Synthesis of peptidoglycan units with UDP at the anomeric position

A series of UDP-disaccharide peptide compounds were synthesized as synthetic substrate analogues or potential inhibitors of glycosyl transferase. Fluorescent compounds have been prepared with the aim of developing a screening method for selecting transglycosylase inhibitors.status: publishe

New chimeric TLR7/NOD2 agonist is a potent adjuvant to induce mucosal immune responses

International audienceBackground: PRR (Pattern Recognition Receptor) agonists have been widely tested as potent vaccine adjuvants. TLR7 (Toll-Like Receptor 7) and NOD2 (nucleotide-binding oligomerization domain 2) are key innate receptors widely expressed at mucosal levels.Methods: Here, we evaluated the immunostimulatory properties of a novel hybrid chemical compound designed to stimulate both TLR7 and NOD2 receptors.Finding: The combined TLR7/NOD2 agonist showed increase efficacy than TLR7L or NOD2L agonists alone or combined in different in vitro models. Dual TLR7/NOD2 agonist efficiently stimulates TLR7 and NOD2, and promotes the maturation and reprogramming of human dendritic cells, as well as the secretion of pro-inflammatory or adaptive cytokines. This molecule also strongly induces autophagy in human cells which is a major intracellular degradation system that delivers cytoplasmic constituents to lysosomes in both MHC class I and II-restricted antigen presentation. In vivo, TLR7/NOD2L agonist is a potent adjuvant after intranasal administration with NP-p24 HIV vaccine, inducing high-quality humoral and adaptive responses both in systemic and mucosal compartments. Use of TLR7/NOD2L adjuvant improves very significantly the protection of mice against an intranasal challenge with a vaccinia virus expressing the p24.Interpretation: Dual TLR7/NOD2L agonist is a very potent and versatile vaccine adjuvant and promote very efficiently both systemic and mucosal immunity

Glycosyl transferase activity of the Escherichia coli penicillin-binding protein 1b: Specificity profile for the substrate

peer reviewedThe glycosyl transferase of the Escherichia coli bifunctional penicillin-binding protein (PBP) 1b catalyzes the assembly of lipid-transported N-acetylglucosaminyl-beta-1,4-N-acetylmuramoyl-L-Ala-gamma-D-Glu-meso-A(2)pm-D-Ala-D-Ala units (lipid II) into linear peptidoglycan chains. These units are linked, at C1 of N-acetylmuramic acid (MurNAc), to a C-55 undecaprenyl pyrophosphate. In an in vitro assay, lipid II functions both as a glycosyl donor and as a glycosyl acceptor substrate. Using substrate analogues, it is suggested that the specificity of the enzyme for the glycosyl donor substrate differs from that for the acceptor. The donor substrate requires the presence of both N-acetylglucosamine (GlcNAc) and MurNAc and a reactive group on C1 of the MurNAc and does not absolutely require the lipid chain which can be replaced by uridine. The enzyme appears to prefer an acceptor substrate containing a polyprenyl pyrophosphate on C1 of the MurNAc sugar. The problem of glycan chain elongation that presumably proceeds by the repetitive addition of disaccharide peptide units at their reducing end is discussed

STING agonists enable antiviral cross-talk between human cells and confer protection against genital herpes in mice

<div><p>In recent years, there has been an increasing interest in immunomodulatory therapy as a means to treat various conditions, including infectious diseases. For instance, Toll-like receptor (TLR) agonists have been evaluated for treatment of genital herpes. However, although the TLR7 agonist imiquimod was shown to have antiviral activity in individual patients, no significant effects were observed in clinical trials, and the compound also exhibited significant side effects, including local inflammation. Cytosolic DNA is detected by the enzyme cyclic GMP-AMP (2’3’-cGAMP) synthase (cGAS) to stimulate antiviral pathways, mainly through induction of type I interferon (IFN)s. cGAS is activated upon DNA binding to produce the cyclic dinucleotide (CDN) 2’3’-cGAMP, which in turn binds and activates the adaptor protein Stimulator of interferon genes (STING), thus triggering type I IFN expression. In contrast to TLRs, STING is expressed broadly, including in epithelial cells. Here we report that natural and non-natural STING agonists strongly induce type I IFNs in human cells and in mice <i>in vivo</i>, without stimulating significant inflammatory gene expression. Systemic treatment with 2’3’-cGAMP reduced genital herpes simplex virus (HSV) 2 replication and improved the clinical outcome of infection. More importantly, local application of CDNs at the genital epithelial surface gave rise to local IFN activity, but only limited systemic responses, and this treatment conferred total protection against disease in both immunocompetent and immunocompromised mice. In direct comparison between CDNs and TLR agonists, only CDNs acted directly on epithelial cells, hence allowing a more rapid and IFN-focused immune response in the vaginal epithelium. Thus, specific activation of the STING pathway in the vagina evokes induction of the IFN system but limited inflammatory responses to allow control of HSV2 infections <i>in vivo</i>.</p></div

