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

Abstract

Invasive 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 enterica serovars 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 vivo 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 msbB and pagP was required to achieve pure penta-acylation. Interestingly, ΔmsbB ΔpagP GMMA 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 Salmonella GMMA. 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 ΔmsbB ΔpagP GMMA was close to that of Shigella sonnei GMMA, which are currently in phase I clinical trials.

Comparative immunogenicity and efficacy of equivalent outer membrane vesicle and glycoconjugate vaccines against nontyphoidal Salmonella

“…GMMA were produced from Δ tolR mutants of the same strains used as the source of OAg for the glycoconjugate vaccine ( 26 ). S. Typhimurium and S. Enteritidis GMMA had similar size and OAg-to-protein weight ratio (0.72 and 0.61, respectively) ( Table 1 ).…”

“…The integrity and attachment of the inner and outer bacterial cell wall membranes of Salmonella can be altered by disruption of the Tol-Pal system, through deletion of the tolR gene ( 26 , 27 ), resulting in the release of large quantities of GMMA. GMMA are outer membrane vesicles of homogeneous size, typically in the range 40–250 nm, released from the surface of genetically mutated Gram-negative bacteria ( 24 ).…”

Protective Immunity Elicited by Oral Immunization of Mice with Salmonella enterica Serovar Typhimurium Braun Lipoprotein (Lpp) and Acetyltransferase (MsbB) Mutants

“…enteritidis (Simon et al, 2011) and S. typhimurium (Simon et al, 2013) as part of efforts to combat iNTS (Strugnell et al, 2014). Most importantly, a vaccine using GMMA (generalized modules for membrane antigens) from msbB and pagP (encoding lipid A palmitoyltransferase) deletion mutants of S. typhimurium and S. enteritidis with reduced potential for in vivo reactogenicity showed a slightly higher stimulatory potential than WT S. typhimurium harboring WT lipid A (Rossi et al, 2016). Therefore, in this regard, the Δ lppAB Δ msbB mutant with modified LPS may have some advantages over the Δ lppAB mutant.…”

“…Flagellin, a TLR-5 agonist, has been recognized and used as an adjuvant for many vaccines (Hayashi et al, 2001; Yin et al, 2013; Gupta et al, 2014). However, a recent study showed that flagellin was not a major factor for GMMA-mediated immune stimulation against salmonellosis (Rossi et al, 2016). Therefore, the immunogenicity of our mutants either should not be significantly influenced by the decreased flagellar-associated proteins or can be further enhanced in conjunction with the flagellin based adjuvants.…”

Intralaboratory evaluation of luminescence based high-throughput Serum Bactericidal Assay (L-SBA) to determine bactericidal activity of human sera againstShigella

“…Several approaches are currently in development to deliver the O-antigen to the immune system, including whole cell attenuated bacteria (6), vaccines in which the O-antigen are chemically-(7) or bio-conjugated to carrier proteins (8), synthetic vaccine conjugates (9), and GMMA based vaccines (10). GMMA are outer membrane exosomes from Gram-negative bacteria, genetically modified to induce hyperblebbing and to reduce the reactogenic potential of lipid A (11, 12). GMMA are easy and inexpensive to produce, and highly immunogenic (10, 13–16).…”