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Conjugation of a therapeutic agent to a polymer for enhanced delivery into target cells followed by its intracellular triggered release has proved to be an effective drug delivery approach. This approach is applied to the delivery of the immune-stimulatory unmethylated cytosine-phosphate-guanine (CpG) oligonucleotide for an anti-tumour immune response after intratumoral administration. On average four CpG-1668 molecules were covalently linked to a 40-kDa amino-functionalised dextran polymer via either a non-reversible (CpG-dextran) or an intracellular redox-responsive disulfide linkage (CpG-SS-dextran). Dynamic light scattering analysis showed that both conjugates had a similar particle size and surface charge of 17 nm and -10 mV, respectively. Agarose gel electrophoresis analysis showed that CpG-SS-dextran was stable in the extracellular low glutathione (GSH) concentration range (i.e. 10-20 μM) and was cleaved at the higher intracellular GSH concentration (5 mM), while CpG-dextran was stable in both GSH concentrations. Uptake and activation assays on bone-marrow-derived dendritic cells showed no significant difference between free CpG, CpG-dextran and CpG-SS-dextran. In a mouse subcutaneous colorectal tumour model the CpG-SS-dextran showed a statistically significantly greater inhibition of tumour growth (p < 0.03) and prolonged survival (p < 0.001) compared to CpG-dextran or free CpG. These results demonstrate that the redox-triggered intracellular release of CpG from a dextran polymer carrier has promise for intratumoral therapeutic vaccination against cancer., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Greg Walker reports equipment, drugs, or supplies was provided by University of Otago. Greg Walker reports a relationship with University of Otago that includes: employment. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

α-Azido ketones and their vinylogous relatives β-alkoxy-γ-azido enones are versatile building blocks for constructing diverse heterocyclic products, but are prone to azide decomposition. Here, we report their condensation with propargylic amines and investigate the fate of the intermediate azido-enamine condensation products, both experimentally and theoretically. Efficient intramolecular cycloaddition was observed for electron-poor azide substrates, and a range of diversely substituted [1,2,3]triazolo[1,5- a ]pyrazine products is reported. For electron-rich substrates, azide decomposition predominated. Computational modeling of possible pathways from the azido-enamine intermediates revealed two alternative mechanisms for azide decomposition, which were consistent with observed side products.

Eliciting an antihapten antibody response to vaccination typically requires the use of constructs where multiple copies of the hapten are covalently attached to a larger carrier molecule. The carrier is required to elicit T cell help via presentation of peptide epitopes on major histocompatibility complex (MHC) class II molecules; as such, attachment to full-sized proteins, alone or in a complex, is generally used to account for the significant MHC diversity in humans. While such carrier-based vaccines have proven extremely successful, particularly in protecting against bacterial diseases, they can be challenging to manufacture, and repeated use can be compromised by pre-existing immunity against the carrier. One approach to reducing these complications is to recruit help from type I natural killer T (NKT) cells, which exhibit limited diversity in their antigen receptors and respond to glycolipid antigens presented by the highly conserved presenting molecule CD1d. Synthetic vaccines for universal use can, therefore, be prepared by conjugating haptens to an NKT cell agonist such as α-galactosylceramide (αGalCer, KRN7000). An additional advantage is that the quality of NKT cell help is sufficient to overcome the need for an extra immune adjuvant. However, while initial studies with αGalCer-hapten conjugate vaccines report strong and rapid antihapten antibody responses, they can fail to generate lasting memory. Here, we show that antibody responses to the hapten 4-hydoxy-3-nitrophenyl acetyl (NP) can be improved through additional attachment of a fusion peptide containing a promiscuous helper T cell epitope (Pan DR epitope, PADRE) that binds diverse MHC class II molecules. Such αGalCer-hapten-peptide tricomponent vaccines generate strong and sustained anti-NP antibody titers with increased hapten affinity compared to vaccines without the helper epitope. The tricomponent vaccine platform is therefore suitable for further exploration in the pursuit of efficacious antihapten immunotherapies.

Background & Aims: Liver diseases resulting from chronic HBV infection are a significant cause of morbidity and mortality. Vaccines that elicit T-cell responses capable of controlling the virus represent a treatment strategy with potential for long-term effects. Here, we evaluated vaccines that induce the activity of type I natural killer T (NKT) cells to limit viral replication and license stimulation of conventional antiviral T-cells., Methods: Vaccines were prepared by conjugating peptide epitopes to an NKT-cell agonist to promote co-delivery to antigen-presenting cells, encouraging NKT-cell licensing and stimulation of T cells. Activity of the conjugate vaccines was assessed in transgenic mice expressing the complete HBV genome, administered intravenously to maximise access to NKT cell-rich tissues., Results: The vaccines induced only limited antiviral activity in unmanipulated transgenic hosts, likely attributable to NKT-cell activation as T-cell tolerance to viral antigens is strong. However, in a model of chronic hepatitis B involving transfer of naive HBcAg-specific CD8 + T cells into the transgenic mice, which typically results in specific T-cell dysfunction without virus control, vaccines containing the targeted HBcAg epitope induced prolonged antiviral activity because of qualitatively improved T-cell stimulation. In a step towards a clinical product, vaccines were prepared using synthetic long peptides covering clusters of known HLA-binding epitopes and shown to be immunogenic in HLA transgenic mice. Predictions based on HLA distribution suggest a product containing three selected SLP-based vaccines could give >90 % worldwide coverage, with an average of 3.38 epitopes targeted per individual., Conclusions: The novel vaccines described show promise for further clinical development as a treatment for chronic hepatitis B., Impact and Implications: Although there are effective prophylactic vaccines for HBV infection, it is estimated that 350-400 million people worldwide have chronic hepatitis B, putting these individuals at significant risk of life-threatening liver diseases. Therapeutic vaccination aimed at activating or boosting HBV-specific T-cell responses holds potential as a strategy for treating chronic infection, but has so far met with limited success. Here, we show that a glycolipid-peptide conjugate vaccine designed to coordinate activity of type I NKT cells alongside conventional antiviral T cells has antiviral activity in a mouse model of chronic infection. It is anticipated that a product based on a combination of three such conjugates, each prepared using long peptides covering clusters of known HLA-binding epitopes, could be developed further as a treatment for chronic hepatitis B with broad global HLA coverage., Competing Interests: None of the authors have any conflict of interest. Please refer to the accompanying ICMJE disclosure forms for further details., (© 2024 The Authors.)

Natural killer T (NKT) cells are a population of innate-like T cells capable of enhancing both innate and adaptive immune responses. Co-delivering an NKT cell agonist and antigen can provide molecular signals to antigen-presenting cells, such as dendritic and B cells, that facilitate strong antigen-specific adaptive immune responses. Accordingly, there has been a significant number of developmental NKT cell-dependent vaccine therapies developed, particularly in the last decade, with many incorporating cancer antigens. In this review, we summarize studies that chemically conjugate the NKT cell agonist and antigen as an effective strategy for agonist-antigen co-delivery to drive antitumor responses.

