## 3 Results
### CoroVaxG.3 Induces Robust and Balanced Antibody Responses against SARS-CoV-2  
Next, we aimed to establish whether fiber exchange might have affected the in vivo immune response induced by CoroVaxG.3. Thus, experimental groups of 6 to 8-week-old BALB/c mice were immunized by intramuscular injection with \(10^{9}\) or \(10^{10}\) viral particles (vp) of CoroVaxG.3, using as a comparator mice vaccinated with CoroVaxG.5 and with the control Ad.C at the same vp dose. Serum samples were collected at different time points after immunization (Figure 3a), and the IgG responses against Spike were evaluated by an ELISA.  
CoroVaxG.3 induced high levels of Spike-specific IgG as early as 2 weeks after a single inoculation, very similar to the levels induced by CoroVaxG.5, while the levels induced by Ad.C were negligible (Figure 3b, \(p<0.05\) of any vaccine vs. same dose of Ad.C at all-time points). No significant differences were observed in antibody levels between the two CoroVaxG vaccines or between doses, starting from 28 days post-vaccination. Moreover, antibody titers remained stable up to 140 days post-vaccination (maximum time point assayed) without evidence of waning. Thus, a single i.m. immunization with CoroVaxG.3 was capable of generating a robust and long-lived S-specific antibody response, with no significant differences in antibody levels between doses. These levels were similar to those  
Figure 1: Luciferase expression following cell transfection or transduction with adenoviral vectors. (**a**) HEK293T cells were transiently transfected with plasmidic vectors encoding for different candidate promoters upstream of luciferase. An empty vector was included as a control. The promoter activity was evaluated by luminescence, using a luciferase gene reporter assay. The relative firefly luciferase units were normalized by renilla and expressed as fold induction over the SV40 promoter. The bars represent means \(\pm\) SEM (\(n=3\)). ***** *  
induced by CoroVaxG.5, showing that fiber exchange did not hamper the immune response induced by CoroVaxG.3.  
To reduce the theoretical risk of vaccine-associated enhanced respiratory disease, associated with a Th2-skewed response [36] we further assessed the concentration of S-specific IgG1 and IgG2a subclasses induced by CoroVaxG.3 as a readout of Th1 and Th2 responses. As a comparator we used sera from mice vaccinated either with CoroVaxG.5 or with the SARS-CoV2-S RBD domain in Freund's adjuvant (FA). RBD + FA vaccinated mice showed a low IgG2a/IgG1 ratio consistent with the production of high levels of IgG1 and a clear skew toward a Th2 phenotype (Figure 3c,d). Interestingly, using sera samples obtained at day 28, we observed that CoroVaxG.3 and CoroVaxG.5 induced a high IgG2a/IgG1 ratio indicative of a Th1-biased response, although the IgG2a/IgG1 ratio was slightly higher for CoroVaxG.5 than for CoroVaxG.3 (Figure 3d, \(p<0.001\)). This difference was mainly due to a higher production of IgG1 by CoroVaxG.3, with similar levels of IgG2a induced by both vaccine candidates and doses (Figure 3c). Similar data were obtained with sera collected 14 days after vaccination (Figure S1).  
Figure 2: Spike expression following in vitro transduction with AdV-vectored vaccines. (**a–c**) WB assay performed on Hs 729T, THP-1 and immature dendritic cells (iDC) after 48 h of transduction with CoroVaxG.5, CoroVaxG.3 and control Ad.C. Spike expression was detected using an anti-Spike antibody. \(\beta\)-actin was used as a loading control. (**d–f**) The bar graphs represent a semi-quantification of the WB assay normalized by \(\beta\) actin. ***\(p<0.001\); one-way ANOVA.