C-type lectin binding molecules, identification and uses thereof

DESCRIPTION

TYPE C LECTIN CONNECTION MOLECULES, IDENTIFICATION AND USE

The invention relates to the field of immunology,

Dendritic cells (DC) are professional antigen presenting cells (APCs) that induce cellular immunity upon recognition of the pathogen. These cells are therefore important in defending against many pathogens.^1;2;3. Immature DCs are distributed throughout peripheral tissues to act as sentinels against invasive pathogens.⁴. Upon capture of the pathogen, DCs are activated, processing the antigen presenting pathogens on the Class II molecules of the Main Histocompatibility Complex secondary lymphoid organs where naif to initiate adaptive immune responses¹'³'’⁴. Depending on the pathogen that is recognized by DC, naif T cell differentiation into Th1 cells is triggered by DC into intracellular microbes, while Th2-mediated ones are generated by DC for extracellularly residing pathogens.³. Thus, DC play an important role in both innate responses and cellular immune responses against tumor antigens as well as pathogens such as viral, bacterial, (MHC) infections and migrate to the T-cell response to eliminate parasitic fungal responses.

1, 5 Knowledge about cell surface receptors in DC that are involved in pathogen recognition is just beginning to emerge, and include Toll-like receptors (TLR)

6; 7 and type C lectins

TLRs recognize specific components derived from pathogens such as lipoproteins, lipopolysaccharides and bacterial DNA, and transmit this information via intracellular signaling cascades that lead to the production of regulatory cytokines and up-regulation of MHC and costimulatory molecules that lead to DC activation / maturation⁶. In contrast, type C lectins recognize carbohydrate structures derived from pathogens and upon binding internalize the pathogens for antigen processing and presentation to T cells.^8-10. In classical calcium-dependent lectins, conserved amino acid residues in the carbohydrate recognition domain (CRD) are involved in calcium binding and sugar specificity.¹¹. An increasing number of type C lectins are described as being specifically expressed by DC. For most of these lectins there is no detailed knowledge about pathogen targets as well as cell ligands, if any, including the identity of the carbohydrate structure they recognize.⁸. DC-specific lectin type C, DC-SIGN (non-dendritic cell-specific ICAM-3 attachment integrin, CD209), is involved in the binding of HIV-1 envelope glycoprotein by DC to improve T-cell infection.¹², while the mannose receptor (MR) is involved in the recognition of mycobacteria and fungus / protozoa. ¹³. Some type C lectins such as DC-SIGN may interact with carbohydrate-bearing glycoproteins (ICAM-2 and ICAM-3) to

14.15 mediate cell adhesion processes

As described herein, a type C lectin may comprise specificity for more than one type of glycoconjugate. This knowledge is useful for a variety of purposes. For example, lectins are instrumental in mediating the binding of pathogens and presenting antigen thereof in dendritic cells. This binding property of Type C lectins is used by a number of pathogens to at least in part facilitate an individual's infection. Therefore, knowledge of the glycoconjugate specificity of such lectins provides an input for the development of medicaments capable of interfering with the ability of the carbohydrates present in the pathogens to interact with lectin and thereby interfering at least in part. , with the infection or its severity in the exposed individual. This disclosure provides a method for inhibiting at least in part binding of a ligand to a type C lectin or a carbohydrate binding moiety thereof, comprising contacting said type C lectin with an isolated and / or recombinant glycoconjugate comprising at least two mannose residues at the α1, 2-bond or a glycoconjugate comprising a fucose residue or a glycoconjugate comprising at least one end having a N-acetylglucosamine or a derivative or multimer thereof. Also provided is the use of a glycoconjugate comprising a fucose residue or a derivative or multimer thereof to inhibit, at least in part, the binding of a ligand to a type C lectin or a carbohydrate binding moiety. same. This method can be used, for example, to study the exact binding properties of lectin. 0 The method is also useful in identifying compounds capable of interfering with an ability of said type C lectin to bind to pathogens. Said method further comprises a cell comprising said type C lectin. Preferably said cell is an antigen presenting cell, more preferably a dendritic or macrophage cell. In the presence of such a cell, the effect of such inhibition may be compared to the function of the antigen presenting cell, for example its ability to present the antigen and / or lymphokine and / or cytokine production. Said type C lectin may comprise DC-SIGN, mSIGNR1, L-SIGN and / or DC-SIGNR. While DC-SIGNR and L-SIGN are expressed predominantly in macrophages / endothelial cells, lymph node cells and sinusoidal endothelial cells in the liver, and DC-SIGN is expressed predominantly in dendritic cells. DC-SIGNR and L-SIGN share the remarkable binding and signaling effects that ManLAM has upon binding to dendritic cells via DC-SIGN. However, while DC-SIGN still includes a particular specificity for glycoconjugates comprising a fucose residue or a derivative or multimer thereof, DC-SIGNR and L-SIGN do not have that particular specificity. Thus, in the above methods there is a preference for DC-SIGN type C lectin.

By the term glycoconjugate comprising a fucose residue is meant a glycoconjugate comprising at least one fucose residue which is attached at 1, 3 or α-1,4 to the glycoconjugate. The binding unit preferably consists of N-acetylglucosamine. The binding unit is preferably coupled to an oligosaccharide or glycan, which in turn may be part of a larger structure (or carrier molecule) comprising subsequent glycoconjugates comprising fucose residues, or other compounds such as mannose in any configuration. . A derivative of said glycoconjugate comprising a fucose residue comprises the same type C lectin binding activity in type but not necessarily in quantity. A derivative may be generated by modifying the fucose residue. Such modifications can be generated in various ways. Preferably, said fucose is a terminal fucose, that is, linked via only a 1, 3 or a 1,4 bond to another molecule or fucose. Preferred examples of glycoconjugates comprising a fucose residue are Lewis Leukocyte blood group antigens.^x, Le^Y, Le^The, Le^Band / or LDNF. Different modifications have different effects on lectin binding properties. Le's sialylation^x (producing sialil-Le^x, a ligand of L-, E- and Pselectin) completely nullifies recognition by DCSIGN, indicating that DC-SIGN has a carbohydrate specificity that is distinct from that of selectins that mediate leukocyte circulation. Sulfation reduces the binding affinity of DC-SIGN to Le^xas well as Le^The(Figure 2a). A glycoconjugate derivative comprising a fucose residue thus comprises a modification wherein said modification allows binding of said glycoconjugate to said type C lectin. Glycoconjugates comprising multiple fucose residues in the configuration of a Lewis blood group antigen typically have a higher binding affinity than a glycoconjugate comprising a single fucose residue.

A glycoconjugate may be mono- or polyvalent for the respective fucose residues. A polyvalent glycoconjugate comprises at least two separate antennas wherein at least two of said antennas comprise said residues. A preferred polyvalent glycoconjugate comprises at least two antennas having a terminal fucose, that is, linked via only an α, β or β 1.4 link to the glycoconjugate or other fucose residue. A preferred polyvalent glycoconjugate comprises at least two antennas having at least two mannose residues at the α 1, 2-bond. A polyvalent glycoconjugate may also comprise at least two antennas having a terminal Nacetylglucosamine, i.e. linked via only one glycoconjugate binding. A multimer is preferably a polyvalent glycoconjugate.

A binding molecule may be specific for a monovalent glycoconjugate or a polyvalent glycoconjugate. The binding of the binding molecule is typically stronger to the polyvalent glycoconjugate.

Typically, the binding of type C lectin and, more particularly, the subsequent action of the type C lectin carrier cell is significantly greater in the case of the binding of a polyvalent glycoconjugate. This is particularly so for glycoconjugates comprising at least one end having an N-acetylglucosamine residue.

Carrier molecules may further comprise a wide variety of different molecules including peptides, proteins, lipids, polysaccharides and also synthetic molecules not expressly mentioned herein. The carrier molecule (s) may of course be part of an even larger structure such as a biological or artificial surface, a virus, a bacterium or a eukaryotic cell.

Binding of ligand to type C lectin may be inhibited by interfering with the ligand binding site, type C lectin binding site or both. Interference with the ligand binding site can be done using a ligand binding molecule capable of specifically binding to the ligand binding portion of the ligand type C. This is preferably done with a protein molecule such as a carbohydrate-specific antibody, preferably specific for a glycoconjugate comprising at least two α, 2-linked mannose residues or a glycoconjugate comprising a fucose residue or a glycoconjugate comprising at least one end having Nacetylglucosamine or a derivative or multimer thereof. The experimental part describes examples of such antibodies. Examples of antibodies comprising a specificity for a glycoconjugate comprising a fucose residue or a derivative or multimer thereof are SMLDN1.1 or SMFG4.1, 6H3, Hpl51, 4D2, 54,1F6A, NAM61-1A2 or SMLDN1.1 . The ligand binding molecule may also be a type C lectin, preferably a soluble derivative thereof, comprising specificity for a glycoconjugate comprising a fucose residue, derivative or multimer thereof. An example of a soluble type C lectin of the invention is a soluble DC-SIGN-Fc chimeric molecule comprising amino acid residues 64-404 fused at the C-terminus with an Fc fragment of human IgG1 as described, for example, in Geijtenbeek et al (Geijtenbeek et al 2002, J. Biol. Chem. 277: 11314-11320). However, soluble type C lectins comprising one or more of the provided specificities may be generated in various ways (e.g., by derivation from DC-SIGNR or L-SIGN). Now that the present disclosure describes a chimeric molecule others may be generated, for example, as described in Fawcett J et al (Fawcett J et al 1992, Nature 360: 481-4).

Of course, interference with the binding site on type C lectin can also be done in a number of ways. Interference with the binding site on the type C lectin may be made using a type C lectin binding molecule capable of specifically binding to the ligand binding part on the type C lectin. This is preferably done with a protein molecule such as a type C lectin-specific antibody or a glycoconjugate comprising a fucose residue or a derivative or multimer thereof, wherein said glycoconjugate or both may be part of a structure. bigger. Of course, binding may be further inhibited by sterically preventing ligand binding to type C lectin. For this it is not absolutely necessary for the interfering molecule itself to bind to the carbohydrate and type C lectin association site. The binding may also be interfered by successful spherical impediment by binding the interfering molecule in the vicinity of the association site. .

Ligand may be any type of structure capable of binding said type C lectin. In general, this will comprise a protein molecule or a carbohydrate. The ligand may be an antibody. It is preferred, but not strictly necessary, that the ligand comprises mannosylated glycans such as ManLAM Mycobacterium cell wall components, or a mannose derivative, lipophosphoglycan such as Leishmania derivative, or a glycoconjugate comprising a fucose residue such as SEA or CD66 or a derivative or multimer thereof. 0 A ligand may have, instead of or in addition to, a glycoconjugate comprising a fucose or a derivative or multimer thereof, different glycoconjugates capable of binding thereto to type C lectin.

The ligand preferably comprises an antigen. As mentioned above, the ligand may comprise a glycoconjugate comprising a fucose residue. The antigen comprises or not said glycoconjugate. In a preferred ligand the antigen comprises said glycoconjugate. The ligand preferably comprises a (tumor) antigen, a pathogen and / or a cell associated receptor. An antigen is used to include peptides or glycolipids and derivatives thereof capable of being presented in the context of MHC class I or class II, or CDlb. At least some type C lectins, and particularly those expressed in dendritic cells, are involved in the process of antigen uptake and presentation by antigen presenting cells. An initial step in this process is the capture of antigen by these lectins. It is possible to interfere, at least in part, with this initial step in the antigen presentation process. Given the importance of antigen presentation in initiating and maintaining antigen-specific immune responses, it is apparent that the method can be favorably used to at least partially decrease the potency of an immune response against said antigen.

The ligand may comprise a pathogen or a type C lectin binding moiety thereof. It has been found that many different pathogens comprise carbohydrates capable of binding to type C lectins. In particular, it has been found that pathogens or parts thereof may bind to said type C lectins via a glycoconjugate. mannose-containing such as a glycoconjugate which comprises a fucose or derivative or multimer thereof. This binding facilitates at least in part the infection of an individual by said pathogen. Interference with the binding of the pathogen or part thereof to type C lectin can thus help, at least in part, an individual to combat a pathogen infection and, in certain cases, even prevent clinically establishing symptoms. visible from it. The latter is useful in vaccination strategies (passive or active). 0 pathogen may be a virus, a fungus, a (medical) bacterium and / or a parasite. The pathogen may comprise a glycoconjugate comprising at least two mannose residues in Al 1, 2 binding or a glycoconjugate comprising a fucose residue or a glycoconjugate comprising at least one Nacetylglucosamine end or a derivative or multimer thereof. Examples of pathogens are human immunodeficiency virus, a helicobacter, a neisseria meningitidis, a leishmania, a schistosoma, a Klebsiella, a probiotic lactobacillus, a hepatitis C virus, a herpes simplex virus or an ebola virus. Inhibition of binding of the specifically mentioned pathogens may be accomplished using a glycoconjugate comprising a fucose residue or a derivative or multimer thereof. Inhibition of binding is accomplished using a ligand binding molecule (pathogen or part thereof) capable of specifically binding to the ligand binding type C ligand. This is preferably done with a protein molecule such as a carbohydrate specific antibody, preferably specific for a mannose carbohydrate such as a glycoconjugate comprising a fucose residue or a derivative or multimer thereof. The experimental part describes examples of such antibodies. The ligand binding molecule may also be a type C lectin, preferably a soluble derivative thereof, comprising specificity for a mannose carbohydrate such as a glycoconjugate comprising a fucose or a derivative or multimer thereof. An example of a soluble type C lectin is a soluble DC-SIGN-Fc as mentioned above. Thus, infection of an individual by said pathogens may be at least in part prevented or treated by administering to said individual a mannose carbohydrate, such as a glycoconjugate comprising a fucose residue, derivative or multimer thereof. . Similarly, a ligand binding molecule is administered to said individual. It can also be used to inhibit, at least in part, binding of the pathogen to a cell expressing type C lectins, thereby inhibiting at least in part contamination or spread of the pathogen in a patient's body. further inhibiting DC maturation and cell adhesion, as also described below.

