## 3 Results  
### Vaccine Construction and In Vitro Studies  
In order to induce Spike immunodominance, the first step in our vaccine design was to select the most appropriate promoter to transcriptionally regulate Spike expression. Other anti-COVID 19 vaccines based on replication-incompetent AdV incorporated different alternatives of the early intermediate CMV promoter to drive Spike transcription [29; 30; 31; 32]. However, it was shown that the CMV promoter can be silenced due to methylation, not only when it is incorporated in viral vectors that integrate to the host genome [33], but also after the in vivo administration of a replication-deficient adenoviral vector in the rat muscle [34]. After an extensive search in the literature, we decided to synthesize different versions of a hybrid promoter that included a CMV enhancer, the chicken \(\beta\)-actin promoter and a chimeric intron that contains the \(5^{\prime}\) splice donor of the chicken \(\beta\)-actin \(5^{\prime}\)UTR and the \(3^{\prime}\) splice acceptor of the minute virus of mice (MVM). This hybrid promoter was shown to drive gene expression in the rat CNS following in vivo transduction with AAV vectors [35]. We designed different versions that differed mainly in the size of the CMV enhancer and the \(\beta\)-actin promoter. In addition, we incorporated stop codons in the three open reading frames after an ATG codon \(3^{\prime}\) splice acceptor of MVM that could interfere with Spike ATG. Two initial versions of the hybrid promoters named Pr1 and Pr2 were cloned into the pShuttle(PS)-IXP-Luc vector. Luciferase expression driven by Pr2-Luc was around 24-fold (\(p<0.0001\)) higher than the control SV40 promoter, compared to a mere 1.6-fold increase over the SV40 promoter induced by Pr1-Luc (Figure 1a). Based on these data, we decided to move on with Pr2 for the design of further constructs. Next, we aimed to establish the adenoviral vector's capacity to transduce target cells following fiber knob exchange. We initially transduced human rhabdomyosarcoma cells Hs 729T (as an example of muscle cells) and human monocytes THP-1, with replication-deficient adenoviruses hAdV5-Luc and the hybrid hAdV5.3-Luc expressing the fiber knob domain of hAdV3. Using luciferase as a surrogate marker, we observed an almost 40-fold induction and more than a 100-fold induction in Hs 729T and THP-1 cells, respectively, with hAdV5.3-Luc compared to hAdV5-Luc (Figure 1b,c). In addition to Pr2, we included the WPRE mRNA stabilizer [26] to post-transcriptionally regulate the Spike expression in our vaccines' constructs. Among different engineered vaccine candidates expressing various Spike sequences, we selected CoroVaxG.3 for further in vitro and in vivo studies. This replication-deficient hAdV-based vaccine candidate encodes the full-length, codon-optimized sequence of the SARS-CoV2 Spike protein (B.1 lineage; D614G) stabilized in its prefusion state (with the exchange of two prolines). CoroVaxG.3 has been additionally modified by engineering the fiber knob domain of hAdV3 instead of the knob domain of hAdV5. As a comparator we used CoroVaxG.5, that retained the native hAdV5 fiber knob domain. A replication deficient hAdV5 vector that carried no transgene was used as control (named Ad.C). In order to establish if exchanging the fiber knob domain in CoroVaxG.3 might indeed improve muscle and dendritic cell transduction, we transduced Hs 729T cells and THP-1 monocytes with CoroVaxG.5 and CoroVaxG.3. We observed a dramatic increase in Spike expression in Hs 729T cells transduced with CoroVaxG.3 compared to CoroVaxG.5 (Figure 2a,d). Moreover, CoroVaxG.3 was able to induce a Spike expression in THP-1 monocytes that was at least six times higher compared to CoroVaxG.5 (Figure 2b,e). Interestingly, CoroVaxG.3 induced a higher increase in Spike expression in THP-1 cells induced to differentiate to immature dendritic cells compared to undifferentiated THP-1 monocytes (compare Figure 2b,c).