Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2005 Mar 30;334(1):74-82.
doi: 10.1016/j.virol.2005.01.034.

Identification of a critical neutralization determinant of severe acute respiratory syndrome (SARS)-associated coronavirus: importance for designing SARS vaccines

Yuxian He  1 , Qingyu Zhu, Shuwen Liu, Yusen Zhou, Baoan Yang, Jiaming Li, Shibo Jiang
Affiliations

Identification of a critical neutralization determinant of severe acute respiratory syndrome (SARS)-associated coronavirus: importance for designing SARS vaccines

Yuxian He et al. Virology. .

Abstract

The spike (S) protein of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is not only responsible for receptor binding, but also a major antigenic determinant capable of inducing protective immunity. In this study, we demonstrated that the receptor-binding domain (RBD) of S protein is an important immunogenic site in patients with SARS and rabbits immunized with inactivated SARS-CoV. Serum samples from convalescent SARS patients and immunized rabbits had potent neutralizing activities against infection by pseudovirus expressing SARS-CoV S protein. Depletion of RBD-specific antibodies from patient or rabbit immune sera by immunoadsorption significantly reduced serum-mediated neutralizing activity, while affinity-purified anti-RBD antibodies had relatively higher potency neutralizing infectivity of SARS pseudovirus, indicating that the RBD of S protein is a critical neutralization determinant of SARS-CoV during viral infection and immunization. Two monoclonal antibodies (1A5 and 2C5) targeting at the RBD of S protein were isolated from mice immunized with inactivated SARS-CoV. Both 1A5 and 2C5 possessed potent neutralizing activities, although they directed against distinct conformation-dependant epitopes as shown by ELISA and binding competition assay. We further demonstrated that 2C5, but not 1A5, was able to block binding of the RBD to angiotensin-converting enzyme 2 (ACE2), the functional receptor on targeted cells. These data provide important information for understanding the antigenicity and immunogenicity of SARS-CoV and for designing SARS vaccines.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1
Measurement of the RBD-specific antibodies in sera from convalescent-phase SARS patients by ELISA. Recombinant RBD-C9 was used for coating plates and the sera from 40 SARS patients and 30 healthy blood donors were tested at 1:50 dilution. Sera were considered positive when the optical density values were above the cutoff value (the mean absorbance at 450 nm of sera from healthy blood donors plus three standard deviations).
Fig. 2
Fig. 2
Isolation of RBD-specific antibodies from sera of convalescent-phase SARS patients by immunoaffinity chromatography. Serum samples were adsorbed to RBD-Fc columns and bound antibodies were eluted at pH 2.5. The patient sera, flowthroughs, and eluates were tested, respectively, against RBD-C9 (A) and S1-C9 (B) at 1:50 dilution by ELISA. One serum sample from a healthy blood donor was used as control.
Fig. 3
Fig. 3
Neutralizing activity of the RBD-specific antibodies isolated from the sera of SARS patients. (A) SARS-A; (B) SARS-B; (C) SARS-C; and (D) SARS-D. Inhibition of SARS pseudovirus infectivity in 293T/ACE2 cells by patient sera, flowthroughs, and eluates, respectively, at a series of 2-fold dilutions was determined and the percentage of neutralization was calculated for each sample.
Fig. 4
Fig. 4
Isolation and characterization of RBD-specific antibodies from rabbit antisera by immunoaffinity chromatography. Reactivities of rabbit antibodies with RBD-Fc (A) and S1-C9 (B) were tested at 1:50 dilution by ELISA. (C) Neutralizing activity of the RBD-specific antibodies isolated from rabbit antisera. Inhibition of SARS pseudovirus infection in 293T/ACE2 cells by rabbit antisera, flowthroughs, and eluates, respectively, at a series of 2-fold dilutions was determined and the percent neutralization was calculated for each sample.
Fig. 5
Fig. 5
Characterization of the RBD-specific MAbs. The reactivities of MAbs against RBD-Fc (A) and S1-C9 (B) were tested by ELISA. MAbs were tested at 10 μg/ml, and control sera were tested at 1:100 dilution. (C) Inhibition of biotinylated MAbs binding to RBD-Fc by RBD-specific MAbs measured by competitive ELISA. Competing MAbs were tested at 100 μg/ml.
Fig. 6
Fig. 6
Inhibition of RBD-Fc binding to ACE2 by MAbs. Inhibitory activity of MAbs on RBD-Fc binding to cell-associated ACE2 expressed on 293T/ACE2 cells was measured by flow cytometry. RBD-Fc and MAbs were used at 1 and 50 μg/ml, respectively.
Fig. 7
Fig. 7
Neutralization of SARS pseudovirus infection in 293T/ACE2 cells by MAbs. Each of the MAbs was tested at a series of 2-fold dilutions and percent neutralization was calculated.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Babcock G.J., Esshaki D.J., Thomas W.D., Jr., Ambrosino D.M. Amino acids 270 to 510 of the severe acute respiratory syndrome coronavirus spike protein are required for interaction with receptor. J. Virol. 2004;78(9):4552–4560. - PMC - PubMed
    1. Bisht H., Roberts A., Vogel L., Bukreyev A., Collins P.L., Murphy B.R., Subbarao K., Moss B. Severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice. Proc. Natl. Acad. Sci. U.S.A. 2004;101(17):6641–6646. - PMC - PubMed
    1. Bonavia A., Zelus B.D., Wentworth D.E., Talbot P.J., Holmes K.V. Identification of a receptor-binding domain of the spike glycoprotein of human coronavirus HCoV-229E. J. Virol. 2003;77(4):2530–2538. - PMC - PubMed
    1. Buchholz U.J., Bukreyev A., Yang L., Lamirande E.W., Murphy B.R., Subbarao K., Collins P.L. Contributions of the structural proteins of severe acute respiratory syndrome coronavirus to protective immunity. Proc. Natl. Acad. Sci. U.S.A. 2004;101(26):9804–9809. - PMC - PubMed
    1. Bukreyev A., Lamirande E.W., Buchholz U.J., Vogel L.N., Elkins W.R., St Claire M., Murphy B.R., Subbarao K., Collins P.L. Mucosal immunisation of African green monkeys (Cercopithecus aethiops) with an attenuated parainfluenza virus expressing the SARS coronavirus spike protein for the prevention of SARS. Lancet. 2004;363(9427):2122–2127. - PMC - PubMed

MeSH terms

-