Type C lectins not only recognize carbohydrate profiles in pathogens but also interact with auto-glycoproteins to mediate cellular processes such as differentiation and migration. Accordingly, a method may also be used to interfere with interaction with one or more auto-glycoproteins and thereby be used to inhibit, at least in part, the cellular processes in which said type C lectins are involved. This can be done by providing the carbohydrate structures as described herein (i.e. mannose containing glycoconjugate, such as a glycoconjugate comprising a fucose residue or derivative or multimer thereof). Preferably, this is accomplished using a ligand binding molecule. Thus, the method is used to inhibit, at least in part, the binding of said type C lectin to an autoglycoprotein preferably a receptor present on the outer membrane of a cell. Preferably said receptor comprises ICAM-2, ICAM-3, CD11b, CD166 or CD66 or a functional, derivative and / or analog moiety thereof. CD166 and CD66 are present in a subset of NK cells and granulocytes. Within the subset of CD66 variants, the receptor preferably comprises CD66a and / or CD66e. Thus, the interaction of these cells with a cell comprising a mentioned type C lectin can be at least in part inhibited using a binding partner specific for a type C lectin mentioned above. Thus, the interaction of preferably dendritic cells and said subset of NK cells and granulocytes may be subject to interference. Through this interaction it is possible to modify an immune response. For example, by inhibiting at least in part the binding of a granulocyte to a dendritic cell by providing type C lectin, it is possible to inhibit, at least in part, the assembly of an effective immune response against antigens by the granulocytes. . On the other hand, it is possible to enhance or induce an effective antigen-specific immune response by providing the antigen presenting cell with a ligand comprising said antigen and a glycoconjugate comprising at least two mannose residues, a fucose residue and / or an end having N-acetylglucosamine. Preferably, said type C lectin comprises DC-SIGN. As a specific binding partner of said type C lectin, a mannose-containing glycoconjugate such as a glycoconjugate comprising a fucose residue or a derivative or multimer thereof may advantageously be used.

The invention thus relates to the use of a glycoconjugate comprising a tumor antigen and a non-sialylated Lewis X antigen capable of binding to DC-SIGN in an antigen presenting cell for the preparation of a composition for stimulating an immune response against the antigen. tumor in an individual. In a preferred embodiment, the tumor antigen is a peptide or glycolipid capable of being presented in the context of class I or class II MHC or Cl b.

In other words, the invention relates to a composition for use in stimulating an immune response against a tumor antigen in an individual, wherein said composition comprises a tumor antigen glycoconjugate and a non-sialylated Lewis X antigen capable of binding. DC-SIGN in an antigen presenting cell. In a preferred embodiment, the tumor antigen is a peptide or glycolipid capable of being presented in the context of class I or class II MHC or Clb.

WO 97/27872 discloses that an antigen comprising the Lewis X antigen has a stimulatory effect on the immune response of S. mansoni infected mice. WO 97/27872 relates to a method for modulating an immune response. The method comprises contacting an immune cell with a stimulating or inhibiting form of a Lewis antigen. 0 The effect of this contact is that the immune cell produces immunostimulatory molecules that enhance an immune response. WO 97/27872 details stimulation of IL-10 production by splenocytes in S. mansoni infected rats. More particularly, WO97 / 27872 describes conjugates between LNFP-III (comprising Lewis X) and human serum albumin (HSA). These conjugates were tested for their ability to stimulate PBMCs from humans suffering from lung and colon cancer. Thus, D5 teaches that the Lewis X antigen is to be conjugated to HSA, which is not a tumor antigen.

The cellular interaction of DC with granulocytes or NK cells is essential in the innate immune response. In particular, granulocytes are involved in the onset of pathogen ingestion and may attract and stimulate DC to participate in pathogen recognition and presentation to T cells. In contrast, NK cells are well known to be involved in cell death. infected. NK cells can kill or activate DCs that allow lysis of DC captured by the pathogen or maturation of DC captured by the pathogen to improve T cell stimulation and immune response. The cellular interaction of granulocytes and NK cells with DC is to improve immunity against pathogens or eliminate pathogen-infected DC.

This disclosure also provides a method for inhibiting, at least in part, the maturation or activation of an antigen presenting cell comprising providing said antigen presenting cell with a DC-SIGN binding glycoconjugate comprising a fucose residue. Using this technique it is possible, for example, to decrease an immune response. Tolerance to antigens may still be induced by this method, for example the antigen may be provided together with the glycoconjugate. In case the antigen presenting cell is activated via a Toll-type receptor pathway, the balance between the activation / inhibition level determines the maturation or activation of the antigen presenting cell. When the TLR pathway is strongly activated at the same time as the type C lectin pathway is not, equilibrium is shifted to activation of the antigen presenting cell while in the reverse situation equilibrium is shifted for toleration and inhibition of maturation or activation.

Human immunodeficiency virus binds to DC-SIGN via a glycoconjugate having at least two mannose residues. Helicobacter, and in particular helicobacter pylori, binds to DC-SIGN via a glycoconjugate having a Lewis antigen.^x. THE

Neisseria meningitidis binds to DC-SIGN via a glycoconjugate having an N-acetylglucosamine residue. Mexican Leishmania binds to DC-SIGN via a glycoconjugate having at least two mannose residues. Schistosoma mansoni binds to DC-SIGN via a glycoconjugate having a Lewis antigen. Klebsiella pneumonea binds to DC-SIGN via a glycoconjugate having at least two mannose residues. 0 hepatitis C virus binds to DC-SIGN via a glycoconjugate having at least two mannose residues. The herpes simplex virus binds to DC-SIGN via a glycoconjugate having at least two mannose residues. The Ebola virus binds to DC-SIGN via a glycoconjugate having at least two mannose residues.

Thus, to alter the immune response in a body against the mentioned pathogens, a binding molecule preferably inhibits, at least in part, the binding of its glycoconjugates to type C lectin.

Immature DCs are highly effective in antigen capture and processing, while mature DCs are specialized in activating the naive T cells required for cellular immune responses. Immature DC such as bacterial (LPS) or inflammatory (TNFα) components mature in response to specific cytokine 'danger' signs

PGE2). It has been found that a glycoconjugate comprising a mannose, a fucose residue or a derivative, a combination or a multimer thereof, does not induce DC maturation. In contrast to the LPS that triggers TLR4, no positively regulated expression of activation markers CD80, CD83, CD86 or HLA-DR was observed. However, when the maturation of immature dendritic cells was studied in the situation where the cells are also activated via a Toll-type receptor pathway, a strong inhibition of maturation induced by particularly when exposed to ManLAM was observed. Thus, receptor dendritic type cells

Toll, such an inhibition decreases Toll-type receptor pathway-stimulated dendritic cell response and thus the ability of an individual to cope with pathogens that are counteracted by activation of the Toll-type receptor pathway. In particular, this reduction in DC activation can be at least in part prevented by inhibiting, at least in part, the binding of a glycoconjugate comprising at least two mannose residues in α 1, 2 binding, but also analogously acting glycoconjugates. for type C lectins on immature dendritic cells. Thus, this disclosure provides a method for determining whether a compound is capable of modulating an activation state of a dendritic cell comprising providing said dendritic cell with a compound capable of specifically binding a type C lectin and determining whether it is modulated. a Toll-type receptor signaling pathway in said dendritic cell. Further provided is a method for modulating the activity of a Toll-like signaling pathway in a cell, wherein said cell comprises a Toll-like receptor and a C-type lectin, wherein said method comprises contacting a Toll-like receptor. said cell having an isolated and / or recombinant type C lectin binding molecule. Preferably, said type C binding molecule comprises glycoconjugate comprising a mannose, a fucose residue or a derivative, a combination or multimer thereof. Activity can be positively modulated by preventing, at least in part, simultaneous stimulation of type C lectin with a glycoconjugate comprising

at least two mannose residues in al? 2 bonding or analogously acting compound. The activity is negatively modulated by simultaneously providing said glycoconjugate comprising at least two mannose residues in α1, 2-binding or analogously acting compound. Analogously acting compounds comprise the same Toll-type signaling interference capability as ManLAM in type, not necessarily in quantity. A preferred analogously acting compound comprises a glycoconjugate comprising a fucose residue or a derivative or multimer thereof. Another level of control is possible using type C lectin binding compounds that interfere, at least in part, with the binding capacity of a glycoconjugate comprising at least two α1-binding mannose residues or a compound which acts analogously. Thus, the activity of the Toll-type receptor pathway can be positively modulated (in the presence of the appropriate Toll-type receptor ligand) even in the presence of a glycoconjugate comprising at least two mannose residues in al, 2-bonding or compound which acts similarly. The latter feature is important in cases where the immune system has problems fighting an infection via the Toll-type receptor pathway, particularly when said infection is accompanied by secretion of a glycoconjugate comprising at least two mannose residues in connection. al, 2 or analogously acting compounds such as mycobacteria and in particular M. tuberculosis and M. Bovis. Thus, this disclosure further provides a method for stimulating the maturation of a dendritic cell that is contacted with a Toll-like receptor ligand and a glycoconjugate comprising at least two α, 2-linked mannose residues or compound which acts as a dye. analogous form, wherein said method comprises providing said dendritic cell with a type C lectin binding molecule capable of blocking the binding of said glycoconjugate to said type C lectin. Type C lectin binding molecules capable of inhibiting, at least in part, the binding of a glycoconjugate comprising at least two α, 2-linked mannose residues or analogously acting compounds are, for example, antibodies. C-type lectin binding moiety or a derivative and / or functional analog part thereof having a binding specificity that blocks or encompasses the ManLAM binding site in said C-type lectin. Preferably, said type C lectin comprises DC-SIGN. Said antibody may be a specific antibody against DC-SIGN. A suitable example of such an antibody is AZN-D1, AZN-D2 or AZN-D3 or a human or humanized analog comprising the same binding specificity in type, not necessarily in quantity. This disclosure also provides a method for stimulating the maturation of a dendritic cell that is contacted with a Toll-like receptor ligand and a glycoconjugate comprising at least two α, 2-bonded mannose residues or compound acting on the other. analogously, wherein said method comprises providing said dendritic cell with a ligand-binding molecule thereby preventing at least in part binding said glycoconjugate comprising at least two mannose residues in Î ± 1, 2-binding, or analogously acting compound, to the dendritic cell.

Also provided herein is the use of a glycoconjugate comprising a fucose residue or a derivative or multimer thereof, a ligand binding molecule and / or a type C lectin binding molecule for the preparation of a medicament. Such medicines may be used for the treatment of an immune system-associated disease or for the treatment of an acquired disease. Preferably, said acquired disease comprises a human immunodeficiency virus infection, a Helicobacter, a Meningitidis neisseria, a leishmania, a schistosoma, a Klebsiella, a probiotic lactobacillus, a hepatitis C virus, a herpes simplex virus or an ebola virus.

Considering the natural role of type C lectins in antigen presenting cells, it is within the scope of the present disclosure to stimulate immune responses in an individual by providing the antigen via a type C lectin receptor in the antigen presenting cell. Of course, this can be achieved when the antigen comprises a glycoconjugate comprising a fucose residue or derivative or multimer thereof. In this method it is preferred that simultaneous activation of the Toll-type signaling pathway is at least partially prevented. Thus, a glycoconjugate comprising an antigen and a glycoconjugate comprising a fucose residue or a derivative or multimer thereof for use in preparing a vaccine is provided. The vaccine may be prophylactic or curative. 0 Antigen may be derived from any source as long as it is capable of being presented through the major histocompatibility complex I, complex II or Clb. In a preferred embodiment said antigen comprises a tumor antigen. The presence of the mentioned carbohydrates in the tumor cells facilitates antigen capture by DC to improve antigen presentation and, as a consequence, immune activation. On the other hand, the immune response against a tumor antigen lacking the mentioned carbohydrates can be significantly stimulated by providing the antigen with one or more of the mentioned carbohydrates thereby stimulating uptake by DC and thus the immune response against the antigen. This is useful in preparing vaccines.

DC-SIGN has been shown to have a carbohydrate specificity for mannose-containing carbohydrates, Lewis antigens and GlcNAc-containing structures (Figure 33, 34, 63-66). We have shown that the carbohydrate specificity of L-SIGN is different as it does not recognize Lewis antigen carbohydrate structures while recognizing structures with high mannose contents (Figures 33 and 34). The murine homologue mSIGNR1 similarly recognizes DC-SIGN antigens with high mannose and Lewis contents, but also has specificity for sialylated Lewis antigens in contrast to DC-SIGN and L-SIGN (Figures 33 and 34).

DC-SIGN is a particular type C lectin highly expressed in dendritic cells. Its function is to recognize the antigen and process it and present it very efficiently in MHC class I and II molecules.

DC-SIGN has long been believed to be a pathogen recognition receptor that recognizes pathogens to activate the immune system, but as it now appears, the long list of pathogens targeting DC-SIGN persist. and escape immunity through different mechanisms. We now have four examples of pathogens that target DC-SIGN to survive in the host.

1. HIV-1 and HCV target DC-SIGN to 'hide' within DC and escape intracellular routing to the lysosomal compartment.

2. ManLam secretion by mycobacteria targets DC-SIGN to induce negative modulation of TLR-induced DC maturation, and IL-10 induction.

3 Helicobacter pylori human gastric pathogen persists: lipopolysaccharide phase variants modulate Th1 / Th2 equilibrium through interaction with dendritic cell lectin DCSIGN.

4 Lactobacilli target DC-SIGN in DC and induce regulatory T cells, inhibition of lactobacilli recognition by DC-SIGN reduces the regulatory state of T cells.

DC-SIGN is thus a receptor that 'normally' recognizes the autoantigen to be tolerated (Annual Review Immunology, 2004). We have demonstrated here that, in fact, targeting lactobacilli to DC-SIGN can induce regulatory T cells. Furthermore, it appears that DC-SIGN may also recognize the CEA tumor antigen (CD66e) in particular Lewis X and Y. These tumor antigens can be secreted and suppress DC activation in a manner similar to ManLam's tuberculosis mycobacteria. The interaction of DC with granulocytes is elucidated by the fact that DC-SIGN recognizes CD66a and CD11b in granulocytes. In particular, the Lewis X antigen is recognized in these molecules. Thus, DC-SIGN acts as a cell adhesion receptor that mediates granulocyte cell interactions with DC. This cellular adhesive function between granulocytes and dendritic cells has never been described but it is likely that granulocyte-DC interaction is essential for bridging the innate immune response with the adaptive immune response such that the granulocyte passes infectious agents to assembly. by DC of an adequate immune response. In particular, granulocyte activates and matures DC, inducing up-regulation of cytokines and costimulatory molecules that are required for DC migration and initiation of the adaptive immune response. The interaction of CD with granulocytes is also observed in vivo for Crohns disease. A new pathogen that interacts with DC-SIGN in DC is Neisseria Meningitidis. Strain variants demonstrate that, in particular, an IgtB strain mutant interacts that contains a GlcNAc residue at the end position. Other experiments (Figure 66) demonstrate that DC-SIGN also recognizes GlcNac.

