[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△8.2(HIV-1 gag/pol construct) and pHR′CMV EGFP(the green fluorescent protein reporter construct).The pseudotyped 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.