PMC

Amino acid sequence alignment and in vitro expression of 3a proteins. (A) The amino acid sequences of full-length 3a from SARS-CoV and SL-CoV were aligned with ClustalX 1.83 and edited using GenDoc. (B) The full-length 3a gene from SARS-CoV or SL-CoV was cloned into pcDNA3.1(+) to make pcDNA3.1H3 or pcDNA3.1B3, respectively. The expression of 3a protein was analyzed at 48 h posttransfection by Western blotting. Lane 1, pcDNA3.1; lane 2, pcDNA3.1H3; lane 3, pcDNA3.1B3.

Specific IFN-γ and IL-4 production in immunized mice. Mice were sacrificed 10 days after the final boost, and the frequency of IFN-γ (A)- or IL-4 (B)-producing cells at the single-cell level was determined by an ELISPOT assay. Data shown are the means ± SD of results from two independent experiments using three animals from each group, with each condition tested in triplicate (*, P values of <0.05 for comparison with control groups).

Frequencies of IFN-γ- and IL-2-positive cells in CD4⁺ or CD8⁺ splenocytes. Splenocytes were isolated from vaccinated mice 10 days after the final immunization. IFN-γ (A)- and IL-2 (B)-positive cells in CD4⁺ or CD8⁺ cell populations were analyzed by flow cytometry. Data shown are the means ± SD of results from two independent experiments using three animals from each group, with each condition tested in triplicate (*, P values of <0.05 for comparison with pcDNA3.1B3).

3a-specific antibody responses in immunized mice. Sera from each group were collected before each immunization. Ten days after the final boost, the mice were sacrificed and the specific antibody responses were examined by ELISA. SARS-CoV 3a protein (A) and SL-CoV 3a protein (B) were used as a coating antigen. Data shown are the means ± SD of results from two independent experiments using five animals from each group, with each condition tested in triplicate (*, P values of <0.05 for comparison with pcDNA3.1B3). OD, optical density; d.p.i., days postimmunization.