Rossi, Omar (2014) MODULATION OF REACTOGENICITY OF GENERALIZED MODULES FOR MEMBRANE ANTIGENS (GMMA)BY GENETIC LIPID A MODIFICATION. [Tesi di dottorato]

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Outer membrane particles from Gram-negative bacteria are attractive vaccine candidates as they present surface antigens in their natural environment and orientation. 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 and contain immune-stimulatory components, especially lipopoly-saccharide (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 S. sonnei and S. flexneri strains. GMMA with resulting penta-acylated lipid A from the msbB mutants showed a 600-fold reduction, GMMA from the S. sonnei ΔhtrB mutant a 60,000-fold reduced ability compared to GMMA with wild-type lipid A to stimulated human Toll-like receptor 4 (TLR4) in a reporter cell line. In contrast, in the S. flexneri ΔhtrB mutant, a compensatory palmitoleoylation occurs resulting in hexa-acylated lipid A with approximately 10-fold higher activity than the penta-acylated lipid A. In human PBMC, GMMA with penta-acylated lipid A showed a marked reduction in induction of inflammatory cytokines (800-fold for S. sonnei ΔhtrB, 300-fold for the msbB mutants) compared to a 50-fold reduction observed for GMMA with palmitoleoylated lipid A from the S. flexneri ΔhtrB strain. We demonstrated that the residual activity of GMMA with penta-acylated lipid A is largely due to non-lipid A related TLR2 activation whereas GMMA with palmitoleoylated hexa-acylated lipid A predominantly activate TLR4. These results identify the relative activation of TLR4 and TLR2 pathways by GMMA.

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