WO2009093007A2

WO2009093007A2 - Immune modulation by regioselectively modified glycosides

Info

Publication number: WO2009093007A2
Application number: PCT/GB2009/000131
Authority: WO
Inventors: Eng-Kiat Lim; Paul Kaye; Dianna Joy Bowles
Original assignee: The University Of York
Priority date: 2008-01-21
Filing date: 2009-01-16
Publication date: 2009-07-30
Also published as: WO2009093007A3; GB0801032D0

Abstract

We describe the use of regioselectively glycosylated glycoside scaffolds in the modulation of the immune system.

Description

Immune Modulation by Reqioselectivelv Modified Glycosides

The invention relates to the use of regioselectively glycosylated aglycones in the modulation of the immune system.

The immune system is in part made up of lymphocytes which are able to recognise specific antigens. B lymphocytes recognise antigens in their native conformation through surface immunoglobulin receptors. T lymphocytes recognise protein antigens that are presented as peptides along with self molecules known as major histocompatibility antigen (MHC)₁ or human leukocyte antigen (HLA) in humans, on the surface of antigen presenting cells. T lymphocytes may be subdivided into CD8⁺ "cytotoxic T lymphocytes", which are able to destroy target cells, and CD4⁺ "T helper lymphocytes". T helper lymphocytes have a regulatory function and are able to help B lymphocytes to produce specific antibodies, or to help macrophages to kill intracellular pathogens. A further class of immune cells are dendritic cells which function to process antigenic material and present it to T lymphocytes of the immune system. They are primarily present in tissues that are in contact with the external environment, for example, skin, nose, lungs, stomach and intestine. Once activated the dendritic cells migrate to the lymph glands and activate T cells and B cells to initiate an adaptive immune response. Thus, dendritic cells play a key role in host defences and a crucial role in putative anti-cancer immune responses.

A yet further class of immune cell is the so called Regulatory T cell ["T regs"] which are a sub-population of T cells that function to suppress activation of the immune system. These cells repress the activity of other immune cells and are involved in suppressing an immune response once it has successfully dealt with the invading agent. T regs are also involved in controlling the immune systems recognition of self that may result in autoimmune disease if T reg function is impaired. Autoimmune diseases result when the immune system is directed to a "normal" tissue component, causing an inflammatory pathology. There are many diseases that are thought to have an autoimmune component. For example, Addison's disease, ankylosing spondylitis, aplastic anaemia, Celiac disease, Crohn's disease, diabetes mellitus typei , Hashimoto's disease, Lupus erythematosus, multiple sclerosis, pernicious anaemia, rheumatoid arthritis, or Sjorgen's syndrome to name but a few. Therefore, in autoimmune diseases an aim is to suppress specific immune responses against the autoantigen by suppressing the immune system. Activated T reg cells are one possible component in this suppression.

Natural products are well known to influence immune function with many products derived from diverse sources including microorganisms and plants now undergoing trials as anti-cancer agents and as immune potentiators. Typically, many of these natural product scaffolds occur in living organisms as glycosides, and indeed, phytochemical studies have demonstrated a vast complexity of small molecule glycosides in higher plants. Often, these scaffolds exist in numerous glycoforms, regioselectively glycosylated at different positions and carrying single sugars or multiple sugars, linked as monosaccharides or as di-, tri- and higher oligosaccharides. As an example, from a total of 5000 different flavonoids, 300 different glycosides of a single flavonol, quercetin, have already been identified in plant extracts.

In the context of immune function, the precise impact of glycosylation on the bioactivity of any particular natural product has been difficult to assess. This has been due to a number of reasons. An extract will often contain a complex mixture of multiple glycosides of a given scaffold, all at low level and difficult to purify to homogeneity. Chemical synthesis of regiospecific glycosides involves multiple steps with procedure complicated by multiple active groups requiring protection and deprotection. An added problem of chemical synthesis is the requirement for heavy metal catalysts which can lead to subsequent contamination of the synthetic glycosides. Also, the availability of characterised biocatalysts for glycosylation has been limited since glycosy transferases (GTs) of small molecule scaffolds are not abundant proteins and have been recalcitrant to traditional biochemical purification.

A class of enzymes involved in carbohydrate metabolism are the glycosyltransferase (GTase) enzymes. GTases are enzymes that transfer sugar residues from an activated nucleotide sugar to monomeric and polymeric acceptor molecules called aglycones (e.g. other sugars, proteins and peptides, lipids and other organic substrates). These glycosylated molecules take part in diverse metabolic pathways and biochemical processes. The transfer of a sugar moiety can alter the acceptor's bioactivity, solubility or transport properties within a cell and between cells. Examples of GTases include glucosyltransf erases, fucosyltransferases, sialyltransferases and galatosyltransferases. The chemical synthesis of glycosides requires glycosyl activation and involves multiple steps of protection/deprotection to control regioselectivity that can often reduce yield of the final product. GTases offer a potential solution to this problem, since the enzymes use unprotected aglycones in aqueous solution and their catalytic activity is chemo-, regio- and enantio-selective.

