[Development and application of a safe SARS-CoV neutralization assay based on lentiviral vectors pseudotyped with SARS-CoV spike protein].
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.
Yan
Ke-Xia
KX
National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 100052, China.
Tan
Wen-Jie
WJ
Zhang
Xiang-Min
XM
Wang
Hui-Juan
HJ
Li
Yan
Y
Ruan
Li
L
D000818:Animals; D000914:Antibodies, Viral / Q000097:blood*; D015153:Blotting, Western; D016086:Lentivirus / Q000235:genetics; D008562:Membrane Glycoproteins / Q000276:immunology*; D051379:Mice; D009500:Neutralization Tests / Q000379:methods*; D010957:Plasmids; D011994:Recombinant Proteins / Q000276:immunology; D012107:Research Design; D045473:Severe acute respiratory syndrome-related coronavirus / Q000276:immunology*; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins / Q000276:immunology*
D000914:Antibodies, Viral; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins; C578557:spike glycoprotein, SARS-CoV; C578553:spike protein, mouse hepatitis virus