Emma Marie Dangerfield

The development of pathogen-associated molecular patterns (PAMPs) that signal via pathogen recognition receptors (PRRs) on innate immune cells is a strategy that is widely adopted in adjuvant research. Less well studied is how covalently linking different PAMPs affects the immune response. Herein, we summarise the research on the effect of PAMP linkage on innate and adaptive immune responses. These covalently linked or “chimeric” PAMPs often lead to immune cell synergies that are greater than those exhibited by the admixed (unconjugated) PAMPs, with several PAMP conjugates exhibiting remarkable adjuvant activity in a variety of disease contexts that include infectious disease, allergy, and cancer immunotherapy. This improvement in immune cell activation is thought to be due to more effective crosstalk between the different PRR signalling pathways, as conjugation ensures that each cell receives each class of PAMP. In addition, PAMP conjugates can form particulates, which has been postulated to lead to improved adjuvanticity, or they may facilitate the targeting of endosomal PRRs via the PRR-mediated endocytosis of the alternative PAMP in the conjugate. PAMP conjugates can also reduce the toxicity of individual PAMPs. However, not all PAMP conjugates are effective, and there are still many aspects of this research platform that are poorly understood, including how linker chemistry affects the immune response and how PRR signalling pathways or PAMP combinations combine to skew the immune response. We will address these and other outstanding questions that relate to the use of PAMP conjugates as vaccine adjuvants.

Dimeric ligands for the macrophage inducible C-type lectin have been synthesised and evaluated in vitro and in vivo . The data provide proof of concept that dimeric Mincle ligands are a new class of potential Mincle-mediated vaccine adjuvants.

We report on a new methodology for the stereoselective synthesis of β-anomeric N-glycosyl amides from unprotected carbohydrates via the reaction of in situ generated carbohydrate oximes with acylsilanes. By variation of the acylsilane, N-glycosyl amides with azide and chloride conjugation handles are prepared in excellent yield and β-selectivity from unprotected sugars.

The Macrophage-inducible C-type lectin (Mincle) is a pattern-recognition receptor (PRR), which has shown much promise as a molecular target for the development of T_H1/T_H17-skewing vaccine adjuvants. In 2009, the first non-proteinaceous Mincle ligands, trehalose dimycolate (TDM) and trehalose dibehenate (TDB), were identified. This prompted a search for other Mincle agonists and the exploration of Mincle agonists as vaccine adjuvants for both preventative and therapeutic (anti-cancer) vaccines. In this review, we discuss those classes of Mincle agonists that have been explored for their adjuvant potential. These Mincle agonists have been used as stand-alone adjuvants or in combination with other pathogen-associated molecular patterns (PAMPs) or immunomodulatory agents. We will also highlight recently identified Mincle ligands with hitherto unknown adjuvanticity. Conjugate vaccines that contain covalently linked adjuvants and/or adjuvant–antigen combinations are also presented, as well as the different formulations (e.g., oil-in-water emulsions, liposomes, and particulate delivery systems) that have been used for the codelivery of antigens and adjuvants. Insofar the reader is presented with a thorough review of the potential of Mincle-mediated vaccine adjuvants, including historical context, present-day research and clinical trials, and outstanding research questions, such as the role of ligand presentation and Mincle clustering, which, if better understood, will aid in the development of the much-needed T_H1/T_H17-skewing vaccine adjuvants.

Herein, we report the modular synthesis and immunological activity of seven bis-aryl triazole trehalolipids (1a-1g) as Brartemicin analogs. The compounds comprised one or two octyloxy (C8) alkyl chains and were synthesized using the venerable CuAAc reaction between the respective aryl acetylenes and a trehalose diazide. A Mincle reporter cell assay revealed that all lipidated analogs activated Mincle. Two compounds, 1c and 1d, produced strong Mincle-dependent immune responses in vitro. The activity was dependent on the degree of alkylation and regiochemistry, with 1c and 1d showing significantly increased IL-1β production in vitro compared to monoalkylated compounds and dialkylated compounds lacking ortho substitution. Molecular docking of 1c positioned the triazole in proximity to Arg-183, which may offer additional interactions that could explain the binding affinity for this class of ligand. These findings demonstrate the capability of triazole-linked Brartemicin analogs as Mincle-mediated Th1/Th17 vaccine adjuvants.

There is a need for improved vaccine adjuvants to augment vaccine efficacy. One way to address this is by targeting multiple immune cell pathogen recognition receptors (PRRs) using chimeric pathogen-associated molecular patterns (PAMPs). Conjugation of the PAMPs will ensure codelivery of the immunostimulatory molecules to the same cell, enhancing adjuvant activity. The macrophage inducible C-type lectin (Mincle) is a promising PRR for adjuvant development; however, no effective chimeric Mincle adjuvants have been prepared. We addressed this by synthesizing Mincle adjuvant conjugates, MDP-C18Brar and MDP-C18Brar-dilipid, which contain PAMPs recognized by Mincle and the nucleotide-binding oligomerization domain 2 (NOD2). The two PAMPs are joined by a pH-sensitive oxyamine linker which, upon acidification at lysosomal pH, hydrolyzed to release the NOD2 ligands. The conjugates elicited the production of Th1 and Th17 promoting cytokines in vitro, and when using OVA as a model antigen, exhibited enhanced T-cell-mediated immune responses and reduced toxicity in vivo, compared to the coadministration of the adjuvants.

The synthesised βG-iGb3 glycolipid, with a terminal 1,3-β linked galactose, induced NKT cell proliferation indicating that the α conformation of the terminal sugar residue of iGb3 is not essential for NKT cell TCR recognition.

The correct diagnosis of multiple sclerosis (MS) remains challenging due to the complex pathophysiological and clinical characteristics of the disease. Consequently, there has been immense interest in finding a non-invasive diagnostic test for MS. Recent studies found that serum anti-α-D-Glcp-(1→4)-α-D-Glcp (GAGA4) IgM antibodies were upregulated in MS patients, and this finding led to the development of a commercial diagnostic test (gMS® Dx test), although the test has poor selectivity and has not been independently validated. Herein, we developed an enzyme-linked immunosorbent assay (ELISA) to evaluate the use and reliability of several anti-glucose IgM antibodies, including those against GAGA4, as diagnostic biomarkers for MS. In contrast to previous studies, our results show that serum anti-GAGA4 IgM antibody levels are not significantly higher in MS patients, which could potentially explain the poor selectivity of the commercial test.