This disclosure also provides for the use of a ligand binding molecule and / or a type C lectin binding molecule for the preparation of a vaccine. A decrease in the immune system due to the effect of a glycoconjugate comprising at least two α, 2-linked mannose residues or analogously acting compounds is reduced by providing a glycoconjugate or antibody capable of inhibiting at least in part the binding of a glycoconjugate comprising at least two mannose residues in Al 1, 2 bond, or compound acting analogously to its type C lectin receptor. Preferably, the antibody comprises SMLDN1.1, SMFG4.1, 6H3 or SMLDN1.1. Thus, the antigen present in said vaccine or separately provided is most effective in stimulating or enhancing an immune response in the presence of a glycoconjugate comprising at least two α, 2-linked mannose residues or analogously acting compound. This is particularly important in patients suffering from a mycobacterial infection such as, but not limited to, M. tuberculosis or M. bovis. Preferably, said vaccine is used to stimulate an antigen-specific immune response in said individual. In a preferred embodiment, the medicament or vaccine is used for treating a subject suffering from cancer.

As used herein the term antibody refers to antibodies derived from humans or other animals. The antibody is preferably produced outside the body. The antibody may also be generated or selected using artificial systems such as phage display selection. Therefore, an antibody that does not have a natural counterpart is also encompassed by the term. Antibodies as used herein also include fragments thereof capable of binding to the same target, such as FAB fragments or even smaller parts. The antigen binding part of an antibody may also be grafted onto another type of molecule to provide that molecule with binding specificity. Modification of the antibody to include human or humanized versions thereof with the same binding specificity in type, not necessarily in quantity, is of course also possible. Also included are single stranded fragments and variants thereof.

As used herein, the term protein molecule refers to a peptide, a polypeptide, protein and the like with or without modification. Such modifications may be synthetic and / or provided by a biological system. Including the latter, for example, post-translational modification such as glycosylation.

Where the invention is described for use in humans, the invention is also functional in other animals, for example livestock and pets. Therefore, these medical and vaccine uses are also part of the invention.

Brief Description of Drawings

Figure 1. Le^x - neoglycoconjugates that bind with high affinity to DC-SIGN. The. Schematic diagram of the covalent structures of the synthetic glycoconjugate glycan chains used in the DC-SIGN binding assay. b and c. Glycoconjugates were coated and binding of recombinant DCSIGN-Fc was measured after incubation with peroxidase labeled goat anti-human Fc. Anti-DC-SIGN AZN-D1 monoclonal antibody was used to block binding, c. Glycoconjugate titration revealed that DC-SIGN binds with high affinity to Le glycoconjugates^x, Le^Y, Le^The, Le^B or LDNF, and alpha 1.3, alpha 1.6 manotriose, sulfo-Lea, while having a lower binding affinity for alpha-mannose, and alpha-L-fucose, and sialyl-Le^x.

Figure 2. Cellular DC-SIGN has a binding specificity similar to soluble DC-SIGN. Binding of DC-SIGN and DC-expressing K562 transfectants derived from immature monocytes with glycoconjugate coated fluorescent beads was measured by FAC scan analysis. Binding was inhibited by the anti-DC-SIGN monoclonal antibody AZN-D2. A representative experiment of three is shown. 0 SD is less than 2%. Built-in: Photograph of Le-coupled fluorescent beads^x that bind to DC expressing DC-SIGN.

Figure 3. DC-SIGN Binds Four New Pathogens

The. Pathogens consisting of Le-rich Helicobacter pylori and Schistosoma mansoni^x, and Mycobacterium tuberculosis mannose-coated lipoarabinanomanan (manLAM) and Leishmania mexican mannose-coated lipophosphoglycan, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococ cus aureus were coated and DC-S-Fc binding was measured. peroxidase-labeled anti-human goat. B. DC-SIGN can bind the mannose-end Klebsiella Leni LPS but does not bind the Klebsiella Len 111 LPS which lack a mannose end indicating that strains containing the DC-SIGN-binding carbohydrates interact with DC-SIGN on DC-SIGN dendritic or transfecting cells.

Figure 4. DCs interact strongly with H. pylori LPS through DC-SIGN. Biotinylated LPS (strains 11637 and M1019) was coated over streptavidin beads and DC adhesion was determined with the fluorescent beads adhesion assay. Specificity was determined using antibodies to DC-SIGN and the mannose receptor. In addition, adhesion was determined in the presence of mannan and EGTA.

Figure 5. DC-SIGN quickly internalizes Le glycans^xfor lysosomal compartments. Le^xSynthetic biotinylated -PAAs were added to K562 cells expressing DC-SIGN or DC derived from immature monocytes for 30 min incubation at 37 ° C. Cells were stained with anti-CD107a (LAMP-1, green) as a lysosomal marker and avidin-Alexa 594 for localization of Le^x after cell permeabilization. Placement of LAMP-1 and Le^x results in a yellow revelation.

Figure 6. DC-SIGN specifically binds ManLAM, a component of the M. tuberculosis cell walls.

a) DC-SIGN interacts with various strains of mycobacteria. DC-SIGN-Fc binding to mycobacteria (5> <10⁵ bacteria) was determined by an Fc specific ELISA. Specificity was determined by measuring binding in the presence of blocking antibodies specific for DCSIGN (AZN-D1 or AZN-D3) and mannan. EGTA was used to determine calcium dependence of DC-SIGN-Fc mediated binding. ICAM-3-Fc binding to mycobacteria was also measured to exclude non-specific binding by the Fc domain. Standard deviation <0.02 OD450. A representative experiment of three is shown. Mycobacterium Smegmatis binding lacking the ManLAM structure does not interact with DC-SIGN (data not shown).

b) The schematic structure of ManLAM. M. tuberculosis lipoarabinomannan (LAM) consists of a glycosylphosphatidyl anchor (GPI), a mannose-rich oligosaccharide nucleus and a branched arabinoside polymer terminating at mannose ends (n = 0-3). AraLAM has a similar structure but does not contain the mannose end (ref).

c) Manosylated mannosylated lipoarabinomannan, in contrast to unmanosylated AraLAM, is specifically bound by DCSIGN. The anti-DC-SIGN AZN-D1 antibody was used to determine specificity. The DCSIGN-Fc binding assay was performed as described in Figure 1a. Standard deviation <0.02 OD450. A representative experiment of three is shown.

d) DC-SIGN does not interact with AraLAM. The DC-SIGN-Fc binding assay was performed as described in Figure 1a. A representative experiment of three is shown.

Figure 7. Cellular DC-SIGN binds strongly to viable mycobacteria and the MyLobacterial component ManLAM through its primary binding site.

a) K562-DC-SIGN transfectants express high levels of DC-SIGN but lack the expression of the other described ManLAM receptors MR, CD11b and CD11c. Transfectants were generated as previously described. Empty histograms represent isotype controls, and solid histograms indicate specific antibody development,

b) DC-SIGN, expressed by K562 transfectants, binds

<td>strongly to the BCG of</td><td>M.</td><td>bovis</td><td>intact and</td><td>to</td><td>component</td>

<td>ManLAM mycobacterial</td><td>but</td><td>no</td><td>to AraLAM.</td><td>THE</td><td>adhesion of</td>

<td>cells to glycans</td><td>LAM</td><td>was</td><td>determined</td><td colspan="2">using the</td>

fluorescent bead adhesion assay. Binding to viable mycobacteria was determined by measuring the binding of K562 transfectants to FITC-conjugated mycobacteria (MOI 20) using flow cytometry. Specificity was determined by measuring binding in the presence of blocking antibodies against DC-SIGN. The standard deviation for the fluorescent bead adhesion assay and the mycobacterial binding assay was &lt;5% and &lt;2%, respectively. A representative experiment of three is shown.

c) The amino acid residue Val351 is not essential for the interaction of DC-SIGN with BCG of M. bovis and ManLAM, similar to HIV-1 gp120, while it is essential for binding of ICAM-3. Binding to the DC-SIGN V351G mutant expressed by K562 cells was measured as described in Figure 2. Specificity was determined by measuring binding in the presence of blocking antibodies against DCSIGN, mannan or EGTA. Standard deviation &lt;5% (fluorescent bead adhesion assay) and &lt;2% (mycobacterial binding assay). A representative experiment of three is shown.

d) The connection of DC-SIGN to ManLAM differential components. Ara-Man, Ara-Man2, Ara-Man3 or Ara6 neoglycoproteins were coated on fluorescent beads and added to cells expressing DC-SIGN. DC-SIGN preferentially binds Man2 and Man3 as demonstrated by the strong binding that can be blocked by the addition of anti-DC-SIGN antibodies (AZN-D1).

Figure 8. DC-SIGN is an important receptor for ManLAM and mycobacteria in DC.

a) Immature DCs express high levels of DC-SIGN and the other described LAM receptors MR, CD11b and CD11c. Empty histograms represent isotype control and solid histograms indicate specific antibody development.

b) Immature DCs bind strongly to ManLAM via DCSIGN. Binding was determined using the fluorescent bead adhesion assay. Specificity was determined by measuring binding in the presence of mannan, EGTA or blocking antibodies against DC-SIGN (AZN-D2), MR (Clone 19), CD11b (bear-1) or CD11c (SHCL3). Standard deviation &lt;5%. A representative experiment of three is shown.

c) DC-SIGN mediates the capture of M. bovis BCG by immature DC. Binding was determined by flow cytometry using FITC-conjugated mycobacteria. Specificity was determined by measuring binding in the presence of antibodies against DC-SIGN (AZN-D1, AZN-D2 and AZN-D3), MR (Clone 19), CD11b (bear-1) and CD11c (SHCL3). Binding was also measured in the presence of C-mannan and EGTA lectin inhibitors, while a known MR ligand, mannose-BSA, was used to determine the contribution of the MR receptor. Standard deviation &lt;2%. A representative experiment of three is shown.

d) DC-SIGN mediates the capture and internalization of M. bovis BCG by K562 cells. K562 transfectants were incubated with FITC-conjugated M. bovis BCG (MOI 20). Cells were washed and surface FITC was deactivated by trypan blue exposure. Phagocytosis was determined by comparing FITC labeling before and after deactivation using flow cytometry. Bacteria bound to the surface are represented by empty bars, internalized by filled bars. Standard deviation &lt;4%. A representative experiment of three is shown.

e) Immature DC rapidly phagocytes mycobacteria through DC-SIGN. Internalization was determined as described in Figure 3b. Bacteria attached to the surface

<td colspan="2">are represented by empty bars</td><td>, at</td><td>internalized</td><td>per</td>

<td>bars to full. Detour</td><td>pattern</td><td> &lt;5%.</td><td>Is shown</td><td>an</td>

<td>representative experience</td><td>of three.</td><td></td><td></td><td></td>

<td>Figure 9. The DC-SIGN</td><td>Medea</td><td>The</td><td>internalization</td><td>in</td>

<td>captured mycobacteria and</td><td>ManLAM</td><td></td><td></td><td></td>

<td colspan="2">(a) M. bovis BCG and ManLAM are</td><td colspan="2">internalized by</td><td>A.D-</td>

SIGN in immature DC and directed to lysosomes. The fate of the captured mycobacteria was followed by analyzing the FITC-conjugated M. bovis pulsed BCG immature DC (MOI 20) for 2 hours using immunofluorescence microscopy (200x magnification). ManLAM was followed by incubating DC with ManLAM (10 mg / mL) for 1 hour. DCSIGN, ManLAM and CD207a / Lamp-1 were developed with AZN-D1, F30.5 and H4A3, respectively. A representative experiment of three is shown.

b) ManLAM induces down-regulation of DC-SIGN, but not MR, CD11b and CD11c. Immature DC were incubated with 15 mg / mL ManLAM or AraLAM for 18 hours and then DC-SIGN expression was determined by flow cytometry. A representative experiment of three is shown.

Figure 10. Mycobacteria induce IL-10 production by DC through ManLAM and direct infection.

a) ManLAM induces the production of LPS-matured DC IL-10. Immature DCs were incubated with 15 mg / mL ManLAM or AraLAM in the presence of LPS (10 ng / mL). Specificity was determined in the presence of blogging antibodies against DC-SIGN (AZN-D2; 20 pg / mL). Supernatants were collected after 18 hours and IL-10 production was measured by ELISA. Values ​​are means ± standard deviations of triplicate determinations. A representative experiment of three is shown.

b) BCG infection of M. bovis of immature DC induces IL-10 production. Immature DCs were infected with M. bovis BCG (MOI 4) and the experiment was performed as described in Figure 5a. Values ​​are means ± standard deviations of triplicate determinations. A representative experiment of three is shown.

Figure 11. ManLAM inhibits LPS-induced DC activation through DC-SIGN binding.

a) ManLAM does not induce the activation of immature DC. Immature DC were incubated with ManLAM, AraLAM or LPS for 18 hours, and activation was determined by measuring expression of CD80, CD86, CD83 and HLA-DR. The dotted lines represent isotype controls, the thin lines indicate immature DC expression levels, and the thick line represents immature DC that have been treated with LPS (10 ng / mL), ManLAM (15 pg / mL) or AraLAM (15 pg / ml). A representative experiment of three is shown.

b) LPS-induced activation of DC is blocked by ManLAM. Immature DCs were co-cultured with LPS alone, or together with ManLAM or AraLAM for 18 hours. Dotted lines represent isotype controls. Thick lines, and mean fluorescence values ​​in histograms, represent expression levels after treatment with LPS alone, or in combination with ManLAM or AraLAM. Fine lines indicate the presence of antibodies to DC-SIGN throughout the incubation. A representative experiment of three is shown.

