Inactivated SARS-CoV vaccine elicits high titers of spike protein-specific antibodies that block receptor binding and virus entry

Abstract

The only severe acute respiratory syndrome (SARS) vaccine currently being tested in clinical trial consists of inactivated severe acute respiratory syndrome-associate coronavirus (SARS-CoV). However, limited information is available about host immune responses induced by the inactivated SARS vaccine. In this study, we demonstrated that SARS-CoV inactivated by beta-propiolactone elicited high titers of antibodies in the immunized mice and rabbits that recognize the spike (S) protein, especially the receptor-binding domain (RBD) in the S1 region. The antisera from the immunized animals efficiently bound to the RBD and blocked binding of RBD to angiotensin-converting enzyme 2, the functional receptor on the susceptible cells for SARS-CoV. With a sensitive and quantitative single-cycle infection assay using pseudovirus bearing the SARS-CoV S protein, we demonstrated that mouse and rabbit antisera significantly inhibited S protein-mediated virus entry with mean 50% inhibitory titers of 1:7393 and 1:2060, respectively. These data suggest that the RBD of S protein is a major neutralization determinant in the inactivated SARS vaccine which can induce potent neutralizing antibodies to block SARS-CoV entry. However, caution should be taken in using the inactivated SARS-CoV as a vaccine since it may also cause harmful immune and/or inflammatory responses.

Fig. 1

Antibody responses against SARS-CoV in the immunized mice (A) and rabbits (B). Mouse and rabbit sera were tested at a series of 4-fold dilutions by commercial diagnostic ELISA kit using a mixture of proteins purified from viral lysates as coating antigens.

Fig. 2

Inactivated SARS-CoV induced high titers of antibodies in mice against full-length S protein (A), S1-C9 (B), and RBD-Fc (C). Mouse sera were tested at a series of 2-fold dilutions by ELISA.

Fig. 3

Inactivated SARS-CoV induced high titers of antibodies in rabbits against full-length S protein (A), S1-C9 (B), and RBD-Fc (C). Rabbit sera were tested at a series of 2-fold dilutions by ELISA.

Fig. 4

Inhibition of RBD-Fc binding to ACE2 by antibodies in the mouse and rabbit antisera. (A) RBD-Fc bound to soluble ACE2 in a dose-dependant manner; mouse (B) and rabbit (C) antisera at 1:50 dilution inhibited binding of RBD-Fc to ACE2.

Fig. 5

Inhibition of S protein-mediated virus entry by antibodies in the mouse antisera. (A) Infection of 293T/ACE2 cells by SARS pseudovirus was determined in the presence of preimmune and antisera at 1:100 dilution. (B) Inhibition of SARS pseudovirus infection by mouse antisera at a series of 2-fold dilutions and percentage inhibition was calculated for each sample.

Fig. 6

Inhibition of S protein-mediated virus entry by antibodies in the rabbit antisera. (A) Infection of 293T/ACE2 cells by SARS pseudovirus was determined in the presence of preimmune and antisera at 1:100 dilution. (B) Inhibition of SARS pseudovirus infection by rabbit antisera at a series of 2-fold dilutions and percentage inhibition was calculated for each sample.