Mode of action of nanoparticles as carriers for bacterial glycoconjugate vaccines

Glycoconjugate vaccines are among the most effective and safe solution for preventing bacterial infections like those caused by Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae type b. These vaccines consist of a capsular polysaccharide, or a shorter chemically synthesized fragment, covalently linked to a carrier protein. The chemical conjugation greatly enhances the immunogenicity of the polysaccharide, which would otherwise be weakly immunogenic on its own, converting the immune response from T-cell independent to T-cell dependent resulting in a more robust immune reaction. However, improvements are needed to address suboptimal immune responses to certain bacterial capsular carbohydrates, and to reduce dosage frequency while increasing antibody persistence. In this context, nanoparticles (NPs) emerge as a promising tool vaccine. The geometric and orderly structure of nanoparticles allows the conjugation of multiple copies of the same antigen or different antigens on these surfaces. This results in a structured matrix with defined orientations that can potentially mimic the repetitiveness, geometry, size, and shape of host-pathogen surface interactions. This behavior also enhances uptake by antigen presenting cells, facilitates B cells cross-linking, increases T cells engagement and leads to durable immune response with high affinity antibodies production. Many studies have been focused on the immune mechanisms of NP-based vaccines for protein antigens. However, further research is needed to understand the role of nanoparticles as carrier for bacterial carbohydrates. The objective of this PhD project was to synthesize nanoparticles displaying saccharide antigens from pathogenic bacteria and to study their immunological mechanism of action through the development of in vivo models and in vitro assays. The research aimed to provide insights into both innate and cellular immune responses to glyco-NPs, elucidating how the physicochemical characteristics of nanoparticles can impact the maturation and persistence of immune responses specific for the different conjugated glycans. To achieve this goal, the optimization of the group B Streptococcus glycoconjugate vaccine developed by GSK Vaccines, was selected as a case study. This vaccine, available in trivalent or hexavalent formulations with capsular polysaccharides conjugated to CRM197, has demonstrated immunogenicity in clinical trials nevertheless individuals naïve to certain serotypes have shown low immune responses, requiring a booster dose. Moreover, this vaccine could be used to induce an antibody response in mothers that can be transferred to the fetus during pregnancy. Therefore, this highlights the necessity of improving certain glycoconjugate vaccines. On this basis, the study proposes replacing the CRM197 carrier with self-assembling protein nanoparticles to enhance the efficacy and duration of the immune response. Specifically, various NPs including ferritin, mI3, 1B5S, and virus-like particles such as Qβ and P22 were studied for their structural properties and impact on the immune response. These NPs were fully physiochemically characterized and conjugated with capsular polysaccharides from Group B Streptococcus serotype II (PSII). When tested in a preclinical mouse model, the PSII- NPs induced robust IgG titers upon a single-dose administration, outperforming the traditional vaccine PSII-CRM197, which requires multiple doses. Specifically, one dose of PSII-NPs elicited IgG titers comparable to titers elicited by two doses of the reference vaccine. Moreover, larger NPs (i.e. PSII-Qβ and PSII-P22) induced a strong differentiation of B cells into plasmablasts and recruited higher frequencies of T follicular helpers in germinal centers. The recruitment of Tfh cells was notably evident for PSII-Qβ, a glyco-NPs characterized by the presence of encapsulated ssRNA, a TLR agonist used as immunomodulator. Additionally, the glyco-NPs showed a strong germinal center expansion which translated into production of antigen-specific memory B cells, maintaining IgM levels out of germinal center for up to 90 days post-immunization. Furthermore, the mechanism of action of these nanoparticles was also characterized in vitro, using human peripheral blood mononuclear cells from healthy donors, stimulated with both glyco-NPs and traditional glycoconjugate. This approach was used to study their interaction with Antigen Presenting Cells (APCs). Multi-parametric flow cytometry demonstrated that glyco-NPs interacted with professional and non-professional antigen presenting cells, such as B cells, compared to the reference vaccine, PSII-CRM197. Further analysis performed on purified human B cells revealed that the nanoparticles predominantly engaged with naïve B cells, specifically CD27- IgM-. The in vitro studies highlighted the potential of NPs to activate B cells, which differ from conventional carrier. Overall, this PhD project lays the groundwork for the next-generation design of multivalent bacterial vaccines and provides encouraging preclinical evidence for the feasibility and effectiveness of a single-dose vaccination strategy. The findings suggest that glyco-NPs with optimized properties can overcome the limitations of traditional glycoconjugates, offering a promising path forward for single-dose vaccine formulations.