Figure 12. M. bovis BCG induces maturation and ManLAM inhibits induced DC activation through DCSIGN binding.

a) M. bovis BCG induces the maturation of immature DC. Immature DCs were incubated with viable M. bovis LPS or BCG (MOI 4, 20 and 100) for 18 hours, and activation was determined by measuring expression of CD80, CD86, CD83 and HLADR. The mean fluorescence intensity is plotted. A representative experiment of three is shown.

b) BCG-induced activation of M. bovis is blocked by ManLAM. Immature DC were infected with M. bovis BCG (MOI 4). Cells were preincubated with 15 pg / ml ManLAM or AraLAM and marker expression was measured after 18 hours as described in Figure 7a. Specificity was determined by preincubating cells with blocking antibodies against DC-SIGN (AZN-D2; 20 pg / mL). A representative experiment of three is shown.

Figure 13. S. mansoni SEA carbohydrate antigen structures, monoclonal antibodies (Mabs) that recognize them, and reactivity of these Mabs with SEA. MAbs reactivity was measured by ELISA using coated SEA at a concentration of 1 pg / mL. Antibodies incubated at a concentration of 1 pg / mL all showed a strong reaction with SEA.

Figure 14. Interaction of SEA with immature human DC.

a) Immature human DCs were cultured from monocytes in the presence of GM-CSF and IL-4. DCs express near high levels of DC-SIGN, MR, CD83, CD86, CD80 and HLA-DR as determined by FACS scan analysis.

b) DC-SIGN, expressed by immature DC, shows an SEA binding similar to that of HIV-1 gp120, a previously defined ligand of DC-SIGN. Adhesion to HIV-1 SEA and gp120 was determined using the fluorescent bead adhesion assay. Mannan and anti-DC-SIGN AZND1 MAb (20 pg / mL), but not clone 19 anti-MR MAb, blocked SEA from adhering to DC. A representative experiment of three using anti-LDN antibodies to couple SEA to fluorescent beads is shown. Similar results are obtained using anti-LDN-DF antibodies (not shown).

Figure 15. Binding of DC-SIGN to S. mansoni SEA. DC-SIGN binds as tightly to SEA as HIV-1 gp120, as determined by an ELISA-based assay using soluble DC-SIGN-Fc. The SEA were coated at a concentration of 5 pg / ml and HIV-1 gp120 at a concentration of 1 pg / ml. Specificity was determined by measuring binding in the presence of anti-DC-SIGN blocking antibody, AZN-D1 (20 pg / ml), or EDTA (5 mM).

Figure 16. DC-SIGN Tightly Connects SEA

Binding of soluble DC-SIGN-Fc to different concentrations of SEA was measured by an anti-IgG-Fc ELISA.

Figure 17. DC-SIGN Binds a SEA Subfraction

The SEAs were separated by SDS-PAGE gel electrophoresis on a 12.5% ​​polyacrylamide gel.

a) Silver-coated SEA (15 pg) polyacrylamide gel to detect all proteins

b) Western blot of polyacrylamide gel following SEA SDS-PAGE (2 pg) using soluble DC-SIGN-Fc to detect DC-SIGN binding glycoproteins.

Figure 18. DC-SIGN Binds to Fucosylated SEA and Al-3-Fucosylated Trisaccharide Le^x

a) SEAs were defucosylated by treatment with mild acid or α3,4-fucosidase. Binding of soluble DC-SIGN-Fc to defucosylated SEA (coated at 1 pg / ml) was measured by an anti-IgG-Fc ELISA. The degree of defucosylation and integrity of non-fucosylated glycans following treatments was established using different anti-glycan MAbs, and goat anti-mouse peroxidase IgM for detection.

b) Competitive inhibition of binding of soluble DC-SIGNFc to HIV SEA and gp120 by anti-glycan mAbs in ELISA. Coated antigens were preincubated with antigenic mAbs prior to the addition of DC-SIGNFc. DC-SIGN Fc binding was measured by an anti-IgGFc ELISA. Preferably the antibodies used are SMLDN1.1 or SMFG4.1 (anti-Le^x) or SMLDN1.1 (anti-LDNF) or anti-DC-SIGN (AZN-D1, D2, AZND3),

c) DC-SIGN is highly bound to neoglycoproteins which have α, 3-fucosylated oligosaccharides Le^x and LDN-F but weakly to neoglycoconjugates having a single α-linked fucose or to LDN-DF. The neoglycoproteins were coated at a concentration of 5 pg / ml. No binding was observed to neoglycoproteins having Galbl, 4GlcNAc or GalNAcbl, 4GlcNAc (LDN) (not shown).

Figure 19. The binding of SEA and Le^x to mutant DC-SIGN and L-SIGN

a) Alignment of the amino acid sequence of part of the DC-SIGN CRDs (AAK20997) with that of the highly homologous L-SIGN type C lectin (AAK20998). The positions of mutations E324A, E347Q and V351G in DC-SIGN are indicated by an arrow.

(b) The bonding of SEA and Le coated beads^x-PAA to wild-type DC-SIGN expressing K562 transfectants and DC-SIGN mutants E324A, E347Q and V351G were measured using the fluorescent bead adhesion assay. To detect SEA, fluorescent beads coupled to the MAb LDN-DF were used. A representative experiment of two is shown.

c) SEA blocks binding of immature human DC-expressed DC-SIGN to HIV-1 ICAM-3 and gp120. In contrast, RNAseB does not block this binding. The adhesion of ICAM-3 and HIV-1 gp120 to DC was determined using the fluorescent bead adhesion assay. Inhibitors were present at a concentration of 20 pg / mL.

d) DC-SIGN, expressed by K562 transfectants, strongly binds to HIV-1 SEA and gp120, while L-SIGN, expressed by K562 transfectants only recognizes HIV-1 gp120. Adhesion was determined using the fluorescent bead adhesion assay. To detect SEA, anti-LDN and anti-LDN-DF coated fluorescent beads were used with similar results. A representative experiment of two using anti-LDN-DF fluorescent beads is shown.

e) K562 transfectants efficiently express DCSIGN, L-SIGN and different mutant DC-SIGNs as determined by FACS scan analysis

Figure 20. Granulocytes interact strongly with DC-SIGN coated beads, partially via ligands containing Le^x.

A. Both mannose and Lewis-X epitopes are highly expressed in granulocyte-expressed glycans.

B. DC-SIGN is highly bound to freshly isolated granulocytes. Freshly isolated granulocytes are incubated with DC-SIGN-Fc coated fluorescent beads. Adhesion is determined by the fluorescent bead adhesion assay. Specificity is determined by measuring adhesion in the presence of mannan, EGTA and antibodies against DCSIGN (AZN-D1, D2 or D3), integrins b2 (NKI-L19) and Le^x (6H3)

Figure 21. DC-SIGN-Fc has a high affinity for granulocytes.

The interaction of DC-SIGN with granulocytes is further investigated by titrating DC-SIGN-Fc and determining binding by measuring bound DC-SIGN-Fc using FITC-conjugated goat anti-human Fc antibodies. Interaction is blocked by antibodies against DC-SIGN (AZN-D1), and mannan and EGTA (A) and by anti-Le^x (3H3, B).

Figure 22. A 66 kDa protein (CD66) is the high affinity ligand of DC-SIGN on granulocytes.

A. Granulocytes are surface labeled with biotin. Cell lysate is incubated overnight at 4 ° C with DC-SIGN-Fc-coated Protein A beads or control antibodies against ICAM-2, ICAM-3 and LFA-1. The are washed and the product on immunoprecipitated SDS-PAGE and beads analyzed is visualized by autoradiography. In particular, a 66kD-sized protein is immunoprecipitated. B. Granulocytes highly express Le66-containing CD66acd and CD66d antigens^x, do not express DC-SIGN ICAM-2 ligand and express ICAM-3.

C. Immunoprecipitation of CD66acd demonstrates strong binding activity for DC-SIGNFc. Anti-CD66acd MAbs coated on an ELISA plate were incubated with a granulocyte lysate. After washing off non-specific proteins, DC-SIGNFc was incubated to demonstrate high affinity ligands. DC-SIGNFc does not recognize granulocyte ICAM-3 by demonstrating that in granulocytes it is not ICAM-3 but CD66acd which is the high affinity ligand for DC-SIGN. CD66acd binding to DC-SIGNFc is completely inhibited by cation removal by EDTA.

Figure 23. Cellular DC-SIGN binds strongly to granulocyte specific protein CD66a.

293T cells are transfected with pIG-CD66a-Fc, pIG-ICAM-3Fc and pIG (simulated). Supernatants are collected and beads are coated with Fc chimeras (CD66a-Fc, ICAM-3-Fc and simulated). Immature DC and DC-SIGN transfected K562 cells are incubated with beads and adhesion is determined in the presence of antibodies against DCSIGN and β2 integrins, and mannan and EGTA.

Figure 24. NK cells interact specifically with DC-SIGN-Fc.

Binding of DC-SIGN-Fc to peripheral blood lymphocyte (PBL) subsets is determined by flow cytometry in the presence of antibodies to DC-SIGN. PBL subsets are distinguished by CD3 and CD56 staining, and adhesion is determined by triple staining with DCSIGN-Fc.

Figure 25. NK cells interact specifically with DCSIGN-Fc in a concentration dependent manner. Binding of DC-SIGN-Fc to peripheral blood lymphocyte (PBL) subsets is determined by flow cytometry in the presence of antibodies to DC-SIGN. NK cells are detected by triple staining with CD3, CD56 and DCSIGN-Fc. CD56 dark and bright cells do not differ in the expression of ICAM-2 or ICAM-3 demonstrating that differential glycosylation is the result of the high affinity binding of DC-SIGNFc to CD56-darkened NK cells.

Figure 26. 0 NK CD5 subset 6^obscuredCDl 6⁺ interacts specifically with DC-SIGN-Fc. Binding of DC-SIGN-Fc to NK subsets is determined by flow cytometry in the presence of antibodies to DC-SIGN. NK subsets are distinguished by CD16 and CD56 staining, and adhesion is determined by triple staining with DC-SIGN-Fc.

Figure 27. A 166 kDa protein and ICAM-2 is recognized by DC-SIGN-Fc in NK cells.

NK cells are surface labeled with radioactive iodine. Cell lysate is incubated overnight at 4 ° C with DCSIGN-Fc coated Protein A beads or control antibodies against ICAM-2, ICAM-3 and LFA-1. The beads are washed and the immunoprecipitated product is analyzed on SDS-PAGE and visualized by autoradiography.

Figure 28. DC maturation by NK cells is mediated by LFA-1.

Immature DCs are co-cultured with NK cells for 24 hours in the presence of antibodies against DC-SIGN (AZN-D1) and LFA-1 (NKI-L19). Maturation is determined by measuring the expression of CD80, CD83, CD86 and HLA-DR. with NK 0 / N cells at 37 ° C. As a control, DCs are matured with LPS (10 ng / mL).

Figure 29. A

<td colspan="2">dependent on</td><td rowspan="2">DC-SIGN. Chromium⁵²</td>

<td>marked</td><td>with</td>

<td>enabled</td><td>per</td><td>IL-2 or</td>

<td>presence</td><td>in</td><td>antibody</td>

<td>(NKI-L19)</td><td>. THE</td><td>lise is c</td>

NK-mediated lysis of immature DC and immature or LPS-matured DC are co-cultured with non-activated NK cells for 4 hours against DC-SIGN (AZN-D1) and LFA-1 determined by measuring the release of

Figure 30. NK-mediated lysis of K562 transfectants is enhanced by DC-SIGN. K562 cells marked with Chromium⁵²they are co-cultured with IL-2-activated or non-activated NK cells for 4 hours in the presence of antibodies to DC-SIGN. Lysis is determined by measuring Cr release⁵².

Figure 31. DC-SIGN has a high affinity for HSV-1 and HSV-2.

HSV is coated on ELISA plates and binding of DC-SIGN-Fc is determined in the presence of antibodies to DC-SIGN.

Figure 32. DC-SIGN tightly binds gV envelope glycoprotein HSB.

A. DC-SIGN transfectants are incubated with HSV envelope glycoprotein-coated beads and DC-SIGN binding is determined in the presence of antibodies to DC-SIGN.

B. L-SIGN tightly binds HSV envelope glycoprotein gB.

L-SIGN transfectants are incubated with HCV envelope glycoprotein-coated beads and DC-SIGN binding is determined in the presence of antibodies to DC-SIGN.

Figure 33A. MSIGNR1 binds mannose-containing carbohydrates, similar to DC-SIGN and L-SIGN. K562 cells expressing human DC-SIGN, human L-SIGN or murine mSIGNR1 bind mannose-containing carbohydrates to a similar degree using the fluorescent bead adhesion assay.

Figure 33B. DC-SIGN, L-SIGN and mSIGNR1 bind differently to Lewis antigens. Transfectants expressing human DC-SIGN, human L-SIGN, or murine mSIGNR1 bind the Lewis antigen coupled to fluorescent beads to a similar degree. DC-SIGN and mSIGNRl, in contrast to L-SIGN, recognize the Lewis X antigen.

Murine mSIGNR1 interacts with sialyl-Lewis X in contrast to human DC-SIGN and L-SIGN.

Figure 33C The carbohydrate specificity of DCSIGN, L-SIGN and mSIGNR1 is different. Lewis X antigen is not recognized by L-SIGN, whereas human DC-SIGN and mSIGNRI recognize it. MSIGNR1 recognizes sialyl-Lewis X in contrast to DC-SIGN and L-SIGN. Both human DC-SIGN and murine mSIGN1 bind sialyl-Lewis A, in contrast to L-SIGN.

Figure 34. DC-SIGN, L-SIGN, and mSIGNR1 bind to yeast-derived mannan, hepatitis C virus E1 / E2, HIV-1 gp120, and mycobacterial ManLAM. Pathogenic ligands were coated onto fluorescent beads and adhesion was measured using the K562 transfected fluorescent beads adhesion assay. Binding of Gp120 to DC-SIGN, L-SIGN and mSIGNR1 was inhibited using yeast derived mannan carbohydrate, demonstrating that these type C lectins also bind yeast derived mannan.

Figure 35. DC-SIGN specifically binds HCV envelope proteins.

