Enhancing T cell responses and tumour immunity by vaccination with peptides conjugated to a weak NKT cell agonist.

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.

A phase I vaccination study with dendritic cells loaded with NY-ESO-1 and α-galactosylceramide: induction of polyfunctional T cells in high-risk melanoma patients.

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 × 105 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.

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.

Synthesis and Activity of 6″-Deoxy-6″-thio-α-GalCer and Peptide Conjugates.

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.

NKT cell-dependent glycolipid-peptide vaccines with potent anti-tumour activity.

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.

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.

Physical characterization of synthetic phosphatidylinositol dimannosides and analogues in binary systems with phosphatidylcholine.

Native phosphatidylinositol mannosides (PIMs) from the cell wall of Mycobacterium bovis (M. bovis) and synthetic analogues have been identified to exert immunostimulatory activities. These activities have been investigated using particulate delivery systems containing native mannosylated lipids or total lipid extracts. Limited work has been carried out examining the incorporation of individual PIM lipids into suitable particulate formulations such as liposomes. The present study explored the possibility of constructing phosphatidylcholine (PC)-based liposomes containing synthetic PIM analogues. A series of six phosphatidylinositol dimannosides (PIM(2)s) and phosphatidylglycerol dimannosides (PGM(2)s) was characterized in this study. Binary Langmuir monolayers are a useful approach for establishing pharmaceutical properties, such as lipid-lipid interactions in mixed monolayers, to facilitate the design of liposome-based delivery systems. In mixed films the phosphoglycolipids were found to be miscible with PC based on evaluation of collapse pressures and deviations of experimental molecular areas from calculated ideal values. Concanavalin A (ConA) agglutination confirmed the presence of mannosylated lipids on the surface of the liposomes. Physicochemical properties of liposomes were affected by the presence of 2% (w/w) of phosphoglycolipids with liposome stability being increased by incorporation of long-chain PIM(2) and PGM2. Overall, while membrane stability of the liposomes was found to be dependent on incorporation of the phosphoglycolipids, all formulations retained proteins in amounts making them suitable for delivery.

Physicochemical and biological characterization of synthetic phosphatidylinositol dimannosides and analogues.

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(+) Vα2Vß5 T cells.

Synthesis and mass spectral characterization of mycobacterial phosphatidylinositol and its dimannosides.

A family of naturally occurring mycobacterial phosphatidylinositol (PI) and its dimannosides (PIM(2), AcPIM(2), and Ac(2)PIM(2)) that all possess the predominant natural 19:0/16:0 phosphatidyl acylation pattern were prepared to study their mass spectral fragmentations. Among these, the first synthesis of a fully lipidated PIM (i.e., (16:0,18:0)(19:0/16:0)-PIM(2)) was achieved from (±)-1,2:4,5-diisopropylidene-D-myo-inositol in 16 steps in 3% overall yield. A key feature of the strategy was extending the utility of the p-(3,4-dimethoxyphenyl)benzyl protecting group for its use at the O-3 position of inositol to allow installation of the stearoyl residue at a late stage in the synthesis. Mass spectral studies were performed on the synthetic PIMs and compared to those reported for natural PIMs identified from a lipid extract of M. bovis BCG. These analyses confirm that fragmentation patterns can be used to identify the structures of specific PIMs from the cell wall lipid extract.

Virus-like particles and α-galactosylceramide form a self-adjuvanting composite particle that elicits anti-tumor responses.

Virus-like particles (VLP) are effective vehicles for delivery of heterologous antigen to antigen-presenting cells. However VLP alone are insufficiently stimulatory to generate the signals required to facilitate effective priming of naïve T cells. We show that the VLP derived from rabbit hemorrhagic disease virus can bind the galactose-containing adjuvant α-galactosylceramide to form a composite particle for co-delivery of antigen and adjuvant to the same antigen-presenting cell. Vaccination with VLP and α-galactosylceramide activated splenic iNKT cells to produce IFN-γ and IL-4, led to the generation of antigen-specific T cells that protected prophylactically against subcutaneous tumor challenge, and was more effective at generating anti-tumor immune responses than either component individually. These data demonstrate a novel method for immunopotentiating VLP to increase their efficacy in the generation of anti-tumor responses via the innate ligand recognition properties of calicivirus-derived nanoparticles.

Phosphatidylinositol di-mannoside and derivates modulate the immune response to and efficacy of a tuberculosis protein vaccine against Mycobacterium bovis infection.

