Shigellosis is the leading cause of diarrheal disease, especially in children of low- and middle-income countries, and is often associated with anti-microbial resistance. Currently, there are no licensed vaccines widely available against Shigella, but several candidates based on the O-antigen (OAg) portion of lipopolysaccharides are in development. We have proposed Generalized Modules for Membrane Antigens (GMMA) as an innovative delivery system for OAg, and a quadrivalent vaccine candidate containing GMMA from S. sonnei and three prevalent S. flexneri serotypes (1b, 2a and 3a) is moving to a phase II clinical trial, with the aim to elicit broad protection against Shigella. GMMA are able to induce anti-OAg-specific functional IgG responses in animal models and healthy adults. We have previously demonstrated that antibodies against protein antigens are also generated upon immunization with S. sonnei GMMA. In this work, we show that a quadrivalent Shigella GMMA-based vaccine is able to promote a humoral response against OAg and proteins of all GMMA types contained in the investigational vaccine. Proteins contained in GMMA provide T cell help as GMMA elicit a stronger anti-OAg IgG response in wild type than in T cell-deficient mice. Additionally, we observed that only the trigger of Toll-like Receptor (TLR) 4 and not of TLR2 contributed to GMMA immunogenicity. In conclusion, when tested in mice, GMMA of a quadrivalent Shigella vaccine candidate combine both adjuvant and carrier activities which allow an increase in the low immunogenic properties of carbohydrate antigens.

Outer membrane vesicles (OMV) represent an innovative platform for the design of polysaccharide based vaccines. Generalized Modules for Membrane Antigens (GMMA), OMV released from engineered Gram-negative bacteria, have been proposed for the delivery of the O-Antigen, key target for protective immunity against several pathogens including Shigella. altSonflex1-2-3 is a GMMA based vaccine, including S. sonnei and S. flexneri 1b, 2a and 3a O-Antigens, with the aim to elicit broad protection against the most prevalent Shigella serotypes, especially affecting children in low-middle income countries. Here we developed an In Vitro Relative Potency assay, based on recognition of O-Antigen by functional monoclonal antibodies selected to bind the key epitopes of the different O-Antigen active ingredients, directly applied to our Alhydrogel-formulated vaccine. Heat-stressed altSonflex1-2-3 formulations were generated and extensively characterized. The impact of detected biochemical changes in in vivo and in vitro potency assays was assessed. The overall results showed how the in vitro assay can replace the use of animals, overcoming the inherently high variability of in vivo potency studies. The entire panel of physico-chemical methods developed will contribute to detect suboptimal batches and will be valuable to perform stability studies. The work on Shigella vaccine candidate can be easily extended to other O-Antigen based vaccines.