A. DC-SIGN binds HCV envelope proteins produced in Hansenula yeast and mammalian cells. The interaction of DC-SIGN with HCV, EI and E2 envelope proteins (2 mg / mL) produced by Hansenula yeast or recombinant vaccinia virus infected mammalian cells was tested in an Fc-based ELISA. Binding specificity was confirmed using DC-SIGN-specific blocking antibody, AZN-D1 (50 mg / mL), mannan (100 mg / mL) or EGTA calcium chelator (5 mM).

B. The interaction of DC-SIGN with HCV EI and E2 is concentration dependent. The EI and E2 produced in mammalian cells were titrated (0-16 or 14 mM resp.) And DC-SIGN-Fc binding was tested as described above. AZN-D1 (50 mg / mL) was used to specifically blog the interaction.

C. DC-SIGN has similar affinity for E1, E2 and gp120. E1 and E2 produced in mammalian (10 nM) and gp120 (2 nM) cells were coated and DC-SIGN-Fc binding was tested as described above. Mannan was titrated (01000 mg / mL) to blog the interaction. The link was expressed as a percentage of the link without blogging.

D. The binding of DC-SIGN to E1, E2 and gp120 is equally calcium dependent. The E1 and E2 produced in mammalian (10 nM) and gp120 (2 nM) cells were coated and DC-SIGN-Fc binding was tested as described above in the presence of a variable amount of calcium (0-5 mM). Binding was expressed as a percentage of binding with 5 mM calcium.

Figure 36. HCV envelope proteins are bound by cellular DC-SIGN and L-SIGN. A. K562 transfectants express similar levels of DC-SIGN and L-SIGN. B. Cellular DCSIGN and L-SIGN bind to both envelope glycoproteins, E1 and E2. DC-SIGN and L-SIGN transfected K562 cells were used to measure HCV envelope protein binding with the fluorescent coated bead adhesion assay. Ç. The DC-SIGN Val351 mutant binds to both HCV envelope proteins, E1 and E2. Binding of DC-SIGN V351G to E1 and E2 glycoprotein-coated beads was investigated and specificity was determined by blogging interaction with the DC-SIGN AZN-D2-specific antibody or EGTA calcium ion gouge. D. Mutagenic analysis showed that similar amino acids within the CRD region of DC-SIGN are involved in the recognition of HCV and HIV-1 and that they are also involved in binding of ICAM-3.

Figure 37. The DC-SIGN internalization pathway is cell-base dependent. A. DC-SIGN and L-SIGN expressed by K562 and THP-1 transfectants similarly bind HCV VLPs. Binding of HCV E1 / E2 VLP to K562 and THP-1 transfectants with DC-SIGN and L-SIGN was measured using the fluorescent bead adhesion assay. BE. 0 DC-SIGN-bound HCV is directed to early endosomes (transferrinat) in THP-1 cells, in contrast to lysosomal targeting (LAMP-1 +) in K562 cells. K-562 (B and C) and THP-1 (D and E) cells expressing DC-SIGN or L-SIGN were incubated overnight with HCV VLPs. HCV was detected using an anti-human HCV antibody and a fluorescent labeled secondary antibody (FITC). 0 Intracellular targeting was determined by staining the rat antibody endosomal compartments with a specific antibody secondary LAMP-1 or

fluorescently labeled (Alexa Fluor 594) lysosomal / endosomal endosomal (B, D), or coinciding cells for 15 minutes with Alexa Fluor 594-labeled transferrin which is specifically transported to the early endosomes and functions as a marker (C, E). The fluorescence microscopy cells.

were analyzed by

Figure 38. Dendritic cells bind tightly to HCV EI and E2 by DC-SIGN. A Immature DCs express high levels of DC-SIGN. Monocyte-derived dendritic cells were isolated as described in materials and methods. DC-SIGN expression was measured by FACS staining with the DCSIGN-specific antibody AZN-D2. B, C Immature DC and mature DC strongly bind to HCV, EI and E2 envelope proteins, and VLPs

HCV Ε1 / Ε2 mixed via DC-SIGN. Binding of immature DC (B) and LPS-matured DC (C) to HCV envelope proteins was determined by a fluorescent bead adhesion assay. Specificity was determined by anti-DC-SIGN antibody, AZN-D2 (50 mg / mL), mannan (100 mg / mL), EGTA (10 mM), and anti-mannose receptor antibody (clone 19) (50 mg / ml).

Figure 39. DC-SIGN in iDC directs HCV VLPs to early endosomes. Immature dendritic cells were incubated with HCV VLPs (30 mg / mL) for 4 hours or overnight. HCV was detected with a human anti-HCV antibody and a FITC-labeled secondary antibody. Intracellular targeting was determined by revealing the endosomal compartments with a mouse antibody to late lysosomal / endosomal specific LAMP-1 or the early endosome specific marker EEA1 and a Alexa Fluor 594-labeled secondary antibody, or co-incubating the cells during 15 minutes with Alexa Fluor 594-labeled transferrin which is specifically transported to early endosomes. B Immature dendritic cells were incubated with HCV VLPs (30 mg / mL) for 4 hours or overnight at 37 or 4 ° C. HCV was detected as described for Figure 5A. The location of DC-SIGN was determined with the DC-SIGN specific antibody, AZN-D2, and an Alexa Fluor 594-labeled secondary antibody.

Figure 40. DC-SIGN in iDCs directs its Lewis X antigen ligand to late endosomes / lysosomes. Immature dendritic cells were incubated with Lewis X (10 mg / mL) for 4 hours. Intracellular targeting was determined by staining the endosomal compartments with a mouse antibody to late lysosomal / endosomal specific LAMP-1 or co-incubating the cells for 15 minutes with Alexa Fluor 594-labeled transferrin which is specifically transported to the early endosomes. The cells were analyzed by fluorescence microscopy.

Figure 41. HCV interacts with L-SIGN-expressing human liver sinusoidal endothelial cells (LSECs) in situ. (A) L-SIGN is expressed by human LSECs as determined by staining liver tissue with a specific antibody against L-SIGN. (B) Binding of HCV VLPs by liver tissue was determined by incubating liver sections with HCV VLPs for 2 hours at 37 ° C. HCV binding was detected using an anti-HCV mouse antibody and Alexa Fluor 594 labeled secondary antibody. (C) Binding of HCV VLP to LSECs was blocked by the calcium chelator EGTA.

Figure 42. Blood group Lewis antigens and some of their related structures bind to DCSIGN. (a) Blood group Lewis antigens expressed by H. pylori. (bc) Carbohydrates representing blood group antigens or their substructures conjugated to polyacrylamide (b) or ceramide (c) were coated and binding of recombinant DC-SIGN-Fc was measured using the DC-SIGN ELISA. -Fc.

Figure 43. H. pylori binding is dependent on Lewis antigen expression. (ac) H. pylori α3-fucosyltransferase mutants (a), strains (b) and phase variants (c) were coated and binding of recombinant DC-SIGN-Fc was measured after incubation with labeled goat anti-human Fc with peroxidase. H. pylori were coated and incubated with antibodies specific for Lewis antigens as indicated and their serotype determined after incubation with peroxidase labeled anti-mouse immunoglobulins.

Figure 44. Phase variation of LPS in H. pylori occurs in vivo.

(a) J223 Lex / y positive phase variants were detected by transfer of colonies with specific mAbs to the indicated Lewis antigens after brief culture directly from the biopsy, followed by a single liquid phase passage and distribution over medium. solid, (b) Segment C sequencing was performed to determine the on and off state of the futA (HP0379) and futB (HP0651) genes. 0 mutant J223.3 futB was generated by natural transformation with a construct containing a chloramphenicol resistance marker cassette inserted into the HP0651 gene, and serotyped as indicated for J223. (c) Consequences of segment C length for the functional expression of futA and futB.

Figure 45. DC-SIGN is expressed in gastric DC and is the major receptor for Le positive H. pylori.

(a) A section of stomach tissue was fixed and stained with anti-DC-SIGN antibodies. Original magnification x 20. Arrows indicate DC-SIGN-positive DC-like cells on the slide itself, (bd) monocyte-derived DC (b), RAW-derived monocyte and macrophage DC, or DC-SIGN transfected K562 cells (d) were incubated with FITC-labeled H. pylori J223.3 or J223.8 and binding was analyzed using flow cytometry. In bed, cells were preincubated with anti-DC-SIGN antibodies, mannan, EDTA, or anti-mannose receptor (MR) antibodies.

Figure 46. H. pylori binding induces a DC-SIGN-dependent increase in IL-10 and IL12 cytokine production, but no changes in IL12p70.

(a) DC were incubated with H. pylori J223.3 or J223.8 at a multiplicity of infection (MOI) of 20 in the presence or absence of anti-DC-SIGN antibodies for 1 h, washed and cultured for 20 h . The supernatant was collected and the amount of IL-10 and IL-12p40 was analyzed by ELISA.

(b) Following DC co-culture with H. pylori J223.3 or J223.8, cells were incubated with CD40L transfected J558 fibroblasts in the absence or presence of IFN-γ for 24 h. The supernatant was collected and the amount of IL-12p70 was analyzed by ELISA.

Figure 47. The binding of H. pylori to DC-SIGN induces a sudden change of naif T cells to Th2.

Following preincubation with anti-DC-SIGN antibodies, DCs were incubated with H. pylori J223.3 or J223.8 at an MOI of 10 for 48 h, washed and subsequently co-cultured with highly CD45RA + CD4 + T cells. purified. Quiescent T cells were again stimulated with PMA and ionomycin and IL-4 and IFN-γ were analyzed on a single cell basis by intracellular flow cytometry.

Figure 48: Lactobacilli induce partial maturation of CD. Immature DC (iDC) were generated as described A) Maturation was induced by addition of LPS (100 ng / mL), 107 bacteria, LPS plus MF (rIL-lb (25 ng / mL) and rTNFa (50 ng / mL) )), or MF plus 1,107 bacteria. After 48 h, mature DC (mDC) was collected, washed and (A) surface expression of CD86, HLA-DR and CD83 was analyzed by flow cytometry. (B) Mature DC (2,104 cells / well) were stimulated with rat CD40L-expressing rat fibroblast cells (J558 cells, 2x104 cells / well) to induce IL-12, IL-10 and IL-6 production. After 24 h, supernatants were collected and IL12p70, IL-10 and IL-6 productions were measured by ELISA.

Figure 49: L. reuteri and L. casei induce the development of regulatory T cells by modulating DC function. IDC generation and maturation conditions are described in the caption of figure 16. (A) Mature DC (5x103 cells / well) were co-cultured with naif helper T cells (Th cells) (2x104 cells / well) and SEB superantigen (10 pg / mL). After 12 days, IFN-g and IL-4 yields per cell were analyzed by intracellular FACS staining after 6 h stimulation with PMA / ionomycin, the last 5 h in the presence of mature Brefeldin A. (B) DC ( 2.5x103 cell / well) were co-cultured with naif Th cells (5x104 cells / well). Cell proliferation was assessed by incorporation of radiolabelled [3 H] -TdR after a 13 KBq / well pulse during the last 16 h of culture 6 days after stimulation as measured by liquid scintillation spectroscopy. (C) Naif Th cells were stimulated as described in part A. After 12 days, the test cells were fluorescently labeled with PKH, and stimulated with suboptimal concentrations of anti-CD3 (1: 5000) and anti-CD28 (1). : 2000). After overnight incubation, fluorescent-labeled target T cells (peripheral CD4 + T cells) (CFSE) were added at a 1: 1 ratio (2.5x10 4 each). After 5 days, PKH and CFSE staining of the cells was analyzed by flow cytometry. The grayscale CFSE profile represents the test conditions (bacteria: 105), while overlap indicates proliferation in the presence of control test cells (MF / LPS). The figure is a representative of 7 independent experiments. (D) The mean fluorescence intensity (MFI) of CFSE-labeled target cells co-cultured in the presence of control test cells (MF / LPS) was set at 100%, which represents the maximum proliferation. The MFI of target T cells co-cultured with other test cells was compared to this value by calculating relative proliferation. Results are expressed as the mean percentages ± SEM of 4-7 independent experiments (Cordicepine: 25 - 12.56.25 pg / mL). Data were analyzed for statistical significance using ANOVA followed by Bonferroni multiple comparison test. * P &lt;0.05, ** P &lt;0.01.

Figure 50: No TLR activity by Lactobacilli.

(A) Stably transfected HEK 293 cells with TLR2 or TLR4 were applied at 0.2x10 6 cells / well and stimulated with 107 bacteria / mL or TNFα. After 24 hours, the supernatant was removed and the IL-8 content was analyzed by ELISA. (B) Human HEK 293 cells were transiently transfected with human TLR1, TLR2, TLR3, TLR4 + MD2, TLR6, TLR7 and TLR9, and were stimulated with 107 bacteria or TNFα. Luciferase activity was determined 6 h after stimulation.

Figure 51: L. reuteri and L. casei bind DC-SIGN in moDC.

(THE). Immature DC were incubated with FITC-labeled bacteria. After washing, the cells were analyzed by flow cytometry. (B) A similar protocol as described in Part A was used to analyze the binding of bacteria to DC-SIGN transfected K562 cells or untransfected K562 cells. Preincubations were performed with TSM + 0.5% BSA, EDTA or anti-DC-SIGN (AZN-D1 and AZN-D2, 20 pg / mL). (C) DC binding was performed as described in part A. Preincubations were performed with TSM + 0.5% BSA, anti-DC-SIGN (AZN-D1 and AZN-D2, 20 pg / mL), anti-MR, Manane or methylglycoside.

Figure 52: Blocking DC-SIGN interaction with L. reuteri and L. casei overrides conditioning for the development of regulatory T cells.

IDC generation is described in the caption of Figure 1. Immature DC were preincubated (30 min, 37 ° C) with isotype control Ab, anti-DC-SIGN (AZN-D1; 20 pg / mL) or Manano . Thereafter, maturation was performed as described in the caption of Fig. 16. Induction of suppressor cell activity was analyzed as described in the caption of Fig. 17.

