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.
Bottom Line: 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.We found that the residual activity of these GMMA is largely due to non-lipid A-related TLR2 activation.The results identify the relative contributions of TLR4 and TLR2 activation by GMMA, which need to be taken into consideration for GMMA vaccine development.
Affiliation: From the Novartis Vaccines Institute for Global Health and.Show MeSH
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Mentions: With the objective of identifying the TLRs that contribute to the residual activation by GMMA with lipid A modification, PBMC were incubated with TLR2 and/or TLR4 blocking antibodies before stimulation with 1 and 10 ng/ml of GMMA, concentrations chosen to give a significant but not saturating increase of IL-6 (Fig. 5). The three GMMA with penta-acylated lipid A, Ss−p − OAg ΔhtrB (Fig. 5D), Ss−p − OAg ΔmsbB (Fig. 5B), and Sf2a−p − OAg ΔmsbB (Fig. 5C), gave similar results as follows: IL-6 production was substantially reduced following incubation with TLR2 blocking antibody (70–90%) but either no reduction or minimal reduction was observed with TLR4 blocking antibody (10–30%) suggesting residual activity was principally due to TLR2 activation. With the Ss−p − OAg ΔmsbB and Sf−p − OAg ΔmsbB GMMA, a small reduction was obtained with the TLR4 blocking antibody alone (Ss−p − OAg ΔmsbB, p = 0.0234; Sf2a−p − OAg ΔmsbB, p = 0.0078 in four experiments) suggesting that there was still residual TLR4 activation. Further reduction was achieved with the combination of TLR2 and TLR4 blocking antibodies compared with TLR2 blocking antibody alone. With the Ss−p − OAgΔhtrB GMMA, no effect from TLR4 blocking was observed.
Affiliation: From the Novartis Vaccines Institute for Global Health and.