Shigella spp. are a major cause of diarrhea and dysentery in children under 5 years old in the developing world. The development of an effective vaccine remains a public health priority, necessitating improved understanding of immune responses to Shigella and identification of protective antigens. We report the development of a core Shigella proteome microarray consisting of 2,133 antigen targets common to all Shigella species. We evaluated the microarray with serum samples from volunteers immunized with either an inactivated whole-cell S. flexneri serotype 2a (Sf2aWC) vaccine or a live attenuated S. flexneri 2a vaccine strain (CVD 1204) or challenged with wild-type S. flexneri 2a (Sf2a challenge). Baseline reactivities to most antigens were detected postintervention in all three groups. Similar immune profiles were observed after CVD 1204 vaccination and Sf2a challenge. Antigens with the largest increases in mean reactivity postintervention were members of the type three secretion system (T3SS), some of which are regarded as promising vaccine targets: these are the invasion plasmid antigens (Ipas) IpaB, IpaC, and IpaD. In addition, new immunogenic targets (IpaA, IpaH, and SepA) were identified. Importantly, immunoreactivities to antigens in the microarray correlated well with antibody titers determined by enzyme-linked immunosorbent assay (ELISA), validating the use of the microarray platform. Finally, our analysis uncovered an immune signature consisting of three conserved proteins (IpaA, IpaB, and IpaC) that was predictive of protection against shigellosis. In conclusion, the Shigella proteome microarray is a robust platform for interrogating serological reactivity to multiple antigens at once and identifying novel targets for the development of broadly protective vaccines.IMPORTANCE Each year, more than 180 million cases of severe diarrhea caused by Shigella occur globally. Those affected (mostly children in poor regions) experience long-term sequelae that severely impair quality of life. Without a licensed vaccine, the burden of disease represents a daunting challenge. An improved understanding of immune responses to Shigella is necessary to support ongoing efforts to identify a safe and effective vaccine. We developed a microarray containing >2,000 proteins common to all Shigella species. Using sera from human adults who received a killed whole-cell or live attenuated vaccine or were experimentally challenged with virulent organisms, we identified new immune-reactive antigens and defined a T3SS protein signature associated with clinical protection.

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