Figure 53. Polymorphic Mononuclear Neutrophils (PMNs) express DC-SIGN ligand Lewisx and bind with high affinity to recombinant DC-SIGN. A. Sialylated Lewisx and Lewisx polyacrylamide glycoconjugates were coated and DC-SIGN-Fc adhesion was measured with goat anti-human labeled peroxidase Fc in DC-SIGNFc ELISA. B. Lewisx expression was determined in CD16-labeled PMNs to discriminate between CD16 + neutrophils and CD16- eosinophils. Ç. Adhesion of ICAM-3-Fc or DC-SIGN-Fc coated beads to PMN was assessed using flow cytometry. Adhesion specificity was obtained using anti-DC-SIGN blocking antibodies (AZN-D1, 20 mg / mL), mannan (50 mg / mL) or EGTA (10 mM). D. Titration of DC-SIGN-Fc for PMNs reveals high binding affinity of DC-SIGN for PMNs. E. Binding of optimal DC-SIGN-Fc concentration (10 mg / mL) to CD16 neutrophil marker-labeled PMNs. Anti-DC-SIGN blocking antibodies (AZN-D1, 50 mg / mL) were used to assess specificity.

Figure 54. PMAC-expressed CEACAM1 is a ligand of DCSIGN and binds through its Lewisx units. A. The complete PMN lysate was analyzed on a 7% polyacrylamide gel for 1.5 h at 120 V, transferred to nitrocellulose transfer and stained with DC-SIGN-Fc, anti-CEACAM1 (anti-CD66) or anti-CD66 antibodies. Lewisx (3 mg / mL) and alkaline phosphatase conjugated secondary antibodies. B. DC-SIGN-Fc coated Protein A (protA) beads were used to immunoprecipitate DC-SIGN ligands from surface biotinylated PMNs. Immunoprecipitates were gel analyzed, transferred for transfer and stained with peroxidase-conjugated streptavidin. Ç. 0 Immunoprecipitated CEACAM1 from PMNs using anti-CEACAM1 antibody coated protein G (protG) beads was revealed on transfer using DC-SIGN-Fc and alkaline phosphatase-conjugated goat anti-human antibodies. D. Incubation with the al-3,4 fucosidase enzyme (5 mU / ml) for 3 days at 37 ° C removes the Lewisx epitope on PMN-derived CEACAM1. AND. Using an ELISA-based system, DC-SIGN-Fc adhesion to PMN CEACAM1 was measured and compared to that after treatment with al-3,4 fucosidase. F. Lewisx-directed antibodies (20 mg / mL) block DC-SIGN-Fc adhesion to PMAC-captured CEACAM1 using anti-CEACAM1 antibody in the adhesion ELISA.

Figure 55. Cellular DC-SIGN expressed in immature K562 and DC transfectants binds native PMN CEACAM1. A. PMN CEACAM1-coated fluorescent beads were incubated with K562 transfectants expressing either wild-type DC-SIGN or mutant DC-SIGN that exhibit a single amino acid substitution in the type C lectin domain crucial for ligand binding. Bead adhesion was measured using FACS analysis. B. The adhesion of CEACAM1 coated beads to the immature DCs was determined. Adhesion specificity was evaluated using anti-DC-SIGN blocking antibodies (20 mg / mL), mannan (50 mg / mL) or EGTA (10 mM).

Figure 56. DC-SIGN is involved in grouping DC and PMN. A. CFSE-labeled PMNs (green) were incubated with fluorescent HE-labeled K562, immature K562-DC-SIGN and DC (red) for 15 min at 37 ° C. Anti-DC-SIGN antibodies (AZN-D1 and AZN-D2, 10 mg / mL) were used to determine the involvement of DC-SIGN. The cell cluster was visualized using fluorescence microscopy and representative photographs were taken. B. Using FACS analysis, cell clustering was followed in time by scoring the percentage of K562 or K562-DC-SIGN that have PMNs bound. C. PMN adhesion to immature DC was ensured by flow cytometry.

Figure 57. Localization of PMN and DC in the colon mucosa of Crohn's disease patients. A. Inflammatory bowel tissue coverslips from Crohn's disease patients were doubly revealed for Lewisx PMN markers (6H3, green fluorescence) and blue fluorescence), green fluorescence)

B.

for

CEACAM1

CEACAM1 (CLB-gran / 10, (CLB-gran / 10, the DC-SIGN DC marker (CSRD, or C. for DC-SIGN (CSRD, red). The anti-CEACAMl antibody produces background revelation in the probably due to the cross reaction with CEA which is expressed in the epithelial cells of the colon.

fluorescence fluorescence), green) and

Lewisx (6H3, fluorescence

Figure 58. PMNs activate immature DC by binding DC-SIGN. A. DC maturation by contact with PMN was determined by measuring expression of DC maturation marker, CD86. Maturation can be inhibited with antiDC-SIGN antibodies (AZN-D1). DC maturation by PMNs was compared with DC maturation with LPS or E.coli. B. Cytokine production of IL-12 and TNFα was analyzed after PMN-DC interaction, and is dependent on binding of DC-SIGN since antibodies against DC-SIGN block cytokine induction.

Figure 59. DC-SIGN binds Lewis X-expressing CD11b and CD66a-d in PMN.

Capture of CD11b and CD66acd from PMN lysate using specific antibodies revealed the presence of Lewis X antigens in these cell membrane molecules. Capture of CD11b and CD66acd from PMN lysate and coupling with fluorescent beads revealed that in particular , PMN-derived CD11b and CD66acd interacted strongly with DC-SIGN-expressing transfectants, indicating that DC-SIGN binds PMN-derived CD11b and CD66acd.

C CD11b / CD18 and CD66 captured from PMN lysate demonstrate high affinity binding to DC-SIGNFc as demonstrated in the DC-SIGN Fc ELISA.

D Immunoblotting studies demonstrate that DC-SIGNFc interacts specifically with PMN CD66acd and CD11b when immunoprecipitated (IP) using anti-CD66acd (lane 9) and CD11b (lane 10) mAbs from a PMN lysate. Cell surface proteins are biotinylated and PMNs are lysed. Lane 1. Negative control using ICAM-3 Fc for IP protein capture. Detection is peroxidase-labeled streptavidin. Track 2. DC-SIGN-Fc is used to capture biotinylated ligands from lysate; Two protein bands are visible, the upper band consists of CD11b and CD66a, while the lower band consists of CD18. Lane 3. Negative control; no band is immunoprecipitated with anti-DC-SIGN antibodies. Lane 4, IP with CD66acd antibodies; A weak band containing CD66a is visible. Lane 5, CD11b IP; two bands are visible; the upper band is CDllc and the lower band is

CD18m, which is co-immunoprecipitated with CD11c. Track 6. IP with DC-SIGN ligands and anti-CD66acd detection; CD66a is captured by DC-SIGN Fc. Range 7. IP with DCSIGN ligands and anti-CDllb detection; CDllb is captured by DCSIGN Fc. Lane 8. Negative control, IP with anti-DC-SIGN antibodies and detection with DC-SIGN-Fc. Range 9. IP with anti-CD66acd and detection with DC-SIGN Fc; CD66a is powered by DC-SIGN on transfer. Track 10. Anti-CDlla IP and DC-SIGN Fc detection; CDlla is bound by DC-SIGN on transfer.

Figure 60

DC-SIGN links Lewis X frameworks to PMN CD11b. Immunoprecipitation (IP) studies have revealed that DCSIGN-Fc binding to CD11b is disrupted when CD11b is treated with α-1, 3,4 Fucosidase which removes the fucose residue present in Lewis X (Lane 7 and 8)

Figure 61

DC-SIGN binds tumor cells expressing the CEA tumor antigen (CD66e) through the interaction of DC-SIGN with CD6 6e.

A. DC-SIGNFc recognizes tumor antigens on various breast carcinomas and adenocarcinomas. Connection can be blocked by anti-DC-SIGN mAbs

B. the SW948 tumor cell line interacts with DC-SIGN expressing cells and with DC and cell adhesion may be blocked by anti-DC-SIGN antibodies (AZN-D1), indicating a function for DC-SIGN in interaction with a cell structure in tumor cells

C. DC-SIGN-Fc binds immunoprecipitated CD66e (CEA). Anti-CD66e (anti-CEA) immunoprecipitation of biotinylated cell surface SW948 tumor cell lysate shows that DC-SIGN-Fc binds the tumor antigen CEA (lane 5).

Figure 62

DC-SIGN recognizes Lewis X and Lewis Y on CD66e from tumor cells.

A. CD66e of tumor cell line SW948 expresses Lewis X structures and Lewis Y carbohydrates determined using specific antibodies.

B. CD66e captured from SW948 lysate is bound by DC-SIGN-Fc

C. DC-SIGN-Fc captures CD66e from a SW948 lysate as detected by an anti-CD66e specific antibody.

D. Immunoprecipitation (IP) studies have revealed that the binding of DC-SIGN-Fc to CD66e is disrupted when CD66e is treated with α-1,4 Fucosidase which removes the fucose residue present in the Lewis X, Y (Strip 3 and 4).

Figure 63.

Schematic figure of different Neisseria Meningitidis mutants with different carbohydrate residues in the final position in their LPS structures.

Figure 64. DC-SIGN binds to Neisseria Meningitidis IgtB and rfaF mutants. The different Neisseria Meningitidis mutants are investigated for their interaction using different C-Fc lectin molecules (DC-SIGN-Fc, DCIR-Fc, BCDA-2-Fc, MGL-Fc Dectin-IB-Fc). ELISA Fc.

Figure 65 Immature DC binds strongly to the Neisseria Meningitidis IgtB mutant via DC-SIGN. Only the fluorescent mutant Neisseria IgtB, which contains a GlcNAc end position, binds tightly to DC as determined by flow cytometry, and the binding can be completely blocked by anti-DC-SIGN antibodies, indicating that DCSIGN is involved in IgtB Neisseria capture. Mannose-BSA and mannan partially inhibit interaction.

Figure 66. DC-SIGN-Fc strongly binds GlcNAc overexpressing CHOP8 cells, indicating that DC-SIGN also binds GlcNAc residues to auto-glycoproteins (CHOP8) and pathogens such as Neisseria (Figure 33).

Examples

Examples not referring to a glycoconjugate comprising a tumor antigen and a non-sialylated Lewis X antigen are not part of the invention.

Methods

Antibodies and proteins. The following monoclonal antibodies (Mab) were used: anti-CD107a (Lamp-1; MabH4A3, BD Pharmingen), anti-MR (Clone 19, BD Pharmingen), CD11b (bear-1)²⁵, CD11c (SHCL3)²⁶, anti-DC-SIGN (AZN-D1, AZN-D2¹⁴, CSRD¹⁰, Blocker CDlla (NKI-L15), Anti-ICAM-1 (Rek-1), Anti-ICAM-2 (12A2), and Anti-ICAM-3 (AZN-IC3 / 1, ICR-2), CD18 Activator (KIM185 ) and PE / FITC conjugated antibodies CD25, CD69, CD80, CD86, HLA-DR (BD Pharmingen), and CD83 and

CD56 (Beckman Coulter). Biotin-conjugated CD3 and CD16 (BD Pharmingen). CLB-T4 (aCD4) was a generous offer from René van Lier. MEM-157 (aCD16) and MEM-97 (aCD20) were kindly provided by Vaclav Horejsi. mAb against ICAM-1 CBR-IC-1/3, mAb against ICAM-2 CBR-IC-2/1, and mAb against ICAM-3 CBR-IC-3/2 were obtained from the Human Leukocyte Differentiation Antigen Workshop. A number of anti-glycan monoclonal antibodies (MAbs) were used: the anti-LDN-DF 114-5B1-A MAb¹⁶, the anti-LDN MAb

SMLDN1.1¹⁷, the anti-LDN-F MAb SMLDNF1¹⁰ and the anti-Le MAb^xCB1 0²⁷anti-Lex mAbs SMLDN1.1 and SMFG4.1 and 6H3 (antiLex). MAb (CLB-gran / 10) was used to reveal and immunoprecipitate CD16.

HSA-Le neoglycoprotein^xcontaining approx. 20-25 oligosaccharide chains per HSA molecule was from Isosep AB, Tullinge, Sweden. The BSA-LDN-DF neoglycoproteins (approx. 12 oligosaccharide chains per BSA molecule) and BSALDN-F (approx. 3-4 oligosaccharides per BSA molecule) were enzymatically synthesized as described by van Remoortere et al.¹⁶, and Nyame et al¹⁷, respectively. Le^xPAA-biotin containing Le^x multivalently coupled biotinylated polyacrylamide was from Syntesome, Munich, Germany.

Purified Saccheromyces cerevisiae mannan (50 pg / mL) and recombinant gp120 (0.50 pg / mL) were obtained from Sigma and the Aidsresource Foundation, respectively. Purified mancose end lipoarabinomannan (manLAM) from Mycobacterium tuberculosis, and M. smegmatis endless LAM (araLAM) were kindly provided by Dr. J. Belisle, Colorado State University through NIH, contract NIAID NO1 AI-75320. Soluble Egg Antigen (SEA) was kindly provided by Dr. AK Nyame, Oklahoma University HSC, USA). Purified lipophosphoglycan from Mexican Leishmania was kindly donated by Dr. M Wiese, Bernard Nocht Inst. Tropical Medicine Hamburg, Germany. Purified Helicobacter pylori lipopolysaccharide was obtained from M. Monteiro, NRC, Ottawa, Canada. An ultrasound-treated sample of bacterial cells from a clinical M. tuberculosis isolate was donated by A. Kolk, KIT, Royal Trop. Inst, Amsterdam. The clinical isolates of

Helicobacter pylori, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus aureus were obtained from VUMC Hospital, Amsterdam. Synthetic glycoconjugates were obtained from Syntesome, Munigue, Germany, and comprise mono- and oligosaccharides multivalently linked to a biotinylated polyacrylamide carrier, MM 40,000.

Soluble DC-SIGN-Fc Adhesion Test. DC-SIGN-Fc consists of the extracellular portion of DC-SIGN (amino acid residues 64-404) fused to the C-terminal end with an Fc fragment of human IgG1. DC-SIGN-Fc was produced in Chinese Hamster Ovary Cells by co-transfecting DC-SIGN-Sig-pIgG1 Fc (20 pg) and pEE14 vector (5 pg). DC-SIGN-Fc concentrations in the supernatant were determined by an anti-IgG1 Fc ELISA. The DC-SIGN-Fc binding assay was performed as follows. The ultrasound glycoconjugates and mycobacteria were coated on ELISA plates at 5 pg / well; intact bacterial cells were coated at 5x10⁷/ ml; Coating occurred for 18 hours at room temperature, followed by blogging with 1% BSA for 30 min at 37 ° C in TSM (20 mM Tris-HCl, pH 7.4 containing 150 mM NaCl, 2 mM CaCl 2 and MgCl 2 2). mM). Soluble DC-SIGN-Fc (approx. 2 pg / ml in TSM buffer) was added and adhesion was performed for 120 min. at TA. Unbound DC-SIGN-Fc was washed off and binding was determined by an anti-IgG1 Fc ELISA using a goat anti-human Fc peroxidase conjugate. Specificity was determined in the presence of 20 pg / ml blogging antibodies, 50 pg / ml mannan or 5 mM EGTA.

