WO2007010084A2

WO2007010084A2 - Immunostimulatory oligo- and polysaccharides and uses thereof

Info

Publication number: WO2007010084A2
Application number: PCT/FI2006/000256
Authority: WO
Inventors: Johannes Savolainen; Kaisa Nieminen
Original assignee: Johannes Savolainen; Kaisa Nieminen
Priority date: 2005-07-15
Filing date: 2006-07-17
Publication date: 2007-01-25
Also published as: WO2007010084A3

Abstract

This invention relates to the use of an immunostimulant selected from the group consisting of mannan polysaccharides comprising β-1,2-linked chains, β -1,2-(D)-mannooligosaccharides, and any combination thereof for the preparation of a medicament for treating a mammal, including human, suffering from or susceptible to a condition which can be improved or prevented by inducing a Th1-type, and/or inhibiting a Th2-type immune response. This invention also relates to an immunostimulatory composition comprising at least one component selected from the group consisting of mannan polysaccharides comprising β -1,2-linked chains and β -1,2-(D)-mannooligosaccharides; and a carrier. This invention further relates to a food comprising at least one component selected from the group consisting of mannan polysaccharides comprising β -1,2-linked chains and β -1,2-(D) mannooligosaccharides and use of mannan polysaccharides comprising ß-1,2-linked chains and β -1,2-(D) mannooligosaccharides as a food additive. This invention also relates to a method for inducing of Th1-type immune response comprising administering to a subject the composition of or the food of the invention in an amount effective to induce a Th1-type immune response and a method for inhibition of Th2-type immune response comprising administering to a subject the composition or the food of the invention in an amount effective to partially or completely inhibit development of Th2-type immune response.

Description

IMMUNOSTIMULATORY OLIGO- AND POLYSACCHARIDES AND USES THEREOF

FIELD OF THE INVENTION

The invention relates to immunostimulatory oligo- and polysaccharides and to their use for modulating T helper (Th) cell mediated-immune responses and in the manufacture of a medicament, pharmaceutical, or nutritional preparation for prevention or treatment of type I immediate atopic allergies, infectious diseases, and cancer.

BACKGROUND OF THE INVENTION

Type I immediate atopic allergies are one of the most common health problems in Western world, and constantly increasing in prevalence (ISAAC 1998). It has been speculated that this increase is associated with the reduced microbial load in the developed societies, which has weakened natural type 1 Th (Thi)-cell responses giving way to enhanced Th2 responses that characterize atopic diseases (Erb 1999, Hopkin 2002).

Th cells (CD4+ T cells) are the main regulators of allergic immune response. Th2 cells that secrete cytokines such as IL-4, IL-5 and IL-13 are important for development of allergic inflammation by inducing IgE-production by B cells, degranulation of mast cells and recruitment of eosinophils. Th1 cells, in contrast, are essential for defence against microbes, in particular intracellular pathogens, and cancer by inducing activation of cytotoxic T (Tc) cells and natural killer (NK) cells. In addition, Th1-type cytokines, including IFN-γ, IL-12 and IL-18, are involved in suppression of allergen-induced Th2-type immune responses (Renz et al. 1992, Li et al. 1998, Yoshimoto et al. 1997, Hofstra et al. 1998). The regulatory cytokine IL-10 secreted by regulatory T (Tr) cells also has an important role in down- regulation of Th2-type responses towards allergens (Bellinghausen et al. 2001 ). Moreover, in allergen vaccination, there is up-regulation of the cytokines IFN-y, IL- 10 and IL-18 indicating their crucial role in suppressing the allergic inflammation also in vivo (Varney et al. 1993, Savolainen et al. 2004).

Recent research has shown that the crucial time for the development of either Th1- or Th2-type immunity is infancy. At birth, there is a weak Th2-type immunity, which in healthy individuals is conversed to a Th1-type response (Holt & Macaubas C 1997). It is thought that the priming of the immune system by microbial components in the environment and gut microflora (endotoxins, lactobacilli, mycobacteria) is essential in this conversion.

Research has been performed on the effects of microbial components on Th1-Th2 balance. A great deal of effort has been directed towards the search of microbial molecules, which could be used in development of more effective and safer vaccines for immunization against infections and in novel therapeutic approaches to treat autoimmune, atopic, and malignant diseases. Among these molecules are 3-deacylated monophosphoryl lipid A (MPL), the non-toxic derivative of lipopolysaccharide of Salmonella minnesota, and immunostimulatory bacterial DNA sequences (CpG-ODN) that generally consist of a central nonmethylated CG dinucleotide. These molecules have been clinically tested and shown to be effective and well-tolerated adjuvants in a number of vaccines for immunization against and treatment of hepatitis B virus (Thoelen et al. 1998, Halperin et al. 2003, Vandepapeliere et al. 2005), human immunodeficiency virus (HIV) (McCormack et al. 2000), influenza A virus (Cooper et al. 2004), and Plasmodium falciparum (Rickman et al. 1991 , Hoffman et al. 1994), as well as in treatment of cancers such as non-Hodgkin lymphoma (Friedberg et al. 2005) and melanoma (Speiser et al. 2005). These molecules have been shown to function by inducing strong Th 1 -type pattern of cytokine production by acting on various Toll-like receptors on antigen-presenting cells such as monocytes, macrophages and dendritic cells (Krieg et al. 2004, Baldridge et al. 2004). Moreover, the Th1- enhancing effects of mycobacteria and bacterial lipopolysaccharide with subsequent suppression of Th2-type response, allergic inflammation, IgE- responses and bronchial hyperreactivity have been shown in several studies (Erb et al. 1998, Wang & Rook 1998, Herz et al. 1998). In addition, probiotic lactobacilli have been shown to reduce the prevalence of atopic eczema at the age of two years when administrated orally during pregnancy and immediately after birth (Kalliomaki et al. 2001 ). Immunotherapy with bacterial CpG-ODN oligonucleotides bound covalently to allergens has also been shown to decrease the nasal inflammatory response in allergic rhinitis patients (Tulic et al. 2004). MPL has been successfully tested as an adjuvant in allergen immunotherapy (Drachenberg et al. 2001 , Drachenberg et al. 2003).

