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

<jats:title>ABSTRACT</jats:title><jats:p>Invasive nontyphoidal<jats:named-content content-type="genus-species">Salmonella</jats:named-content>(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<jats:named-content content-type="genus-species">Salmonella enterica</jats:named-content>serovars Typhimurium and Enteritidis. A vaccine using GMMA (generalized modules for membrane antigens) from<jats:italic>S.</jats:italic>Typhimurium and<jats:italic>S.</jats:italic>Enteritidis containing lipid A modifications to reduce potential<jats:italic>in vivo</jats:italic>reactogenicity 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<jats:italic>msbB</jats:italic>and<jats:italic>pagP</jats:italic>was required to achieve pure penta-acylation. Interestingly, Δ<jats:italic>msbB</jats:italic>Δ<jats:italic>pagP</jats:italic>GMMA from<jats:italic>S.</jats:italic>Enteritidis had a slightly higher stimulatory potential than those from<jats:italic>S.</jats:italic>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<jats:named-content content-type="genus-species">Salmonella</jats:named-content>GMMA. No relevant contribution to the stimulatory potential of GMMA was detected even when the flagellin protein FliC from<jats:italic>S.</jats:italic>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<jats:italic>S.</jats:italic>Typhimurium and<jats:italic>S.</jats:italic>Enteritidis Δ<jats:italic>msbB</jats:italic>Δ<jats:italic>pagP</jats:italic>GMMA was close to that of<jats:named-content content-type="genus-species">Shigella</jats:named-content><jats:italic>sonnei</jats:italic>GMMA, which are currently in phase I clinical trials.</jats:p>