In our co-pending application WO2007/034190 we describe glycosyltransferases that regioselectively modify plant intermediate metabolites such as daidzein and resveratrol. In WO2004/106508 we describe glycosyltransferases that regioselectively modify flavonoids such as quercetin, luteolin, and the catechin, epicatechin.

This disclosure relates to natural plant scaffolds, for example quercetin and resveratrol. These have been investigated as examples of aglycones (non-glycosylated) and as regioselectively glycosylated glucosides. The activity of quercetin was compared to that of the 7,3'-di-0-glucoside and the activity of resveratrol to that of two individual mono glucosides, the 3-0- and 4'-O-glucoside. In this way the regioselective glycosylation of two natural products could be examined through their ability to direct the functional differentiation of immune cells, for example bone marrow-derived dendritic cells (DCs) in vitro.

We disclose that regiospecific glycosylation alters the functional activity of natural plant products such as quercetin and resveratrol. Both molecules had the ability to induce DC activation, as measured by altered expression of the co-stimulatory molecules CD40 and CD80 and release of IL-6.

According to an aspect of the invention there is provided a regioselectively glycosylated aglycone scaffold for use as an adjuvant.

In a preferred embodiment of the invention said glycosylated aglycone is a flavonoid.

A flavonoid is a plant secondary metabolite and can be classified into flavonoids, isoflavonoids and neoflavonoids. They are well known for their anti-oxidant activity and are synthesized by the phenylpropanoid metabolic pathway. An adjuvant is a substance or procedure which augments specific immune responses to antigens by modulating the activity of immune cells. Examples of adjuvants include, by example only, agonsitic antibodies to co-stimulatory molecules, Freunds adjuvant, muramyl dipeptides, liposomes. An adjuvant is therefore an immunomodulator. In a preferred embodiment of the invention said flavonoid is quercetin.

In a preferred embodiment of the invention quercetin is modified with glucose.

In an alternative preferred embodiment of the invention quercetin is modified with rhamnose.

In a preferred embodiment of the invention said quercetin glycoside is selected from the group consisting of: 3-O-glucoside, 7-O-glucoside, 3'-O-glucoside, 3,7-di-O-glucoside, 7,3'-di-O-glucoside, 3-O-rhamnoside, 7-O-rhamnoside, 3,7-di-O-rhamnoside.

In a preferred embodiment of the invention said quercetin glycoside is 3,7-di-O-glucoside or 7,3'-di-O-glucoside.

In an alternative preferred embodiment of the invention said aglycone scaffold is a stilbene.

A stilbene is the alkene ethene with two phenyl groups on either carbon of a parent chain. Stilbene derivatives are produced by plants.

In a preferred embodiment of the invention said stilbene is resveratrol.

In a preferred embodiment of the invention said resveratrol glycoside is selected from the group consisting of: 3-O-glucoside, 4'-O-glucoside, 3,4'-di-O-glucoside, 3-O- rhamnoside, 4'-O-rhamnoside.

In a preferred embodiment of the invention said resveratrol glycoside is 3-O-glucoside or ^'-O-glucoside.

In a further alternative embodiment of the invention said aglycone scaffold is a terpenoid, for example a monoterpenoid.

Plant terpenoids, also called isoprenoids are products derived from a five carbon isoprene unit. Terpenoids are classified with reference to the number of isoprene units that comprise the particular terpenoid. For example a monoterpenoid comprises two isoprene units; a sesquiterpene^ comprises three isoprene units and a di-terpenoid four isoprene units. Polypterpnoids comprise multiple isoprene units.

In a preferred embodiment of the invention said terpenoid is the O-glycoside of geraniol.

In a further preferred embodiment of the invention said terpenoid is the O-glycoside of perillyl alcohol.

According to a further aspect of the invention there is provided a regioselectively glycosylated aglycone scaffold for use in the activation of immune cells.

Preferably said immune cells are myeloid cells.

In a preferred embodiment of the invention said myeloid cells are selected from the group consisting of: macrophages, monocytes, leukocytes, eosinophils, basophils, dendritic cells

In a preferred embodiment of the invention said immune cells are dendritic cells, for example myeloid dendritic cells or lymphoid dendritic cells.

In an alternative preferred embodiment of the invention said immune cells are regulatory T cells.

Preferably said immune cells are non-haematopoietic stromal cells.

In a preferred embodiment of the invention said immune cell is human.

Methods to isolate and characterize immune cells such as dendritic cells and regulatory T cells are known in the art. For example see reference 1-5.

According to a further aspect of the invention there is provided the use of a regioselectively glycosylated aglycone scaffold in the treatment of an autoimmune disease.

In a preferred embodiment of the invention said autoimmune disease is selected from the group selected from: multiple sclerosis, type 1 diabetes, autoimmune thyroid disease, autoimmune hepatitis, rheumatoid arthritis, autoimmune colitis, Crohns disease, celiac disease, autoimmune nephritis, autoimmune neuropathy (guillan Barre), encephalopathy (Rasmussen), fibrosing alveolitis, ankylosing spondylitis, aplastic anaemia, Hashimoto's disease, Lupus erythematosus, pernicious anaemia, rheumatoid arthritis, or Sjorgen's disease.

