Project summary Salmonella typhi (S. typhi) is the major cause of typhoid fever which causes 9.2 million cases of the disease and 110 thousand deaths as reported in 2019. Vaccines against S. typhi have been developed, including Vi polysaccharide vaccine and more recently, Vi conjugate vaccines. They are based on the Vi polysaccharide of S. typhi which is a linear alpha 1–4 linked polygalacturonic acid (PGA) that is N-acetylated at C2 and O-acetylated at C3 of the galacturonic acid (Gal UA) residue. The O-acetyl group at C3 is the dominant determinant of immunogenicity and potency. The Vi conjugate vaccines, overcoming the T-independence of Vi polysaccharide vaccine, are T-dependent and effective in protection against typhoid fever in people > 6 months. However, because of the high molecular weight of Vi polysaccharide, these conjugate vaccines could potentially still induce a partial T-independent response and hyporesponsiveness after booster immunization in people under 2 years. Thus, conjugate vaccines with fragmented or low molecular weight Vi (~50 kDa) are being developed as the potential fully T-dependent and more effective vaccines. Production of Vi vaccines are complex and challenging due to reliance on bacterial fermentation and processing difficulties and low yield of the Vi polysaccharide. Thus, synthetic Vi polysaccharides have been generated and evaluated through an extensive series of studies initiated by scientists of NICHD (National Institute of Child Health and Human Development). It is produced by O-acetylation of plant polygalacturonic acid (PGA) which shares the same backbone as the Vi polysaccharide and are readily and abundantly available. The resulting O-acetylated PGA (AcPGA) thus carries the dominant O-acetyl group epitope of Vi polysaccharide. A phase I clinical trial with a synthetic Vi conjugate vaccine has been successfully conducted by the NICHD team which showed that it is safe and immunogenic, thus providing a critical clinical proof-of- concept for the synthetic Vi conjugate vaccine. Such a synthetic vaccine could provide significant advantages over current Vi conjugate vaccine by eliminating the complex and challenging bacteria-based production system. However, development of the synthetic Vi vaccines does face a significant challenge of potential variation of plant PGA raw materials, especially in molecular weight, which in turn could lead to variation in AcPGA or the synthetic Vi antigen. To overcome this challenge and also produce a more effective T- dependent synthetic Vi polysaccharide, we have developed a simple and efficient process to generate low molecular weight (LMW) AcPGA through controlled hydrolysis of AcPGA. The resulting LMW AcPGA has a low molecular weight of 30-60 kDa and a high degree of O-acetylation (DOAc) which exceeds its specification for Vi polysaccharide. We have further generated a LMW AcPGA conjugate with a novel nanoparticle carrier protein (Dps, DNA-binding protein from starved cell...