title: Modulation of endotoxicity of Shigella generalized modules for membrane antigens (GMMA) by genetic lipid A modifications: relative activation of TLR4 and TLR2 pathways in different mutants.
abstract: Outer membrane particles from Gram-negative bacteria are attractive vaccine candidates as they present surface antigens in their natural context. We previously developed a high yield production process for genetically derived particles, called generalized modules for membrane antigens (GMMA), from Shigella. As GMMA are derived from the outer membrane, they contain immunostimulatory components, especially lipopolysaccharide (LPS). We examined ways of reducing their reactogenicity by modifying lipid A, the endotoxic part of LPS, through deletion of late acyltransferase genes, msbB or htrB, in GMMA-producing Shigella sonnei and Shigella flexneri strains. GMMA with resulting penta-acylated lipid A from the msbB mutants showed a 600-fold reduced ability, and GMMA from the S. sonnei ΔhtrB mutant showed a 60,000-fold reduced ability compared with GMMA with wild-type lipid A to stimulate human Toll-like receptor 4 (TLR4) in a reporter cell line. In human peripheral blood mononuclear cells, GMMA with penta-acylated lipid A showed a marked reduction in induction of inflammatory cytokines (S. sonnei ΔhtrB, 800-fold; ΔmsbB mutants, 300-fold). We found that the residual activity of these GMMA is largely due to non-lipid A-related TLR2 activation. In contrast, in the S. flexneri ΔhtrB mutant, a compensatory lipid A palmitoleoylation resulted in GMMA with hexa-acylated lipid A with ∼10-fold higher activity to stimulate peripheral blood mononuclear cells than GMMA with penta-acylated lipid A, mostly due to retained TLR4 activity. Thus, for use as vaccines, GMMA will likely require lipid A penta-acylation. The results identify the relative contributions of TLR4 and TLR2 activation by GMMA, which need to be taken into consideration for GMMA vaccine development.
authors.affiliation: From the Novartis Vaccines Institute for Global Health and.
authors.forename: Omar
authors.initials: O
authors.lastname: Rossi
authors.affiliation: From the Novartis Vaccines Institute for Global Health and.
authors.forename: Isabella
authors.initials: I
authors.lastname: Pesce
authors.affiliation: From the Novartis Vaccines Institute for Global Health and.
authors.forename: Carlo
authors.initials: C
authors.lastname: Giannelli
authors.affiliation: Novartis Vaccines, 53100 Siena, Italy.
authors.forename: Susanna
authors.initials: S
authors.lastname: Aprea
authors.affiliation: From the Novartis Vaccines Institute for Global Health and.
authors.forename: Mariaelena
authors.initials: M
authors.lastname: Caboni
authors.affiliation: From the Novartis Vaccines Institute for Global Health and.
authors.forename: Francesco
authors.initials: F
authors.lastname: Citiulo
authors.affiliation: Novartis Vaccines, 53100 Siena, Italy.
authors.forename: Sara
authors.initials: S
authors.lastname: Valentini
authors.affiliation: Novartis Vaccines, 53100 Siena, Italy.
authors.forename: Ilaria
authors.initials: I
authors.lastname: Ferlenghi
authors.affiliation: From the Novartis Vaccines Institute for Global Health and.
authors.forename: Calman Alexander
authors.initials: CA
authors.lastname: MacLennan
authors.affiliation: Novartis Vaccines, 53100 Siena, Italy.
authors.forename: Ugo
authors.initials: U
authors.lastname: D'Oro
authors.affiliation: From the Novartis Vaccines Institute for Global Health and.
authors.forename: Allan
authors.initials: A
authors.lastname: Saul
authors.affiliation: From the Novartis Vaccines Institute for Global Health and christiane.gerke@novartis.com.
authors.forename: Christiane
authors.initials: C
authors.lastname: Gerke
mesh_terms: D000215:Acylation / Q000276:immunology; D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism; D000942:Antigens, Bacterial / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D016207:Cytokines / Q000276:immunology / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D057809:HEK293 Cells; D006801:Humans; D008050:Lipid A / Q000032:analysis / Q000276:immunology* / Q000378:metabolism; D046529:Microscopy, Electron, Transmission; D009000:Monocytes / Q000276:immunology / Q000378:metabolism; D009154:Mutation; D012760:Shigella / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D012763:Shigella flexneri / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D012764:Shigella sonnei / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D015398:Signal Transduction / Q000276:immunology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D051195:Toll-Like Receptor 2 / Q000276:immunology* / Q000378:metabolism; D051197:Toll-Like Receptor 4 / Q000276:immunology* / Q000378:metabolism
chemical_list: D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D016207:Cytokines; D008050:Lipid A; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4; D000217:Acyltransferases
keywords: Cytokine; Endotoxin; GMMA; Lipopolysaccharide (LPS); Outer Membrane Vesicles; Shigella; Toll-like Receptor (TLR); Vaccine; htrB; msbB