Abstract Streptococcus pneumoniae (Spn) is a major respiratory pathogen that causes a spectrum of non-invasive (otitis media, sinusitis, pneumonia) as well as invasive (sepsis, meningitis) diseases. Despite a decline in the overall Spn disease burden caused by currently available vaccines (Spn/pneumococcal conjugate vaccines: PCVs), Spn diseases continue to occur and remain a significant medical problem. Due to the emergence of new, replacement serotypes (STs), Spn protein-based vaccines that aim to protect against Spn diseases in a serotype-independent manner are considered to be the best solution. However, there is a pressing need to develop novel adjuvants that can enhance antibody response and its biological function directed against Spn proteins antigens, given the target populations of these vaccines are children, elderly, and immunocompromised individuals. We developed a trivalent protein vaccine involving two well-known Spn vaccine candidates, N-terminal pneumococcal surface protein A (PspA) and domain 4 of pneumolysin (PlyD4), genetically fused with diphtheria The purpose of choosing N-PspA and PlyD4 is based on their prior characterization in animal models of vaccination with demonstratable efficacy against Spn colonization and sepsis. Therefore, genetic fusion of CRM197 to proven Spn candidates will help compare the CRM197 driven enhancement of immunogenicity and protective efficacy with published work and to expand the platform to include other promising vaccine candidates subsequently. detoxified antigen CRM197 (CRM197-N-PspA-PlyD4). Our preliminary data shows that the genetic fusion of CRM197 significantly enhanced (several folds) the antibody titers against N-terminal PspA. Additionally, the genetic fusion (trivalent vaccine) led to a significant increase in the binding of antisera to Spn bacteria, a measure of antibody functionality. Furthermore, the incubation of Spn bacteria with trivalent antisera significantly reduced Spn adherence to A549 cells. Based on our preliminary data, we hypothesize that “Genetic fusion of CRM197 enhances the memory T-cell response to Spn antigens, leading to enhanced antibody functionality and protection against Spn colonization and disease.” This hypothesis will be tested in two structured aims that will establish the role of CRM197 in enhancing the immunogenic and protective efficacy of Spn antigens, N-PspA and PlyD4. The findings of the proposed work could lead to a broader utilization of the adjuvant effect of CRM197 in enhancing the immune response to Spn as well as non-Spn protein antigens, leading to a more robust T-cell dependent immune response and protective effect.