The therapeutic use of all these microbial compounds face problems. LPS is a toxin and as such unfit for therapeutic use. CpG-ODN is a gene and, in addition to being expensive for large-scale synthesis, implies potential problems with the public opinion (such as gene manipulated food products). MPL adjuvant is not a single chemical entity, but a mixture of analogues, with differences reflected in the number and length of fatty acid chains. Probiotic lactobacilli are live bacteria, comprising safety problems with respect to possible infections, and furthermore are difficult to standardize. Also mycobacterial lysates are crude mixtures of bacterial components out of range of biological standardization. An ideal therapeutic microbial component for allergy treatment would be a naturally occurring, non-toxic, safe purified molecule with as small molecular size as possible.

In WO 02/09728 complex carbohydrates are presented for prevention and treatment of a variety of over 80 diseases. In this publication carbohydrates are defined as any polymer comprising more than two sugar moieties and including such classes of compounds as polysaccharides (that include mucopolysaccharides and mannans) and oligosaccharides [that are comprised of branched oligosaccharides such as sialylated sugars including milk sugars; the key milk sugars (also called hexaoses) incorporated in the general class of complex carbohydrates being difucosyllacto-N-hexaose a and b, disialyl-monofucosyllacto- N-hexaose and monofucosyllacto-N-hexaose I, Il and II]. The diseases listed include conditions associated with allergies; individually mentioned are anaphylaxis, asthma and itching associated with allergies and hypersensitivity and are based on e.g. inhibition of white cell adhesion. US 2002/0054886 and US 2002/016009 disclose a vaccine, comprising β-1 , 2- linked straight chain oligo-mannosyl residues for the treatment of candidiasis. The oligo-mannosyl residues are used as an epitope to elicit a protective antibody response against candidiasis or to prevent Candida albicans adhesion to mammalian cells.

OBJECT AND SUMMARY OF THE INVENTION

One object of the present invention is to provide an immunostimulant for the preparation of a medicament for treating a mammal, including human, suffering from or susceptible to a condition which can be improved or prevented by inducing a Th 1 -type, and/or inhibiting a Th2-type immune response.

Another object of the present invention is to provide an immunostimulatory composition.

A further object of the present invention is to provide a immunostimulatory food.

Yet another object of the present invention is to provide for use of an immunostimulatory compound as a food additive.

A yet further object of the present invention is to provide a method for inducing of Th1-type immune response in a subject and a method for inhibiting of a Th2-type immune response in a subject.

The present invention provides use of an immunostimulant selected from the group consisting of mannan polysaccharides comprising β-1 ,2-Iinked chains, β-1 ,2-(D)-mannooligosaccharides, and any combination thereof for the preparation of a medicament for treating a mammal, including human, suffering from or susceptible to a condition which can be improved or prevented by inducing a Th1- type, and/or inhibiting a Th2-type immune response.

The present invention also provides an immunostimulatory composition comprising at least one component selected from the group consisting of mannan polysaccharides comprising β-1 ,2-Iinked chains and β-1 ,2-(D)-manno- oligosaccharides; and a carrier. The present invention further provides a food comprising at least one component selected from the group consisting of mannan polysaccharides comprising β-1 ,2- linked chains and β-1 ,2-(D)-mannooligosaccharides.

The present invention also provides use of mannan polysaccharides comprising β-1 ,2-linked chains, β-1 ,2-(D)-mannooligosaccharides and/or any combination thereof as a food additive.

The present invention also provides a method for inducing of Th1-type immune response comprising administering to a subject the composition of the invention or the food of the invention in an amount effective to induce a Th1-type immune response, and a method for inhibition of Th2-type immune response comprising administering to a subject the composition of the invention or the food of the invention in an amount effective to partially or completely inhibit development of Th2-type immune response.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 illustrates the chemical structure of β-1 ,2-(D)-mannotriose.

Figure 2 illustrates the chemical structure of β-1 ,2-(D)-mannobiose.

Figures 3a and 3b depict the proliferation of (3a) and IFN-γ production in (3b) human white blood cell cultures following stimulation with 200 μg/ml of Candida albicans mannan hydrolyzed at mild acidic conditions for 15, 30, or 60 minutes.

Figures 4a, 4b and 4c depict the expression of Th2-attenuating cytokines IFN-γ (4a), IL-10 (4b) and IL-18 (4c) in human white blood cells following stimulation with 0, 0.2, 2 and 20 μg/ml of β-1 ,2-(D)-manno-oligosaccharides, from β-1 ,2-(D)-mannohexaose to β-1 ,2-(D)-mannobiose. The y-axis indicates fold increase in mRNA expression in comparison to culture without oligomannoside.

Figure 5 illustrates the effect of 0, 0.2, 2 and 20 μg/ml of β-1 ,2-(D)-mannotriose on production of interleukin-4 (Th2 cytokine) in allergen (birch) stimulated white blood cell cultures of eleven patients with allergic rhinoconjunctivitis. The y-axis indicates IL-4 mRNA production as the percentage of allergen-induced response.