According to a further aspect of the invention there is provide the use of a regioselectively glycosylated aglycone scaffold in the reconstitution of the immune system in subjects that are immune deficient.

In a preferred embodiment of the invention said immune deficiency is the result of chemotherapy for example cancer chemotherapy.

In a preferred embodiment of the invention said immune deficiency is neutropenia.

In an alternative preferred embodiment of the invention said immune deficiency is the result of infection for example HIV infection

According to an aspect of the invention there is provided a composition comprising regioselectively glycosylated resveratrol and an adjuvant.

According to a further aspect of the invention there is provided a vaccine composition comprising a regioselectively glycosylated aglycone scaffold and at least one antigen to which an immune response is desired.

The compositions of the invention can be administered by any conventional route, including injection or by gradual infusion over time. The administration may be, for example, oral, intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous, transdermal or as an aerosol.

The compositions of the invention are administered in effective amounts. An "effective amount" is that amount of a composition that alone, or together with further doses, produces the desired response. This may involve only slowing the progression of the disease temporarily, although more preferably, it involves halting the progression of the disease permanently. This can be monitored by routine methods. Such amounts will depend, of course, on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation.

It is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment.

The compositions used in the foregoing methods preferably are sterile and contain an effective amount of glycosylated aglycone for producing the desired response in a unit of weight or volume suitable for administration to a patient. The response can, for example, be measured by measuring the physiological effects of the composition, such as regression of a tumour, decrease of disease symptoms, modulation of apoptosis, etc.

The doses of glycosylated aglycone administered to a subject can be chosen in accordance with different parameters, in particular in accordance with the mode of administration used and the state of the subject. Other factors include the desired period of treatment. In the event that a response in a subject is insufficient at the initial doses applied, higher doses (or effectively higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits.

Other protocols for the administration of glycosylated aglycone will be known to one of ordinary skill in the art, in which the dose amount, schedule of injections, sites of injections, mode of administration (e.g., intra-tumoural) and the like vary from the foregoing. Administration of glycosylated aglycone compositions to mammals other than humans, (e.g. for testing purposes or veterinary therapeutic purposes), is carried out under substantially the same conditions as described above. A subject, as used herein, is a mammal, preferably a human, and including a non-human primate, cow, horse, pig, sheep, goat, dog, cat or rodent.

When administered, the compositions of the invention are applied in pharmaceutically- acceptable amounts and in pharmaceutically-acceptable compositions. The term

"pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. Such preparations may routinely contain salts, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents. Pharmacologically and pharmaceutically-acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic, and the like. Also, pharmaceutically-acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts.

The compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well-known in the art of pharmacy. All methods include the step of bringing the active agent into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the active compound into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.

Compositions suitable for oral administration may be presented as discrete units, such as capsules, tablets, lozenges, each containing a predetermined amount of the active compound. Other compositions include suspensions in aqueous liquids or non-aqueous liquids such as syrup, elixir or an emulsion.

The vaccine composition according to the invention may further comprise a carrier.

The term carrier is construed in the following manner. A carrier is an immunogenic molecule which, when bound to a second molecule augments immune responses to the latter. Some antigens are not intrinsically immunogenic yet may be capable of generating antibody responses when associated with a foreign protein molecule such as keyhole-limpet haemocyanin or tetanus toxoid. Such antigens contain B-cell epitopes but no T cell epitopes. The protein, moiety of such a conjugate (the "carrier" protein) provides_. T-cell epitopes which stimulate helper T-cells that in turn stimulate antigen-specific B- cells to differentiate into plasma cells and produce antibody against the antigen. Helper T-cells can also stimulate other immune cells such as cytotoxic T-cells, and a carrier can fulfil an analogous role in generating cell-mediated immunity as well as antibodies.

Antigens may be derived from pathological organism, for example a virus, a bacterial pathogen, a fungal pathogen e.g. Candida albicans, a parasite. The antigen can be protein or peptide e.g. tumour rejection antigen. Tumour rejection precursors are known in the art and are predominantly expressed by cancer cells. For example, the tumour rejection antigens MAGE, BAGE, GAGE and DAGE families of tumour rejection antigens, see Schulz et al Proc Natl Acad Sci USA, 1991, 88, pp991- 993). These precursor proteins are expressed by cancer cells and are typically processed to smaller peptides that act as tumour rejection antigens. These typically are nonapeptides but can vary in size from 8-30 amino acids.

The antigen could be or carbohydrate based e.g. capsular polysaccharide. The capsule is a protective structure that surrounds the surface of many bacteria. The polysaccharides that form the capsule are variable in structure, for example Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae or Group B Streptococcus). These capsular polysaccharides are major antigenic determinants.

Further no limiting examples of antigens are HIV antigen [e.g. gp120], a tuberculosis antigen [e.g Ag85]; or a leishmanial antigen [e.g. HASPB1].

The antigen could be lipid based e.g. glycosphingolipid (ganglioside).

In a preferred embodiment of the invention said composition further comprises at least one additional adjuvant.

