ABSTRACTShigellais responsible for high burdens of diarrhea and dysentery globally. Children living in areas of endemicity are the most affected, and currently, there are no licensed vaccines to prevent shigellosis. Vaccine approaches have traditionally targeted the bacterial lipopolysaccharide as a protective antigen.ShigellaO-polysaccharide (OPS) conjugated to recombinantPseudomonas aeruginosaexotoxin A (rEPA) or tetanus toxoid (TT) is advanced in clinical evaluation. Adequate efficacy of these vaccines, particularly in the infant target group, remains to be demonstrated. A major limitation of the OPS-glycoconjugate concept is its limited coverage, since immunity to the O antigen is serotype specific, and there are multiple disease-causing serotypes. Another concern is the use of protein carriers already included in multiple other childhood vaccines. This study reports a novelShigellaOPS conjugate vaccine that uses theShigellainvasion plasmid antigen B (IpaB) as the carrier protein. IpaB is a virulence factor component of theShigellatype III secretion system and highly conserved amongShigellaserotypes. It is robustly immunogenic and a protective antigen. IpaB and IpaB containing nonnative amino acids (nnAA) were produced at large scale using cell-free protein synthesis. Incorporation of nnAA enabled site-specific conjugation of IpaB toShigella flexneri2a OPS using click chemistry, yielding OPS-IpaB glycoconjugate. Parenteral immunization of mice with the OPS-IpaB vaccine resulted in high levels of OPS- and IpaB-specific serum IgG and robust protection against lethalS. flexneri2a orShigella sonneichallenge. The OPS-IpaB vaccine is a promising new vaccine candidate with the capacity to confer broad protection against clinically relevantShigellaserotypes.IMPORTANCEDiarrhea caused byShigellaspecies results in long-term disability and mortality globally, disproportionally affecting younger children living in poor countries. Although it is treatable by antibiotics, the rapid and widespread emergence of resistant strains and the highly contagious nature of the disease compel the development of preventive tools. Currently, severalShigellaOPS conjugate vaccines are being evaluated in clinical studies, but these rely exclusively on immunity against the bacterial O antigen, which limits their coverage to only the immunizing serotype; multivalent vaccines are needed to protect against the most prevalent serotypes. This is the first report of a novelShigellaOPS-conjugate vaccine that usesShigellaIpaB as a carrier and protective antigen. This vaccine, administered parenterally, elicited robust immunity and protected mice against lethal infection byS. flexneri2a orS. sonnei. The OPS-IpaB vaccine is a promising candidate for evaluation in vulnerable populations.