Regan J. Anderson

Total synthesis of variolin B

Abstract The total synthesis of the marine alkaloid variolin B has been achieved in eight steps, starting from commercially available 4-chloro-2-methylthiopyrimidine. The key reaction involves the tandem deoxygenation and cyclization of a triarylmethanol using a combination of triethylsilane and trifluoroacetic acid.

A self-adjuvanting vaccine induces cytotoxic T lymphocytes that suppress allergy

Epitope-based peptide vaccines encompass minimal immunogenic regions of protein antigens to allow stimulation of precisely targeted adaptive immune responses. However, because efficacy is largely determined by the functional status of antigen-presenting cells (APCs) that acquire and present peptides to cells of the adaptive immune system, adjuvant compounds are needed to enhance immunogenicity. We present here a vaccine consisting of an allergen-derived peptide conjugated to a prodrug of the natural killer-like T (NKT) cell agonist α-galactosylceramide, which is highly effective in reducing inflammation in a mouse model of allergic airway inflammation. Unlike other peptide-adjuvant conjugates that directly activate APCs through pattern recognition pathways, this vaccine encourages third-party interactions with NKT cells to enhance APC function. Therapeutic efficacy was correlated with marked increases in the number and functional activity of allergen-specific cytotoxic T lymphocytes (CTLs), leading to suppression of immune infiltration into the lungs after allergen challenge in sensitized hosts.

Analysis of the CD1 Antigen Presenting System in Humanized SCID Mice

CD1 molecules are glycoproteins that present lipids and glycolipids for recognition by T cells. CD1-dependent immune activation has been implicated in a wide range of immune responses, however, our understanding of the role of this pathway in human disease remains limited because of species differences between humans and other mammals: whereas humans express five different CD1 gene products (CD1a, CD1b, CD1c, CD1d, and CD1e), muroid rodents express only one CD1 isoform (CD1d). Here we report that immune deficient mice engrafted with human fetal thymus, liver, and CD34+ hematopoietic stem cells develop a functional human CD1 compartment. CD1a, b, c, and d isoforms were highly expressed by human thymocytes, and CD1a+ cells with a dendritic morphology were present in the thymic medulla. CD1+ cells were also detected in spleen, liver, and lungs. APCs from spleen and liver were capable of presenting bacterial glycolipids to human CD1-restricted T cells. ELISpot analyses of splenocytes demonstrated the presence of CD1-reactive IFN-γ producing cells. CD1d tetramer staining directly identified human iNKT cells in spleen and liver samples from engrafted mice, and injection of the glycolipid antigen α-GalCer resulted in rapid elevation of human IFN-γ and IL-4 levels in the blood indicating that the human iNKT cells are biologically active in vivo. Together, these results demonstrate that the human CD1 system is present and functionally competent in this humanized mouse model. Thus, this system provides a new opportunity to study the role of CD1-related immune activation in infections to human-specific pathogens.

Studies toward the total synthesis of the variolins: rapid entry to the core structure

The pyrido[3′,2′:4,5]pyrrolo[1,2-c]pyrimidine core structure of the variolins has been synthesized in three steps from commercially available materials. The key reaction involves the deoxygenation and concomitant cyclization of a triarylmethanol using the combination of triethylsilane and trifluoroacetic acid. Introduction of amine functionality as required for the natural products has been achieved in two steps.

Synthetic TRP2 long-peptide and α-galactosylceramide formulated into cationic liposomes elicit CD8+ T-cell responses and prevent tumour progression

The lipid antigen α-galactosylceramide (α-GalCer) is a potent activator of invariant natural killer T-cells (iNKT cells) and can stimulate cytotoxic and anti-tumour immune responses. However optimal responses appear to be induced by α-GalCer when cell-based vaccines are delivered intravenously. Here we investigated if co-delivery of protein and peptide antigens along with α-GalCer in a liposomal formulation could stimulate therapeutic anti-tumour immune responses. Cationic liposomes were inherently immune-stimulatory and induced cytotoxic immune responses when delivered both by intravenous and subcutaneous injection. However, only vaccine delivered intravenously stimulated therapeutic anti-tumour immune responses to a peptide antigen. Surface modification with polyethylene glycol (PEG) did not improve immune responses to either intravenously or subcutaneously delivered vaccines. Immune responses to short and long peptide sequences (CD8 and CD4 epitopes) of the self-antigen tyrosinase-related protein 2 (TRP2) as a vaccine antigen, co-delivered with α-GalCer in either cationic liposomes or PBS were further examined. Enhanced production of IFN-γ, increased cytotoxic T-cell responses and tumour survival were observed when a long TRP2-peptide was delivered with α-GalCer in cationic liposomes.

