Fig 2: Immunogenicity of Adenovirus-Vectored CCHFV Vaccines in Balb/c Mice Following Prime-Boost Immunization. This figure illustrates the humoral and cellular immune responses elicited by the various recombinant adenovirus vaccine candidates against CCHFV, as assessed through a prime-boost immunization regimen. (A) Schematic representation of the immunization and sampling schedule in Balb/c mice. Animals received a priming immunization on Day 0 and a booster immunization on Day 28. Serological assessments were conducted on Days 0, 28, and 35. Comprehensive immunogenicity profiling, including virus neutralization testing, cytokine profiling, ELISPOT, and cellular immunity characterization (CD4 + and CD8 + T cell phenotyping), was performed on Day 35. (B) Kinetics of seroconversion rates across all experimental groups at Day 0 (baseline), Day 28 (post-prime), and Day 35 (post-boost). Following prime immunization at Day 28, differential seroconversion patterns emerged, with most groups showing 80–100% seroconversion. By Day 35, most groups maintained or achieved 100% seroconversion rates. (C) Mean neutralizing antibody titers at Day 35 (post-boost) for each experimental group. Groups 1, 5, and 6 exhibited the highest mean neutralization titers, ranging from approximately 80 to 160 arbitrary units. Groups 9 and 10 showed the lowest neutralizing responses, with mean titers around 30–65 units. Statistical analysis revealed significant differences between groups, with Group 5 showing significantly higher neutralization titers compared to several other groups. (D) Fold changes in IFN-γ production (obtained from RTqPCR), a key marker of Th1-type cellular responses. Group 6 demonstrated the highest IFN-γ response with fold changes exceeding 30-fold compared to baseline. Group 6 showed highly significant differences compared to most other groups. (E) Fold changes in IL-2 production (obtained from RTqPCR), indicative of T cell activation and proliferative capacity. Groups 2 and 5 exhibited the highest IL-2 responses with fold changes reaching approximately 3.5–4.0-fold above baseline. Highly significant differences were observed between high-responding groups and most other groups, with additional significant differences between intermediate and low responders. (F) Comparative ELISpot analysis of IFN-γ and IL-2 production presented as percentages of responding splenocytes. Data represent cytokine-producing cells per total splenocytes analyzed from each experimental group. (G) Flow cytometric analysis of T cell subsets, displaying the percentages of CD3 + , CD4 + , and CD8 + lymphocytes. CD3 + T cell frequencies showed slight elevations in Groups 6, 7, 8, and 9 (p < 0.0001, *p < 0.05). CD4 + T helper cells showed significantly elevated frequencies in Groups 4, 5, 6, 7, 8, and 9 (***p < 0.0001, ***p < 0.001, **p < 0.01). CD8 + cytotoxic T lymphocyte frequencies showed modest variation, with Groups 1, 9, and 10 showing slightly lower frequencies (around 15–20%) compared to others. (H) Kinetics of anti-adenovirus serotype 5 (AdV5) neutralizing antibody development in immunized BALB/c mice. Serum samples collected at baseline (day 0) and post-boost (day 35) were assessed for neutralizing antibodies against the AdV5 vector using virus neutralization testing. Each symbol represents a different experimental group (corresponding to Groups 1–10 as defined in the main study), with individual data points shown and error bars indicating standard error of the mean. (I) Representative flow cytometry plots illustrating the systematic gating strategy employed for T cell subset analysis. Initial gating identified the total lymphocyte population, followed by singlet discrimination, and then identification of CD3 + T cells, which were further subdivided into CD4 + T helper and CD8 + T cytotoxic cells.