An adjuvant is a substance or procedure which augments specific immune responses to antigens by modulating the activity of immune cells. Examples of adjuvants include, by example only, agonsitic antibodies to co-stimulatory molecules, Freunds adjuvant, muramyl dipeptides, liposomes. An adjuvant is therefore an immunomodulator. A carrier is an immunogenic molecule which, when bound to a second molecule augments immune responses to the latter. The term carrier is construed in the following manner. A carrier is an immunogenic molecule which, when bound to a second molecule augments immune responses to the latter. Some antigens are not intrinsically immunogenic yet may be capable of generating antibody responses when associated with a foreign protein molecule such as keyhole-limpet haemocyanin or tetanus toxoid. Such antigens contain B-cell epitopes but no T cell epitopes. The protein moiety of such a conjugate (the "carrier" protein) provides T-cell epitopes which stimulate helper T-cells that in turn stimulate antigen-specific B-cells to differentiate into plasma cells and produce antibody against the antigen. Helper T-cells can also stimulate other immune cells such as cytotoxic T-cells, and a carrier can fulfil an analogous role in generating cell-mediated immunity as well as antibodies. Certain antigens which lack T-cell epitopes, such as polymers with a repeating B-cell epitope (e.g. bacterial polysaccharides), are intrinsically immunogenic to a limited extent. These are known as T-independent antigens. Such antigens benefit from association with a carrier such as tetanus toxoid, under which circumstance they elicit much stronger antibody responses.

In a preferred embodiment of the invention said adjuvant is selected from the group consisting of: cytokines selected from the group consisting of GM CSF, interferon gamma, interferon alpha, interferon beta, interleukin 12, interleukin 23, interleukin 17, interleukin 2, interleukin 1 , TGF, TNFα, and TNFβ.

In a further alternative embodiment of the invention said adjuvant is a TLR agonist such as CpG oligonucleotides, flagellin, monophosphoryl lipid A, poly I:C and derivatives thereof.

In a preferred embodiment of the invention said adjuvant is a bacterial cell wall derivative such as muramyl dipeptide (MDP) and/or trehelose dycorynemycolate (TDM)

Preferably said additional adjuvant is a ToI Like Receptor [TLR] agonist.

In a preferred embodiment of the invention said TLR agonist is selected from the group presented in Table 2.

According to a further aspect of the invention there is provided a method for the activation of immune cells and optionally treatment of a subject suffering from or with a predisposition to immune deficiency comprising: i) removing from said subject a cell sample comprising immune cells; ii) contacting said cell sample with a preparation comprising a regioselectively glycosylated aglycone scaffold; optionally iii) monitoring the activation of said immune cells in said cell sample; and optionally iv) administering said activated immune cells to a subject in need of treatment for immune deficiency. In a preferred method of the invention said immune cells are dendritic cells or regulatory T cells.

According to an aspect of the invention there is provided a preparation comprising an activated immune cells and a regioselectively glycosylated aglycone scaffold for use in the treatment of immune deficiency.

In a preferred embodiment of the invention said immune cells are dendritic cells or T regulatory cells.

According to a further aspect of the invention there is provided a screening method for determining the adjuvant effect of a regioselectively glycosylated aglycone scaffold comprising: i) forming a preparation comprising a regioselectively glycosylated aglycone scaffold and an immune cell; ii) determining the adjuvant activity of said glycoside by measuring at least one determinant of immune cell activation compared to a control preparation.

Activation of immune cells can be measured by a number of parameters which include by example, suppression of anergy, expression of pro-inflammatory cytokines e.g. IL 6, 1L-12, and receptor activation e.g. CD40, CD80.

Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", means "including but not limited to", and is not intended to (and does not) exclude other moieties, additives, components, integers or steps.

Throughout the description and cjaims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

An embodiment of the invention will now be described by example only and with reference to the following figures:

Figure 1a illustrates that bone marrow dendritic cells respond differentially to flavanoid aglycones and their glycosides; Figure 1b illustrates DC responses to TLR2 agonist Pam3CSK4 in the presence or absence of resveratrol or modified resveratrol; Figure 1c illustrates DC responses to TLR3 agoinst Poly (I₁C) in the presence or absence of resveratrol or modified resveratrol; and Figure 1c illustrates DC responses to TLR9 agoinst ODN 1668 in the presence or absence of resveratrol or modified resveratrol;

Figure 2 is the nucleic acid and amino acid sequence of UGT71C1;

Figure 3 is the nucleic acid and amino acid sequence of UGT73B2;

Figure 4 is the nucleic acid and amino acid sequence of UGT73C5;

Figure 5 is the nucleic acid and amino acid sequence of UGT74D1 ;

Figure 6 is the nucleic acid and amino acid sequence of UGT76E12;

Figure 7 is the nucleic acid and amino acid sequence of UGT78D1 ;

Figure 8 is the nucleic acid and amino acid sequence of UGT78D2;

Figure 9 is the nucleic acid and amino acid sequence of UGT88A1 ;

Figure 10 is the nucleic acid and amino acid sequence of UGT89C1 ;

Figure 11 is the amino acid sequence of amino acid sequence of UGT71 B;

Figure 12 is the amino acid sequence of amino acid sequence of UGT72B1 ;