DETAILED DESCRIPTION OF THE INVENTION

The mechanism of the present invention is believed to be β-1 ,2-(D)-manno- oligosaccharide -mediated modulation of Th-immune response as presented in Scheme 1. In the context of this application the term β-1 ,2-(D)-mannooligo- saccharide refers to a carbohydrate consisting of two to nine (D)-mannose units comprising at least one β-1 ,2-glycosidic link, preferably two or more β-1 ,2- glycosidic linkages and most preferably only β-1 ,2-glycosidic linkages. The term polysaccharide refers to a corresponding carbohydrate consisting of at least ten monosaccharides. Allergic inflammation is characterized by IgE antibody production, mast cell degranulation and eosinofilic inflammation. These responses are mediated by allergen-specific Th2-type immune cells that secrete cytokines such as IL-4 and IL-5. The Th1 cells that secrete cytokines, including IFN-γ are involved in suppression of allergen-induced Th2-type immune responses. The regulatory cytokine IL-10 is also important in down-regulation of Th2-type immune responses. Based on the experiments described in examples 2 and 3, the discovery that β-1 ,2-(D)-manno-oligosaccharides stimulate the IFN-γ, IL-10 and IL- 18 in human white blood cells suggests these cytokines have a role in β-1 ,2-(D)- manno-oligosaccharide-mediated suppression of Th2 cell response, and thus allergic inflammation.

The present invention describes immunostimulatory β-1 ,2-(D)-manno- oligosaccharides, and the use of such molecules or their prodrugs for modulation of Th-mediated immune responses and in the manufacture of a medicament, pharmaceutical and nutritional preparation for prevention or treatment of type I atopic allergies, infectious diseases, and cancer in a subject. The term immunostimulant refers to a biologically active substance whose activities affects or plays a role in the functioning of the host immune system by stimulating T helper type 1 and regulatory type T cell responses. The term subject refers to a mammal, including human. Scheme 1

β-1 ,2-manno- oligosaccharides

Allergic

Inflammation

Protective Immune Response to Microbes and

Cancer

A composition according to the invention typically includes at least one component selected from the group consisting of β-1 ,2-(D)-mannooligosaccharides, preferably β-1 ,2-(D)-mannotriose and/or β-1 ,2-(D)-mannobiose, which have the chemical formulas depicted in Fig. 1 and Fig. 2, respectively, β-1 ,2-(D)- mannotriose thus refers to a structure: {Man(beta.1-2)Man(beta.1-2)Man} (Fig. 1) and β-1 ,2-(D)-mannobiose to a structure: {Man(beta.1-2)Man} (Fig. 2). It should be noted that the although Fig. 1 and Fig. 2 disclose the reducing end in β-hemiacetal form, in solution the reducing end can also be in α-hemiacetal form or in open chain form. The composition can also consist of or comprise prodrugs of β-1 ,2-(D)- mannooligosaccharides, preferably β-1 ,2-(D)-mannotriose and β-1 ,2-(D)- mannobiose, e.g. a mannan polysaccharide comprising β-1 ,2-linked chains, β-1 ,2- (D)-mannooligosaccharides, preferably β-1 ,2-(D)-mannotriose and β-1 ,2-(D)- mannobiose, can be prepared from mannan of Candida albicans yeast or other fungi, synthesized chemically, or produced enzymatically by any method known in the art. Mannan of Candida albicans yeast typically comprises about 10 % β-1 ,2- linked chains and its hydrolysis results in an amount of β-1 ,2-(D)-mannotriose and β-1 ,2-(D)-mannobiose that corresponds to about 1 % of the amount of the mannan. The invention also encompasses a method for inducing a Th1-type immune response. The method involves administrating to a subject compositions according to the invention in an effective amount to induce the synthesis of TM -type cytokines. In preferred embodiments, the method involves, but is not limited to, the induction of IFN-γ synthesis in T cells. In a further aspect of the invention, the method involves the induction of IL-12 and IL-18 production in antigen-presenting cells. In preferred embodiments, the antigen-presenting cell is a macrophage, monocyte or dendritic cell. In addition, the method involves activation of cytolytic activity of Tc cells and NK cells.

The invention further involves a method for inhibiting a Th2-type immune response. The method involves administrating to a subject, the composition according to the invention in an effective amount to partially, or completely inhibit the development of Th2-type immune response to an allergen. In preferred embodiments, the mechanisms of inhibition include, but are not limited to, induction of 1L-10 production in T cells. The method can also involve suppression of Th2-type immune response by inhibition or suppression of the function of Th2- type T cells, mast cells and eosinophil and basophil granulocytes.

The present invention also provides a method for modulating a Th-mediated immune response. Preferably, the immune response stimulated according to the invention is biased toward the Th1-type response and away from the Th2-type response. In one aspect, the method involves administrating to a subject, said composition in an effective amount to stimulate the production of Th1-type cytokines. In preferred embodiments, the method involves, but is not limited to, the induction of lFN-γ synthesis in T cells. In further embodiments, the method involves, but is not restricted to, the induction IL-12 and IL-18 production in antigen-presenting cells. The term antigen-presenting cell used herein denotes to a macrophage, monocyte or dendritic cell. Typically the method also involves activation of Tc and NK cells. In other aspect, the method involves administrating to a subject said composition in an effective amount to partially, or completely inhibit the development of Th2-type immune response to allergen. In preferred embodiments, the mechanisms of inhibition include, but are not limited to, induction of IL-10 production in T cells. The method can also involve suppression of Th2-type immune response by inhibition or suppression of the function of Th2- type T cells, mast cells and eosinophil and basophil granulocytes.

