Purification, Characterisation and Synthesis of Glycerolipids Extracted from L. plantarum and B. longum subsp. infantis

Glycolipids from the cell wall of Gram-positive bacteria have been the topic of my PhD. It is well known that many bacterial glycolipids (e.g. LPS, TDMs and PIMs) have profound immunological effects, and therefore the characterisation, biological testing and synthesis of gram-positive bacterial glycolipids is of interest. The first part of this thesis includes a description of the extraction and characterisation of glycolipids from gut bacteria including Bifidobacterium and Lactobacillus genus and the second part focussed on the chemical synthesis of Streptococcus sp. DSM 8747 glycolipids and lipoteichoic acid analogues (LTA). Members of the genus Lactobacillus are common in the gut microbiota and are often used as probiotics. As lactobacilli are known to have benefits to human health, compounds on its surface are of high interest. To date, the structures of the glycolipids from L. plantarum have not been conclusively assigned. Thus, for the first time, the full characterisation of the four principal glycolipids of the L. plantarum cell wall was reported using sugar, linkage and FAME analysis, as well as ESI-MS/MS and 1D- and 2D-NMR spectroscopy. The major glycolipids were identified as: α-D-Glcp-diglyceride, α-D-Galp-(1→2)-α-D-Glcp-diglyceride, β-D-Glcp-(1→6)-α-D-Galp-(1→2)-6-O-acyl-α-D-Glcp-diglyceride and β-D-Glcp-(1→6)-α-D-Galp-(1→2)-α-D-Glcp-diglyceride. These glycolipids showed weak activation of murine bone marrow macrophages in an initial biological screen. After having identified the structures of the glycolipids from L. plantarum, the glycolipids from Bifidobacterium, a dominant member of the gut microbiota in infants, were extracted. Bifidobacteria are considered to be important in the development of a healthy immune system and they are believed to exhibit anticancerous properties, alleviate the symptoms of irritable bowel syndrome, and are thought to reduce atopic disease. Despite this, the chemical nature of immunomodulatory compounds on the surface of bifidobacteria has not been well documented. Thus, glycolipids were extracted from B. longum subsp. infantis, fractionated chromatographically and analyzed using NMR spectroscopy, constituent sugar and linkage analysis, and fatty acid analysis. These analyzes revealed a novel glycolipid, containing an unprecedented mixed acetal moiety and a galactofuranose moiety as a head group. However, like L. plantarum glycolipids, bifidobacterial glycolipids were shown only to induce little macrophage activity when tested. Having successfully characterised a novel glycolipid present in bifidobacteria, analogues of this glycolipid as well as poly(glycerophosphate) lipotechoic acids analogues (LTAs) were then synthesised. Much debate still remains about the role of LTAs during Gram-positive bacterial infection. This is partly due to differences in the biological activities of extracted versus synthesised LTAs and highlights the need for structurally defined non-contaminated LTAs when investigating the effect of these glycolipids on the innate immune response. An efficient synthesis of the core lipoteichoic acid (LTA) anchor of the Streptococcus species DSM 8747, and derivatives thereof, was achieved. These Streptococcus glycolipids contain a galactofuranose moiety and thus have similarities to the novel glycolipid that was found in bifidobacteria. The syntheses, which commence with readily available D-galactose, are short (7-9 steps), convergent, and high-yielding (33-37% overall yield). In total 11 different targets were synthesised. The biological activity of these compounds was also investigated, with several analogues (particularly the sn-1,2-di-acylglycerol LTA anchors) found to induce macrophage activation

Attribution et caractéristiques de liaison du domaine tandem PDZ2/3 de PTP-BL par RMN

PTP-BL = Protein-tyrosine phosphatase basophil-like.PDZ = PSD-95; Postsynaptic density-95, disc-large and zonulin-1

Use of stable CHO pools and CHO TGE to accelerate SARS-CoV-2 vaccine development

Please click Additional Files below to see the full abstract

Archaeal glycolipid adjuvanted vaccines induce strong influenza-specific immune responses through direct immunization in young and aged mice or through passive maternal immunization.

