PMC

a, The structure of the cDNAs used. b, Expression of these constructs, determined by western blot analysis with antisera reactive with SARS-CoV S, was evaluated after transfection of the indicated plasmid expression vectors in 293T cells. Arrows indicate specific SΔCD (upper) and SΔTM (lower) bands.

Immunization and challenge were performed in mice as described previously¹⁸, and viral replication (mean log₁₀TCID₅₀ per g tissue with standard error) in the lower (a) and upper (b) respiratory tract after challenge with SARS-CoV was measured for five immunized animals inoculated with SΔCD, SΔTM or empty plasmid vector control. The lower limit for detection of SARS-CoV replication was 1.5 TCID₅₀ per g in the lungs and 1.8 TCID₅₀ per g in the nasal turbinates. A non-parametric two-tailed t-test (Mann–Whitney) was used for statistical analysis. Log-transformed virus titres were compared, and statistical significance was assigned to the differences, both with a P value of 0.0079.

a, Monoclonal rat anti-mouse anti-CD4, CD8 or CD4/CD8/CD90 were used to deplete T cells in SΔCD and control vaccinated mice (n = 4) as described previously²³. Mice were then challenged with SARS-CoV 48 h later. Viral replication in the lungs was measured as described in Fig. 3. Data represent mean ± s.e., and no statistically significant difference was observed between groups depleted as shown. b, Lack of protection against SARS-CoV replication in the lungs after adoptive T-cell transfer from vaccinated mice. Each naive mouse (n = 4) received 3 × 10⁷ purified T cells from a donor mouse confirmed to respond to the vaccine (immune) or from a non-immune mouse (control). The recipient mice were challenged 24 h after adoptive T-cell transfer. Viral titre in lungs was measured as described in Fig. 3. There was no statistically significant difference between these two groups using the non-parametric Mann–Whitney two-tailed t-test (P = 0.49). c, Protection against SARS-CoV replication in the lungs after passive transfer of immune IgG from vaccinated mice. Purified IgG from SΔCD or control vaccinated donor mice (n = 4) was passively transferred into recipient naive mice (n = 4). Serum from recipient mice was collected one day before challenge, and neutralization was confirmed using the S pseudotyped lentiviral vector (left panel). Recipient mice (n = 4 per group) were challenged 24 h after IgG transfer with 10⁴ TCID₅₀ SARS-CoV and SARS-CoV replication was measured as described in Fig. 3. The non-parametric two-tailed t-test (Mann–Whitney) revealed a statistically significant difference of P = 0.0286 between these two groups (right panel).

a, Intracellular cytokine staining was performed to quantify the percentage of activated T cells that produce either IFN-γ or TNF-α in response to stimulation with overlapping S peptide pools in CD4 (left) or CD8 (right) lymphocytes from mice (n = 5 per group) immunized with empty plasmid vector (control) or mice (n = 5 per group) immunized with the indicated plasmid at weeks 0, 3 and 6. Immune responses were measured 10 days after the final boost. Non-stimulated cells gave responses similar to those of the control subjects, at background levels. Symbols indicate the response of each individual animal, and the median value is shown (horizontal bar). b, Antibody responses induced by plasmid DNA vaccination against the SARS-CoV S protein. End-point dilution ELISA titres of SARS-CoV S-specific antibodies (left panel) in serum of vaccinated animals collected 10 days after the final boost were determined by optical density as described in the Methods. Neutralization by antisera from mice immunized with the relevant SARS-CoV S mutant or no insert (control) plasmid DNA vectors at the indicated concentrations was measured using the luciferase assay with S pseudotyped lentiviral vectors (middle panel). Reduction of gene transfer was observed with immune sera in a dose-dependent fashion. Twofold dilutions of heat-inactivated sera were tested in a microneutralization assay for the presence of antibodies that neutralized the infectivity of 100 TCID₅₀ of SARS-CoV in Vero cell monolayers, using four wells per dilution on a 96-well plate (right panel). The presence of viral cytopathic effect (cpe) was read on days 3 and 4. The dilution of serum that completely prevented cpe in 50% of the wells was calculated by the Reed Muench formula. Data are presented as the mean ± s.e. for each group. A non-parametric two-tailed t-test (Mann–Whitney) was used for statistical analysis, and the relevant P values are indicated (b, right panel).