Neutralizing antibody and protective immunity to SARS coronavirus infection of mice induced by a soluble recombinant polypeptide containing an N-terminal segment of the spike glycoprotein

Abstract

A secreted, glycosylated polypeptide containing amino acids 14 to 762 of the SARS coronavirus (SARS-CoV) spike protein and a polyhistidine tag was expressed in recombinant baculovirus-infected insect cells. Mice received the affinity-purified protein with either a saponin (QS21) or a Ribi (MPL + TDM) adjuvant subcutaneously and were challenged intranasally with SARS-CoV. Both regimens induced binding and neutralizing antibodies and protection against SARS-CoV intranasal infection. However, the best results were obtained with QS21 and protein, which provided the highest antibody as well as complete protection of the upper and lower respiratory tract.

Fig. 1

Expression and characterization of SARS-CoV nS glycoprotein. (A) Schematic representation of pMelBacB-based baculovirus transfer vector. Abbreviations: P_PH, polyhedrin promoter; HBM, DNA encoding honeybee melittin signal sequence; nS, DNA segment encoding amino acids (aa) 14–762 of the SARS-CoV S protein; His₆, DNA encoding 6 histidine residues. (B) Purified nS analyzed by SDS polyacrylamide gel electrophoresis and Coomassie Blue staining (lane 1), silver staining (lane 2) and Western blot analysis with anti-His mAb (lane 3) or anti-SARS CoV S polyclonal antibody (lane 4). (C) Purified nS protein was (+) or was not (−) treated with peptide N-glycosidase F and was analyzed by SDS polyacrylamide gel electrophoresis and western blotting with anti-His mAb and anti-SARS-CoV S polyclonal antibody. Molecular masses of marker proteins in kDa are shown on the left.

Fig. 2

Binding of antibodies from mice immunized with nS to full-length membrane-bound S. HeLa cells were uninfected (A–B), infected with non-recombinant MVA (C–D) or MVA expressing S (E–H) for 18 h. After fixation, the unpermeabilized cells were stained with pooled sera from mice immunized three times with nS and MPL + TDM (E–F) or nS and QS21 (A–D, G–H) followed by Alexa 594-conjugated anti-mouse IgG and viewed by visible (A, C, E, G) or fluorescence (B, D, F, H) light microscopy.

Fig. 3

ELISA and neutralizing antibody responses to nS. Groups of 7 BALB/c mice were immunized subcutaneously with 10 μg of purified nS and QS21 or MPL + TDM adjuvant at 4-week intervals (arrows) and challenged intranasally with 10⁵ TCID₅₀ SARS-CoV on day 82 (arrow head). Control mice were immunized at the same times with purified soluble vaccinia virus L1R protein. (A) End-point ELISA titers of pooled sera collected on days indicated were measured using nS as the capture antigen. The absorbance obtained with serum from mice immunized with L1R was subtracted. (B) Dilution of serum that completely prevented cytopathic effects of SARS-CoV in 50% of wells containing Vero cells was calculated. Assays were performed on pooled serum collected on days 28 and 56 days and on individual mouse serum collected on day 78. Standard error bars are shown for the latter.

Fig. 4

Protection against SARS-CoV replication in immunized mice. Groups of 7 BALB/c mice were immunized and challenged with SARS-CoV as described in the legend to Fig. 3. Two days after the challenge, the virus titers (mean log₁₀TCID₅₀ per g tissue with standard error) were measured in the lower (A) and upper (B) respiratory tract. The dotted line represents the lower limit of detection corresponding to 1.8 log₁₀TCID₅₀ per g tissue for upper respiratory tract (A) and 1.5 log₁₀TCID₅₀ per g tissue for lower respiratory tract (B).