ABSTRACT
Replication incompetent human Adeno (hAd)-based vectors are able to induce potent adaptive immune responses in animal models and humans, being of interest for both infectious diseases and cancer therapy or prevention. Different Adenovirus-based vaccines have been approved for human use, including those developed to contrast Covid-19 pandemic. Immunological potency of hAdvectors based on different serotypes can be influenced by the extent of pre-existing host immunity, different cell tropism and intracellular trafficking, also impacting the extent of innate immunity induction. Vaccines based on species C hAd are among the most potent, yet significant seroprevalence in humans limits their application into the clinic, with pre-existing neutralizing antibodies negatively affecting vectored vaccine immunogenicity and efficacy. Error prone PCR represents an efficient method to introduce random mutations by reducing the DNA polymerase fidelity, allowing directed evolution and improvement of target proteins. Since the majority of hAd neutralization determinants are located in the hypervariable regions (HVR) of the hexon, the major capsid protein, we generated seven different hexon-variant libraries with mutations in single or multiple HVRs from Group C hAd, through error prone PCR. Such libraries have been used to produce complex repertoires of hAd vectors potentially including variants with novel properties such as lower recognition by anti-hAd antibodies present in humans or reduced unwanted side effects in the vaccinees. These libraries will be surveyed with different screening strategies to identify novel variants with improved properties for both prophylactic and therapeutic vaccine applications.