Toll-Like Receptor Activation by Generalized Modules for Membrane Antigens from Lipid A Mutants of Salmonella entericaSerovars Typhimurium and Enteritidis

ABSTRACTInvasive nontyphoidal Salmonella(iNTS) disease is a neglected disease with high mortality in children and HIV-positive individuals in sub-Saharan Africa, caused primarily by Africa-specific strains of Salmonella entericaserovars Typhimurium and Enteritidis. A vaccine using GMMA (generalized modules for membrane antigens) from S.Typhimurium and S.Enteritidis containing lipid A modifications to reduce potential in vivoreactogenicity is under development. GMMA with penta-acylated lipid A showed the greatest reduction in the level of cytokine release from human peripheral blood monocytes from that for GMMA with wild-type lipid A. Deletion of the lipid A modification genes msbBand pagPwas required to achieve pure penta-acylation. Interestingly, ?msbB?pagPGMMA from S.Enteritidis had a slightly higher stimulatory potential than those from S.Typhimurium, a finding consistent with the higher lipopolysaccharide (LPS) content and Toll-like receptor 2 (TLR2) stimulatory potential of the former. Also, TLR5 ligand flagellin was found in SalmonellaGMMA. No relevant contribution to the stimulatory potential of GMMA was detected even when the flagellin protein FliC from S.Typhimurium was added at a concentration as high as 10% of total protein, suggesting that flagellin impurities are not a major factor for GMMA-mediated immune stimulation. Overall, the stimulatory potential of S.Typhimurium and S.Enteritidis ?msbB?pagPGMMA was close to that of ShigellasonneiGMMA, which are currently in phase I clinical trials.