## 3 Results
### CoroVaxG.3 Induces a Potent and Durable T Cell Response  
The generation and persistence of memory T cells provides life-long protection against pathogens and, in particular, the induction of virus-specific CD8+ T cell responses has the potential to improve the efficacy of vaccination strategies [37; 38]. In order to characterize the cellular immune response induced by a single immunization with CoroVaxG.3, we assessed the IFN-\(\gamma\) production by isolated splenocytes after a specific ex vivo re-stimulation with Spike peptide pools, using CoroVaxG.5 again as a comparator. We observed an early strong primary immune response in vaccinated mice at 14 days post-immunization, showing a similar range of IFN-\(\gamma\) secreting cells in both vaccinated groups (Figure 4a). Remarkably, IFN-\(\gamma\) secretion was induced up to 140 days following vaccination, with no significant differences observed between the two CoroVaxG vaccines (Figure 4b). The administration of the Ad.C vector did not induce IFN-\(\gamma\) production at any of the assessed time points (Figure 4a,b). Figure 3: Immunogenicity and long-term humoral response induced by Ad-vectored vaccines. (**a**) Schedule of immunization and sample collection. Six-week-old BALB/c mice (\(n=5\)/group) received immunizations with \(10^{9}\) or \(10^{10}\) vp of an Ad-vectored vaccine. Sera were collected and assayed at the indicated time points (t.p.v. = time post vaccination). Animals were euthanized at 20 weeks post-vaccination (long term). Serum samples were used to (**b**) titer specific anti-S IgG, and (**c**,**d**) to assess the IgG1 and IgG2a anti-S concentration 28 days post-vaccination. The blue symbols correspond to CoroVaxG.5; the red symbols to CoroVaxG.3; and the grey and yellow symbols to Ad.C; the light symbols correspond to \(10^{9}\) vp and the dark symbols to \(10^{10}\) vp. (**b**) Data are means \(\pm\) SEM (standard error of the mean). * \(p\) \(<\) 0.05; two-way ANOVA with Bonferroni correction (**c**,**d**). The box plots show the median, 25 th and 75 th percentiles, and the whiskers show the range. *** \(p\) \(<\) 0.001; one-way ANOVA with Brown–Forsythe test. The identification of distinct memory T cells is usually based on the differential cell surface expression levels of CD44 and CD62L. Effector-memory T cells (TEM) located in secondary lymphoid organs can be identified as CD44 high CD62 Llow while, splenocated central-memory T cells (TCM) are identified as CD44 high CD62 Lhigh [39; 40; 41; 42]. We observed a remarkable induction of CD44 high CD62 Lhigh CD8+ cells in splenocytes of vaccinated groups compared to control groups at day 140 after a single-shot vaccination (Figure 4c,d). The proportion of CD44 high CD62 Llow CD8+ did not change in vaccinated mice at this time point. Ex vivo stimulated splenocytes in the Ad.C group showed levels of CD8+ cells expressing CD44 high CD62 Lhigh similar to those observed in naive mice or unstimulated splenocytes (Figure 4c,d). Collectively, the data show that mice vaccinated with both vaccines developed an effective long-lasting memory T-cell response against SARS-CoV-2. Figure 4: Cellular immune response induced by Ad-vectored vaccines. BALB/c mice received \(10^{9}\) or \(10^{10}\) vp of an Ad-vectored vaccine (blue: CoroVaxG.5; red: CoroVaxG.3; grey: Ad.C; green: Naïve) and were sacrificed after 14 days (\(10^{10}\) vp) or 140 days (\(10^{9}\) vp). Cells secreting IFN-\(\gamma\) per million of splenocytes were determined by ELISPOT at **(a)** 14 days. **(b)** 140 days post-immunization. The samples were analyzed in duplicates. The results of each group are expressed as the mean of spot-forming units (SFU). For FACS analysis, splenocytes were stained with anti-CD8\(\alpha\), anti-CD62L and anti-CD44 fluorochrome-conjugated antibodies. Stained splenocytes were subjected to flow cytometry analysis to quantify memory T cells (TCM: CD44 high CD62 Lhigh and TEM: CD44 high CD62 Llow) **(c,d)**. **(c)** Data are expressed as percentage of total CD8+ cells; unstimulated controls (black dots) were included.