## 3 Results
### CoroVaxG.3 Induces Neutralizing Antibody Responses against VOC  
To evaluate the functional quality of vaccine-generated Spike-specific antibodies, we used a pseudovirion-based neutralization assay (PBNA) to test the ability of sera from immunized mice to neutralize the entry of the pseudovirus-bearing Spike on their surface. Sera collected from mice at days 14, 28 and 140 after vaccination were tested for the presence of SARS-CoV-2-specific neutralizing antibodies (nAbs). NAbs against SARS-CoV-2 were detected in mice immunized by both CoroVaxG.3 and CoroVaxG.5. The resulting SARS-CoV-2-neutralizing activity at all-time points assayed was statistically significant (\(p<0.0001\)) compared to the undetectable nAbs in the Ad.C control group, with no significant differences between the vaccines. Remarkably, this neutralizing effect was observed even after 140 days of the single-dose vaccination (Figure 5a).  
To evaluate the potential resistance of new variants to the neutralization elicited by CorovaxG.3, we assessed the neutralization activity against SARS-CoV-2-pseudotyped viruses containing the Spike protein of the reference B.1 strain (wild type; D614G), as well as two prevalent circulating variants in our region, B.1.1.7 (alpha, first identified in the UK), P.1 (gamma, first identified in Manaos, Brazil) and B.1.617.2 (delta, first identified in India). Sera from eight mice were obtained at day 28 after vaccination with \(10^{10}\) vp of CoroVaxG.3, and sera from four naive mice were used as a control. All naive sera showed undetectable levels of neutralization against the tested SARS-CoV-2 variants (IC50 \(<25\)). The neutralizing antibody titers elicited against the wild-type strain (GMT = 870.7, CI 95% 384.8-1970) showed a slight decrease (1.4 fold, \(p<0.01\)) versus B.1.117 lineage (GMT = 627.1, CI 95% 402.9-976.2) and a larger but still moderate decrease versus the P.1 (2.6 fold, \(p<0.01\); GMT = 334, CI 95% 232.5-480) and B.1.617.2 lineages (3.5 fold, \(p<0.01\); GMT = 246.3, CI 95% 166.4-364.4) (Figure 5b). The assessment of the same sera samples from day 28 used for the PBNA studies confirmed that CoroVaxG.3 was able to elicit neutralizing antibodies against the authentic P.1/gamma VOC (Figure 5c).  
Figure 5: Elicitation of neutralizing antibodies by Ad-vectored vaccines. **(a)** Sera from animals vaccinated with the higher dose were used to measure SARS-CoV2 neutralizing antibodies by a Pseudovirus-Based Neutralization Assay. The box plots show the median, 25 th and 75 th percentiles, and the whiskers show the range. Comparisons were performed by a Kruskal-Wallis test, followed by [PERSON]’s multiple comparisons. **(b)** Changes in reciprocal serum neutralization IC50 values of CoroVaxG.3 vaccinated mouse sera (28 days post-vaccination) against SARS-CoV-2 variants of concern. The Fold change in Geometric Mean Titers relative to the WT is written above the p values. The statistical analysis was performed using a Wilcoxon matched-pairs signed rank test. Two-tailed p values are reported. **(c)** Neutralization against the authentic P.1/gamma VOC of CoroVaxG.3-vaccinated mouse sera (28 days post-vaccination). The box plots show the median, 25 th and 75 th percentiles, and the whiskers show the range. \({}^{**}p<0.01\), \({}^{***}p<0.0001\); two-tailed Mann-Whitney U test.