Cells. Immature DC were cultured from monocytes in the presence of IL-4 and GM-CSF (500 and 800 U / mL, respectively; Schering-Plow; Brussels, Belgium²⁸. On day 7, the phenotype of cultured DC was confirmed by flow cytometric analysis. CD expressed high levels of MHC class I and II, αΜβ2 (CDllb), αΧβ2 (CDllc) and ICAM-1, moderate levels of LFA-1 and CD80, and low levels of CD14. Wild-type DC-SIGN expressing K562 transfectants were generated by transfecting K562 cells with 10 pg of plasmid pRc / CMVDC-SIGN by electroporation as previously described.

Stable IC5-expressing K562 transfectants were obtained by electroporation of pCDM8-ICAM-3 and pGK-HYG. K562 cells were cultured in RPMI 10% FCS, while K562-ICAM-3 cells were cultured in RPMI 10% FCS: Iscove 5% FCS 3: 1 containing 0.5 mg / mL hygromycin to maintain expression. of ICAM-3.

Naif NK cells were isolated from the leukocyte layers of healthy donors. Briefly, the fraction of PBMC obtained by Ficoll centrifugation was sequentially depleted of CD14 cells.⁺, and CD3 cells⁺, CD4⁺ and CD20⁺ using MACS classification. CD14 cells⁺ were depleted by CD14 microspheres (Miltenyi Biotec) on an LS column (Miltenyi Biotec), and CD3 cells⁺, CD4⁺ and CD20⁺ were labeled by mAbs T3B (aCD3), CLB-T4 (aCD4), MEM97 (aCD20), and thereafter depleted by rat goat microspheres (Miltenyi Biotec) on an LD column (Miltenyi Biotec). The NK cells thus obtained were routinely tested for CD16 (75% to 90% expression), CD56 (80 to 95% expression) NK cell markers, CD3, CD4, CD14 and CD20 non-lineage markers (all but 1% expression), and the

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CD5 6^obscured and CD5 6^bright were isolated from NK cells obtained from MACS by FACS classification in low and high expression of CD56, respectively. NK CD5 g cell populations^obscured and CD5g^bright were more than 95% pure as evaluated by CD16 and CD56 developed flow cytometry.

DC activation. Immature DC (2x10⁶ cells / mL) were cultured for 24 hours in the presence of IL-4 (500 U / mL, Schering-Plow, Brussels, Belgium), GM-CSF (800 U / m; Schering-Plow, Brussels, Belgium) and LPS ( 10 ng / mL) or LAM glycolipids (15 pg / mL). The effect of LAM on LPS-induced activation was determined by preincubating immature DC (300,000 cells) with AZN-D2 (40 pg / mL) for 30 minutes, and subsequently with LPS in the presence of LAM (15 pg / mL) for 30 minutes. 18 hours. LAM glycolipids were obtained from J. Belisle (Colorado State University and NIH (contract NO1 AI-75320)) and contained &lt;5 ng / mg endotoxin. Activation was determined by surface expression of MHC class II cells (HLA-DR) and costimulatory molecules CD80, CD83 and CD86 using PE-conjugated antibodies.

Fluorescent bead adhesion test. Carboxylate modified TransFluorSpheres (488/645 nm, 1.0 pm; Molecular Probes, Eugene, OR) were coated with HIV-1 gp120 and ICAM-3 as described. Streptavidma was covalently coupled to beads as described and streptavidin coated beads were incubated with biotinylated PAA-linked glycoconjugates (50 pMol; Syntesome, Munich, Germany). The fluorescent bead adhesion assay was performed as described¹⁴. Ligand-coated fluorescent beads (20 beads / cell) were added to the cells for 45 minutes at 37 ° C, washed and analyzed by flow cytometry (FACScan, Becton Dickinson, Oxnard, CA), measuring the percentage of cells that had bound beads. fluorescent. LAM coated beads were generated by incubating streptavidin coated beads were incubated with biotinylated goat anti-mouse IgG F (ab ') 2 fragment (6 pg / mL; Jackson Immunoresearch), followed by one day incubation for the another with anti-LAM rat antibody (F30.5) at 4 ° C. The beads were washed and incubated with 250 ng / ml purified LAM glycolipid (obtained from J. Belisle, Colorado State University and NIH (contract NO1 AI-75320)) overnight at 4 ° C. SEA-coated beads were generated by incubating streptavidin-coated beads with biotinylated goat anti-mouse IgG F (ab ') 2 fragment (6 pg / ml; Jackson Immunoresearch), followed by overnight incubation at 4 ° C with anti-LDN MAb or anti-LDNDF MAb. The beads were washed and incubated with 1pg / ml SEA overnight at 4 ° C. Essentially 50x10³Cells were preincubated in adhesion buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM CaC12, 2 mM MgC12, 0.5% BSA) with or without blocking MAbs (20 pg / mL ) or mannan (50 pg / mL) for 10 minutes at room temperature. Ligand-coated fluorescent beads (20 beads / cell) were added to the cells and the suspension was incubated for 45 minutes at 37 ° C. Cells were washed and adhesion was determined using flow cytometry (FACScan, Becton Dickinson, Oxnard, CA) by measuring the percentage of cells that had fluorescent beads attached. HIV-1 gp120 fluorescent beads were prepared as previously described¹⁹.

DC-SIGN-Fc adhesion. Cells were incubated with DCSIGN-Fc for 30 minutes at 37 ° C under saturation conditions (concentration: 10 pg / mL), and subsequently with FITC-conjugated goat-secondary antibodies to monitor DC-SIGN adhesion. -Fc. Prior to cell incubation, DC-SIGN-Fc was preincubated for 10 min at RT with medium, aDC-SIGN (AZN-D1, 50 pg / mL), mannan (50 pg / mL) or EGTA (10 mM ) to determine the specificity of DC-SIGN-Fc adhesion. DCSIGN-Fc adhesion was determined by flow cytometry (FACS Calibur, Beckman Coulter).

Mycobacteria. M. bovis (Pasteur) and M. tuberculosis H37Ra BCG strains were offered by A. Kolk (Royal Tropical Institute, Amsterdam). M. bovis BCG was cultivated cultured in vitro using Middelbrook 7H9 broth supplemented with 0.05% Tween 80 and albumin dextrosecatalase. Glycolipids ManLAM and AraLAM were obtained from J. Belisle, Colorado State University and NIH (contract NO1 AI-75320). The DCs were infected with mycobacteria by culturing them at an appropriate multiplicity of infection (MOI) as indicated in the figure captions.

Fluorescent mycobacterial binding assay. Catch and internalization of mycobacteria by cells were evaluated using fluorescein isothiocyanate-conjugated M. bovis BCG (FITC). The Bacteria (10⁹/ mL) were labeled by incubating 0.5 mg FITC per mL in phosphate buffered saline (pH 7.4) at room temperature for 1 hour. FITC-pulsed bacteria were washed three times to remove unbound FITC. Capture was determined by measuring the percentage of cells that bound FITC-conjugated bacteria using flow cytometry (FACScalibur, Becton Dickinson Immunocytometry, San Jose, CA). Phagocytosis was determined using a fluorescence deactivation technique as previously described.²⁹. In summary, inactivated membrane bound FITC-conjugated M. bovis BCG deactivation was achieved by treating the cells with 0.05% trypan blue for 5 minutes. H. pylori binding was assessed by labeling the bacteria with FITC and binding to DC was investigated similarly to the bead assay.

Cytokine production. For cytokine detection, culture supernatants were harvested on day 1 and frozen at -80 ° C until analysis. Supernatants were analyzed for the presence of IL-10 and IL-12p40 by ELISA (Biosource International, CA).

Defucosylation of SEA. SEAs were defucosylated by incubating the antigens at 100 ° C for 1 hour in 0.1 M TFA. After neutralization, the defucosylated antigen was coated on ELISA plates. Enzymatic defucosylation was performed by incubating the SEA in 50 mM sodium phosphate, pH = 5.0 with 1, 3/4-fucosidase (0.4 mU / pg SEA) (Calbiochem) overnight at 37 ° C. Ç. The degree of antigen defucosylation was assessed by their ability to bind MAbs that specifically recognize the fucosylated glycan epitopes LDN-DF and Le^x, while the integrity of other non-fucosylated glycan epitopes was assessed by measuring reactivity with anti-LDN MAb.

SDS-PAGE, Western blot and silver staining. SEAs were separated by SDS-PAGE under reducing conditions on a 12.5% ​​polyacrylamide gel using the Mini-Protean II (BioRad) system, and the proteins visualized by silver staining. For Western blotting, proteins were transferred to a nitrocellulose membrane (Schleicher and Schuell). The membrane was blocked in a 5% BSA solution in TSM for 2 h followed by incubation in 2 pg / ml DC-SIGN-Fc in TSM buffer containing 1% BSA for 1 h. After washing, the membrane was subsequently incubated for 1h in peroxidase-conjugated goat anti-human IgGl and the reactive bands were visualized by CN / DAB substrate detection (Pierce).

Cytotoxicity. 0 release test⁵¹Cr at 4 hours standard was used to evaluate NK cell mediated cytotoxicity. Briefly, 1 * 10⁶ target cells were hour at 37

C, free Cr, and mDC) or 1000 marked 100 pCi of ⁵¹Cr was thoroughly washed to remove resuspended at 2500 cells / well (iDC cells / well (K562 and K562-DC-SIGN), and incubated with NK cells for 4 hours at 37 ° C in the indicated proportions and conditions. After 4 hours scintillation liquid (Perkin Elmer) was added to the supernatants, and the release of ⁵¹Cr was determined on a micro-b counter (PerkinElmer).

NK cell mediated DC maturation. Resting and activated NK cells were obtained by overnight incubation in medium and IL-2 (1000 U / mL), respectively. Thereafter, resting and activated NK cells were incubated overnight with immature DC in a 96-well U-bottom plate (Costar) in RPMI with 10% FCS. NK cells were preincubated for 10 min. at RT with oIFA-1 blocking medium or mAbs (NKI-L15, 50 pg / mL), while DC were preincubated with aDC-SIGN blocking medium or mAbs (AZN-D2, 50 pg / mL). As a positive control, DCs were incubated overnight in the presence of LPS (2 pg / mL). DC maturation was assessed by flow cytometry (FACS Scan, Beckman Coulter) for CD80, CD83, CD86, and HLA-DR maturation markers by PE-conjugated or FITC-conjugated mAbs.

DC-induced NK cell activation. Resting NK cells were incubated for 2 days with immature or mature DC (obtained by LPS maturation) in a U-bottom 96-well plate (Costar) in RPMI with 10% FCS. LFA-1 dependent DC-induced NK cell activation was determined by a 10 min preincubation. mAbs against aLFA-1 mAbs (NKI-L15, 50 pg / mL), while DC-SIGN dependence was assessed by a 10 min preincubation. DC with anti-DC-SIGN mAbs (AZN-D1 and AZN-D2, 50 pg / mL). As a positive control, NK cells were incubated for 2 days with IL-2 (1000 U / ml). NK cell activation was assessed by flow cytometry (FACS Calibur, Beckman Coulter) for the early activation marker CD69 by FITC-conjugated mAbs.

Immunoprecipitation. NK cells were surface iodinated with 1 mCi of I, or biotmilated and subsequently lysed in lysis buffer (1% Triton-X-100, 10 mM TEA, 150 mM NaCl, CaCl₂ 1 mM, MgCl₂ 1 mM, 1 mM PMSF, 20 pg / ml trypsin inhibitor, 20 pg / ml leupeptin and 20 pg / ml aprotinin). DCSIGN, ICAM-2, ICAM-3, and LFA-1 ligands were immunoprecipitated from prot A-pretreated NK cell lysate by DC-SIGN-Fc, aICAM-2 covalently linked prother beads ( 12A2), aICAM-3 (AZN-IC-3/1) and aLFA-1 (NKI-L15). Immunoprecipitates were reduced in sample buffer (containing 4% SDS and 5% b-mercaptoethanol), heated for 5 min at 95 ° C, and analyzed in 5-15% gradient on polyacrylamide gel (SDS-PAGE). .

Alternatively, specific antibodies against CD66acd or ICAM-3 or LFA-1 were coated on an ELISA plate, subsequently lysed and incubated for 1 h at 4 ° C to retract specific proteins. DC-SIGNFc was incubated for specific protein binding activity and developed with anti-human Goat PO Fc and developed.

Experimental preparation.

Dendritic cells (DC) are instrumental in handling pathogens for processing and presentation to T cells, thus eliciting an appropriate immune response. Type C lectins expressed by the function of DC as pathogen recognition receptors; however their specificity for carbohydrate structures in pathogens is not fully understood. Here we look at the carbohydrate specificity of DC-SIGN / CD209, the newly documented HIV-1 receptor in DC. Our studies show that DC-SIGN binds with high affinity to mannose and fucose-containing synthetic glycoconjugates. These carbohydrate structures are abundantly expressed by pathogens as demonstrated by DC-SIGN affinity for natural glycans on the surface of human pathogens Mycobacterium tuberculosis, Helicobacter pylori, Leishmania mexicana and Schistosoma mansoni. Surprisingly, these pathogens target DC-SIGN to infect DC but also to modulate DC-mediated immune responses.

This analysis expands our knowledge of the specificity for DC-SIGN carbohydrates and pathogens, and identifies this lectin as being nucle3ar in pathogen-DC interactions.

Example 1: New Carbohydrate Specificity for DC-SIGN

For in vitro binding studies, we generated a chimeric DC-SIGN protein with a human IgG1 Fc marker, which we used to screen in an ELISA format for reactivity with a synthetic glycoconjugate panel containing mannose or fucose residues and their derivatives in multimeric form.