Malaria is caused by Plasmodium species transmitted by Anopheles mosquitoes. Following a mosquito bite, Plasmodium sporozoites migrate from skin to liver, where extensive replication occurs, emerging later as merozoites that can infect red blood cells and cause symptoms of disease. As liver tissue-resident memory T cells (Trm cells) have recently been shown to control liver-stage infections, we embarked on a messenger RNA (mRNA)-based vaccine strategy to induce liver Trm cells to prevent malaria. Although a standard mRNA vaccine was unable to generate liver Trm or protect against challenge with Plasmodium berghei sporozoites in mice, addition of an agonist that recruits T cell help from type I natural killer T cells under mRNA-vaccination conditions resulted in significant generation of liver Trm cells and effective protection. Moreover, whereas previous exposure of mice to blood-stage infection impaired traditional vaccines based on attenuated sporozoites, mRNA vaccination was unaffected, underlining the potential for such a rational mRNA-based strategy in malaria-endemic regions., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

mRNA vaccines have recently generated significant interest due to their success during the COVID-19 pandemic. Their success is due to advances in mRNA design and encapsulation into ionizable lipid nanoparticles (iLNPs). This has highlighted the potential for the use of mRNA-iLNPs in other settings such as cancer, gene therapy, or vaccines for different infectious diseases. Here, we describe the production of mRNA-iLNPs using commercially available reagents that are suitable for use as vaccines and therapeutics. This article contains detailed protocols for the synthesis of mRNA by in vitro transcription with enzymatic capping and tailing and the encapsulation of the mRNA into iLNPs using the ionizable lipid DLin-MC3-DMA. DLin-MC3-DMA is often used as a benchmark for new formulations and provides an efficient delivery vehicle for screening mRNA design. The protocol also describes how the formulation can be adapted to other lipids. Finally, a stepwise methodology is presented for the characterization and quality control of mRNA-iLNPs, including measuring mRNA concentration and encapsulation efficiency, particle size, and zeta potential. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Synthesis of mRNA by in vitro transcription and enzymatic capping and tailing Basic Protocol 2: Encapsulation of mRNA into ionizable lipid nanoparticles Alternate Protocol: Small-scale encapsulation of mRNA using preformed vesicles Basic Protocol 3: Characterization and quality control of mRNA ionizable lipid nanoparticles., (© 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.)

Aim: We have previously reported that polyfunctional T cell responses can be induced to the cancer testis antigen NY-ESO-1 in melanoma patients injected with mature autologous monocyte-derived dendritic cells (DCs) loaded with long NY-ESO-1-derived peptides together with α-galactosylceramide (α-GalCer), an agonist for type 1 Natural Killer T (NKT) cells., Objective: To assess whether inclusion of α-GalCer in autologous NY-ESO-1 long peptide-pulsed DC vaccines (DCV + α-GalCer) improves T cell responses when compared to peptide-pulsed DC vaccines without α-GalCer (DCV)., Design, Setting and Participants: Single-centre blinded randomised controlled trial in patients ≥ 18 years old with histologically confirmed, fully resected stage II-IV malignant cutaneous melanoma, conducted between July 2015 and June 2018 at the Wellington Blood and Cancer Centre of the Capital and Coast District Health Board., Interventions: Stage I. Patients were randomised to two cycles of DCV or DCV + α-GalCer (intravenous dose of 10 × 10 6 cells, interval of 28 days). Stage II. Patients assigned to DCV + α-GalCer were randomised to two further cycles of DCV + α-GalCer or observation, while patients initially assigned to DCV crossed over to two cycles of DCV + α-GalCer., Outcome Measures: Primary: Area under the curve (AUC) of mean NY-ESO-1-specific T cell count detected by ex vivo IFN-γ ELISpot in pre- and post-treatment blood samples, compared between treatment arms at Stage I. Secondary: Proportion of responders in each arm at Stage I; NKT cell count in each arm at Stage I; serum cytokine levels at Stage I; adverse events Stage I; T cell count for DCV + α-GalCer versus observation at Stage II, T cell count before versus after cross-over., Results: Thirty-eight patients gave written informed consent; 5 were excluded before randomisation due to progressive disease or incomplete leukapheresis, 17 were assigned to DCV, and 16 to DCV + α-GalCer. The vaccines were well tolerated and associated with increases in mean total T cell count, predominantly CD4 + T cells, but the difference between the treatment arms was not statistically significant (difference - 6.85, 95% confidence interval, - 21.65 to 7.92; P = 0.36). No significant improvements in T cell response were associated with DCV + α-GalCer with increased dosing, or in the cross-over. However, the NKT cell response to α-GalCer-loaded vaccines was limited compared to previous studies, with mean circulating NKT cell levels not significantly increased in the DCV + α-GalCer arm and no significant differences in cytokine response between the treatment arms., Conclusions: A high population coverage of NY-ESO-1-specific T cell responses was achieved with a good safety profile, but we failed to demonstrate that loading with α-GalCer provided an additional advantage to the T cell response with this cellular vaccine design., Clinical Trial Registration: ACTRN12612001101875. Funded by the Health Research Council of New Zealand., (© 2023. The Author(s).)

Protective immune responses against respiratory pathogens, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza virus, are initiated by the mucosal immune system. However, most licensed vaccines are administered parenterally and are largely ineffective at inducing mucosal immunity. The development of safe and effective mucosal vaccines has been hampered by the lack of a suitable mucosal adjuvant. In this study we explore a class of adjuvant that harnesses mucosal-associated invariant T (MAIT) cells. We show evidence that intranasal immunization of MAIT cell agonists co-administered with protein, including the spike receptor binding domain from SARS-CoV-2 virus and hemagglutinin from influenza virus, induce protective humoral immunity and immunoglobulin A production. MAIT cell adjuvant activity is mediated by CD40L-dependent activation of dendritic cells and subsequent priming of T follicular helper cells. In summary, we show that MAIT cells are promising vaccine targets that can be utilized as cellular adjuvants in mucosal vaccines., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Synthetic vaccines that induce T cell responses to peptide epitopes are a promising immunotherapy for both communicable and noncommunicable diseases. Stimulating strong and sustained T cell responses requires antigen delivery to appropriately activated antigen presenting cells (APCs). One way this can be accomplished is by chemically conjugating immunogenic peptide epitopes with α-galactosylceramide (α-GalCer), a glycolipid that acts as an immune adjuvant by inducing stimulatory interactions between APCs and type I natural killer T (NKT) cells. Here we investigate whether increasing the ratio of antigen:adjuvant improves antigen-specific T cell responses. A series of conjugate vaccines was prepared in which one, two, four, or eight copies of an immunogenic peptide were covalently attached to a modified form of α-GalCer via the poly(ethoxyethylglycinamide) dendron scaffold. Initial attempts to synthesize these multivalent conjugate vaccines involved attaching the bicyclo[6.1.0]non-4-yne (BCN) group to the adjuvant-dendron structure followed by strain-promoted azide-alkyne cycloaddition of the peptide. Although this approach was successful for preparing vaccines with either one or two peptide copies, the synthesis of vaccines requiring attachment of four or eight BCN groups suffered from low yields due to cyclooctyne degradation. Instead, conjugate vaccines containing up to eight peptide copies were readily achieved through oxime ligation with adjuvant-dendron constructs decorated with the 8-oxo-nonanoyl group. When evaluating T cell responses to vaccination in mice, we confirmed a significant advantage to conjugation over admixes of peptide and α-GalCer, regardless of the peptide to adjuvant ratio, but there was no advantage to increasing the number of peptides attached. However, it was notable that the higher ratio conjugate vaccines required lower levels of NKT cell activation to be effective, which could be a safety advantage for future vaccine candidates.