Rational design of adjuvants targeting the C-type lectin Mincle

The advances in subunit vaccines development have intensified the search for potent adjuvants, particularly adjuvants inducing cell-mediated immune responses. Identification of the C-type lectin Mincle as one of the receptors underlying the remarkable immunogenicity of the mycobacterial cell wall, via recognition of trehalose-6,6'-dimycolate (TDM), has opened avenues for the rational design of such molecules. Using a combination of chemical synthesis, biological evaluation, molecular dynamics simulations, and protein mutagenesis, we gained insight into the molecular bases of glycolipid recognition by Mincle. Unexpectedly, the fine structure of the fatty acids was found to play a key role in the binding of a glycolipid to the carbohydrate recognition domain of the lectin. Glucose and mannose esterified at O-6 by a synthetic alpha-ramified 32-carbon fatty acid showed agonist activity similar to that of TDM, despite their much simpler structure. Moreover, they were seen to stimulate proinflammatory cytokine production in primary human and murine cells in a Mincle-dependent fashion. Finally, they were found to induce strong Th1 and Th17 immune responses in vivo in immunization experiments in mice and conferred protection in a murine model of Mycobacterium tuberculosis infection. Here we describe the rational development of new molecules with powerful adjuvant properties

STING agonists induces type I IFN expressing <i>in vivo</i>.

<p>Equimolar (1.687x10^-7 mol) doses of STING agonists 2’3’-cGAMP (121 μg/mouse), 3’3’c-diAMP (119 μg/mouse), 2’3’-cGAM(PS)₂ (125 μg/mouse), 3’3’-cAIMP (111 μg/mouse) or DMXAA (95 μg/mouse, STING activation by DMXAA requires two molecules (3.374x10^-7 mol)) were administrated to mice i.p. Samples were collected 6 hours later for further analysis. (<b>A</b>) Levels of phosphorylated STAT1 (pSTAT1), STING, viperin and ISG15 were determined by Western blotting of spleen, vagina and brain tissues. Vinculin was used as loading control. n = 5, two representative samples are shown. (<b>B</b>) IFNα and IFNβ in the serum determined by ELISA. n = 3–5. * = p<0.05 compared to mock. (<b>C</b>) Expression for <i>Ifnb</i> and <i>Mx1</i> mRNA in tissues samples from vagina, spleen and brain, normalized to GAPDH. n = 3–5. * = p<0,05 compared to mock. <b>(D</b>) Mice were perfused prior to isolation of brain samples and gene expressions of <i>Ifnb</i> and <i>Mx1</i> mRNA were measured. n = 3–5. * = p<0,05 compared to mock. Statistics, (<b>B-D</b>) Kruskal-Wallis test with Dunn’s multiple comparisons test.</p

Systemic treatment with STING agonists confers protection against genital HSV2 infection.

<p>Wildtype and <i>cGas</i>^-/- mice were treated with 2’3’-cGAM(PS)₂ (Rp/Sp) (125 μg/mouse) and infected intravaginally with HSV2 (6.7×10⁴ p.f.u.). (<b>A</b>) Illustration of the timeline for the treatment regimens. (<b>B, C</b>) Overall survival for HSV2-infected and treated wildtype (<b>B</b>) or <i>cGas</i>^<i>-</i>/- (<b>C</b>) mice. n = 6–10. * = p<0,05 compared to mock. <b>(D</b>) HSV2 titer (TCID50) in vaginal washes collected 48 hours post infection. n = 6–10. * = p<0,05 compared to mock in the same genotype. Statistics, (<b>B</b>, <b>C</b>) Log-rank test with Holm-Bonferroni correction. (<b>D</b>) One-way ANOVA of log₁₀-transformed data with Dunnett’s multiple comparisons test.</p

STING agonists induce IFN responses in the vaginal epithelium faster and more efficiently than TLR agonists.

<p>(<b>A</b>) HaCaT cells were treated with imiquimod (1μg/ ml), ODN2216 (1μg/ ml), and 3’3’-cAIMP (100μg/ ml) for 24 h. Levels of ISG15 and viperin were determined in the cell lysate by Western blotting. (<b>B</b>) Mice were treated intravaginally with imiquimod or ODN1826 (25 μg per mouse) 12 h prior to infection with HSV2. Vaginal washes were collected 48 h p.i. and viral load was determined. n = 5 per group. (<b>C-D</b>) Mice were anesthetized for 30 min and imiquimod, ODN1826, or 3’3’-cAIMP was applied to the vagina. Tissues were isolated (<b>C</b>) 6 h or (<b>D</b>) 36 h after treatment. Paraffin sections of the vaginal tissues were stained for viperin (red). DAPI (blue) marks the nuclei and the dotted white lines mark basal membrane between the epithelium and stroma. White arrows highlight examples of viperin positive cells. L = lumen, E = epithelium, S = stroma. n = 4. One representative picture is shown for each staining. (<b>E-G</b>) RNA was isolated from vaginal tissue treated as indicated for 6 h, and levels of <i>Ifnb</i>, <i>Mx1</i>, and <i>Tnfa</i> mRNA were determined by RT-qPCR. n = 4–5. mRNA levels were normalized to <i>Gapdh</i> and shown as relative levels of expression compared to mock-treated mice. (<b>B, E-G</b>) Statistics, Kruskal-Wallis test with Dunn’s multiple comparisons test. * = p<0,05 compared to mock treated.</p