AdV-based vectors have shown remarkable success in immunization against SARS-CoV-2 and Ebola virus in recent years. Beyond commonly used types like HAdV-C5 and -D26, the vast number of AdV types brings diverse possibilities to expand the vector library for future pandemic preparedness. When screening for suitable AdV types for immunization purposes, immunogenicity is an important consideration, referring to their ability to elicit an immune response in the host, including the activation of innate immunity and the initiation of downstream cellular immune responses through the involvement of dendritic cells. In the first study presented in this thesis, a systematic analysis was conducted of 40 AdV types, comprising 39 human AdV types and one animal-derived AdV type (ChAdOx), to evaluate their effects on the activation of murine bone marrow-derived dendritic cells (bmDCs) and CD8+ T cell proliferation. The findings revealed that AdV transduction significantly altered the immunophenotype and cytokine profiles of bmDCs, evidenced by substantial variations in the expression patterns of 11 bmDC surface markers and 13 secreted cytokines across different AdV types. Additionally, significant differences were observed in the effects of various AdV types on CD8+ T cell expansion, with some types (e.g., HAdV-F41, -A31, -C6, -E4, and -C2) showing minimal impact, whereas others (e.g., HAdV-D10, -D24, -D80, and -A18) exhibited strong inhibitory effects. Ultimately, HAdV-D8, -B7, -D33, -A31, and -D65 were identified as the most favorable AdV types. They are capable of inducing a favorable level of innate immune responses and did not impair CD8+ T cell proliferation, while exhibiting low seroprevalence and weak PF4 binding affinity as demonstrated in other studies. As such, these AdVs represent promising candidates for the development of future vaccine vectors.

AdV seroprevalence, alongside immunogenicity, is another metric for selecting AdV types for immunization. While AdV seroprevalence has been extensively explored in previous studies, longitudinal serum samples from the same cohort to dynamically monitor antibody responses to a broad range of AdV types have not been used. To address this, the second study included in this thesis obtained serial serum samples from a group of regular blood donors, with each donor providing two samples annually over a five-year period from 2018 to 2022. BAbs against 39 human AdV types were tested. The data showed that HAdV-specific antibody levels in longitudinal serum samples remained generally stable over time at the cohort level, although fluctuations were frequently observed at the individual level. In addition, antibody responses to certain clusters of AdVs exhibited highly similar reaction patterns, most notably for bAbs targeting HAdV-D25, -D26, and -D27, as well as HAdV-D24, -D73, -D75, and -D80. Correlation analysis suggested extensive cross-reactivity among AdV-specific antibodies.

AAV vector development for gene therapy has made remarkable strides in recent years, with seven AAV-based therapies approved for clinical use to date. Neuromuscular disorders (NMDs) represent a diverse group of conditions affecting various cell populations along the neuromuscular axis and encompassing multiple subgroups. Given the lack of effective alternative treatments and the predominantly monogenic nature of most NMD subgroups, AAV-based gene therapy presents a promising therapeutic option. However, pre-existing immunity against AAV can neutralize the vector during systemic administration, significantly limiting the sustained expression of therapeutic genes and thereby compromising the overall efficacy of gene therapy. Building on this premise, the third study included in the thesis examined the levels of binding and neutralizing antibodies against 18 AAV types, including 11 with wild-type capsids, 5 with peptide-modified capsids, and 2 with shuffled capsids, in serum samples from NMD patients. The analysis revealed that antibodies against AAV5, AAV9, AAV12, AAV7P2, AAV9A2, and AAV9P1 had the lowest overall seroprevalence. It was also observed that the effects of capsid peptide modifications and capsid shuffling on AAV-specific antibodies were inconsistent, and immune recognition and humoral neutralization were altered in a serotype-specific manner. Among the factors examined for their potential influence on the levels of AAV-specific antibodies, age emerged as the most prominent, with nAb levels increasing with age. In contrast, sex, the presence of NMD, and NMD subgroups showed no significant impact on AAV seroprevalence.