The racemic title compound, C 34 H 32 OS 2 , comprises an atropisomeric binaphthyl di-thio-acetal substituted at the methyl-ene carbon atom with a chiral benzyl alcohol. The two naphthalene ring systems are additionally substituted at the 3,3'-position with isopropyl groups. The overall stereochemistry is defined as aS,R and aR,S . The hydroxyl group forms an intra-molecular O-H⋯S hydrogen bond to one of the sulfur atoms. The crystal structure contains weak C-H⋯π inter-actions that link the mol-ecules into extended arrays., (© Beare et al. 2023.)

The closely related title compounds, 1-(di-naphtho-[2,1- d :1',2'- f ][1,3]dithiepin-4-yl)-2,2-di-methyl-propan-1-ol, C 26 H 24 OS 2 , 1 and 2-(di-naphtho-[2,1- d :1',2'- f ][1,3]dithiepin-4-yl)-3,3-di-methyl-butan-2-ol, C 27 H 26 OS 2 , 2 , both comprise an atrop-isomeric binaphthyl di-thio-acetal unit substituted at the methyl-ene carbon atom with a chiral neopentyl alcohol grouping. The overall stereochemistry of the racemate in each case is defined as aS,R and aR,S . In 1 , the hydroxyl group generates inversion dimers via pairwise inter-molecular O-H⋯S hydrogen bonds whereas in 2 , the O-H⋯S link is intra-molecular. Weak C-H⋯π inter-actions link the mol-ecules into extended arrays in both structures., (© Beare et al. 2023.)

MR1 is a highly conserved microbial immune-detection system in mammals. It captures vitamin B-related metabolite antigens from diverse microbes and presents them at the cell surface to stimulate MR1-restricted lymphocytes including mucosal-associated invariant T (MAIT) cells. MR1 presentation and MAIT cell recognition mediate homeostasis through host defense and tissue repair. The cellular mechanisms regulating MR1 cell surface expression are critical to its function and MAIT cell recognition, yet they are poorly defined. Here, we report that human MR1 is equipped with a tyrosine-based motif in its cytoplasmic domain that mediates low affinity binding with the endocytic adaptor protein 2 (AP2) complex. This interaction controls the kinetics of MR1 internalization from the cell surface and minimizes recycling. We propose MR1 uses AP2 endocytosis to define the duration of antigen presentation to MAIT cells and the detection of a microbial metabolic signature by the immune system., (© 2022 Lim et al.)

The effect of dextran molecular weight on the in vitro physicochemical and immune properties of cytosine-phosphate-guanine (CpG) oligodeoxynucleotide-amino-dextran conjugates is investigated. CpG-1668 was conjugated at the 3'-end to amino-dextran of differing molecular weight (20, 40, 70 or 110-kDa) via a stable bis-aryl hydrazone linkage. Conjugate formation was confirmed by agarose gel electrophoresis and dynamic light scattering measured the size and surface charge of conjugates. Uptake and immune-stimulatory activity of CpG-dextran by antigen-presenting cells was evaluated by flow cytometry and confocal microscopy. Degradation by DNase I was monitored by loss of the fluorescent signal from labelled CpG and changes in size and zeta potential. Hydrazone bond formation (UV 354 nm) showed on average four CpG molecules conjugated per polymer. CpG-dextran prepared from 20 or 40-kDa dextran had a size of 17 nm while 70 or 110-kDa was 30 nm. CpG-dextran was preferentially taken up by dendritic cells, followed by macrophages and then B-cells. Only the 20-kDa dextran conjugate significantly enhanced uptake by bone-marrow derived dendritic cells (BMDCs) compared to free CpG. Confocal microscopy showed that CpG and CpG-dextran accumulates in the endo-lysosomal compartment of BMDCs at 24 h. All conjugates upregulated activation markers (CD40, CD80 or CD86) of BMDCs to a similar level as for free CpG. CpG-dextran 40-kDa produced highest levels of cytokines (TNF-α, IL-6, and IL-12p70) secreted by BMDCs. Enzymatic protection assays showed that the conjugate made from dextran 20-kDa provided no protection for CpG while the higher molecular weight conjugates reduced degradation by DNase I. The 40-kDa dextran conjugate produced the greatest in vitro immune activity, this was due to the conjugate being relatively small in size for cell uptake while sufficiently large enough to protect CpG from nuclease attack. These in vitro studies identify the need to consider the molecular weight of the carrier in bioconjugate design., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier B.V.)

Objectives: Metastasis is the principal cause of breast cancer mortality. Vaccines targeting breast cancer antigens have yet to demonstrate clinical efficacy, and there remains an unmet need for safe and effective treatment to reduce the risk of metastasis, particularly for people with triple-negative breast cancer (TNBC). Certain glycolipids can act as vaccine adjuvants by specifically stimulating natural killer T (NKT) cells to provide a universal form of T-cell help., Methods: We designed and made a series of conjugate vaccines comprising a prodrug of the NKT cell-activating glycolipid α-galactosylceramide covalently linked to tumor-expressed peptides, and assessed these using E0771- and 4T1-based breast cancer models in vivo . We employed peptides from the model antigen ovalbumin and from clinically relevant breast cancer antigens HER2 and NY-ESO-1., Results: Glycolipid-peptide conjugate vaccines that activate NKT cells led to antigen-presenting cell activation, induced inflammatory cytokines, and, compared with peptide alone or admixed peptide and α-galactosylceramide, specifically enhanced CD8 + T-cell responses against tumor-associated peptides. Primary tumor growth was delayed by vaccination in all tumor models. Using 4T1-based cell lines expressing HER2 or NY-ESO-1, a single administration of the relevant conjugate vaccine prevented tumor colonisation of the lung following intravenous inoculation of tumor cells or spontaneous metastasis from breast, respectively., Conclusion: Glycolipid-peptide conjugate vaccines that activate NKT cells prevent lung metastasis in breast cancer models and warrant investigation as adjuvant therapies for high-risk breast cancer., Competing Interests: IH and GP are inventors on patents licensed to or owned by biotech start‐up Avalia Immunotherapies Limited., (© 2022 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.)

Full-spectrum flow cytometry is now routinely used in many laboratories internationally, and the demand for this technology is rapidly increasing. With capacity to use larger and more complex staining panels, standardized protocols are required for optimal panel design and analysis. Importantly, for ex vivo analysis, tissue preparation methods also need to be optimized to ensure samples are truly representative of tissues in situ. This is particularly relevant given the recent interest in adaptive immune cells that form residency in specific organs. Here we provide optimized protocols for tissue processing and phenotyping of memory T cells and natural killer T (NKT) cell subsets from liver, lung, spleen, and lymph node using full-spectrum flow cytometry. We provide a 21-color antibody panel for identification of different memory subsets, including tissue-resident memory T (T RM ) cells, which are increasingly regarded as important effectors in adaptive immunity. We show that processing procedures can affect outcomes, with liver T RM cells particularly sensitive to heat, such that accurate evaluation requires fast processing at defined temperatures. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Processing mouse liver for flow cytometric analysis of memory T and NKT cell subsets Basic Protocol 2: Processing mouse spleen for flow cytometric analysis of memory T and NKT cell subsets Basic Protocol 3: Processing mouse lungs for flow cytometric analysis of memory T and NKT cell subsets Basic Protocol 4: Processing mouse lymph nodes for flow cytometric analysis of memory T and NKT cell subsets Basic Protocol 5: Staining and flow cytometric analysis of samples for memory T and NKT cell subsets Support Protocol: Obtaining cell counts from flow cytometry data., (© 2022 Wiley Periodicals LLC.)