Figure 13 is the amino acid sequence of amino acid sequence of UGT72E2; Figure 14 is the amino acid sequence of amino acid sequence of UGT73B3;

Figure 15 is the amino acid sequence of amino acid sequence of UG774B1 ;

Figure 16 is the amino acid sequence of amino acid sequence of UGT74F1 ;

Figure 17 is the amino acid sequence of amino acid sequence of UGT74F2;

Figure 18 is the amino acid sequence of amino acid sequence of UGT75B1 ;

Figure 19 is the amino acid sequence of amino acid sequence of UGT76C1 ;

Figure 20 is the amino acid sequence of amino acid sequence of UGT84A2;

Figure 21 is the amino acid sequence of amino acid sequence of UGT84A2;

Figure 22 is the amino acid sequence of amino acid sequence of UGT84A3;

Figure 23 is the amino acid sequence of amino acid sequence of UGT84B1 ;

Figure 24 is the amino acid sequence of amino acid sequence of UGT85A1 ; and

Figure 25 is the amino acid sequence of amino acid sequence of UGT89B1.

Materials and Methods

Preparation of glycosides Whole-cell biotransformation - A 3 L glass autoclavable fermenter system (Applikon Biotechnology Ltd.) was used for the whole-cell biotransformation. Escherichia colt BL21 overnight culture (10 ml) expressing UGT was transferred into the fermenter system containing 1 L of sterilized minimal medium made from 2 g/L KH₂PO₄, 5.79 g/L K₂HPO₄, 0.5 /L (NH₄)₂SO₄, 5 g/L glucose, 0.31 g/L MgSO₄JH₂O, 1.25 ml trace element solution (27 g/L FeCI₃.6H₂O, 2 g/L Na₂MoO₄.2H₂O, 1.9 g/L CuSO₄.5H₂0, 1.4 g/L CaCI₂.2H₂O, 1.3 g/L ZnCI₂ , 0.5 g/L H₃BO₃, 100 ml/L HCI) and 1.25 ml vitamin solution (6 g/L nicotinic acid, 5.4 g/L pantothenic acid, 1.4 g/L pyridoxine, 0.42 g/L riboflavin, 0.06 g/L biotin and 0.04 g/L folic acid. The fermenter was previously equilibrated at 30 ⁰C, pH 7.4. The dissolved O₂ level was maintained at 95% for 1 h and was then decreased to 50%. When glucose was depleted (decrease in oxygen demand, after 5-6 h), the culture was fed with Feed 1 (600 g/L glucose, 12g/L MgSO₄, 6 g/L (NH₄J₂SO₄, 15 ml/L trace element solution and 15 ml/L vitamin solution) at a rate 200 ml/day until it reached OD -50. The culture was then induced with 1 mM IPTG for recombinant protein expression at 25^βC and 25% dissolved O₂. After 6 h of induction, 5 g/L of aglycone substrate were added to start the biotransformation process. Four hours after the addition of the substrate, the feed was switched to Feed 2 (4% glycerol, 12g/L MgSO₄, 15 ml/L trace element solution and 15 ml/L vitamin solution). The fermentation was terminated at appropriate time point and the culture medium was harvested for purification of the glycosylated products.

Glycoside purification - The culture medium harvested from the fermenter was centrifuged at 8,000 rpm, 30 min to remove the cells. The fermentation broth was then flocculated with 50 g/L CaCI₂ and 50 g/L K₂HPO₄ and centrifuged at 8,000 rpm, 30 min to remove the cell debris. The clarified fermentation broth was incubated with conditioned SP-207 beads for 2 h. The broth was then decanted and the beads were incubated with iso-propanol overnight under gentle stirring to elute the glycosylated products from the beads. The products were dried using rotary evaporation and re-dissolved in DMSO for preparative chromatography using an Akta purifier system 10 (Amersham Biosciences) equipped with a 250x30 mm Columbus 10 μ C18 column (Phenomenex).

Analysis of Glycoside Adjuvant activity

Bone marrow cells were flushed from the femurs of young adult C57BL/6 mice, washed in RPMI medium and the cultured in 90mm tissue culture grade Petri dishes for 9 days in

RPMI medium supplemented with 10% heat inactivated FCS, 100μg/ml streptomycin,

100U/ml penicillin, 2mM sodium pyruvate, 2mM L-glutamine, 50μM β2-mercaptoethanol and 10v/v supernatant from a myeloma line transfected with murine GM-CSF cDNA.

Medium was replenished every 3 days. On day 8 of culture, non-adherent and loosely adherent DC were recovered and re-plated in 24 well costar plates or 96 well micrtitre plates. Test compounds were added at the appropriate concentrations for 24h in the presence of GM-CSF supplemented medium. After 24h supernatants were removed and stored at -20 for cytokine analysis. Cells were stained by conventional techniques using flurochrome-labeled anti-CD40 and anti-CD80 mAbs and analysed using a BD FACSArray. IL-6 was measured using a CBA assay kit for detectionof murine IL-6 and data evaluated using a BD FACSArray. Example 1

We have investigated two plant scaffolds, quercetin and resveratrol, analysing DC activation in terms of cell surface phenotypic changes and secretion of IL-6. IL-6 was chosen for these early studies given its role in cross-tolerance of DC following TLR ligation in the development of Treg cells (which may dampen vaccine-induced immunity), our own data showing a critical role for this cytokine during DC-based immunotherapy for visceral leishmaniasis.

