The bacterial pathogen Streptococcus pneumoniae kills more than 1 million people each year. Although pneumococcal vaccines exist, current vaccines only protect against a small subset of the 100+ known serotypes. We have developed a new serotype-independent whole cell-inactivated pneumococcal vaccine. Central to the success of this vaccine approach is the expression of antigens that reflect those expressed during disease. Throughout the course of a pneumococcal infection, metal ions are restricted by host metal binding proteins. In the present study, we aimed to investigate whether sequestration of metal ions during vaccine manufacture improved the immunogenicity of the inactivated vaccine. The S. pneumoniae Rx1∆lytAPdT attenuated strain was cultured with or without a metal chelator (EDTA) and the metal content was assessed by inductively coupled plasma-mass spectrometry. RNA sequencing was performed to understand the broad changes in gene expression induced by EDTA. The vaccine was generated by inactivation of the bacteria with gamma irradiation and mice were immunised by intramuscular injection. Antibody responses were extensively profiled by ELISA, flow cytometry, and against a multi-antigen microarray of pneumococcal proteins. S. pneumoniae cultured with EDTA displayed reduced manganese and zinc abundance, which was associated with changes in the corresponding metal-responsive antigenic genes. EDTA-treated vaccines showed improved immunogenicity in mice. Antibodies from mice immunised with the EDTA-treated vaccine displayed increased binding to the bacterial surface and exhibited differences in antigen recognition. This work highlights the potential to apply molecular knowledge of host-pathogen interactions to improve vaccine antigen profile via simple changes to the manufacturing process.