Intratumoural administration of unmethylated cytosine-phosphate-guanine motifs (CpG) to stimulate toll-like receptor (TLR)-9 has been shown to induce tumour regression in preclinical studies and some efficacy in the clinic. Because activated natural killer T (NKT) cells can cooperate with pattern-recognition via TLRs to improve adaptive immune responses, we assessed the impact of combining a repeated dosing regimen of intratumoural CpG with a single intratumoural dose of the NKT cell agonist α-galactosylceramide (α-GalCer). The combination was superior to CpG alone at inducing regression of established tumours in several murine tumour models, primarily mediated by CD8 + T cells. An antitumour effect on distant untreated tumours (abscopal effect) was reliant on sustained activity of NKT cells and was associated with infiltration of KLRG1 + NKT cells in tumours and draining lymph nodes at both injected and untreated distant sites. Cytometric analysis pointed to increased exposure to type I interferon (IFN) affecting many immune cell types in the tumour and lymphoid organs. Accordingly, antitumour activity was lost in animals in which dendritic cells (DCs) were incapable of signaling through the type I IFN receptor. Studies in conditional ablation models showed that conventional type 1 DCs and plasmacytoid DCs were required for the response. In tumour models where the combined treatment was less effective, the addition of tumour-antigen derived peptide, preferably conjugated to α-GalCer, significantly enhanced the antitumour response. The combination of TLR ligation, NKT cell agonism, and peptide delivery could therefore be adapted to induce responses to both known and unknown antigens., Competing Interests: KK Prasit, IF Hermans and GF Painter are named inventors on a provisional patent application related to data and synthetic conjugate compounds described here. IF Hermans and GF Painter are founding scientists and shareholders of the biotech start-up Avalia Immunotherapies Limited. DI Godfrey is a member of the scientific advisory board, and a shareholder of Avalia Immunotherapies Limited., (© 2022 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Self-adjuvanting vaccines consisting of peptide epitopes conjugated to immune adjuvants are a powerful way of generating antigen-specific immune responses. We previously showed that a Plasmodium -derived peptide conjugated to a rearranged form of α-galactosylceramide (α-GalCer) could stimulate liver-resident memory T (T RM ) cells that were effective killers of liver-stage Plasmodium berghei ANKA (Pba)-infected cells. To investigate if similar or even superior T RM responses can be induced by modifying the α-GalCer adjuvant, we created new conjugate vaccine cadidates by attaching an immunogenic Plasmodium -derived peptide antigen to 6″-substituted α-GalCer analogues. Vaccine synthesis involved developing an efficient route to α-galactosylphytosphingosine (α-GalPhs), from which the prototypical iNKT cell agonist, α-GalCer, and its 6″-deoxy-6″-thio and -amino analogues were derived. Attaching a cathepsin B-cleavable linker to the 6″-modified α-GalCer created pro-adjuvants bearing a pendant ketone group available for peptide conjugation. Optimized reaction conditions were developed that allow for the efficient conjugation of peptide antigens to the pro-adjuvants via oxime ligation to create new glycolipid-peptide (GLP) conjugate vaccines. A single dose of the vaccine candidates induced acute NKT and Plasmodium -specific CD8 + T cell responses that generated potent hepatic T RM responses in mice. Our findings demonstrate that attaching antigenic peptides to 6″-modifed α-GalCer generates powerful self-adjuvanting conjugate vaccine candidates that could potentially control hepatotropic infections such as liver-stage malaria., Competing Interests: The authors declare the following competing financial interest(s): G. F. P. and I. F. H. are the chief technical officer and chief scientific officer (respectively) of Avalia Immunotherapies Limited, and W. R. H. is a member of its Scientific Advisory Board. Avalia holds exclusive, worldwide license to patents related to aspects of the chemical design reported here. R. J. A., I. F. H., G. F. P., and B. J. C. are inventors on a granted patent (U.S. patent no. 10046046, granted 14 August 2018). B.J.C., R.J.A., I.F.H., G. F. P., L. E. H., and W. R. H. are named inventors on a PCT application (PCTNZ2020050048) submitted by Victoria University of Wellington, Malcorp Biodiscoveries Limited, and Victoria Link Limited that covers the production of tissue-resident memory T cells with glycolipid-peptide vaccines., (This journal is © The Royal Society of Chemistry.)

Prostate cancer is the second most common cancer in men worldwide. Despite an abundance of prostate-specific antigens, immunotherapies have yet to become a standard of care, potentially limited by T-cell dysfunction. Up to 10% of human circulating T-cells, and a significant fraction in the urogenital tract, are mucosal-associated invariant T (MAIT) cells. MAIT cells express stereotyped T-cell receptors that recognize riboflavin metabolites derived from microbes presented by MR-1. We evaluated the number, phenotype and function of circulating MAIT cells, alongside two other innate-like T (ILT) -cell subsets, in men with prostate cancer and age- and sex-matched controls. MAIT cells in men with prostate cancer circulated at similar frequencies to controls, but their cytokine production and proliferation was impaired. In contrast, the function of two other ILT-cell populations (natural killer T-cells and Vγ9Vδ2 T-cells) was not impaired. In both patients and controls, MAIT cells expressed high levels of the immune checkpoint molecule PD-1 at rest, while upregulation of PD-1 in response to the MR-1 ligand 5-amino-6D-ribitylaminouracil (5-A-RU) was greater in patients. 5-A-RU also induced upregulation of PD-L1 and -L2 RNA in primary mononuclear cells. We confirmed that circulating MAIT cell number and function were preserved before and during anti-PD1 therapy with pembrolizumab in a cohort of patients with melanoma. In vitro , 5-A-RU enhanced mononuclear cell cytotoxicity against the PD-L1 positive prostate cancer cell line PC3 in an MR-1-dependent manner. Addition of pembrolizumab enhanced this cytotoxicity, and was associated with increased MAIT cell expression of CD107a and IFN-γ. We conclude that prostate cancer is associated with MAIT-cell dysfunction, and that this might be overcome through the application of potent MR-1 ligands with PD-1 blockade. These findings may have implications for the development of cancer immunotherapies that exploit MAIT cells., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Jarvis, Collings, Authier-Hall, Dasyam, Luey, Nacey, Painter, Delahunt, Hermans and Weinkove.)

Natural killer T (NKT) cells are innate-like T cells capable of enhancing both innate and adaptive immune responses. When NKT cells are stimulated in close temporal association with co-administered antigens, strong antigen-specific immune responses can be induced, prompting the study of NKT cell agonists as novel immune adjuvants. This activity has been attributed to the capacity of activated NKT cells to act as universal helper cells, with the ability to provide molecular signals to dendritic cells and B cells that facilitate T cell and antibody responses, respectively. These signals can override the requirement for conventional CD4 + T cell help, so that vaccines can be designed without need to consider CD4 + T cell repertoire and major histocompatibility complex Class II diversity. Animal studies have highlighted some drawbacks of the approach, namely, concerns around induction of NKT cell hyporesponsiveness, which may limit vaccine boosting, and potential for toxicity. Here we highlight studies that suggest these obstacles can be overcome by targeted delivery in vivo . We also feature new studies that suggest activating NKT cells can help encourage differentiation of T cells into tissue-resident memory cells that play an important role in prophylaxis against infection, and may be required in cancer therapy., (© The Author(s) 2021. Published by Oxford University Press.)

