Polysaccharide-based vaccines are widely used to protect against Streptococcus pneumoniae (S. pneumoniae) infections in infants and the elderly. However, their use is limited by strain specificity, which restricts both their geographical and economical utility. There is an urgent need for protein-based vaccines that are likely to provide broader, more economical protection against the global burden of pneumococcal disease. In this paper, we describe the pre-clinical development of a multi-subunit protein vaccine that can be manufactured efficiently and economically to meet this need. Genetically engineered Streptococcus pneumoniae TIGR4 B7.1 PlyD6 cell substrate was constructed to deliver non-toxic Ply.