As shown in Figure 1a, both aglycones influenced DC activation. More importantly, regiospecific glucosylation resulted in selective alterations in function. Thus, whereas quercetin treatment increased expression of CD40 but not CD80 and dose-dependently inhibited basal IL-6 production, quercetin 7,3'-di-O-glucoside selectively enhanced CD80 expression and IL-6 production, whilst retaining equivalent activity towards CD40 as the aglycone.

In the case of resveratrol, the aglycone increased expression of both CD40 and CD80, without influencing IL-6 production. Resveratrol 3-O-glucoside had increased bioactivity at low doses (for CD40 and CD80), whereas the 4'-O-glucoside induced a reciprocal response. These differential activities of the resveratrol monoglucosides were also observed for IL-6 secretion, though over a more limited and bell-shaped dose response. Our data indicate for the first time that regiospecific glycosylation has selective effects on the bioactivity of these small molecule natural product immune modulators.

Example 2

Murine bone marrow derived DC were exposed to the TLR2 agoinst Pam3CSK4 in the absence or presence of increasing concentrations of resveratrol or resveratrol 3-0 glucoside. The 3-0 glucoside increased levels of IL-6 production over a 2 log dose response, whereas the aglycone was inhibitory, see Figure 1 b.

TLR2 is engaged by fungal pathogens including Candida albicans, and manipulation of TLR2 responses is known to affect the outcome of candidiasis in experimental models. Thus: 1. manipulation of TLR2 signalling by resveratrol may influence the outcome of systemic fungal infections and 2: resveratrol may modify response to other anti-fungal agents or drugs targeting TLRs and other pattern recognition receptors involved in antifungal immunity e.g. dectin-1 Example 3

Murine bone marrow derived DC were exposed to the TLR3 agoinst Poly (I₁C) in the absence or presence of increasing concentrations of resveratrol or resveratrol 3-0 glucoside. The 3-0 glucoside increased levels of IL-6 production over a 2 log dose response, whereas the aglycone was inhibitory, see Figure 1c.

TLR3 mediated responses are important in anti-viral immunity e.g in the response to Hepatiis B, respiratory syncytial virus, or influenza virus. TLR3 may induce anti-viral immunity through the induction of Type I inetrefreons or may contribute to viral pathogenicity by regulating pro-inflammatory cytokine production (as seen for influenza virus). Manipulation of TLR3 induced cytokines may play a role in fine tuning the activity of vaccine adjuvants or immune stimulants for viral disease

Example 4

Murine bone marrow derived DC were exposed to the TLR9 agoinst ODN 1668 in the absence or presence of increasing concentrations of resveratrol or resveratrol 3-0 glucoside. The aglycone was inhibitory for IL-6 and IL-10 production whereas the 3-0 glucoside had minimal activity, see Figure 1d.

Synthetic natural and non-natural oligodeoxynucleotides with specific motifs around a CpG dinucleotide and similar motifs contained within many naked DNA vaccines are TLR9 agonists. TLR9 agonists are immune modulators with well demonstrated activity as vaccine adjuvants. IL-10 has been shown to reduce vaccine efficacy. Coadministration of resveratrol with a TLR9 targeted adjuvant e.g. in a vaccine comprising HASPB for leishmaniasis or Ag85 for tuberculosis may therefore selectively improve vaccine efficacy by reducing unwanted IL-10. IL-10 may also hinder therapeutic benefit of TLR9 agonists when used as direct monotherapies for viral infection e.g. hepatitis C

References 1. Bellinghausen, I., B. Konig, I. Bottcher, J. Knop, and J. Saloga. 2006. Inhibition of human allergic T-helper type 2 immune responses by induced regulatory T cells requires the combination of interleukin-10-treated dendritic cells and transforming growth factor-beta for their induction. CHn Exp Allergy 36:1546. 2. Hubert, P., N. Jacobs, J. H. Caberg, J. Boniver, and P. Delvenne. 2007. The cross-talk between dendritic and regulatory T cells: good or evil? J Leukoc Biol 82:781.

3. Larche, M. 2007. Regulatory T cells in allergy and asthma. Chest 132:1007.

4. Wilczynski, J. R., J. Kalinka, and M. Radwan. 2008. The role of T-regulatory cells in pregnancy and cancer. Front Biosci 13:2275.

