Development and application of a safe SARS-CoV neutralization assay based on lentiviral vectors pseudotyped with SARS-CoV spike protein.
- Author:
Ke-Xia YAN
1
;
Wen-Jie TAN
;
Xiang-Min ZHANG
;
Hui-Juan WANG
;
Yan LI
;
Li RUAN
Author Information
1. National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 100052, China.
- Publication Type:Journal Article
- MeSH:
Animals;
Antibodies, Viral;
blood;
Blotting, Western;
Lentivirus;
genetics;
Membrane Glycoproteins;
immunology;
Mice;
Neutralization Tests;
methods;
Plasmids;
Recombinant Proteins;
immunology;
Research Design;
SARS Virus;
immunology;
Spike Glycoprotein, Coronavirus;
Viral Envelope Proteins;
immunology
- From:
Chinese Journal of Virology
2007;23(6):440-446
- CountryChina
- Language:Chinese
-
Abstract:
The severe acute respiratory syndrome-associated coronavirus (SARS-CoV) spike protein (S) is a major target for neutralizing antibody. To develop and apply a safe neutralization assay for SARS-CoV, lentiviral SARS-CoV S pseudotypes had been constructed based on a three plasmid system, which contained pVRC8304 (harboring codon optimized full-length SARS-CoV S protein), pCMV delta 8. 2 (HIV-1 gag/pol construct) and pHR'CMV EGFP (the green fluorescent protein reporter construct). The pseudo-typed lentiviral particles were used to develop an in vitro microneutralization assay that was both sensitive and specific for SARS-CoV neutralizing antibody. We used this assay to determine the titers of the neutralizing antibodies (Nabs) in serum samples from mice immunized with various rVVs expressing different S fragments of SARS-CoV. The serum antibodies derived from S and various segments of S1 region neutralized SARS-CoV in vitro. No cross-neutralization occurred with the goat antiserum prepared with inactivated HCoV-OC43 or HCoV-229E. Neutralization titers measured by this assay were highly parallel with those measured by the assay using live SARS-CoV. Because the pseudotype assay does not require handling live SARS virus, it is a useful tool to determine serum neutralizing titers during natural infection and the preclinical evaluation of candidate vaccines.
