Design of SARS-CoV-2 subunit vaccines via rational understanding of their immunological mechanism: Long-term SARS-CoV-2 immune memory and evaluation of RBD as a carrier for antibacterial glycoconjugate vaccines

The COVID-19 pandemic generated an unprecedented global effort to develop new vaccination strategies and produced extensive clinical evidence on the durability and qualitative features of immune memory induced by Spike-based antigens, including the SARS-CoV-2 receptor binding domain (RBD). These data support the presence of CD4+ T-cell epitopes within Spike/RBD that can provide T-cell help, a prerequisite for T-cell-dependent antibody responses. Since only a limited repertoire of carrier proteins is licensed for use in bacterial glycoconjugate vaccines, there is a rationale to explore alternative carriers able to provide robust CD4+ T-cell help to polysaccharide-specific B cells. This thesis aimed to evaluate the SARS-CoV-2 RBD as a candidate carrier protein for bacterial polysaccharides and to identify immune readouts relevant to benchmark the quality of carrier-driven responses. Two complementary approaches were integrated. First, two longitudinal observational cohort studies were analyzed to define design-relevant endpoints for carrier benchmarking. Homologous and heterologous vaccination regimens elicited durable immunity up to 21 months after immunization, including persistent anti-Spike IgG, class-switched Spike-specific memory B cells and functional CD4+ and CD8+ T cells, supporting the idea that SARS-CoV-2 derived antigens can be evaluated as candidate carriers in new glycoconjugates vaccines with the ability to efficiently stimulate a cellular response. These studies also highlighted the impact of prior infection on long-term immune features, including modulation of CD8+ cytokine responses in heterologous primary vaccination regimens. Second, an experimental workflow was established to enable controlled development and future benchmarking of RBD-based glycoconjugates Salmonella Typhimurium O-antigen (O-Ag) and Klebsiella pneumoniae K64 capsular polysaccharide (K-Ag) were purified and quality-controlled to analytical standards, showing low residual contaminants and supporting reproducible downstream conjugation. As a proof-of-principle for RBD-based feasibility, an O-Ag-RBD glycoconjugate was generated using CDAP-mediated activation and subsequently purified and characterized. Finally, a functional antibody assay was implemented as a mechanism-based endpoint by establishing a preliminary serum bactericidal assay (SBA) on pooled sera from glycoconjugate-immunized mice, measuring complement-mediated killing of K. pneumoniae K64. This functional framework provides the basis for future direct benchmarking of RBD against established carrier proteins under matched glycan loading and standardized input quality. Overall, this thesis links long-term immunophenotyping of Spike-based immune memory with an analytically controlled glycoconjugate development pipeline, proposing immune readouts and experimental benchmarks to evaluate whether SARS-CoV-2 RBD can serve as a novel carrier protein for anti-bacterial glycoconjugate vaccines.