## 4 Discussion  
In the present study we provide evidence of a novel, replication-deficient hAdV-based vaccine, aimed at achieving Spike immunodominance to skew the immune response toward the transgene and reducing the vector-targeted immune response that will occur despite the vector used. The immunodominance of Spike was tackled from different angles: (i) optimizing Spike expression using a potent promoter and an mRNA stabilizer; (ii) stabilizing Spike in a prefusion conformation to enhance Spike immunogenicity [43; 44]; (iii) engineering the hAdV5 fiber to express the fiber knob domain of hAdV3 to enhance the transduction of muscle and dendritic cells. We observed a dramatic enhancement of Spike expression in human muscle cells as well as in human monocytes and dendritic cells with CoroVaxG.3 compared to CoroVaxG.5. Since in both vaccine candidates Spike expression was under the regulation of Pr2 and the WPRE mRNA stabilizer, we concluded that the enhanced Spike expression in human muscle derived cells, monocytes and dendritic cells was due to the fiber knob exchange, as demonstrated also through the studies using replication-deficient vectors expressing luciferase. CoroVaxG.3 was able to induce strong humoral and cellular immune responses similar to the levels previously reported in other preclinical animal studies, including the production of anti-SARS-CoV2 neutralizing antibodies [43; 44; 45]. Despite the significant difference in Spike expression in the human targeted cells in vitro, the in vivo potency of both vaccines in mice was quite similar, although CoroVaxG.3 induced higher levels of IgG1 (see below). A likely explanation is that CoroVaxG.3 uses alternative receptors in mice to those used to transduce human cells. Indeed, murine DSG-2 is not recognized by hAdV3 or vectors derived from this hAdV serotype [46]; thus, murine cells' transduction in vivo likely occurred through alternative receptors. Moreover, although the hAdV expressing the serotype 3 fiber were shown to efficiently transduce human monocytes and skin resident DC (Langerhans cells and skin DC), the transduction occurred through binding to the B7-family members CD80/CD86 without affecting subsequent T cell stimulation [47], suggesting that CoroVaxG.3 can transduce target cells through alternative receptors. In this regard, it has been shown that the development of protective cellular immunity induced by adenoviral-based vaccines correlated with the magnitude and persistence of the transgene expression [48]. Thus, even if we were unable to see remarkable differences with CoroVaxG.3 and G.5 vaccines in the immune response in the mice model, it is likely that a persistently high Spike expression in vaccinated individuals following immunization with CoroVaxG.3 would enable the induction of a sustained and enriched immune response. One of the main initial concerns regarding vaccine development was the possibility of vaccine-related diseases, either induced by antibodies (antibody-dependent enhanced disease, ADE), which is associated with the presence of non-neutralizing antibodies, or vaccine-associated enhanced respiratory disease (VAERD), linked to inflammation induced by Th2-skewed immune responses [49; 50]. On the other hand, the induction of an unbalanced inflammatory Th1-biased response by some Ad vectors, due to an exacerbated production of type I interferons, has been suggested to impair transgene expression levels, dampening subsequent humoral immune responses [51]. Therefore, the induction of a balanced immune response is desirable. Interestingly, and despite the fact that both vaccine candidates induced a robust and long-lasting humoral and cellular immune response, CoroVaxG.3 elicited higher levels of IgG1, and hence a more balanced Th1/Th2 ratio, evidencing an optimal immune profile in mice. The difference in the subclass profile between the two vaccines might be related to the differential tropism observed in the in vitro studies.