The invention further relates to the use of a composition according to the invention for the manufacture of a medicament, pharmaceutical, or nutritional preparation for prevention or treatment of type I immediate atopic allergies. In preferred embodiments, the type I immediate atopic allergy is selected from the group consisting of atopic eczema/dermatitis syndrome (AEDS), allergic asthma, allergic urticaria, food allergy, venom allergy, and allergic rhinoconjunctivitis. In further embodiments, the composition of the invention can be used for prevention and treatment of infectious diseases and cancer. The term prevent used herein refers to inhibiting completely or partially the development of the disorder in a subject that has, or is at high risk of developing, said disorder. The term treating is defined as administering a subject an effective amount of the said composition to prevent the onset of, alleviate the symptoms of, or stop the progression of the disorder. The term effective amount used herein refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. The therapeutically effective amount typically varies from about 1 μg to several grams depending on the composition and especially the mode of administration. E.g. the typical amount for parenteral administration of β-1 ,2~(D)-mannooligosaccharide, preferably β-1 ,2-(D)-mannotriose and/or β-1 ,2-(D)-mannobiose, is from 1 μg to 100 μg, preferably 2 μg to 30 μg, most preferably 3 μg to 10 μg, and for oral administration the typically amount can be much higher from a few mg up to several grams. If a prodrug is administered the effective amount depends on how much β-1 ,2-(D)-mannotriose and/or β-1 ,2-(D)-mannobiose it can result in and how much of is actually released.

In further embodiments, the medicament or pharmaceutical preparation of the invention may be administered to a subject by any known route in art, including enteral, mucosal, parenteral and topical routes. The enteral routes include oral and any route involving absorption from the gastrointestinal tract. The mucosal routes include, but are not restricted to, oral, nasal, sublingual, buccal, pulmonary, transdermal and ocular routes. The parenteral routes include, but are not restricted to, intravenous, intradermal, intramuscular, and subcutaneous routes.

In some embodiments, the composition of the invention is used in conjugation with a pharmaceutically acceptable carrier. The term pharmaceutically acceptable carrier refers to a carrier substance with which the active ingredient is combined to facilitate the application to a subject and that is physiologically acceptable to the recipient. The pharmaceutically acceptable carrier can be selected from the group consisting of, but not restricted to, transdermal carriers, transmucosal carriers, oral carriers and parenteral carriers based upon the intended route of administration. In enteral routes of administration, a mannan polysaccharide comprising β-1 ,2-linked chains, can optionally serve as the natural carrier of immunostimulatory β-1 ,2-(D)- mannotriose and β-1 ,2-(D)-mannobiose.

In further embodiments, the pharmaceutical composition of the invention may include another therapeutic compound. The term therapeutic compound used herein is preferentially an allergy medicament, an asthma medicament, an anticancer medicament, or antimicrobial agent. In other embodiments, the pharmaceutical composition of the invention comprises an antigen. The term antigen broadly includes any type of molecule (e.g. protein, peptide, polysaccharide, glycoprotein, nucleic acid, or combination thereof) that is recognized by a host immune system and is capable of eliciting a specific immune response. In some embodiments the antigen is an allergen preparation for specific allergen immunotherapy (allergen vaccination or sublingual immunotherapy). The term allergen used herein refers to a substance that can induce an allergic or asthmatic response in a susceptible subject, and includes but is not limited to pollens, insect venoms, animal dander, fungal spores and house dust mite. The term specific allergen immunotherapy refers to treatment of a subject with atopic disorders by administrating gradually increasing amounts of allergen by any of the known routes to induce tolerization to the allergen to prevent further allergic reactions. Alternatively, the pharmaceutical composition of the invention comprises an antigen preparation for cancer vaccination or immunization against microbial infections. Cancer antigens used herein are compounds associated with tumour or cancer cell surface, and include but are not limited to antigens that are prepared by crude extracts of cancer cells, by recombinant technology, or by de novo synthesis of known antigens. The term microbial antigens used herein include intact microorganisms, as well as natural isolates and fragments or derivates thereof, and also synthetic compounds, which are identical to or similar to natural microbial antigens. The term infectious disease refers to a disease arising from the presence of foreign microorganisms or infectious pathogens in the body. The term infectious pathogens, i.e. microbes, refers to viruses, bacteria and parasites. As such, the term infectious pathogens also includes normal flora, which is not desirable. In one aspect, the combined administration of the pharmaceutical composition of the invention and cancer or microbial antigen is useful for stimulating enhanced immune response to malignant cells and pathogens. In yet other embodiments, the pharmaceutical composition of the invention comprises antibodies or antibody fragments which specifically bind or recognize cancer or microbial antigens.

The present invention also relates to use of immunostimulatory composition comprising of β-1 ,2-(D)-mannooligosaccharide, preferably β-1 ,2-(D)-mannotriose, β-1 ,2-(D)-mannobiose and/or prodrugs, e.g. mannan oligo- or polysaccharides comprising β-1 ,2-linked chains, thereof in the manufacture of a medicament, pharmaceutical, or nutritional preparation for prevention or treatment of type I immediate atopic allergy. In preferred embodiments, the type I immediate atopic allergies, include, but are not limited to, atopic eczema/dermatitis syndrome (AEDS), allergic asthma, allergic urticaria, food allergy, venom allergy and allergic rhinoconjunctivitis.

