Shigella flexneri is one of the leading causes of diarrheal disease in children under five in developing countries, yet currently there is no licensed vaccine to prevent disease. The primary aim of this study was to assess two new vaccine candidates in combination with CVD 1208S as a multi-component broad-spectrum vaccine. Additionally, the development of model systems for studying Shigella pathogenesis and profiling of host and bacterial responses served to advance our capacity to characterize the vaccine candidates. Host responses to the vaccines were measured using macrophage cytotoxicity assays, epithelial cell invasion assays, qRT PCR for transcriptional activity and enzyme-linked immunosorbent assays for cytokine secretion. The CVD 1208S vaccine was further evaluated for its impact on host gene expression using RNA sequencing. In addition to in vitro experiments, in vivo guinea pig and infant mouse studies were performed to assess Shigella specific antibody responses and protection against wild type challenge following immunization with each vaccine individually and as a trivalent formulation. In vitro studies demonstrated the new vaccine strains have a similar profile as the well-studied CVD 1208S vaccine. Guinea pig immunization studies revealed a robust induction of Shigella serotype specific IgA and IgG antibodies following immunization with each individual vaccine strain and a combined inoculum of all three. Challenge using the guinea pig Sereny test demonstrated protection against wild type S. flexneri serotypes 2a, 3a and 6 following immunizations with a mixed immunization including CVD 1208S, CVD 1213 and CVD 1215. Furthermore, the vaccines were safe and immunogenic in infant mice and experiments are ongoing to assess protection against wild type challenge. CVD 1208S was also used to evaluate three-dimensional and ex vivo models for Shigella pathogenesis. These data indicate that CVD 1213 and CVD 1215 are viable candidates for the creation of a multivalent vaccine that could confer broad-spectrum protection against S. flexneri infections. Moreover, global transcriptional analysis provides a broader view of host response to the vaccines as well as the effects of vaccine attenuation on bacterial gene expression. This data contributes to a broader profile of Shigella pathogenesis for future vaccine development.