Severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice

Abstract

The spike protein (S), a membrane component of severe acute respiratory syndrome coronavirus (SARS-CoV) is anticipated to be an important component of candidate vaccines. We constructed recombinant forms of the highly attenuated modified vaccinia virus Ankara (MVA) containing the gene encoding full-length SARS-CoV S with and without a C-terminal epitope tag called MVA/S-HA and MVA/S, respectively. Cells infected with MVA/Sor MVA/S-HA synthesized a 200-kDa protein, which was recognized by antibody raised against a synthetic peptide of SARS-CoV S or the epitope tag in Western blot analyses. Further studies indicated that S was N-glycosylated and migrated in SDS polyacrylamide gels with an apparent mass of approximately 160 kDa after treatment with peptide N-glycosidase F. The acquisition of resistance to endoglycosidase H indicated trafficking of S to the medial Golgi compartment, and confocal microscopy showed that S was transported to the cell surface. Intranasal or intramuscular inoculations of BALB/c mice with MVA/S produced serum antibodies that recognized the SARS S in ELISA and neutralized SARS-CoV in vitro. Moreover, MVA/S administered by either route elicited protective immunity, as shown by reduced titers of SARS-CoV in the upper and lower respiratory tracts of mice after challenge. Passive transfer of serum from mice immunized with MVA/S to naïve mice also reduced the replication of SARS-CoV in the respiratory tract after challenge, demonstrating a role for antibody to S in protection. The attenuated nature of MVA and the ability of MVA/S to induce neutralizing antibody that protects mice support further development of this candidate vaccine.

Fig. 1.

Expression of SARS-CoV S by recombinant MVA. (A) Diagram of selected portion of MVA/S. The GFP and S ORFs were inserted into a deletion site (del III) of the MVA genome. The early/late mH5 and late P11 vaccinia virus promoters regulate S and GFP, respectively. MVA/S-HA has an identical structure except for the presence of a short segment of DNA encoding the influenza virus HA tag at the C terminus of S. (B) Western blot analysis of SARS-CoV S protein. HeLa cells were uninfected (control) or infected with MVA or MVA/S-HA. After 18 h, the cells were harvested, the cleared cell lysates were analyzed by SDS/PAGE, and the proteins were transferred to a nitrocellulose membrane and detected with anti-HA mAb (lanes 1, 2, and 3) or anti-SARSCoV S polyclonal antibody (lanes 4, 5, and 6). The molecular masses of marker proteins in kDa are shown on the left and the position of SARS-CoV S protein is indicated by an arrow on the right.

Fig. 2.

Characterization of SARS-CoV S glycoprotein. (A) Endo H and PNGase F sensitivity. HeLa cells were uninfected (lanes 1 and 5), or infected with MVA (lanes 2 and 6) or MVA/S-HA (lanes 3, 4, 7, and 8). After 18 h, the cells were lysed, cleared by centrifugation, and incubated with anti-HA affinity matrix. The bound proteins were treated with endo H or PNGase F, as indicated by plus signs and were analyzed by SDS/PAGE and Western blotting with anti-HA mAb. The positions of two glycosylated forms of S and a nonglycosylated (ngS) form are shown by arrows. (B) Kinetics of endo H sensitivity. HeLa cells at 8 h after infection with MVA/S-HA were pulse-labeled with [³⁵S]methionine and [³⁵S]cysteine for 10 min and were then washed and chased for 0, 20, 40, 60, and 80 min in medium supplemented with unlabeled cysteine and methionine. Cells were lysed immediately after the pulse or chase and the S was captured with anti-HA affinity matrix, subjected to endo H digestion, resolved by SDS/PAGE, and visualized by autoradiography. The molecular masses of marker proteins in kDa are shown on the left.

Fig. 3.

Cellular localization of SARS-CoV S. Unfixed and unpermeabilized CEF (A-F) that had been infected with MVA (A and B), MVA/S (C and D), and MVA/S-HA (E and F) for 18 h were stained with anti-SARS-CoV mouse serum (A-D) or anti-HA mAb (E and F), followed by Alexa 594-conjugated anti-mouse IgG and viewed by confocal microscopy. CEF infected with MVA/S-HA (G and H) were fixed, permeabilized, and stained with anti-HA mAb, followed by Alexa 594-conjugated anti-mouse IgG. Shown are GFP (Left) and Alexa 594 (Right) fluorescence, respectively.

Fig. 4.

Antibody responses after immunization with recombinant MVA/Sby an i.n. or i.m. route. (A) End point ELISA titers of pooled serum (n = 8), taken before (pre-bleed) or after immunizations, were determined by using insect cell-expressed S1 domain of the SARS-CoV S as the capture antigen. Sera from two mice were pooled after challenge and analyzed. Thin and thick arrows depict times of immunizations and challenge with SARS-CoV, respectively. (B) Prechallenge SARS-CoV neutralization titers of pooled serum were determined. The dilution of serum that completely prevented SARS-CoV cytopathic effect in 50% of the wells was calculated.

Fig. 5.

Protection of immunized mice from subsequent challenge with SARS-CoV. Groups of eight BALB/c mice were mock-vaccinated or vaccinated with MVA or MVA/S by the i.n. or i.m. route at time 0 and 4 weeks, and were challenged 4 weeks later with 10⁴ TCID₅₀ of SARS-CoV administered by the i.n. route. Two days later, the titers of SARS-CoV in the lungs and nasal turbinates of four mice in each group were determined. Virus titers are expressed as log₁₀ TCID₅₀ per g of tissue. Statistical comparison of MVA/S titers to unvaccinated controls was performed by using a Mann-Whitney U nonparametric analysis; ^*, P = 0.02.