In yet other embodiments, the composition of the invention is used as a food additive. The composition of the invention can also be a nutritional preparation. The nutritional composition is preferentially enterally administrable, e.g. powder, tablet, capsule, a liquid concentrate, solid product, or ready-to drink beverage. Alternatively, the composition of the embodiment is used as an additive of usual food products. In other embodiments, the nutritional composition of the invention is used for enrichment of infant formulas and other functional food products. A further embodiment comprising the composition of the invention could be a chewing gum.

EXAMPLES

Materials and methods

CTAB-mannan

CTAB-mannan was prepared according to Nakajima and Ballou (1974). Briefly, fungi were autoclaved for 2 hours at 14O⁰C in neutral citrate buffer (0.02 M CeH₅Na₃O₇ x 2 H₂O). The mannan was isolated from the primary extract by methanol precipitation (4 days in 70 % methanol) and with a final precipitation with CTAB(cetyltrimethylammonium bromide, Sigma, MO, USA) (2.7 % CTAB and 2.7 % lyophilised methanol precipitate in distilled water overnight).

Mild acid hydrolysis

The long β-1 ,2-linked polysaccharide side chains of Candida albicans mannan were degraded by subjecting CTAB-mannan to mild acid hydrolysis (20 mg/ml of lyophilized mannan in 0.1 N HCI) for 60 minutes at 100 ⁰C.

Human PBMC

The human PBMC were isolated from heparinized blood samples with Ficoll (Ficoll-Paque, Amersham Pharmacia Biotech, Sweden) density gradient centrifugation. The PBMC were washed twice with Hanks' balanced salt solution (HBSS) and suspended in RPMI medium supplemented 5 % autologous serum, 2 mM L-glutamine (Fluka Biochemica, Germany) and 100 μg/ml gentamycin (Biological Industries, Israel). The cell suspension was diluted in the RPMI-based culture medium to 10 cells/ml and applied on 48-well culture plates (Costar, Cambridge, MA, USA), 400 μl/well in duplicate for cytokine assays and on 96-well culture plates (Costar), 100 μl suspension/well in triplicate for proliferation assays. The cultures were incubated at 37 ⁰C, in humidified atmosphere with 5 % CO₂ for 5-7 days. In the proliferation assay, tritium-labelled thymidine (Amersham Pharmacia Biotech) was used in the culture medium during the last 18 hours and the incorporated radioactivity was measured from the cells with liquid scintillation counter.

RNA extraction

PBMC were harvested and lysed in 500 μl of Trizol-solution (Gibco, Life Technologies, Scotland, UK) and stored at -70 ⁰C until RNA extraction. Total RNA was isolated according to Trizol instructions with the exception that prior to isopropanol precipitation, one μl of glycogen (20 mg/ml, Boehringer Mannheim,

Germany) was added into the reaction. Extracted RNA was stored in 200 μl of

75 % ethanol at -20 ⁰C. Prior to RT reaction, extracted RNA was suspended to 20 μl of diethylpyrocarbonate (DEPC)-treated water (Promega, Wl, USA).

RT-PCR

The reverse transcription (RT) reaction was performed with First Strand cDNA Synthesis Kit (Amersham Pharmacia Biotech) using poly(dT)12-18 primers, and cDNA was stored at -70 ⁰C. The amplification of β-actin, IFN-γ, IL-10, IL-18 and IL-4 cDNAs were performed in MicroAmp^® optical 96-well reaction plate (Applied Biosystems, CA, USA). The 25 μl reaction in each well contained 1 μl of total cDNA, 300 nM of sequence specific primers and 200 nM of dual-labelled fluorogenic probe in 1x Taqman^® Universal PCR master mix (Applied Biosystems). The primer and probe sequences were designed using Primer Express software (Applied Biosystems) and were labelled with FAM (6-carboxyfluorescein) at the 5'-end and with TAMRA (6-carboxytetramethylrhodamine) at the 3'-end. A negative PCR control without template and a positive PCR control with a template of known amplification were included in each assay. The reaction was performed in ABI PRISM 7700 Sequence Detection System (Applied Biosystems) according to manufacturer's instructions. The Ct values of the PCRs (the cycle number at which the detected fluorescence exceeds the threshold) for each amplification product were determined using a threshold value of 0.03. IFN-γ, IL-10, IL-18 and IL-4 specific signals were normalized by constitutively expressed β-actin signals using formula (I).

o-ΔCt _ 2"(Ct,β-actin - Ct,IFN-γ, IL-10, IL-18 or IL-4)

(I) lFN-γ, IL-10, IL-18 and IL-4 stimulation indexes were thereafter calculated by the formula (II) as described in ABI PRISM 7700 SDS relative quantitation of gene expression-protocol by Applied Biosystems.

o-ΔΔCt _ r_j-(ΔCt for stimulated culture- ΔCt for unstimulated culture) ....

ELISA

Production of IFN-γ to culture supernatants by PBMCs was measured by cytokine- specific ELISA in 96-well flat-bottomed microtitre plates (Nunc-lmmuno Plate Maxisorp, Roskilde, Denmark) using commercially available antibody pairs and standard kits for ELISA (Pharmingen, San Diego, CA, USA).