Cytosine-phosphate-guanine (CpG) oligonucleotides are commonly-used vaccine adjuvants to promote the activation of antigen-presenting cells (APCs). To mount an effective immune response, CpG needs to be internalized and bind to its endosomal Toll-like receptor 9 (TLR-9) inside the APCs. Using flow cytometry and fluorescence microscopy, this article presents the cellular uptake data of the amino-dextran nanoparticle (aDNP) and aDNP loaded with CpG immobilized on its surface by either electrostatic adsorption or covalent conjugation. The uptake of fluorescently-labelled aDNPs by murine splenic dendritic cells and macrophages was determined by flow cytometry and uptake by murine bone-marrow-derived dendritic cells was evaluated by fluorescence microscopy. The data presented in this paper correlates with the in vitro immune-stimulatory activity observed for the two different CpG loading methods in the research article " Nanoparticle system based on amino-dextran as a drug delivery vehicle: immune-stimulatory CpG-oligonucleotide loading and delivery " (Nguyen et al ., 2020) [1]. The data provide experimental evidence for a better understanding how the nanoparticle surface loading method of CpG influences the uptake of these nanoparticles by antigen-presenting cells as a step guide in the design of more effective vaccine formulations., Competing Interests: The authors declare that there are no conflicts of interest regarding the publication of this paper., (© 2021 The Authors. Published by Elsevier Inc.)

The capacity of α-galactosylceramide (α-GalCer) to act as an anti-cancer agent in mice through the specific stimulation of type I NKT (iNKT) cells has prompted extensive investigation to translate this finding to the clinic. However, low frequencies of iNKT cells in cancer patients and their hypo-responsiveness to repeated stimulation have been seen as barriers to its efficacy. Currently the most promising clinical application of α-GalCer, or its derivatives, is as stimuli for ex vivo expansion of iNKT cells for adoptive therapy, although some encouraging clinical results have recently been reported using α-GalCer pulsed onto large numbers of antigen presenting cells (APCs). In on-going preclinical studies, attempts to improve efficacy of injected iNKT cell agonists have focussed on optimising presentation in vivo, through encapsulation in particulate vectors, making structural changes that help binding to the presenting molecule CD1d, or injecting agonists covalently attached to recombinant CD1d. Variations on these same approaches are being used to enhance the APC-licencing function of iNKT cells in vivo to induce adaptive immune responses to associated tumour antigens. Looking ahead, a unique capacity of in vivo-activated iNKT cells to facilitate formation of resident memory CD8 + T cells is a new observation that could find a role in cancer therapy., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The aim of this study is to prepare and characterize an amino-dextran nanoparticle (aDNP) platform and investigate two loading strategies for unmethylated cytosine-phosphate-guanine (CpG) oligonucleotide. aDNP was prepared by desolvation of amino-dextran followed by the chemical crosslinking of amino groups. Size, surface charge, and surface morphology of aDNP was determined by dynamic light scattering and transmission electron microscopy. CpG was either loaded onto aDNP by adsorption (CpG-adsorbed-aDNP) or conjugated to aDNP (CpG-conjugated-aDNP). In vitro cytokine production by bone marrow-derived dendritic cells (BMDCs) was measured by flow cytometry. aDNPs size and zeta potential could be controlled to produce uniform particles in the size range of 50 to 300 nm, surface charge of -16.5 to +14 mV, and were spherical in shape. Formulation control parameters investigated included the anti-solvent, water-to-anti-solvent ratio, level of amine functionality of dextran, and the molar ratio of glutaraldehyde to amine. aDNP could be lyophilized without additional cryoprotectant. Unloaded cationic aDNP (+13 mV) showed acceptable in vitro hemolysis. Unloaded and CpG-loaded aDNPs showed no cytotoxicity on BMDCs. CpG-loaded nanoparticles stimulated cytokine production by BMDCs, the level of cytokine production was higher for CpG-conjugated-aDNP compared to CpG-absorbed-aDNP. aDNP is a promising new drug delivery platform as its offers versatility in loading and tuning of particle properties.

Methylglyoxal (MGO) is the main antimicrobial determinant associated with using Mānuka Honey as a topical dressing. While direct mechanisms of Mānuka honey MGO's antimicrobial activity have been demonstrated, such as disruption of bacterial fimbria and flagella, no interaction of Mānuka honey-derived MGO with antimicrobial effector cells of the immune system, such as mucosal-associated invariant T cells (MAIT cells), has yet been reported. MAIT cells are an abundant subset of human T cells, critical for regulating a diverse range of immune functions, including antimicrobial defense mechanisms but also mucosal barrier integrity. MAIT cells become activated by recognition of an important microbial metabolite, 5-amino-6-d-ribitylaminouracil (5-A-RU), which is produced by a wide range of microbial pathogens and commensals. Recognition is afforded when 5-A-RU condenses with mammalian-cell derived MGO to form the potent MAIT cell activator, 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil (5-OP-RU). Formation of 5-OP-RU and its subsequent presentation to MAIT cells by major histocompatibility (MHC)-related molecule 1 (MR1) facilitates host-pathogen and host-commensal interactions. While MGO is a metabolite naturally present in mammalian cells, it is unclear whether exogenous dietary MGO sources, such as those obtained from Mānuka honey intake, can contribute to 5-OP-RU formation and enhance MAIT cell activation. In this work, we report that endogenous MGO is the rate-limiting substrate for converting microbial 5-A-RU to 5-OP-RU and that Mānuka honey-derived MGO significantly enhances MAIT cell activation in vitro. Our findings posit a novel mechanism by which intake of a food item, such as Mānuka honey, can potentially support immune homeostasis by enhancing MAIT cell-specific microbial sensing.

Liver resident-memory CD8 + T cells (T RM cells) can kill liver-stage Plasmodium -infected cells and prevent malaria, but simple vaccines for generating this important immune population are lacking. Here, we report the development of a fully synthetic self-adjuvanting glycolipid-peptide conjugate vaccine designed to efficiently induce liver T RM cells. Upon cleavage in vivo, the glycolipid-peptide conjugate vaccine releases an MHC I-restricted peptide epitope (to stimulate Plasmodium -specific CD8 + T cells) and an adjuvant component, the NKT cell agonist α-galactosylceramide (α-GalCer). A single dose of this vaccine in mice induced substantial numbers of intrahepatic malaria-specific CD8 + T cells expressing canonical markers of liver T RM cells (CD69, CXCR6, and CD101), and these cells could be further increased in number upon vaccine boosting. We show that modifications to the peptide, such as addition of proteasomal-cleavage sequences or epitope-flanking sequences, or the use of alternative conjugation methods to link the peptide to the glycolipid improved liver T RM cell generation and led to the development of a vaccine able to induce sterile protection in C57BL/6 mice against Plasmodium berghei sporozoite challenge after a single dose. Furthermore, this vaccine induced endogenous liver T RM cells that were long-lived (half-life of ~425 days) and were able to maintain >90% sterile protection to day 200. Our findings describe an ideal synthetic vaccine platform for generating large numbers of liver T RM cells for effective control of liver-stage malaria and, potentially, a variety of other hepatotropic infections., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The synthesis of the invariant natural killer (iNK) T cell agonist β-mannosylceramide along with a series of fatty amide analogues is reported. Of the six β-glycosylation protocols investigated, the sulfoxide methodology developed by Crich and co-workers proved to be the most effective where the reaction of a mannosyl sulfoxide and phytosphingosine derivative gave a key glycolipid intermediate as a 95 : 5 mixture of β- to α-anomers in high yield. A series of mannosyl ceramides were evaluated for their ability to activate D32.D3 NKT cells and induce antitumour activity.

