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. 2013 Jan 22;110(4):1464-9.
doi: 10.1073/pnas.1218080110. Epub 2013 Jan 7.

Modulating the innate immune response by combinatorial engineering of endotoxin (V体育官网)

Brittany D Needham  1 , Sean M Carroll, David K Giles, George Georgiou, Marvin Whiteley, M Stephen Trent
Affiliations

Modulating the innate immune response by combinatorial engineering of endotoxin (VSports最新版本)

Brittany D Needham et al. Proc Natl Acad Sci U S A. .

Abstract

Despite its highly inflammatory nature, LPS is a molecule with remarkable therapeutic potential. Lipid A is a glycolipid that serves as the hydrophobic anchor of LPS and constitutes a potent ligand of the Toll-like receptor (TLR)4/myeloid differentiation factor 2 receptor of the innate immune system. A less toxic mixture of monophosphorylated lipid A species (MPL) recently became the first new Food and Drug Administration-approved adjuvant in over 70 y. Whereas wild-type Escherichia coli LPS provokes strong inflammatory MyD88 (myeloid differentiation primary response gene 88)-mediated TLR4 signaling, MPL preferentially induces less inflammatory TRIF (TIR-domain-containing adaptor-inducing IFN-β)-mediated responses. Here, we developed a system for combinatorial structural diversification of E. coli lipid A, yielding a spectrum of bioactive variants that display distinct TLR4 agonist activities and cytokine induction. Mice immunized with engineered lipid A/antigen emulsions exhibited robust IgG titers, indicating the efficacy of these molecules as adjuvants VSports手机版. This approach demonstrates how combinatorial engineering of lipid A can be exploited to generate a spectrum of immunostimulatory molecules for vaccine and therapeutics development. .

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Combinatorial engineering of lipid A anchors to generate diverse immune responses. A schematic is shown illustrating how the outer surface of E. coli strains varies in LPS structure (indicated by different colors) when plasmids are expressed that contain combinations of up to five lipid A-modifying enzymes. The altered LPS molecules bind and activate the TLR4/MD2 complex differentially, altering the nature of downstream cytokine production, represented by shapes that indicate different types and quantity of cytokines released.
Fig. 2.
Fig. 2.
Modification machinery used for generation of diversified lipid A molecules in whole bacteria. Lipid A structures of wild-type E. coli K12, BN1, and BN2 are shown with the names of the six lipid A-modifying enzymes represented in color next to the group that each enzyme modifies: LpxR (green), PagL (blue), LpxE (brown), and LpxF (purple) all remove the corresponding group. LpxO (orange) and PagP (red) transfer the group onto the molecule (4). The attachment site for the remaining polysaccharide is indicated at the 6′ position of each molecule (A). The organism source, activity, and active site topology of each of the six enzymes (B) and the 61 combinatorial strains (C) are presented. Combinatorial strains were generated by transformation of BN1 and BN2 with a pQLinkN plasmid expressing combinations of the six lipid A-modifying enzymes. Each enzyme is abbreviated by its final letter and ordered alphabetically in the plasmid name (i.e., LpxE is abbreviated E, LpxF is F, LpxR is R, PagP is P, PagL is L, and LpxO is O).
Fig. 3.
Fig. 3.
Analysis of engineered lipid A molecules. TLC of 32P-labeled isolated lipid A from combinatorial strains is shown to illustrate the diversity within the collection (A). This method allows species separation, identification, and quantification based upon hydrophobicity-mediated migration. MS of isolated lipid A from selected strains allows further identification of lipid A species (B–D). BN1 pE produces a major peak at m/z 1,716.8, consistent with the expected removal of one phosphate group (B). BN2 pLR produces a major peak at m/z 1,133.9, corresponding to the mass of a triacylated lipid A molecule (C). This is contrasted with BN1 pELR (D), which produces a predominant peak at m/z 1,053.6, corresponding to the dephosphorylation of the major peak seen in BN1 pLR. Minor peaks in both of these strains are similar. Peaks at m/z ∼1,360 and ∼1,570 correspond to masses of lipid A resulting from a single deacylation by either LpxR or PagL, respectively. The peak at m/z ∼1,796 corresponds to residual unmodified BN1 lipid A. In BN1 pLR, there is a slight loss of the labile 1-phosphate group from the major species, yielding a peak at m/z 1,054.0.
Fig. 4.
Fig. 4.
TLR4 stimulation by whole bacterial cells and LPS. Stimulation of TLR4 following incubation of whole bacterial cells with HEK-Blue cells expressing TLR4, MD2, and CD14 is depicted (A). The TLR4 responses to whole cells are shown for all strains. Colors were assigned based on the TLR4 stimulation results in the BN1 strain. The rationale for colorimetric designations is displayed in SI Appendix, Fig. S4. The positive control is E. coli K12 strain W3110, the parent strain of the mutants used in this study. The negative control for this assay is strain CMR300, an E. coli strain that produces only lipid IVA, a tetraacylated TLR4 antagonist (35). *P < 0.05 at 104 cfu per well. HEK-Blue-TLR4-MD2-CD14 cells were also incubated with increasing concentrations of LPS from 13 of the 61 engineered strains (B). E. coli K-12 LPS was used as a positive control and R. sphaeroides LPS, a known TLR4 antagonist, served as a negative control. *P < 0.05 at 1.0 ng/mL; °P < 0.05 10 ng/mL.
Fig. 5.
Fig. 5.
Engineered strains induce diverse stimulation and cytokine production in vitro and high IgG titers in vivo. THP1-XBlue monocytes expressing all TLRs, Nod1, Nod2, MD2, and CD14 were incubated with whole bacterial cells, and overall innate immune receptor activation was measured (A). The graph of representative samples illustrates that in the range of 103 to 105 cfu per well, the activation of the THP1 cells was reduced, and all samples were significantly different from BN1 at 104 cfu per well (P < 0.001) (A). Production of TRIF pathway cytokines (G-CSF, RANTES, and MCP-1) and MyD88 pathway cytokines (TNF-α, IL-6, IL-1β, and IL-8) by wild-type THP1 monocytes differentiated into macrophage-like cells when incubated with 100 ng/mL LPS (B and C). Cytokine levels are presented as percentages of the BN1 level. BALB/cJ mice were immunized with 50 µL of an emulsion of 30 µg of lysozyme from chicken egg white (HEL) with 6 pM purified lipid A, and serum was analyzed by ELISA (D). All lipid A adjuvants tested (BN1 pELP, BN1 pPR, BN2 pEP, and BN1 pLPR) induced a high IgG response, and only BN2 pEP was significantly lower than the MPL control (P = 0.0009).
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References

    1. Pfeiffer R. Untersuchungen uber das Choleragift. Z Hygeine. 1892;11:393–412. German.
    1. Raetz CR, Whitfield C. Lipopolysaccharide endotoxins. Annu Rev Biochem. 2002;71:635–700. - "V体育安卓版" PMC - PubMed
    1. Park BS, et al. The structural basis of lipopolysaccharide recognition by the TLR4-MD-2 complex. Nature. 2009;458(7242):1191–1195. - PubMed
    1. Raetz CR, Reynolds CM, Trent MS, Bishop RE. Lipid A modification systems in gram-negative bacteria. Annu Rev Biochem. 2007;76:295–329. - V体育官网入口 - PMC - PubMed
    1. Casella CR, Mitchell TC. Putting endotoxin to work for us: Monophosphoryl lipid A as a safe and effective vaccine adjuvant. Cell Mol Life Sci. 2008;65(20):3231–3240. - PMC - PubMed

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