Receptor-binding domain of severe acute respiratory syndrome coronavirus spike protein contains multiple conformation-dependent epitopes that induce highly potent neutralizing antibodies

J Immunol. 2005 Apr 15;174(8):4908-15. doi: 10.4049/jimmunol.174.8.4908.

Abstract

The spike (S) protein of severe acute respiratory syndrome associated coronavirus (SARS-CoV) is a major antigenic determinant capable of inducing protective immunity. Recently, a small fragment on the SARS-CoV S protein (residues 318-510) was characterized as a minimal receptor-binding domain (RBD), which mediates virus binding to angiotensin-converting enzyme 2, the functional receptor on susceptible cells. In this study, we demonstrated that a fusion protein containing RBD linked to human IgG1 Fc fragment (designated RBD-Fc) induced high titer of RBD-specific Abs in the immunized mice. The mouse antisera effectively neutralized infection by both SARS-CoV and SARS pseudovirus with mean 50% neutralization titers of 1/15,360 and 1/24,737, respectively. The neutralization determinants on the RBD of S protein were characterized by a panel of 27 mAbs isolated from the immunized mice. Six groups of conformation-dependent epitopes, designated as Conf I-VI, and two adjacent linear epitopes were identified by ELISA and binding competition assays. The Conf IV and Conf V mAbs significantly blocked RBD-Fc binding to angiotensin-converting enzyme 2, suggesting that their epitopes overlap with the receptor-binding sites in the S protein. Most of the mAbs (23 of 25) that recognized the conformational epitopes possessed potent neutralizing activities against SARS pseudovirus with 50% neutralizing dose ranging from 0.005 to 6.569 microg/ml. Therefore, the RBD of SARS S protein contains multiple conformational epitopes capable of inducing potent neutralizing Ab responses, and is an important target site for developing vaccines and immunotherapeutics.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Antibodies, Monoclonal
  • Antibodies, Viral / biosynthesis
  • Antigens, Viral / chemistry*
  • Antigens, Viral / genetics
  • Binding Sites
  • Cell Line
  • Chlorocebus aethiops
  • Epitopes / chemistry
  • Epitopes / genetics
  • Humans
  • In Vitro Techniques
  • Membrane Glycoproteins / chemistry*
  • Membrane Glycoproteins / genetics
  • Membrane Glycoproteins / immunology*
  • Membrane Glycoproteins / metabolism
  • Mice
  • Mice, Inbred BALB C
  • Molecular Sequence Data
  • Neutralization Tests
  • Peptide Fragments / chemistry
  • Peptide Fragments / genetics
  • Peptide Fragments / immunology
  • Protein Conformation
  • Receptors, Virus / metabolism
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / immunology
  • SARS Virus / genetics
  • SARS Virus / immunology*
  • SARS Virus / pathogenicity
  • Spike Glycoprotein, Coronavirus
  • Vero Cells
  • Viral Envelope Proteins / chemistry*
  • Viral Envelope Proteins / genetics
  • Viral Envelope Proteins / immunology*
  • Viral Envelope Proteins / metabolism

Substances

  • Antibodies, Monoclonal
  • Antibodies, Viral
  • Antigens, Viral
  • Epitopes
  • Membrane Glycoproteins
  • Peptide Fragments
  • Receptors, Virus
  • Recombinant Fusion Proteins
  • Spike Glycoprotein, Coronavirus
  • Viral Envelope Proteins
  • spike glycoprotein, SARS-CoV
  • spike protein, mouse hepatitis virus