Aim: The efficacy of anti-lymphoma vaccines that exploit the cellular adjuvant properties of activated natural killer T (NKT) cells were examined in mouse models of CNS lymphoma. Materials & methods: Vaccines were prepared by either loading the NKT cell agonist, α-galactosylceramide onto irradiated and heat-shocked B- and T-lymphoma cells, or chemically conjugating α-galactosylceramide to MHC-binding peptides from a lymphoma-associated antigen. Vaccine efficacy was analyzed in mice bearing intracranial tumors. Results: Both forms of vaccine proved to be effective in preventing lymphoma engraftment through activity of T cells that accessed the CNS. Established lymphoma was harder to treat with responses constrained by Tregs, but this could be overcome by depleting Tregs prior to vaccination. Conclusion: Simply designed NKT cell-activating vaccines enhance T-cell responses and have the potential to protect against CNS lymphoma development or prevent CNS relapse. To be effective against established CNS lymphoma, vaccines need to be combined with Treg suppression.

The immune system plays a significant role in controlling systemic metabolism. Innate-like T (ILT) cells in particular, such as mucosal-associated invariant T (MAIT) cells, invariant natural killer T (iNKT) cells and γδ T cell receptor expressing cells, have been reported to promote metabolic homeostasis. However, these different ILT cell subsets have, to date, been generally studied in isolation. Here we conducted a pilot study assessing the phenotype and function of circulating MAIT, iNKT, and Vδ2 + T cells in a small cohort of 10 people with obesity and type 2 diabetes (T2D), 10 people with obesity but no diabetes, and 12 healthy individuals. We conducted phenotypic analysis by flow cytometry ex vivo , and then functional analysis after in vitro stimulation using either PMA/ionomycin or synthetic agonists, or precursors thereof, for each of the cell-types; use of the latter may provide important knowledge for the development of novel therapeutics aimed at activating human ILT cells. The results of our pilot study, conducted on circulating cells, show clear dysfunction of all three ILT cell subsets in obese and obese T2D patients, as compared to healthy controls. Importantly, while both iNKT and Vδ2 + T cell dysfunctions were characterized by diminished IL-2 and interferon-γ production, the distinct dysfunctional state of MAIT cells was instead defined by skewed subset composition, heightened sensitivity to T cell receptor engagement and unchanged production of all measured cytokines., (Copyright © 2020 Li, Woods, Parry-Strong, Anderson, Capistrano, Gestin, Painter, Hermans, Krebs and Gasser.)

Mucosal-associated invariant T (MAIT) cells are antibacterial effector T cells that react to pyrimidines derived from bacterial riboflavin synthesis presented by the monomorphic molecule MR1. A major challenge in MAIT cell research is that the commonly used MAIT agonist precursor, 5-amino-6-d-ribitylaminouracil (5-A-RU), is labile to autoxidation, resulting in a loss of biological activity. Here, we characterize two independent autoxidation processes by LCMS. To overcome the marked instability, we report the synthesis of a 5-A-RU prodrug generated by modification of the 5-amino group with a cleavable valine-citrulline- p -aminobenzyl carbamate. The compound is stable in prodrug form, with the parent amine (i.e., 5-A-RU) released only after enzymatic cleavage. Analysis of the prodrug in vitro and in vivo showed an enhanced MAIT cell activation profile compared to 5-A-RU, which was associated with preferential loading within recycling endosomes, a route used by some natural agonists. This prodrug design therefore overcomes the difficulties associated with 5-A-RU in biological studies and provides an opportunity to explore different presentation pathways.

iNKT cells are CD1d-restricted T cells recognizing lipid antigens. The prototypic iNKT cell-agonist α-galactosylceramide (α-GalCer) alongside compounds with similar structures induces robust proliferation and cytokine production of iNKT cells and protects against cancer in vivo . Monoclonal antibodies (mAbs) that detect CD1d-α-GalCer complexes have provided critical information for understanding of antigen presentation of iNKT cell agonists. Although most iNKT cell agonists with antitumor properties are α-linked glycosphingolipids that can be detected by anti-CD1d-α-GalCer mAbs, β-ManCer, a glycolipid with a β-linkage, induces strong antitumor immunity via mechanisms distinct from those of α-GalCer. In this study, we unexpectedly discovered that anti-CD1d-α-GalCer mAbs directly recognized β-ManCer-CD1d complexes and could inhibit β-ManCer stimulation of iNKT cells. The binding of anti-CD1d-α-GalCer mAb with β-ManCer-CD1d complexes was also confirmed by plasmon resonance and could not be explained by α-anomer contamination. The binding of anti-CD1d-α-GalCer mAb was also observed with CD1d loaded with another β-linked glycosylceramide, β-GalCer (C26:0). Detection with anti-CD1d-α-GalCer mAbs indicates that the interface of the β-ManCer-CD1d complex exposed to the iNKT cell TCR can assume a structure like that of CD1d-α-GalCer, despite its disparate carbohydrate structure. These results suggest that certain β-linked monoglycosylceramides can assume a structural display similar to that of CD1d-α-GalCer and that the data based on anti-CD1d-α-GalCer binding should be interpreted with caution., (Copyright © 2019 Clark, Yau, Bloom, Wang, Venzon, Suzuki, Pasquet, Compton, Cardell, Porcelli, Painter, Zajonc, Berzofsky and Terabe.)

Activated NKT cells can stimulate antigen-presenting cells leading to enhanced peptide antigen-specific immunity. However, administration of potent NKT cell agonists like α-galactosylceramide (α-GalCer) can be associated with release of high levels of cytokines, and in some situations, hepatotoxicity. Here we show that it is possible to provoke sufficient NKT cell activity to stimulate strong antigen-specific T cell responses without these unwanted effects. This was achieved by chemically conjugating antigenic peptides to α-galactosylphytosphingosine (α-GalPhs), an NKT cell agonist with very weak activity based on structural characterisation and biological assays. Conjugation improved delivery to antigen-presenting cells in vivo, while use of a cathepsin-sensitive linker to release the α-GalPhs and peptide within the same cell promoted strong T cell activation and therapeutic anti-tumour responses in mice. The conjugates activated human NKT cells and enhanced human T cell responses to a viral peptide in vitro. Accordingly, we have demonstrated a means to safely exploit the immunostimulatory properties of NKT cells to enhance T cell activation for virus- and tumour-specific immunity.

