RESEARCH SUMMARY Shigella causes a high burden of dysentery globally. Children younger than 5 years of age, particularly toddlers 2-3 years old, living in impoverished areas lacking clean water and sanitation are the most affected. Repeated infection results in lifelong health impairment and disability. Shigella also causes outbreaks in industrialized countries (daycare centers and medical institutions) and is a serious threat due to its multi-drug resistance. There is no approved vaccine. Most of the existing candidates rely on immunity to the Shigella O-antigen, which is serotype-specific (there are 4 species and >50 diarrheagenic Shigella serotypes). Shigella O-polysaccharide (OPS) conjugates (e.g., SF2a-TT15 and Flexyn2a) are the most clinically advanced. These vaccines are limited in their use of irrelevant (non-Shigella) proteins as carriers. SF2a-TT15 uses tetanus toxoid, which is included in many vaccines given to children of the target age and is known to suppress responses to other vaccine components. Flexyn2a uses rEPA as carrier but had modest efficacy in a recent challenge study (did not meet study endpoints). An earlier S. sonnei OPS-rEPA was effective in older children but not in toddlers (target age). In response to RFA-AI-22-037, the University of Maryland (UMB), in partnership with Vaxcyte, proposes to develop a bivalent (Shigella flexneri 2a and Shigella sonnei) OPS conjugate vaccine using Shigella IpaB, a Type III secretion protein that is highly conserved among all Shigella spp., as carrier. IpaB is exceptionally immunogenic and is a known broadly protective antigen; serum IgG (IgG1) levels and IpaB antibody function were found to be positively associated with reduced disease in controlled human challenge studies. For the first time, we have produced soluble, immunoreactive IpaB at a high (industrial) yield using Vaxcyte’s cell-free protein expression system. IpaB is conjugated to S. flex 2a and S. sonnei OPS using Vaxcyte’s site- specific conjugation technology. S. flex 2a OPS-IpaB given to mice intramuscularly with Alum on two occasions 28 days apart, elicited robust immune responses and afforded 78% protection against S. flex 2a (homologous) and 56% against S. sonnei (heterologous) lethal pulmonary Shigella challenge. Vaccine efficacy against S. flex 2a was higher as compared with S. flex 2a OPS-conjugated to CRM197 (a diphtheria toxoid mutant). This proposal consists of three aims to optimize process development, formulation, and scale-up production of S. flex 2a- and S. sonnei OPS-IpaB conjugates (Aim 1), evaluate the immunogenicity and vaccine efficacy in two animal models: mice (pulmonary challenge) and guinea pigs (rectocolitis infection) (Aim 2), and identify immune operatives associated with protective immunity through passive transfer experiments and a suite of in vitro functional assays (Aim 3). A bivalent Shigella-OPS-IpaB vaccine is expected to be well tolerated and to have enhanced protective capacity than exis...