Characterization of polysaccharide-based vaccines against invasive nontyphoidal Salmonella disease (iNTS)

iNTS is a leading cause of death and morbidity in developing countries and no vaccines are available. The most common pathogens are Salmonella Typhimurium (STm) and Enteritidis (SEn). The O-antigen (OAg) portion of the lipopolysaccharide (LPS) is target of protective immunity and two OAg-based vaccine strategies have been evaluated at GVGH: bivalent formulation of STm and SEn Generalized Modules for Membrane Antigens (GMMA) and conjugation of STm and SEn OAg to CRM197 carrier protein. The first part of the project focused on the characterization of GMMA produced by STm and SEn mutated strains, with particular attention to the OAg component, in a process of screening to identify the most suitable GMMA candidate vaccines. GMMA are outer membrane vesicles naturally released by Gram-negative bacteria. STm and SEn strains were engineered to increase GMMA production and detoxify the lipid A moiety of LPS. I found that genetic mutations can impact expression of OAg chains and their structural characteristics, like chain length and level and position of O-acetylation. However, when tested in mice, all GMMA induced high levels of anti-OAg-specific IgG functional antibodies, despite variation in density and OAg structural modifications. Methods to check quality, consistency of production, stability of GMMA vaccines are of fundamental importance. Not only it is important to characterize the key antigens displayed on GMMA surface and presented to the immune system, but also to characterize GMMA as particles. In this context, I compared Dynamic Light Scattering (DLS), Multi-Angle Light Scattering (MALS) and Nanoparticle Tracking Analysis (NTA) for size distribution determination and for verifying integrity and eventual aggregation of GMMA particles. Each methodology resulted to have strengths and weaknesses, but all the three techniques provided complementary information allowing a more complete evaluation of GMMA size. Unlike the MALS diameter, the DLS diameter correlated with the number of OAg chains per GMMA particle. NTA allows real time visualization and simultaneously tracking and counting of individual particles, but it is deeply operator dependent, making more difficult to obtain consistent results. Second part of my PhD project focused on the in depth characterization of OAg-CRM197 glycoconjugates. OAg was extracted by direct acid hydrolysis of bacteria and conjugated to CRM197 through reductive amination of the terminal reducing sugar, which is expected to be 3-Deoxy-D-manno-oct-2-ulosonic acid (KDO) of the LPS core region. KDO structural rearrangements after acid hydrolysis have been reported in literature and its high reactivity in reaction of reductive amination has been attributed to these chemical modifications. Here, analysis by MS and NMR of the core oligosaccharide (OS) extracted from STm bacteria showed the presence of KDO in its native form with no structural changes. Core OS was fully characterized and conjugated to CRM197, through insertion of ADH and SIDEA as linkers. KDO was the sugar involved in the first step, with kinetic of reductive amination with ADH faster than KDO monosaccharide, due to the linkage in position 5 to the sugar chain. The step of SIDEA introduction was not selective as the linker reacted also with the PPEtN group present in the core region, even in the presence of ADH and by performing the reaction at low pH. Glycosylation sites investigation confirmed that the conjugation process used is highly random involving many lysine residues of CRM197, with preference for those surface exposed. The method also allowed investigation of the regioselectivity of the reaction confirming that OAg chains linkage to the protein happened through both SIDEA on PPEtN group and on ADH. These studies contributed to better characterize the Salmonella antigens and help to define and improve manufacturing processes and analytical methods for OAg-based vaccines in general.