Example 1

Th1 -inducing capacity of mild acid hydrolysis products of Candida albicans mannan

To study the Th1 -inducing capacity of different size components of Candida albicans mannan, CTAB-mannan was hydrolyzed with 0.1 M HCI at 100 ⁰C for 15, 30, 45, or 60 min, and the lyophilized fractions were used for stimulation of human PBMC as described above. As illustrated in Fig. 3a, prolonged incubation of CTAB-mannan at mild acidic conditions resulted in a progressive degradation of mannan polysaccharide according to decrease in optic density on TLC (thin layer chromatogram). In contrast, there was no decrease of proliferation induction and a clear increase of IFN-γ production (mRNA and protein) following prolonged hydrolysis of CTAB-mannan at mild acidic conditions(Fig. 3b). Lymphoproliferation and IFN-γ production were induced by low molecular weight β-1 ,2-linked oligomannosidic side chains released by mild acid hydrolysis. Example 2

Production of Th2-attenuating cytokines by human PBMC in response to β-1 ,2-(D)-mannotriose and β-1 ,2-(D)-mannobiose

β-1 ,2-linked side chains of mannan polysaccharide were fractionated with Bio-Gel P-2 column (Bio-Rad Laboratories, CA, USA) chromatography. The potency of the isolated β-1 ,2-(D)-manno-oligosaccharides to induce production of the Th2-attenuating cytokines IFN-γ, IL-10 and IL-18 were thereafter tested in vitro in PBMC cultures of allergic subjects. Following stimulation for 3 days in the presence of 0, 0.2, 20 or 200 μg/ml of β-1 ,2-(D)-manno-oligosaccharides, from β- 1 ,2-(D)-mannohexaose to β-1 ,2-(D)-mannobiose, the expression IFN-γ, IL-10 and IL-18 mRNA were measured by real-time RT-PCR as described above. As illustrated in Figs. 4a^4c, strong inductions of IFN-γ, IL-10 and IL-18 production were elicited by β-1 ,2-(D)-manno-oligosaccharides.

Example 3

Effect of β-1 ,2-(D)-mannotriose on allergen-induced immune response

β-1 ,2-(D)-mannotriose and β-1 ,2-(D)-mannobiose induced strong Th1-type cytokine responses in human PBMC, are naturally occurring, non-toxic, safe, purified molecules with small molecular size, and thus ideal therapeutic microbial components for allergy treatment. To show that the invention was valid, the effect of β-1 ,2-(D)-mannotriose on the production of IL-4, the main Th2-type cytokine promoting allergic inflammation, was studied in an allergen stimulated PBMC culture of eleven patients with allergic rhinitis. The PBMC were isolated and cultured as described above. Birch whole allergen extract was used at a concentration of 50 μg/ml and β-1 ,2-(D)-mannotriose in concentrations 0.2, 2 and 20 μg/ml. The cultures were incubated for 3 days. Following stimulation, the expression of IL-4 and IFN-γ mRNA were measured by real-time RT-PCR. In the cultures of both atopic patients β-1 ,2-(D)-mannotriose effectively suppressed the birch allergen induced IL-4 response in a dose-dependent manner (Fig. 5). The p- values in Fig. 5 refer to a non-parametric paired test (Wilcoxon rank sum test). REFERENCES

Baldridge JR, McGowan P, Evans JT, Cluff C, Mossman S, Johnson D, Persing D (2004) Taking a Toll on human disease: Toll-like receptor 4 agonists as vaccine adjuvants and monotherapeutic agents. Expert Opin Biol Ther 4:1129-38.

Bellinghausen I, Knop J, Saloga J (2001 ) The role of interleukin 10 in the regulation of allergic immune responses, lnt Arch Allergy Immunol 126:97-101.

Cooper CL, Davis HL, Morris ML, Efler SM, Krieg AM, Li Y, Laframboise C, Al Adhami MJ, Khaliq Y, Seguin I, Cameron DW (2004) Safety and immunogenicity of CPG 7909 injection as an adjuvant to Fluarix influenza vaccine. Vaccine 22:3136-43.

Drachenberg KJ, Heinzkill M, Urban E, Woroniecki SR (2003) Efficacy and tolerability of short-term specific immunotherapy with pollen allergoids adjuvanted by monophosphoryl lipid A (MPL) for children and adolescents. Allergol lmmunopathol 31 :270-7.

Drachenberg KJ, Wheeler AW, Stuebner P, Horak F (2001 ) A well-tolerated grass pollen-specific allergy vaccine containing a novel adjuvant, monophosphoryl lipid A, reduces allergic symptoms after only four preseasonal injections. Allergy 56:498-505.

Erb K (1999) Atopic disorders: a default pathway in the absence of infection. Immunol Today 20:317-22.

Erb KJ, Holloway JW, Sobeck A, Moll H, Le Gros G (1998) Infection of mice with Mycobacterium bovic-Bacillus Calmette-Guerin (BCG) suppresses allergen- induced airway eosinophilia. J Exp Med 187:561-9.

Friedberg JW, Kim H, McCauley M, Hessel EM, Sims P, Fisher DC, Nadler LM, Coffman RL, Freedman AS (2005) Combination immunotherapy with a CpG oligonucleotide (1018 ISS) and rituximab in patients with non-Hodgkin lymphoma: increased interferon-alpha/beta-inducible gene expression, without significant toxicity. Blood 105:489-95. Halperin SA, Van Nest G, Smith B, Abtahi S, Whiley H, Eiden JJ (2003) A phase I study of the safety and immunogenicity of recombinant hepatitis B surface antigen co-administered with an immunostimulatory phosphorothioate oligonucleotide adjuvant. Vaccine 21 :2461-7.

Hertz U, Gerhold K, Grϋber C, Braun A, Wahn U, Renz H, Paul K (1998) BCG infection suppresses allergic sensitization and development of increased airway reactivity in an animal model. J Allergy Clin Immunol 102:867-74.