Background: A key challenge in the manufacturing of polymeric colloids is producing nanoparticles with good batch-to-batch consistency., Objective: Develop a robust microfluidics method for the preparation of PEG-PLGA nanoparticles using dimethyl sulfoxide (DMSO) as the organic phase solvent for the encapsulation of DMSO soluble agents., Methods: Microfluidic process parameters, total flow rate (10 mL/min), flow rate ratio (1:1) of the aqueous phase and the organic polymer solution, and polymer concentration (5 mg/ml). Polyvinyl alcohol (PVA) or human serum albumin (HSA) was included in the aqueous phase. Dynamic light scattering and transmission electron microscopy were used to investigate the size and morphology of particles., Results: PLGA nanoparticles made using DMSO with the aqueous solvent containing PVA (2%) had an average size of 60 nm while PLGA-PEG nanoparticles made with and without PVA (2%) had an average size of 70 and 100 nm, respectively. PLGA-PEG nanoparticles generated with or without PVA had a high batch-to-batch coefficient of variation for the particle size of 20% while for PLGA nanoparticles with PVA it was 4%. HSA added to the aqueous phase reduced the size and the zeta potential of PEG-PLGA nanoparticles as well the batch-to-batch coefficient of variation for particle size to < 5%. Nanoparticles were stable in solution and after lyophilized in the presence of sucrose., Conclusion: Albumin was involved in the self-assembly of PEG-PLGA nanoparticles altering the physicochemical properties of nanoparticles. Adding protein to the aqueous phase in the microfluidic fabrication process may be a valuable tool for tuning the properties of nanoparticles and improving batch-to-batch consistency., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Vaccines that elicit targeted tumor antigen-specific T-cell responses have the potential to be used as adjuvant therapy in patients with high risk of relapse. However, the responses induced by vaccines in cancer patients have generally been disappointing. To improve vaccine function, we investigated the possibility of exploiting the immunostimulatory capacity of type 1 Natural killer T (NKT) cells, a cell type enriched in lymphoid tissues that can trigger improved antigen-presenting function in dendritic cells (DCs). In this phase I dose escalation study, we treated eight patients with high-risk surgically resected stage II-IV melanoma with intravenous autologous monocyte-derived DCs loaded with the NKT cell agonist α-GalCer and peptides derived from the cancer testis antigen NY-ESO-1. Two synthetic long peptides spanning defined immunogenic regions of the NY-ESO-1 sequence were used. This therapy proved to be safe and immunologically effective, inducing increases in circulating NY-ESO-1-specific T cells that could be detected directly ex vivo in seven out of eight patients. These responses were achieved using as few as 5 × 10 5 peptide-loaded cells per dose. Analysis after in vitro restimulation showed increases in polyfunctional CD4 + and CD8 + T cells that were capable of manufacturing two or more cytokines simultaneously. Evidence of NKT cell proliferation and/or NKT cell-associated cytokine secretion was seen in most patients. In light of these strong responses, the concept of including NKT cell agonists in vaccine design requires further investigation.

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.

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.

d-chiro-inositol (DCI, 1) evokes therapeutic actions in diabetes and insulin resistance but has sub-optimal pharmacokinetic profiles. To investigate what positions on the DCI cyclohexanol ring may be amenable to modification to improve pharmaceutical formulations, a series of analogues based on DCI were synthesised. These compounds were then evaluated for their ability to stimulate glucose transport using 3T3-L1 adipocytes as a model system. Positional analyses indicate that the hydroxyl group at position 1 is not essential for activity and can be modified without affecting glucose uptake. Removal of the hydroxyl at position 3 also had minimal effect on activity but this group is sensitive to modification. By comparison, the oxygen at position 2 is crucial to the potency of DCI, although this group can withstand modification without fundamentally affecting activity. These data reveal that positions 1 and 2 on the cyclohexanol ring of DCI offer further scope for modification to develop DCI analogues with desirable pharmacokinetic profiles for the potential treatment of metabolic disease., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

A major challenge in the development of highly defined synthetic vaccines is the codelivery of vaccine components (i.e., antigen and adjuvant) to secondary lymphoid tissue to induce optimal immune responses. This problem can be addressed by synthesizing vaccines that comprise peptide antigens covalently attached to glycolipid adjuvants through biologically cleavable linkers. Toward this, a strategy utilizing previously unreported 6″-deoxy-6″-thio analogues of α-GalCer that can undergo chemoselective conjugation with peptide antigens is described. Administration of these conjugate vaccines leads to enhanced priming of antigen specific T cells. This simple vaccine design is broadly applicable to multiple disease indications such as cancer and infectious disease.

RFamide-related peptide-3 (RFRP-3) is a recently discovered neuropeptide that has been proposed to play a role in the stress response. We aimed to elucidate the role of RFRP-3 and its receptor, neuropeptide FF (NPFF1R), in modulation of stress and anxiety responses. To achieve this, we characterized a new NPFF1R antagonist because our results showed that the only commercially available putative antagonist, RF9, is in fact an agonist at both NPFF1R and the kisspeptin receptor (KISS1R). We report here the identification and pharmacological characterization of GJ14, a true NPFFR antagonist. In in vivo tests of hypothalamic-pituitary-adrenal (HPA) axis function, GJ14 completely blocked RFRP-3-induced corticosterone release and neuronal activation in CRH neurons. Furthermore, chronic infusion of GJ14 led to anxiolytic-like behavior, whereas RFRP-3 infusion had anxiogenic effects. Mice receiving chronic RFRP-3 infusion also had higher basal circulating corticosterone levels. These results indicate a stimulatory action of RFRP-3 on the HPA axis, consistent with the dense expression of NPFF1R in the vicinity of CRH neurons. Importantly, coinfusion of RFRP-3 and GJ14 completely reversed the anxiogenic and HPA axis-stimulatory effects of RFRP-3. Here we have established the role of RFRP-3 as a regulator of stress and anxiety. We also show that GJ14 can reverse the effects of RFRP-3 both in vitro and in vivo. Infusion of GJ14 causes anxiolysis, revealing a novel potential target for treating anxiety disorders.

Herein, we report on a highly efficient synthesis of a crystalline protected Lewis(X) trisaccharide that was converted to Lewis(X) following global deprotection. The trisaccharide was prepared in a highly convergent synthesis (seven steps, longest linear sequence) and in a 38% overall yield using a strategy that involved the regioselective glycosylation of a GlcNAc acceptor with a galactose thioglycoside donor, followed by fucosylation of the remaining free GlcNAc hydroxyl as key steps. The core trisaccharide also has the potential to be converted to other members of the Type-2 Lewis family of antigens due to the orthogonal nature of the protecting groups employed., (Copyright © 2015. Published by Elsevier Ltd.)

It is known that T cells can eliminate tumour cells through recognition of unique or aberrantly expressed antigens presented as peptide epitopes by major histocompatibility complex (MHC) molecules on the tumour cell surface. With recent advances in defining tumour-associated antigens, it should now be possible to devise therapeutic vaccines that expand specific populations of anti-tumour T cells. However there remains a need to develop simpler efficacious synthetic vaccines that possess clinical utility. We present here the synthesis and analysis of vaccines based on conjugation of MHC-binding peptide epitopes to α-galactosylceramide, a glycolipid presented by the nonpolymorphic antigen-presenting molecule CD1d to provoke the stimulatory activity of type I natural killer T (NKT) cells. The chemical design incorporates an enzymatically cleavable linker that effects controlled release of the active components in vivo . Chemical and biological analysis of different linkages with different enzymatic targets enabled selection of a synthetic vaccine construct with potent therapeutic anti-tumour activity in mice, and marked in vitro activity in human blood.

