Figure 2. Immune responses to SARS-CoV DNA vaccination in BALB/c mice.
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).