Vaccine induced responses are often weaker in those individuals most susceptible to infection, namely the very young and the elderly, highlighting the need for safe and effective vaccine adjuvants. Herein we evaluated different archaeosome formulations as an adjuvant to the H1N1 influenza hemagglutinin protein and compared immune responses (anti-HA IgG and hemagglutination inhibition assay titers) as well as protection to an influenza A virus (strain A/Puerto Rico/8/1934 H1N1) homologous challenge to those generated using a squalene-based oil-in-water nano-emulsion, AddaVax™ in a murine model. The impact of age (young adult vs aged) on vaccine induced immune responses as well as the protection in pups due to the transfer of maternal antibodies was measured. Overall, we show that archaeal lipid based adjuvants can induce potent anti-HA responses in young and aged mice that can also be passed from vaccinated mothers to pups. Furthermore, young and aged mice immunized with archaeal lipid adjuvants as well as pups from immunized mothers were protected from challenge with influenza. In addition, we show that a simple admixed archaeosome formulation composed of a single sulfated glycolipid namely sulfated lactosylarchaeol (SLA; 6′-sulfate-β-D-Galp-(1,4)-β-D-Glcp-(1,1)-archaeol) can give equal or better protection compared to AddaVax™ or the traditional antigen-encapsulated archaeosome formulations

Alternate synthesis to D-​glycero-​β-​D-​manno-​heptose 1,​7-​biphosphate

d-glycero-β-d-manno-heptose 1,7-biphosphate (HBP) is an enzymatic intermediate in the biosynthesis of the heptose component of lipopolysaccharide (LPS), and was recently revealed to be a pathogen-associated molecular pattern (PAMP) that allows detection of Gram-negative bacteria by the mammalian immune system. Cellular detection of HBP depends upon its stimulation of a cascade that leads to the phosphorylation and assembly of the TRAF-interacting with forkhead-associated domain protein A (TIFA), which activates the transcription factor NF-κB. In this note, an alternate chemical synthesis of HBP is described and its biological activity is established, providing pure material for further assessing and exploiting the biological activity of this compound

Optimization of the Synthesis and Conjugation of the Methyl Rhamnan Tip of Pseudomonas aeruginosa A‑Band Polysaccharide and Immunogenicity Evaluation for the Continued Development of a Potential Glycoconjugate Vaccine

Pseudomonas aeruginosa is an antimicrobial-resistant bacterium that has no vaccine approved for human use. Additionally, it has been identified by the World Health Organization as a priority pathogen for novel vaccines and therapeutic development. We previously developed a synthetic mimic of the A-band polysaccharide tip that showed promise in terms of immunogenicity for use as a glycoconjugate vaccine. In this current manuscript, we improve upon the previous work to continue the development of this glycoconjugate vaccine. Herein, we report a higher-yielding synthesis of mimics containing a handle and a spacer that improved conjugation efficiency, resulting in better carbohydrate-to-protein ratios and also good immunogenicity of these conjugates in mice and rabbits. The data suggested that perhaps only a tetrasaccharide was required to induce an immune response capable of recognizing whole cells of P. aeruginosa

Simplifying glycan monitoring of complex antigens such as the SARS-CoV-2 spike to accelerate vaccine development

Abstract Glycosylation is a key quality attribute that must be closely monitored for protein therapeutics. Established assays such as HILIC-Fld of released glycans and LC-MS of glycopeptides work well for glycoproteins with a few glycosylation sites but are less amenable for those with multiple glycosylation sites, resulting in complex datasets that are time consuming to generate and difficult to analyze. As part of efforts to improve preparedness for future pandemics, researchers are currently assessing where time can be saved in the vaccine development and production process. In this context, we evaluated if neutral and acidic monosaccharides analysis via HPAEC-PAD could be used as a rapid and robust alternative to LC-MS and HILIC-Fld for monitoring glycosylation between protein production batches. Using glycoengineered spike proteins we show that the HPAEC-PAD monosaccharide assays could quickly and reproducibly detect both major and minor glycosylation differences between batches. Moreover, the monosaccharide results aligned well with those obtained by HILIC-Fld and LC-MS

Discovery of Lipids from B. longum subsp. <i>infantis</i> using Whole Cell MALDI Analysis

Bifidobacteria are dominant members of the microbial community in the intestinal tract of infants, and studies have shown that glycolipids extracted from the cell surface of these bacteria elicit beneficial immune responses. Accordingly, the identification and structural characterization of glycolipids from the cell wall of bifidobacteria is the first step in correlating glycolipid structure with biological activity. Using whole cell MALDI as a screening tool, we herein present for the first time the identification and structural elucidation of the major polar lipids from Bifidobacterium longum subs. <i>infantis</i>. The lipids identified include an unprecedented plasmenyl cyclophosphatidic acid and a mixed acetal glycolipid, with the latter subsequently being isolated and found to suppress the innate immune response