5. Zhou, Y. 2008. Regulatory T cells and viral infections. Front Biosci 13:1152.

Table 1 Glycosylated scaffolds:

Scaffolds Glycosides GTs

Polyphenol scaffolds -

Quercetin 3-O-glucoside 78D2

7-O-glucoside 73B2

3'-O-glucoside 71 C1

3,7-di-O-glucoside 76E12

7,3'-di-O-glucoside 71 C1

3-O-rhamnoside 78D1

7-O-rhamnoside 89C1

3,7-di-O-rhamnoside

3-O-galactoside

7-O-galactoside

3,7-di-O-galactoside

Resveratrol 3-O-glucoside 88A1

4'-O-glucoside 74B1

3,4'-di-O-glucoside 73C5

3-O-rhamnoside

4'-O-rhamnoside

3-O-galactoside

4'-O-galactoside

Geraniol 0-glucoside 73C5

Perillyl alcohol O-glucoside 73C5

Table 2

Table 3

Aglycone Glycoside Glycosyltransferase

Abscisic acid Abscisic acid glucose ester 84B1

Artemisinic acid Atremisinic acid glucose ester 84B1

Benzoic acid Benzoylglucose 74F1

2-hydroxybenzoic acid 2-O-glucosylbenzoic acid 74F1

2-hydroxybenzoylglucose 74F2

3-hydroxybenzoic acid 3-O-glucosylbenzoic acid 72B1

3-hydroxybenzoylglucose 75B1

4-hydroxy-benzoic acid 4-O-glucosylbenzoic acid 89B1

4-hydroxybenzoylglucose 75B1

2,3-dihydroxybenzoic acid 2-hydroxy-3-O-glucosylbenzoic acid 72B1

2,4-dihydroxybenzoic acid 2-hydroxy-4-O-glucosylbenzoic acid 89B1

2,5-dihydroxybenzoic acid 2-hydroxy-5-0-glucosylbenzoic acid 71B1

3,4-dihydroxybenzoic acid 3-hydroxy-4-O-glucosylbenzoic acid 71B1

3-O-glucoyl-4-hydroxybenzoic acid 72B1

3,4-dihydroxybenzoylglucose 75B1

Borneol Borneol glucoside 73C5

Caffeic acid Caffeic acid-3-O-glucoside 71C1

Caffeic acid-4-O-glucoside 71B1

Caffeoylglucose 84A1

Casticin Casticin-3'-0-glucoside 71C1

Cinnamic acid Cinnamoylglucose 84A3 cis-zeatin cis-zeatin-7-N-glucoside 76C1 cis-zeatin-9-N-glucoside 76C1 cis-zeatin-O-glucoside 85A1

Citronellol Citronellol glucoside 73C5

Coniferyl alcohol Coniferyl alcohol-4-O-glucosie 72E2

Daidzein Daidzein-7-O-glucoside 73B2

Daidzein-4'-O-g lucoside 73C4

Dihydrozeatin Dihydrozeatin-7-N-glucoside 76C1

Dihydrozeatin-9-N-glucoside 76C1

Dihydrozeatin-0-glucoside 85A1

Esculetin Esculetin-6-O-glucoside 72B1

Esculetin-7-O-glucoside 71C1

Farnesol Farnesol glucoside 85A1

Ferulic acid Ferulic acid-4-O-glucoside 72E2

Feruloylglucose 84A3

Geraniol Geraniol glucoside 85A1 lndo-3-acetic acid Indole-acetylglucose 84B1

Kinetin Kinetin-7-N-glucoside 76C1

Kinetin-9-N-glucoside 76C1

Linalool Linalool glucoside 73C5

Luteolin Luteolin-7-O-glucoside 73B2

Luteolin-3'-O-glucoside 71C1

Luteolin-7,3'-di-O-glucoside 71C1

Menthol Menthol glucoside 73C5

N6-benzyladenine N6-benzyladenine-7-N-glucoside 76C1

N6-benzyladenine-9-N-glucoside 76C1 N6-isopenteny laden ine N6-isopentenyladenine-7-N- 76C1 glucoside

N6-isopentenyladenine-9-N- 76C1 glucoside p-coumaric acid p-coumaroylglucose 84A1

Perillyl alcohol Perillyl alcohol 85A1

Quercetin Quercetin-3-O-rhamnoside 78D1

Quercetin-7-O-rhamnoside 89C1

Quercetin-3,7-di-O-rhamnoside 78D1 + 89C1

Quercetin-3-O-glucoside 73B3

Quercetin-7-O-glucoside 73B2

Quercetin-3'-O-glucoside 71C1

Quercetin-3,7-di-O-glucoside 76E12

Quercetin-7,3'-di-O-glucoside 71C1

Retinoic acid Retinoylglucose 84B1

Scopoletin Scopoletin-7-O-glucoside 71C1

Sinapic acid Sinapic acid-4-O-glucoside 72E2

Sinapoylglucose 84A2

Sinapyl alcohol Sinapyl alcohol-4-O-glucoside 72E2

Teφineol Terpineol glucoside 85A1 trans-resveratrol trans-resveratrol-3-O-glucoside 88A1 trans-resveratrol-4'-0-glucoside 74B1 trans-zeatin trans-zeatin-7-N-glucoside 76C1 trans-zeatin-7-N-glucoside 76C1 trans-zeatin-O-g lucoside 85A1

Claims

1 Regioselectively glycosylated resveratrol for use as an adjuvant.

2. Use according to claim 1 wherein resveratrol is modified with glucose.

3. Use according to claim 1 wherein resveratrol is modified with rhamnose.

4. Use according to claim 2 or 3 wherein said resveratrol glycoside is selected from the group consisting of: 3-O-glucoside, 4'-O-glucoside, 3,4'-di-O-glucoside, 3-O- rhamnoside, 4'-O-rhamnoside.

