Figure 4. Stronger antigen-specific IFN-γ-secreting CD4 T cell responses elicited by SIVgag-LC3b fusion protein compared to SIVgag antigen alone in mice.

A, Immunization schedule to evaluate the immunogenicity of the SIVgag-LC3b fusion antigen. C57BL/6 female mice were divided into four groups with 8–10 mice per group. Each mouse was intramuscularly injected 50 μg of the appropriate DNA plasmids at weeks 0 and 2, then boosted intramuscularly with 1×10⁹ vp of corresponding adenoviral vectors at weeks 4 and 6. To assess the immune responses, mice were sacrificed at weeks 4, 6 and 8 after the first immunization to collect splenocytes and serum for analysis of cellular and humoral immune responses. The symbol “↓” represents the time-point of injection; the symbol “Δ” represents the time-point of sacrifice and sample collection. The SIVgag-specific cellular immune responses, as assessed using the IFN-γ ELISPOT assay following stimulation with SIVgag peptide, were shown after DNA-based constructs immunization at week 4 (B) and after adenoviral-based constructs immunization at week 6 (C). The SIVgag-specific cellular immune responses, as assessed using the intracellular IFN-γ cytokine staining assay, were shown after DNA-based constructs immunization at week 4 (D) and after adenoviral-based constructs immunization at week 8 (E). Data were analyzed using the Student’s t-test, and a two-tailed p-value of less than 0.05 was considered statistically significant. These data were expressed as the mean±SEM from four mice samples (*: p<0.05;**: p<0.01; ***: p<0.001). Two independent experiments for the animal immunization were repeated.