Mycobacterium bovis infects a wide range of hosts, including domestic livestock, wildlife, and humans. Development of an effective vaccine protecting against bovine tuberculosis would provide a cost-effective tuberculosis control strategy. The objective of this study was to investigate the ability of phosphatidylinositol di-mannoside (PIM(2)) and its derivatives to modulate cell-mediated immunity in vivo in a bovine tuberculosis mouse model in response to a relevant antigen, namely a fusion protein of mycobacterial proteins Ag85A and ESAT-6. The addition of synthetic PIM(2) to the vaccine resulted in a significant reduction in lung bacterial counts and a cytokine profile indicating a Th 1 type immune response. The addition of the other PIM(2) derivatives to the vaccine or the fusion protein alone did not result in reduced lung bacterial counts; moreover, the addition of PIM(2)ME appeared to negate the induction of an antigen-specific interferon-γ response and protection against tuberculosis. In conclusion, this study provides further evidence that PIMs can function as potent adjuvants for protein or sub-unit vaccines, but subtle structural differences among PIMs can markedly alter the type of immune response induced.

Synthesis and Toll-like receptor 4 (TLR4) activity of phosphatidylinositol dimannoside analogues.

A series of five PIM(2) analogues were synthesized and tested for their ability to activate primary macrophages and modulate LPS signaling. Structural changes included replacement of the fatty acid esters of the phosphatidyl moiety of PIM(2) with the corresponding ether or amide. An AcPIM(2) analogue possessing an ether linkage was also prepared. The synthetic methodology utilized an orthogonally protected chiral myo-inositol starting material that was conveniently prepared from myo-inositol in just two steps. Important steps in the synthetic protocols included the regio- and α-selective glycosylation of inositol O-6 and introduction of the phosphodiester utilizing phosphoramidite chemistry. Replacement of the inositol core with a glycerol moiety gave compounds described as phosphatidylglycerol dimannosides (PGM(2)). Biological testing of these PIM compounds indicated that the agonist activity was TLR4 dependent. An ether linkage increased agonist activity. Removal of the inositol ring enhanced antagonist activity, and the presence of an additional lipid chain enhanced LPS-induced cytokine production in primary macrophages. Furthermore, the interruption of the LPS-induced 2:2 TLR4/MD-2 signaling complex formation by PIM(2) represents a previously unidentified mechanism involved in the bioactivity of PIM molecules.

Chemical synthesis and immunosuppressive activity of dipalmitoyl phosphatidylinositol hexamannoside.

Phosphatidylinositol mannosides (PIMs) isolated from mycobacteria have been identified as an important class of phosphoglycolipids with significant immune-modulating properties. We present here the synthesis of dipalmitoyl phosphatidylinositol hexamannoside (PIM(6)) 1 and the first reported functional biology of a synthetic PIM(6). Key steps in the synthetic protocol included the selective glycosylation of an inositol 2,6-diol with a suitably protected mannosyl donor and construction of the glycan core utilizing a [3 + 4] thio-glycosylation strategy. The target 1 was purified by reverse phase chromatography and characterized by standard spectroscopic methods, HPLC, and chemical modification by deacylation to dPIM(6). The (1)H NMR spectrum of synthetic dPIM(6) obtained from 1 matched that of dPIM(6) obtained from nature. PIM(6) (1) exhibited dendritic cell-dependent suppression of CD8(+) T cell expansion in a human mixed lymphocyte reaction consistent with the well established immunosuppressive activity of whole mycobacteria.

A PIM2 analogue suppresses allergic airway disease.

Two approaches for the synthesis of a phosphatidylinositol dimannoside (PIM2) analogue 4 that mimics the suppressive activity of natural PIMs and also synthetic PIM2 have been developed. This analogue, where the inositol core was replaced by glycerol, was tested for its ability to suppress cellular inflammation in a mouse model of allergic asthma and shown to be effective in suppressing airway eosinophilia. Suppression of all inflammatory cells monitored was observed, indicating a general blockade of cellular activity. These data indicate that the inositol core is not essential for this suppressive activity.

MIS416, a non-toxic microparticle adjuvant derived from Propionibacterium acnes comprising immunostimulatory muramyl dipeptide and bacterial DNA promotes cross-priming and Th1 immunity.

Propionibacterium acnes was modified using biochemical extraction methods generating a suspension of microparticles (MIS416) comprising a minimal cell wall skeleton rich in immunostimulatory crosslinked muramyl dipeptide repeats and native bacterial DNA fragments, each which have known adjuvant activity. In vitro studies demonstrated that MIS416 was readily internalized by human myeloid and plasmacytoid DC inducing cytokine secretion and cell activation/maturation. Vaccination studies in mice using OVA as a model antigen demonstrated that MIS416 acts as a Th1 adjuvant, promoting cross-priming of cytotoxic CD8(+) T cell responses and enhanced anti-tumour immunity. Covalent attachment of OVA to MIS416 enabling simultaneous delivery of antigen and adjuvant to the antigen presentation system resulted in a dose-sparing vaccine formulation. Preclinical GLP toxicology studies demonstrated that MIS416 has a favorable safety profile in mouse and rabbit supporting its use in human vaccine formulations.