5. Use according to claim 4 wherein said resveratrol glycoside is 3-O-glucoside or 4'-O-glucoside.

6. Regioselectively glycosylated resveratrol for use in the activation of immune cells.

7. Use according to claim 6 wherein said immune cells are myeloid cells.

8. Use according to claim 7 wherein said myeloid cells are selected from the group consisting of: macrophages, monocytes, leukocytes, eosinophils, basophils, dendritic cells

9. Use according to claim 8 wherein said immune cells are dendritic cells.

10. Use according to claim 9 wherein said dendritic cells are myeloid dendritic cells.

11. Use according to claim 9 wherein said dendritic cells are lymphoid dendritic cells.

12. Use according to claim 6 wherein said immune cells are regulatory T cells.

13. Use according to claim 6 wherein said immune cells are non-haematopoietic stromal cells.

14. Use according to any of claims 6-13 wherein said immune cells are human.

15. The use of a regioselectively glycosylated resveratrol in the treatment of an autoimmune disease.

16. Use according to claim 15 wherein said autoimmune disease is selected from the group selected from: multiple sclerosis, type 1 diabetes, autoimmune thyroid disease, autoimmune hepatitis, rheumatoid arthritis, autoimmune colitis, Crohns disease, celiac disease, autoimmune nephritis, autoimmune neuropathy (guillan Barre), encephalopathy fibrosing alveolitis, ankylosing spondylitis, aplastic anaemia, Hashimoto's disease, Lupus erythematosus, pernicious anaemia, rheumatoid arthritis, or Sjorgen's disease.

17. The use of regioselectively glycosylated resveratrol in the reconstitution of the immune system in subjects that are immune deficient.

18. Use according to claim 17 wherein said immune deficiency is the result of chemotherapy.

19. Use according to claim 18 wherein said immune deficiency is neutropenia.

20. Use according to claim 17 wherein said subject is immune deficient as a result of infection.

21. Use according to claim 20 wherein said immune deficiency is the result of HIV infection.

22. A composition comprising regioselectively glycosylated resveratrol and an adjuvant.

23. A composition according to claim 22 wherein said adjuvant is selected from the group consisting of: cytokines selected from the group consisting of GM CSF, interferon gamma, interferon alpha, interferon beta, interleukin 12, interleukin 23, interleukin 17, interleukin 2, interleukin 1 , TGF, TNFα, and TNFβ.

24. A composition according to claim 22 wherein said additional adjuvant is a ToI Like Receptor [TLR] agonist.

25. A composition according to claim 24 wherein said TLR agonist is selected from the group presented in Table 2.

26. A composition according to claim 22 wherein said additional adjuvant is a bacterial cell wall derivative.

27. A composition according to claim 26 wherein said bacterial cell wall derivative is a muramyl dipeptide (MDP) and/or trehelose dycorynemycolate (TDM).

28. A composition according to any of claims 22-27 wherein said composition is a vaccine composition and further includes at least one antigen and optionally a carrier.

29. A composition according to claim 28 wherein said antigen is an HIV antigen.

30. A composition according to claim 29 wherein said HIV antigen is gp120.

31. A composition according to claim 28 wherein said antigen is a tuberculosis antigen.

32. A composition according to claim 28 wherein said tuberculosis antigen is Ag85.

33. A composition according to claim 28 wherein said antigen is a leishmanial antigen.

34. A composition according to claim 33 wherein said antigen is HASPB1.

35. A method for the activation of immune cells and optionally treatment of a subject suffering from or with a predisposition to immune deficiency comprising: i) removing from said subject a cell sample comprising immune cells; ii) contacting said cell sample with a preparation comprising regioselectively glycosylated resveratrol ; optionally iii) monitoring the activation of said immune cells in said cell sample; and optionally iv) administering said activated immune cells to a subject in need of treatment for immune deficiency.

36. The method according to claim 35 wherein said immune cells are myeloid cells.

37. The method according to claim 36 wherein said myeloid cells are selected from the group consisting of: macrophages, monocytes, leukocytes, eosinophils, basophils, dendritic cells.

38. The method according to claim 37 wherein said immune cells are dendritic cells.

39. The method according to claim 38 wherein said dendritic cells are myeloid dendritic cells.

40. The method according to claim 38 wherein said dendritic cells are lymphoid dendritic cells.

41. The method according to claim 35 wherein said immune cells are regulatory T cells.

42. The method according to claim 35 wherein said immune cells are non- haematopoietic stromal cells.

43. A method according to any of claims 35-42 wherein said cells are human.

44. A preparation comprising activated immune cells and regioselectively glycosylated resveratrol scaffold for use in the treatment of immune deficiency.

45. A regioselectively glycosylated glycoside for use as an adjuvant.

46. Use according to claim 45 wherein said glycoside is selected from the group represented in Table 1.

47. Use according to claim 45 wherein said glycoside is selected from the group represented in Table 3.