The function of dendritic cells (DCs) can be modulated through multiple signals, including recognition of pathogen-associated molecular patterns, as well as signals provided by rapidly activated leukocytes in the local environment, such as innate-like T cells. In this article, we addressed the possibility that the roles of different murine DC subsets in cross-priming CD8(+) T cells can change with the nature and timing of activatory stimuli. We show that CD8α(+) DCs play a critical role in cross-priming CD8(+) T cell responses to circulating proteins that enter the spleen in close temporal association with ligands for TLRs and/or compounds that activate NKT cells. However, if NKT cells are activated first, then CD8α(-) DCs become conditioned to respond more vigorously to TLR ligation, and if triggered directly, these cells can also contribute to priming of CD8(+) T cell responses. In fact, the initial activation of NKT cells can condition multiple DC subsets to respond more effectively to TLR ligation, with plasmacytoid DCs making more IFN-α and both CD8α(+) and CD8α(-) DCs manufacturing more IL-12. These results suggest that different DC subsets can contribute to T cell priming if provided appropriately phased activatory stimuli, an observation that could be factored into the design of more effective vaccines., (Copyright © 2015 by The American Association of Immunologists, Inc.)

The synthesis of a number of N-glycosyl-N-alkyl-methoxyamine bifunctional linkers is described. The linkers contain an N-methoxyamine functional group for conjugation to carbohydrates and a terminal group, such as an amine, azide, thiol, or carboxylic acid, for conjugation to the probe of choice. The strategy for the linker synthesis is rapid (3-4 steps) and efficient (51-96% overall yield), and many of the linkers can be synthesized using a three-step one-pot strategy. Moreover, the linkers can be conjugated to glycans in excellent yield and they show excellent stability toward hydrolytic cleavage.

TLRs are key innate immune receptors that recognize conserved features of biological molecules that are found in microbes. In particular, TLR2 has been reported to be activated by different kinds of microbial ligands. To advance our understanding of the interaction of TLR2 with its ligands, the recombinant human TLR2 ectodomain (hTLR2ED) was expressed using a baculovirus/insect cell expression system and its biochemical, as well as ligand binding, properties were investigated. The hTLR2ED binds synthetic bacterial and mycoplasmal lipopeptides, lipoteichoic acid from Staphylococcus aureus, and synthetic lipoarabinomannan precursors from Mycobacterium at extracellular physiological conditions, in the absence of its co-receptors TLR1 and TLR6. We also determined that lipopeptides and glycolipids cannot bind simultaneously to hTLR2ED and that the phosphatidyl inositol mannoside 2 (Pim2) is the minimal lipoarabinomannan structure for binding to hTLR2ED. Binding of hTLR2ED to Pim4, which contains a diacylglycerol group with one of its acyl chains containing 19 carbon atoms, indicates that hTLR2ED can bind ligands with acyl chains longer than 16 carbon atoms. In summary, our data indicate that diacylglycerol is the ligand moiety of microbial glycolipids and lipoproteins that bind to hTLR2ED and that both types of ligands bind to the same binding site of hTLR2ED., (© The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.)

Orthogonally protected chiral myo-inositol derivatives are important intermediates for higher order myo-inositol-containing compounds. Here, the use of the immobilized enzyme Novozym 435 to efficiently catalyze the acetylation of the 5R configured enantiomer of racemic 1,2-O-isopropylidene-myo-inositols possessing chemically and sterically diverse protecting groups at O-3 and O-6 is described. The resolutions were successful with allyl, benzyl, 4-bromo-, 4-methoxy-, 4-nitro-, and 4-(3,4-dimethoxyphenyl)benzyl, propyl, and propargyl protection at O-6 in combination with either allyl or benzyl groups at O-3. Bulky protecting groups slow the rate of acetylation. No reaction was observed for 3,6-di-O-triisopropylsilyl-1,2-O-isopropylidene-myo-inositol. The utility of this methodology was demonstrated by the first reported synthesis of an Ac2PIM1 (9), which used both enantiomers of the resolved 3-O-allyl-6-O-benzyl-1,2-O-isopropylidene-myo-inositol in a convergent synthesis.

Acute leukemias with adverse prognostic features carry a high relapse rate without allogeneic stem cell transplantation (allo-SCT). Allo-SCT has a high morbidity and is precluded for many patients because of advanced age or comorbidities. Postremission therapies with reduced toxicities are urgently needed. The murine acute leukemia model C1498 was used to study the efficacy of an intravenously administered vaccine consisting of irradiated leukemia cells loaded with the natural killer T (NKT)-cell agonist α-galactosylceramide (α-GalCer). Prophylactically, the vaccine was highly effective at preventing leukemia development through the downstream activities of activated NKT cells, which were dependent on splenic langerin(+)CD8α(+) dendritic cells and which led to stimulation of antileukemia CD4(+) and CD8(+) T cells. However, hosts with established leukemia received no protective benefit from the vaccine, despite inducing NKT-cell activation. Established leukemia was associated with increases in regulatory T cells and myeloid-derived suppressor cells, and the leukemic cells themselves were highly suppressive in vitro. Although this suppressive environment impaired both effector arms of the immune response, CD4(+) T-cell responses were more severely affected. When cytarabine chemotherapy was administered prior to vaccination, all animals in remission posttherapy were protected against rechallenge with viable leukemia cells., (© 2014 by The American Society of Hematology.)

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.

A reliable reagent system for the cleavage of 4-(3,4-dimethoxyphenyl)benzyl (DMPBn) ethers under acidic conditions has been established. Treatment of DMPBn-protected mono- and pseudodisaccharides with TFA in anhydrous CH2Cl2 and 3,4-(methylenedioxy)toluene as a cation scavenger resulted in the selective cleavage of the DMPBn ether giving the corresponding deprotected products in moderate to high yields. Examples are reported which show that allyl, benzyl, and p-bromobenzyl ethers, esters, and glycosidic linkages are stable to these reaction conditions. The selective cleavage of allyl, p-bromobenzyl, and PMB ethers in protected carbohydrates containing DMPBn ethers are also demonstrated. This work establishes the 4-(3,4-dimethoxyphenyl)benzyl ether as an effective and robust alternative to p-methoxybenzyl as a protecting group for alcohols.

Native phosphatidylinositol mannosides (PIMs), isolated from the cell wall of Mycobacterium bovis, and synthetic PIM analogues have been reported to offer a variety of immunomodulating properties, including both suppressive and stimulatory activity. While numerous studies have examined the biological activity of these molecules, the aim of this research was to assess the physicochemical properties at a molecular level and correlate these characteristics with biological activity in a mouse model of airway eosinophilia. To accomplish this, we varied the flexibility and lipophilicity of synthetic PIMs by changing the polar headgroup (inositol- vs glycerol-based core) and the length of the acyl chains of the fatty acid residues (C0, C10, C16, and C18). A series of six phosphatidylinositol dimannosides (PIM2s) and phosphatidylglycerol dimannosides (PGM2s) were synthesized and characterized in this study. Langmuir monolayer studies showed that surface pressure-area (π-A) isotherms were greatly influenced by the length of the lipid acyl chains as well as the steric hindrance and volume of the headgroups. In aqueous solution, lipidated PIM2 and PGM2 compounds were observed to self-assemble into circular aggregates, as confirmed by dynamic light scattering and transmission electron microscopic investigations. Removal of the inositol ring but retention of the three-carbon glycerol unit maintained biological activity. We found that the deacylated PGM2, which did not show self-organization, had no effect on the eosinophil numbers but did have an impact on the expansion of OVA-specific CD4(+) Vα2Vβ5 T cells.