Regioselective Approach to Phosphatidylinositol 3,5-Bisphosphates: Syntheses of the Native Phospholipid and Biotinylated Short-Chain Derivative

A selective bis-silylation of 1D-O-TBDPS-myo-inositol leads to a 1,3,5-trisubstituted inositol, which can be advanced to the headgroup of phosphatidylinositol-3,5-bisphosphate [PI(3,5)P(2)]. A mild, regioselective method for construction of the diacylglycerol moiety containing differing fatty acid chains, including the naturally occurring lipids, was developed. Their union in the synthesis of the cell-signaling molecule PI(3,5)P(2) containing the sn-1-stearoyl and sn-2-arachidonoyl groups is described. The methodology was also used to generate dioctanoyl-PI(3,5)P(2) and a previously unreported biotin-PI(3,5)P(2) conjugate, which was coupled to neutravidin beads and used to pull down PI(3,5)P(2)-binding proteins from the cytosolic extract of adrenal neurosecretory cells. We report the specific pull-down of the PI(3,5)P(2)-binding protein svp1p, a known PI(3,5)P(2) effector involved in membrane trafficking.

Activation of Human Mucosal-Associated Invariant T Cells Induces CD40L-Dependent Maturation of Monocyte-Derived and Primary Dendritic Cells

Mucosal-associated invariant T (MAIT) cells are innate T cells that recognize intermediates of the vitamin B2 biosynthetic pathway presented by the monomorphic MR1 molecule. It remains unclear whether, in addition to their cytolytic activity that is important in antimicrobial defense, MAIT cells have immune-modulatory functions that could enhance dendritic cell (DC) maturation. In this study, we investigated the molecular mechanisms dictating the interactions between human MAIT cells and DCs and demonstrate that human MAIT cells mature monocyte-derived and primary DCs in an MR1- and CD40L-dependent manner. Furthermore, we show that MAIT cell–derived signals synergize with microbial stimuli to induce secretion of bioactive IL-12 by DCs. Activation of human MAIT cells in whole blood leads to MR1- and cytokine-dependent NK cell transactivation. Our results underscore an important property of MAIT cells, which can be of translational relevance to rapidly orchestrate adaptive immunity through DC maturation.

Glycolipid-peptide conjugate vaccines enhance CD8 + T cell responses against human viral proteins

An important goal of vaccination against viruses and virus-driven cancers is to elicit cytotoxic CD8+ T cells specific for virus-derived peptides. CD8+ T cell responses can be enhanced by engaging help from natural killer T (NKT) cells. We have produced synthetic vaccines that induce strong peptide-specific CD8+ T cell responses in vivo by incorporating an NKT cell-activating glycolipid. Here we examine the effect of a glycolipid-peptide conjugate vaccine incorporating an NKT cell-activating glycolipid linked to an MHC class I-restricted peptide from a viral antigen in human peripheral blood mononuclear cells. The vaccine induces CD1d-dependent activation of human NKT cells following enzymatic cleavage, activates human dendritic cells in an NKT-cell dependent manner, and generates a pool of activated antigen-specific CD8+ T cells with cytotoxic potential. Compared to unconjugated peptide, the vaccine upregulates expression of genes encoding interferon-γ, CD137 and granzyme B. A similar vaccine incorporating a peptide from the clinically-relevant human papilloma virus (HPV) 16 E7 oncoprotein induces cytotoxicity against peptide-expressing targets in vivo, and elicits a better antitumor response in a model of E7-expressing lung cancer than its unconjugated components. Glycolipid-peptide conjugate vaccines may prove useful for the prevention or treatment of viral infections and tumors that express viral antigens.

Augmenting Influenza-Specific T Cell Memory Generation with a Natural Killer T Cell-Dependent Glycolipid–Peptide Vaccine

The development of a universal vaccine for influenza A virus (IAV) that does not require seasonal modification is a long-standing health goal, particularly in the context of the increasing threat of new global pandemics. Vaccines that specifically induce T cell responses are of considerable interest because they can target viral proteins that are more likely to be shared between different virus strains and subtypes and hence provide effective cross-reactive IAV immunity. From a practical perspective, such vaccines should induce T cell responses with long-lasting memory, while also being simple to manufacture and cost-effective. Here we describe the synthesis and evaluation of a vaccine platform based on solid phase peptide synthesis and bio-orthogonal conjugation methodologies. The chemical approach involves covalently attaching synthetic long peptides from a virus-associated protein to a powerful adjuvant molecule, α-galactosylceramide (α-GalCer). Strain-promoted azide-alkyne cycloaddition is used as a simple and efficient method for conjugation, and pseudoproline methodology is used to increase the efficiency of the peptide synthesis. α-GalCer is a glycolipid that stimulates NKT cells, a population of lymphoid-resident immune cells that can provide potent stimulatory signals to antigen-presenting cells engaged in driving proliferation and differentiation of peptide-specific T cells. When used in mice, the vaccine induced T cell responses that provided effective prophylactic protection against IAV infection, with the speed of viral clearance greater than that seen from previous viral exposure. These findings are significant because the vaccines are highly defined, quick to synthesize, and easily characterized and are therefore appropriate for large scale affordable manufacture.