Immune mediators of innate and specific resistance to Shigella flexneri infection

Abstract

Shigella sp. are Gram-negative facultative intracellular bacteria responsible for the majority of bacillary dysentery cases worldwide. The hallmark pathological feature of bacillary dysentery is an acute inflammatory reaction in the infected large intestine. A protective vaccine has been sought to prevent morbidity and mortality due to Shigella infection. Epidemiological and experimental data demonstrate that partial immunity develops following natural or experimental infections. Consequently, many live attenuated Shigella vaccine strains have been constructed and evaluated for safety and efficacy following the oral route of inoculation. The major limitation to these current vaccine strains is that they are too reactogenic when evaluated in humans. Thus, the focus of Shigella vaccine research has been to construct more attenuated vaccine strains; however the immune response and the mediators of protective immunity to Shigella infections have not been characterized.;Immune mediators of innate resistance to S. flexneri infection were examined by evaluating the susceptibility of mice with targeted deletions in each of specific aspects of the immune system to primary infection. Interferon-{dollar}\gamma{dollar} deficient mice exhibited greater than 5 orders of magnitude increased susceptibility, while mice deficient in subsets of B or T lymphocytes, or all B and T lymphocytes demonstrate no difference in susceptibility. The cellular source of interferon-{dollar}\gamma{dollar} for innate immunity was determined to be natural killer cells. The protective effects of interferon-{dollar}\gamma{dollar} could be reproduced in vitro by activating S. flexneri infected cells with this cytokine. For primary macrophages, a macrophage cell line, and a fibroblast cell line, decreased intracellular S. flexneri persistence was observed in interferon-{dollar}\gamma{dollar} activated cells.;The expression of the cytochrome bd terminal oxidase in S. flexneri was correlative with virulence as assessed in both in vitro and in vivo assays. S. flexneri mutants which overexpressed the cytochrome bd terminal oxidase formed larger bacterial plaques and had a 10-fold decreased lethal dose for intranasally infected mice; while a S. flexneri mutant defective in cytochrome bd expression (strain SSW202) formed smaller bacterial plaques and had a 100-fold increased lethal dose for intranasally infected mice. Prior inoculation with SSW202 conferred greater than 90% protective efficacy to subsequent homologous challenge in immunocompetent mice.;Mice vaccinated with SSW202 were used to study the mediators of specific immunity to S. flexneri infection. Depletion of all T lymphocytesin vaccinated mice or adoptive transfer of splenocytes from vaccinated mice to naive mice did not alter the susceptibility of these mice to S. flexneri infection, while passive transfer of serum from vaccinated mice to naive mice was able to transfer protection. The inability of vaccination to confer protection in B lymphocyte deficient mice and the ability of vaccination to confer protection in T lymphocyte deficient mice confirms the requirement for antibody, and independence of T lymphocytes, for protective immunity. Furthermore, we demonstrate that while interferon-{dollar}\gamma{dollar} is essential for innate resistance to Shigella infection, it is not required for specific immunity. These data will be useful in the future development and evaluation of Shigella vaccines.