The ability of genetically detoxified lipopolysaccharide (LPS) to stimulate adaptive immune responses is an ongoing area of investigation with significant consequences for the development of safe and effective bacterial vaccines and adjuvants. One approach to genetic detoxification is the deletion of genes whose products modify LPS. The msbB1 and msbB2 genes, which encode late acyltransferases, were deleted in the Shigella flexneri 2a human challenge strain 2457T to evaluate the virulence, inflammatory potential, and acquired immunity induced by strains producing underacylated lipid A. Consistent with a reduced endotoxic potential, S. flexneri 2a msbB mutants were attenuated in an acute mouse pulmonary challenge model. Attenuation correlated with decreases in the production of proinflammatory cytokines and in chemokine release without significant changes in lung histopathology. The levels of specific proinflammatory cytokines (interleukin-1 [IL-1], macrophage inflammatory protein 1␣ [MIP-1␣], and tumor necrosis factor alpha [TNF-␣]) were also significantly reduced after infection of mouse macrophages with either single or double msbB mutants. Surprisingly, the msbB double mutant displayed defects in the ability to invade, replicate, and spread within epithelial cells. Complementation restored these phenotypes, but the exact nature of the defects was not determined. Acquired immunity and protective efficacy were also assayed in the mouse lung model, using a vaccination-challenge study. Both humoral and cellular responses were generally robust in msbB-immunized mice and afforded significant protection from lethal challenge. These data suggest that the loss of either msbB gene reduces the endotoxicity of Shigella LPS but does not coincide with a reduction in protective immune responses.Shigellosis, or bacillary dysentery, is an acute colitis caused by Shigella flexneri, a gram-negative enteroinvasive bacterium that is transmitted to humans via the fecal-oral route. Shigella triggers its uptake into the M cells of the lower intestine, where they are taken up by the underlying antigen-presenting cells (macrophages and dendritic cells) (18). Shigella bacteria are released from macrophages after inducing cell death (12,29) and invade the surrounding enterocytes, where they begin to multiply and spread to adjacent cells. Effector proteins secreted through a molecular-needle-like complex called the type III secretion system (TTSS) mediate the processes of macrophage cytotoxicity, enterocyte invasion, and modulation of the host cell immune response. The TTSS and associated effector proteins are encoded on a large virulence plasmid that is present in all invasive strains of Shigella (46). During replication and dissemination in host cells, components of the bacterial cell wall (lipopolysaccharide [LPS] and peptidoglycan) are released, inducing proinflammatory cytokines and chemokines which activate the innate immune response (reviewed in reference 31). Although the immune mechanisms of protection remain relatively undef...