. Woo,et al.  SARS coronavirus spike polypeptide DNA vaccine priming with recombinant spike polypeptide from Escherichia coli as booster induces high titer of neutralizing antibody against SARS coronavirus , 2005, Vaccine.

[56]  A. Roberts,et al.  A single immunization with a rhabdovirus-based vector expressing severe acute respiratory syndrome coronavirus (SARS-CoV) S protein results in the production of high levels of SARS-CoV-neutralizing antibodies. , 2005, The Journal of general virology.

[57]  B. Moss,et al.  Neutralizing antibody and protective immunity to SARS coronavirus infection of mice induced by a soluble recombinant polypeptide containing an N-terminal segment of the spike glycoprotein , 2005, Virology.

[58]  Y. Guan,et al.  Intranasal immunization with inactivated SARS-CoV (SARS-associated coronavirus) induced local and serum antibodies in mice , 2004, Vaccine.

[59]  S. Morikawa,et al.  A subcutaneously injected UV-inactivated SARS coronavirus vaccine elicits systemic humoral immunity in mice , 2004, International immunology.

[60]  B. Murphy,et al.  Mucosal immunisation of African green monkeys (Cercopithecus aethiops) with an attenuated parainfluenza virus expressing the SARS coronavirus spike protein for the prevention of SARS , 2004, The Lancet.

[61]  Y. Guan,et al.  Coronavirus as a possible cause of severe acute respiratory syndrome , 2003, The Lancet.

[62]  J. Peiris,et al.  Medical reviews. Coronaviruses. , 1974, The Yale journal of biology and medicine.

[63]  David H. L. Bishop,et al.  The International Committee on Taxonomy of Viruses , 1995 .