Hoffman SL, Edelman R, Bryan JP, Schneider I, Davis J, Sedegah M, Gordon D, Church P, Gross M, Silverman C, et al. (1994) Safety, immunogenicity, and efficacy of a malaria sporozoite vaccine administered with monophosphoryl lipid A, cell wall skeleton of mycobacteria, and squalane as adjuvant. Am J Trap Med Hyg 51 :603-12.

Hofstra CL, Van Ark I, Hofman G, Kool M, Nijkamp FP, Van Oosterhout AJ (1998) Prevention of Th2-like cell responses by coadministration of IL-12 and IL-18 is associated with inhibition of antigen-induced airway hyperresponsiveness, eosinophilia, and serum IgE levels. J Immunol 161 :5054-60.

Holt PG, Macaubas C (1997) Development of long-term tolerance versus sensitisation to environmental allergens during the perinatal period. Curr Opin Immunol 9:782-7.

Hopkin JM (2002) The rise of atopy and links to infection. Allergy 57(Suppl 72):5-9.

The International Study of Asthma and Allergies in Childhood (ISAAC) Steering Committee (1998) Worldwide variations in the prevalence of symptoms of asthma, allergic rhinoconjunctivitis and atopic eczema. Lancet 351 :1225-32.

Kalliomaki M, Salminen S, Arvilommi H, Kero P, Koskinen P, lsolauri E (2001) Probiotics in primary prevention of atopic disease: a randomised placebo- controlled trial. Lancet 357:1076-9.

Krieg AM, Efler SM, Wittpoth M, Al Adhami MJ, Davis HL (2004) Induction of systemic TH1-like innate immunity in normal volunteers following subcutaneous but not intravenous administration of CPG 7909, a synthetic B-class CpG oligodeoxynucleotide TLR9 agonist. J lmmunother 27:460-71.

Li Y, Simons FE, HayGIass KT (1998) Environmental antigen-induced IL-13 responses are elevated among subjects with allergic rhinitis, are independent of IL-4, and are inhibited by endogenous IFN-gamma synthesis. J Immunol 161 :7007-14.

McCormack S, Tilzey A, Carmichael A, Gotch F, Kepple J, Newberry A, Jones G, Lister S, Beddows S, Cheingsong R, Rees A, Babiker A, Banatvala J, Bruck C, Darbyshire J, Tyrrell D, Van Hoecke C, Weber J (2000) A phase i trial in HIV negative healthy volunteers evaluating the effect of potent adjuvants on immunogenicity of a recombinant gp120W61 D derived from dual tropic R5X4 HIV- 1ACH320. Vaccine 18:1166-77.

Nakajima T, Ballou CE (1974) Characterization of the carbohydrate fragments obtained from Saccharomyces cerevisiae mannan by alkaline degradation. J Biol Chem 23:7679-84

Renz H, Jujo K, Bradley KL, Domenico J, Gelfand EW, Leung DY (1992) Enhanced IL-4 production and IL-4 receptor expression in atopic dermatitis and their modulation by interferon-gamma. J Invest Dermatol 99:403-8.

Rickman LS, Gordon DM, Wistar R Jr, Krzych U, Gross M, Hollingdale MR, Egan JE, Chulay JD, Hoffman SL (1991 ) Use of adjuvant containing mycobacterial cell- wall skeleton, monophosphoryl lipid A, and squalane in malaria circumsporozoite protein vaccine. Lancet 337:998-1001.

Savolainen J, Laaksonen K, Rantio-Lehtimaki A, Terho EO (2004) Increased expression of allergen-induced in vitro interleukin-10 and interleukin-18 mRNA in peripheral blood mononuclear cells of allergic rhinitis patients after specific immunotherapy. Clin Exp Allergy 34:413-9.

Speiser DE, Lienard D, Rufer N, Rubio-Godoy V, Rimoldi D, Lejeune F, Krieg AM, Cerottini JC, Romero P (2005) Rapid and strong human CD8+ T cell responses to vaccination with peptide, IFA, and CpG oligodeoxynucleotide 7909. J Clin Invest 115:739-46.

Thoelen S, Van Damme P, Mathei C, Leroux-Roels G, Desombere I, Safary A, Vandepapeliere P, Slaoui M, Meheus A (1998) Safety and immunogenicity of a hepatitis B vaccine formulated with a novel adjuvant system. Vaccine 16:708-14.

Tulic MK, Fiset P-O, Christodoulopoulos P, Vaillancourt P, Desrosiers M, Lavigne F, Eiden J, Hamid Q (2004) Amb a-immunomodulatory oligodeoxynucleotide conjugate immunotherapy decreases the nasal inflammatory response. J Allergy Clin Immunol 113:235-41.

Vandepapeliere P, Rehermann B, Koutsoukos M, Moris P, Garcon N, Wettendorff M, Leroux-Roels G (2005) Potent enhancement of cellular and humoral immune responses against recombinant hepatitis B antigens using AS02A adjuvant in healthy adults. Vaccine 23:2591-601.

Varney VA, Hamid QA, Gaga M, Ying S, Jacobson M, Frew AJ, Kay AB, Durham SR (1993) Influence of grass pollen immunotherapy on cellular infiltration and cytokine mRNA expression during allergen-induced late-phase cutaneous responses. J Clin Invest 92:644-51.

Wang C-C, Rook GAW (1998) Inhibition of an established allergic response to ovalbumin in BALB/c mice by killed Mycobacterium vaccae. Immunology 93:307- 13.

Yoshimoto T, Okamura H, Tagawa Yl, Iwakura Y, Nakanishi K (1997) lnterleukin 18 together with interleukin 12 inhibits IgE production by induction of interferon- gamma production from activated B cells. Proc Natl Acad Sci USA 94:3948-53.