Mannosylated saponins based on oleanolic and glycyrrhizic acids. Towards synthetic colloidal antigen delivery systems.

Immunostimulatory saponin based colloidal antigen delivery systems show promise as adjuvants for subunit vaccines. For this reason, allyl oleanolate was glycosylated at the 3-position using trichloroacetimidate donors to give monodesmodic saponins following deprotection. Bisdesmodic saponins were synthesized by double glycosylation at the 3- and 28-positions of oleanolic acid. When formulated together with cholesterol and phospholipids, ring-like, helical and rod-like nanostructures were formed depending on the saponin concentrations used. As an indication of adjuvant activity, the ability of these formulations, and the saponins by themselves, to induce dendritic cell maturation was measured, but no significant activity was observed.

Phosphatidylinositol mannoside ether analogues: syntheses and interleukin-12-inducing properties.

Phosphatidylinositol mannosides (PIMs) isolated from mycobacteria have been identified as an important class of glycolipids with significant immune modulating properties. We present here the syntheses of phosphatidylinositol dimannoside ether analogues 2 and 3 and evaluate their interleukin-12 (IL-12)-inducing properties along with dipalmitoyl PIM2 (1) in an in vitro bovine dendritic cell assay. Both synthetic PIM analogues and synthetic dipalmitoyl PIM2 (1) were effective at enhancing IL-12 production by immature bovine dendritic cells. Unexpectedly, ether analogue 2 was significantly more active than dipalmitoyl PIM2 (1) which indicates that modified PIM compounds can be strongly immunoactive and may have significant adjuvant activities.

Novel synthesis of alpha-galactosyl-ceramides and confirmation of their powerful NKT cell agonist activity.

alpha-Galactosyl-ceramide (1) has been identified as a powerful modulator of immunological processes through its capacity to bind CD1d molecules and specifically activate invariant natural killer (NK)-like T cells (iNKT cells). This paper describes the synthesis of 1, the analogous alpha-galactosyl-ceramide 3, and its short chain analogue 'OCH' (2), by use of the 4,6-di-O-tert-butylsilylene (DTBS) protecting group to produce a powerful alpha-galactosylating agent. In vivo experiments confirmed these compounds to be potent and selective activators of iNKT cells in a CD1d-dependent manner, each inducing a unique profile of cytokine release. This synthesis strategy will permit the generation of novel derivatives for use in the study of the mechanism of iNKT cell activation.

Phosphatidylinositol mannosides: synthesis and adjuvant properties of phosphatidylinositol di- and tetramannosides.

Phosphatidylinositol mannosides (PIMs) isolated from mycobacteria have been identified as an important class of glycolipids with significant immune modulating properties. We present here the syntheses of phosphatidylinositol dimannoside (PIM2, 1) and phosphatidylinositol tetramannoside (PIM4, 2) and evaluate their adjuvant properties in a transgenic mouse model. The key step in the synthetic methodology for the synthesis of 2 relies on the selective glycosylation of diol 3 with mannosyl donor 11. Both synthetic PIMs were effective at enhancing IFN-gamma when given as adjuvants with a model antigen, with PIM2 being the more active. These data suggest that in this assay the PIM core structure is responsible for the observed biological activity.

A stereoselective synthesis of 6,6,6-trifluoro-L-daunosamine and 6,6,6-trifluoro-L-acosamine.

A short synthesis of 6,6,6-trifluoro-L-acosamine 15 and 6,6,6-trifluoro-L-daunosamine 19 has been accomplished. The pyranose ring system of these carbohydrate analogues was formed by a hetero-Diels-Alder reaction of vinylogous imide 11 and ethyl vinyl ether which gave adduct 12a in 40% yield. Hydroboration gave 13 and subsequent hydrogenolytic removal of the (R)-2-phenylethyl chiral auxiliary gave ethyl 6,6,6-trifluoro-L-acosaminide 14. Acid hydrolysis furnished target 15. Glycoside 13 was N-trifluoroacetylated to give 16, the structure was confirmed by single crystal X-ray diffraction. The C-4 stereochemistry of 16 was inverted by Swern oxidation of the 4-OH group, and subsequent borohydride reduction to give 17. Hydrogenolytic removal of the auxiliary gave ethyl-6,6,6-trifluoro-L-daunosaminide 18. Acid hydrolysis provided 19.