The Human Cytomegalovirus (HCMV) is a ubiquitously distributed member of the Herpesviridae family that establishes lifelong infection in humans. Seroprevalence in the human adult population is superior to 70% worldwide. HCMV is responsible for high morbidity and mortality in immunocompromised patients, and is the leading viral cause of congenital birth defects, affecting 0.7% of newborns with permanent sequelae such as mental retardation and hearing loss. The development of a HCMV vaccine has been deemed a national priority by the US National Academy of Medicine since 1999. Several clinical trials targeting different HCMV antigens, such as the glycoprotein (g)B, the pentameric complex or the phosphoprotein 65 (pp65), have been conducted. However, none of those conferred full sterilizing immunity and up to date there is no vaccine or monoclonal antibody (mAb) approved for clinical use. The employment of neutralizing antibodies to identify protective antigens, in association with structural vaccinology to stabilize an antigen of interest and scaffold presentation, to enhance immunogenicity, are required to target challenging pathogens such as HCMV. With a general methodology for rational vaccine design, combining identification of prevailing antigenic domains (ADs) with orthogonal technologies, we aimed at generating a HCMV candidate vaccine with an increased immunogenicity. Applying our approach to the immunogenic gB fusogen, we have not only characterized the immunogenicity of its individual ADs, but also demonstrated that gB domain I (AD5) is the main target of neutralizing antibodies. Through sophisticated antigen trimerization and stabilization techniques, to ensure proper antigen conformation, we were able to focus the antibody response to the key protective domain of the HCMV fusion glycoprotein. To cope with the ambitious purpose of further increase the immunogenicity of our candidate vaccine, we employed the multivalent antigen presentation properties of nanoparticle scaffolds. Indeed, presentation of multiple copies of an antigen in a repetitive array is known to drive a more robust humoral immune response than its soluble counterpart. Fusion of our stabilized antigen with different carriers allowed the generation of two HCMV candidate vaccines displaying multiple copies of the trimeric gB-AD5 antigen. We showed that multimeric AD5-Nanoparticles elicited up to 100-fold higher sera neutralization titer in mice, compared to the most studied recombinant gB vaccines. Collectively, these results illustrate with a medically relevant example, a general approach combining antigen discovery, protein engineering and scaffold presentation for modern development of subunit vaccines against complex pathogens.