Claims

1. Use of an immunostimulant selected from the group consisting of mannan polysaccharides comprising β-1 ,2-linked chains, β-1 ,2-(D)-mannooligosac- charides, and any combination thereof for the preparation of a medicament for treating a mammal, including human, suffering from or susceptible to a condition which can be improved or prevented by inducing a Th 1 -type, and/or inhibiting a Th2-type immune response.

2. The use according to claim 1 characterized in that the immunostimulant is β-1 ,2-(D)-mannobiose and/or β-1 ,2-(D)-mannotriose, preferably β-1 ,2-(D)- mannotriose.

3. The use according to claim 1 or 2 characterized in that a Th1-type immune response is induced, and said response comprises production of a) IFN-Y in T cells, and/or b) IL-12 and/or IL18 in antigen-presenting cells.

4. The use according to claim 1 or 2 characterized in that a Th2-type immune response is inhibited by a) induction of IL-10 production in T-cells and/or b) inhibition or suppression of the function of Th2-type T cells, mast cells, eosinophil granulocytes and/or basophil granulocytes.

5. Use according to any of claims 1 to 4, characterized in that the condition is type I immediate atopic allergy.

6. Use according to claim 5, characterized in that the condition is selected from the group consisting of a) atopic eczema/dermatitis syndrome (AEDS), b) allergic asthma, c) allergic urticaria, d) food allergy, e) venom allergy, and f) allergic rhinoconjunctvitis.

7. Use according to claim 1, 2 or 3, characterized in that the condition is infectious disease or cancer.

8. An immunostimulatory composition comprising at least one component selected from the group consisting of mannan polysaccharides comprising β-1 ,2-linked chains and β-1 ,2-(D)-mannooligosaccharides; and a carrier.

9. The immunostimulatory composition according to claim 8 characterized in that composition comprises β-1 ,2-(D)-mannobiose and/or β-1 ,2-(D)-mannotriose, preferably β-1 ,2-(D)-mannotriose.

10. The composition according to claim 8 or 9, characterized in that the carrier is selected from the group consisting of transdermal carriers, transmucosal carriers, oral carriers and parenteral carriers.

11. The composition according to any of claims 8 to 10, characterized in that the carrier is a transmucosal carrier for sublingual and/or buccal administration.

12. The composition according to any one of claims 8 to 11 , characterized in that it further comprises an allergen preparation for specific allergen immunotherapy; and/or an additional allergy or asthma medicament.

13. The composition according to any one of claims 8 to 11 , characterized in that it further comprises a cancer specific antigen for vaccination against cancer; and/or an anticancer agent.

14. The composition according to any one of claims 8 to 11 , characterized in that it further comprises a microbe-specific antigen for vaccination against infectious disease; and/or an antimicrobial agent.

15. A food comprising at least one component selected from the group consisting of mannan polysaccharides comprising β-1 ,2-linked chains and β~1 ,2-(D)-mannooligosaccharides.

16. A food according to claim 15 characterized in that it comprises β-1 ,2-(D)- mannobiose and/or β-1 ,2-(D)-mannotriose, preferably β-1 ,2-(D)-mannotriose.

17. Use of mannan polysaccharides comprising β-1 ,2-linked chains, β-1 ,2-(D)-mannooligosaccharides and/or any combination thereof as a food additive.

17. The use according to claim 16 characterized in that β-1 ,2-(D)-mannobiose and/or β-1 ,2-(D)-mannotriose, preferably β-1 ,2-(D)-mannotriose is used.

18. A method for inducing of Th1-type immune response comprising administering to a subject the composition of any of claims 8 to 14 or the food of claim 15 or 16 in an amount effective to induce a Th1-type immune response.

19. The method according to claim 18 wherein the response comprises production of a) IFN-Y in T cells, and/or b) IL-12 and/or IL18 in antigen-presenting cells

20. A method for inhibition of Th2-type immune response comprising administering to a subject the composition of any of claims 8 to 14 or the food of claim 11 or 16 in an amount effective to partially or completely inhibit development of Th2-type immune response. 22

15. A food comprising at least one component selected from the group consisting of mannan polysaccharides comprising β-1 ,2-linked chains and β-1 ,2-(D)-mannooligosaccharides.

18. The use according to claim 17 characterized in that β-1 ,2-(D)-mannobiose and/or β-1 ,2-(D)-mannotriose, preferably β-1 ,2-(D)-mannotriose is used.

19. A method for inducing of Th1-type immune response comprising administering to a subject the composition of any of claims 8 to 14 or the food of claim 15 or 16 in an amount effective to induce a Th 1 -type immune response.

20. The method according to claim 19 wherein the response comprises production of a) IFN-Y in T cells, and/or b) IL-12 and/or IL18 in antigen-presenting cells

21. A method for inhibition of Th2-type immune response comprising administering to a subject the composition of any of claims 8 to 14 or the food of claim 11 or 16 in an amount effective to partially or completely inhibit development of Th2-type immune response. 23

21. The method according to claim 20 wherein the Th2-type immune response is inhibited by a) induction of IL-10 production in T-cells and/or b) inhibition or suppression of the function of Th2-type T cells, mast cells, eosinophil granulocytes and/or basophil granulocytes.

23

22. The method according to claim 21 wherein the Th2-type immune response is inhibited by a) induction of IL-10 production in T-cells and/or b) inhibition or suppression of the function of Th2-type T cells, mast cells, eosinophil granulocytes and/or basophil granulocytes.