As described above, DC-SIGN-Fc binds to purified yeast derived mannan and HIV-1 gp120 containing high mannose contents but also to less complex mannose containing glycoconjugates ie mannose and al-&gt; 3, al&gt; 6manotriosis (Table I, Figure 1). Surprisingly, DC-SIGN binds to the Lewis blood group antigens (Le^x, Le^Y, Le^The, Le^B), glycan comprising at least one terminal fucose linked at 1, 3 or 1, 4 to Nacetylglucosamine (Le^x, Le^Y Le^The, Le^B, LDNF), or D-1.2 to Galactose (Le^Y, Le^B) (Table I, Figure 1). Le's sialylation^x (producing sialil-Le^x, a ligand of L-, E- and Pselectin) completely nullifies recognition by DCSIGN, indicating that DC-SIGN has a carbohydrate specificity that is distinct from that of selectins that mediate leukocyte circulation. Sulfation reduced binding affinity of DC-SIGN for Le^xas well as Le^The (Figure 1c). To further compare the binding affinity of DC-SIGN with Le^x and alpha 1&gt; 3, alpha 1&gt; 6-manotriose, titration studies were performed with the different DC-SIGN binding glycoconjugates (Figure 1c). Surprisingly, DC-SIGN binds with much greater affinity to the fucose-containing carbohydrate Le^x than to manotriosis. The binding activity of DC-SIGN-Fc to these glycan structures was specific as anti-DC-SIGN antibodies blocked the interaction (Figure 1c).

To determine if DC-SIGN-Fc exhibits a carbohydrate recognition profile similar to DC-SIGN expressed on cell surface, both DC-SIGN and monocyte-derived DC transfectants were studied for the binding activity of carbohydrates using a fluorescent bead adhesion assay with different glycoconjugates (al-&gt; 3, al-&gt; 6manotriose, Le^xand sulfo-Le^The) (Figure 2). In fact, DC-SIGN expressed by K562 transfectants bound similarly to glycoconjugates than DC-SIGN-Fc and binding was completely inhibited by anti-DC-SIGN antibodies (Figure 2). Although DC expresses many other type C lectins on their cell surface, our data demonstrate that Le-containing glycoconjugates^x and al-&gt; 3, al&gt; 6manotriose are preferably linked by DC-SIGN. The interaction is specific as anti-DCSIGN antibodies almost completely inhibited binding activity. This illustrates that DC-SIGN is the primary receptor in DC for these carbohydrate structures. The Sulfo-Le Link^The DC could only be partially blocked by anti-DC-SIGN antibodies, indicating that other type C lectins in DC compete with DC-SIGN for sulfoLe binding.^The. Our data show that DC-SIGN recognizes a wider range of glycan structures, including Lewis blood group antigens, than hitherto understood. Thus, DC-SIGN may be an important receptor for recognition of new biologically relevant targets expressed by the host, or alternatively by human pathogens.

Example 2. New carbohydrate specificity (Le^xand high mannose content) identifies new pathogens (Helicobacter pylori, Schistosoma mansoni, Mycobacterium tuberculosis, Leishmania mexicana) that interact with DCSIGN

We subsequently investigated the binding of DC-SIGN to human pathogens expressing mannose or fucose containing glycans. Gram negative bacterium Helicobacter pylori, which induces peptic ulcer and gastric carcinoma²⁰, and the parasitic worm (the causative agent of squamous^{x 21}. At H. pylori, Le^xis present in the lipopolysaccharide located on the surface while in S. mansoni Le^x is expressed for all stages of the parasite, including soluble egg antigen (SEA)²¹. DC-SIGN-Fc binding to Le positive H. pylori lysate^x and to extract from S. mansoni was strong and was completely inhibited by anti-DCSIGN antibodies (Figure 3); When analyzing two different forms of Klebsiella LPS, one containing mannose end and one without (Leni and Lenlll, respectively) (Figure 3B). DCSIGN expressed by DCs also bound to purified H. pylori LPS (Figure 4). Binding of DC to H. pylori cells and purified LPS could be completely blogged with anti-DC-SIGN Mab (Figure 4). Similarly, the glycans present in Mycobacterium tuberculosis, the causative agent of tuberculosis, are linked by DC-SIGN (Figure 3). The mannose-end surface glycan, lipoarabinomannan from Mycobacterium tuberculosis probably contains the recognition site for DCSIGN. This is further supported by the fact that DC-SIGN also bound to the mannose end surface lipophosphoglycan (LPG) expressed by a unicellular parasite that causes leishmaniasis (Figure 3). The connection of DC-SIGN to Leishmania has been described very recently.³⁰ but we demonstrated above that LPG is the structure in Leishmania which is recognized by DC-SIGN (Figure 3). No binding of DC-SIGN to three clinically relevant Gram-negative human bacterial pathogens (Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa) or to Gram-positive Staphylococcus aureus was observed. However, other clinical isolates of Klebsiella gue contain a mannose-end LPS binding to DC-SIGN. These findings indicate that the binding of DC-SIGN to pathogens is selective, and that DC-SIGN's carbohydrate specificity governs a broader recognition of pathogens than only viruses such as HIV-1 pc and Ebola virus. '

Example 3 DC-SIGN Interacts with M. tuberculosis via ManLAM Glycolipids

Whole mycobacteria such as M. tuberculosis strain H37Ra and M. bovis bacillus Calmette-Guérin (BCG) were coated and the interaction of DC-SIGN with these pathogens was analyzed using the DC-SIGN-Fc binding assay. M. bovis BCG is a tuberculosis strain that is almost non-pathogenic but retains the immunological properties of tuberculosis. DC-SIGN-Fc interacted specifically with M. tuberculosis H37Ra and M. BCG bovis, since the interaction was inhibited with antibodies

<td>specific</td><td>blockers</td><td>d</td><td>and DC-SIGN</td><td>(Figure 6a).</td><td>M.</td>

<td colspan="3">smegmatis, a strain not</td><td>virulent</td><td>which does not contain</td><td>an</td>

<td>far end</td><td>mannose,</td><td>no</td><td>turn on the</td><td>DC-SIGN (data</td><td>no</td>

<td>presented)</td><td> •</td><td></td><td></td><td></td><td></td>

<td>Furthermore,</td><td>a chimera</td><td>in</td><td colspan="2">Fc irrelevant, ICAM-3-Fc,</td><td>no</td>

interacted with mycobacteria (Figure 6a). The interaction is mediated by the DC-SIGN type C lectin domain, since binding to M. tuberculosis and M. bovis BCG was inhibited by EGTA, mannan and oo-specific antibody.

DC-SIGN, AZN-D1, which recognizes the lectin domain (Figure 6a).

We then investigated the binding of DC-SIGN to purified mycobacterial lipoarabinomanane (LAM), since DC-SIGN has a high affinity for mannose-containing carbohydrates and LAM is the main mannose-containing component of the mycobacterial cell wall.³¹. The LAM comprises a mannose-rich polysaccharide core containing highly branched arabinofuranosyl side chains and a GPI anchor (Figure 6b). LAM isolated from M. tuberculosis contains mannose residues consisting exclusively of D- Dmanoses mono-, di- and trimers directly attached to the arabinofuranosyl end and is called ManLAM, whereas LAM isolated from M. smegmatis de fast growing has no mannose extremity and is called AraLAM³¹ (Figure 6b). Surprisingly, purified ManLAM was efficiently bound by DC-SIGN, in contrast to AraLAM (Figure 6c), demonstrating that DC-SIGN interacts specifically with ManLAM's α-mannose mono-, di- and trimers. Even at high concentrations, DC-SIGN did not bind AraLAM, demonstrating a high specificity for ManLAM and its mannose extremity (Figure 6d). The interaction of DC-SIGN with ManLAM is specific, since binding was inhibited by antibodies against DC-SIGN, whereas an irrelevant Fc chimera did not interact with ManLAM (Figure 6c). DC-SIGN interacts similarly with ManLAM and whole M. bovis BCG indicating that DCSIGN binds mycobacteria via ManLAM (Figure 6a and c).

Example 4 Both mycobacteria and ManLAM interact with the DC-SIGN primary binding site.

We used K562 transfectants that stably express DC-SIGN to investigate cell surface-expressed DC-SIGN binding to M. bovis BCG and the mycobacterial component ManLAM. These cells do not express the previously described mycobacterial receptors Manose Receptor (MR), CD11b and CD11c (Figure

7a). K562 transfectants express high levels of DCSIGN (Figure 7a) and bind strongly to M. bovis and ManLAM BCG, in contrast to simulated transfected K562 cells (Figure 7b). The interaction is blocked by antibodies specific for DC-SIGN (Figure 7b). The interaction of DC-SIGN with BCG from M. bovis and ManLAM is similar to that of the other DC-SIGN ICAM-3 and HIV-1 ligands (Figure 7b). Thus, cellular DC-SIGN specifically binds to M. BCG bovis and ManLAM, as observed with recombinant DC-SIGN-Fc (Figure 6c).

DC-SIGN's Type C lectin domain contains two calcium ions, and amino acid residues that are in close contact with Ca at site 2 (Glu, Asn, Glu and Asn³⁶⁵) form the nucleus of ligand binding sites²³. The change in DC-SIGN of Glu³⁴⁷ for Gin (E347Q), or gaqg &lt;n

Asn and Asn for Asp resulted in complete loss of binding to whole mycobacteria and ManLAM (Figure 7b and data not shown), similarly as previously demonstrated for both ICAM-3 and HIV1 gp120 (Figure 7b). The CA²⁺ at site 1, the so-called helper site coordinates the correct positioning of the primary p_gpq binding site, and the loss of this Ca by mutation of Asp, Glu (E324A), Asn or Asp to Ala residues resulted in complete loss of binding. bovis and ManLAM BCG (Figure 7b and results not shown).

We have recently demonstrated that the DC-SIGN binding site to its ICAM-3 cell ligand is distinct from that of HIV-1 gp120, since a specific mutation in DC-SIGN (V351G) disrupts ICAM-3 binding. 3, but not HIV-1 gp120 (Figure 7c). Surprisingly, the DC-SIGN V351G mutant also interacts with M. bovis BCG as well as ManLAM (Figure 7c), demonstrating that both the

HIV-1 as mycobacteria bind similarly to DCSIGN at a site distinct from the ICAM-3 cell ligand. The similar binding of both M. bovis BCG and ManLAM to DC-SIGN mutants further support our observations that DC-SIGN interacts specifically with ManLAM on whole mycobacteria. Binding of fluorescent beads coated with distinct ManLAM neoglycoproteins consisting of Arabinose, Arabinose-alfal, 5 mannose, Arabinose-alfal, 5Man-alpha 1,2 Man, Ara-alpha, 1, 5Man-alpha 1, 2Man-alpha 1 , 2Man or Ara6 demonstrate that DCSIGN particularly recognizes in ManLam a di-mannose (Man alpha 1.2 component) or mannosetriosis (Man alpha 1.2 component) (Figure 7D).

Example 5 DC-SIGN is an important receptor for mycobacteria in DC.

Immature DCs express, in addition to high levels of DC-SIGN, high levels of the MR, CD11b and CD11c receptors (Figure 8a), which have previously been described as mediating macrophage binding of mycobacteria. We used blocking antibodies against these receptors to evaluate their contributions to ManLAM binding by DC. Immature DCs bind strongly to ManLAM, but not AraLAM, and the interaction was inhibited by the DC-SIGN specific antibody, but surprisingly not by any of the antibodies against MR, CD11b or CD11c (Figure 8b). Both EGTA and type C lectin-specific inhibitor mannan block ManLAM binding by DC to a degree similar to DCSIGN-specific antibodies, demonstrating that DC-SIGN is the major ManLAM-binding type C lectin in immature DCs. turn on ManLAM (Figure 8b).

whole mycobacteria.

strongly interacted with BCG (Figure 8c).

DC-SIGN's main contribution to the interaction of immature DC with ManLAM led us to investigate whether DC-SIGN could be important in the interaction of immature DC with M. bovis immature DC

Surprisingly, DC-SIGN is an important receptor for M. bovis BCG, since antibodies against DC-SIGN strongly inhibited M. bovis BCG immature DC infection (Figure 8c). Antibodies against MR, CD11b, and CD11c do not inhibit infection, whereas type C lectin inhibitor mannan blocked the infection to a level similar to that of DC-SIGN antibodies (Figure 8c). Moreover, the mannose-BSA MR ligand did not inhibit the interaction of DC with M. BCG. bovis (Figure 8c) demonstrating that MR domains of type C lectins are not involved in DC infection by M. bovis BCG. Both anti-MR antibody and mannose-BSA are inhibitors of MR function as they block the binding of another MR ligand, dextran, to DC (data not shown). These results demonstrate that DC-SIGN is the major type C lectin in DC that

<td>acts as</td><td>one</td><td colspan="2">recipient for BCG</td><td>in</td><td>M.</td><td>bovis. Others</td>

<td>receivers</td><td>in</td><td>type</td><td>non lectin</td><td colspan="2">may</td><td>participate in</td>

<td>interaction,</td><td>an</td><td>turn</td><td>that the infection</td><td>no</td><td>was</td><td>completely</td>

inhibited by antibodies against DC-SIGN (Figure 8c).

Example 6 DC-SIGN facilitates the capture and internalization of M. bovis BCG by immature DCs by ligating the mycobacterial cell wall component ManLAM.

Next, we investigated whether DC-SIGN mediates the internalization of M. bovis BCG using trypan blue to deactivate FITC-conjugated mycobacteria on the surface. Simulated transfected K562 cells do not phagocyte M. bovis BCG, while 50% of DC-SIGN-expressing K562 transfectors internalized M. bovis BCG in less than 45 minutes (Figure 8d). Both mannan and DC-SIGN antibodies blocked the internalization of M. bovis BCG (Figure 8d).

Immature DC are highly phagocytic cells, and in fact, in less than 45 minutes more than 90% of the dendritic cells that bound M. bovis BCG had internalized mycobacteria (Figure 8e). Similar to that observed for M. bovis BCG binding by DC (Figure 8c), M. BCG phagocytosis bovis is partially blocked by antibodies against DC-SIGN while anti-MR antibodies and MR mannose-BSA ligand did not inhibit the observed phagocytosis (Figure 8e). These results demonstrate that DC-SIGN facilitates the capture and internalization of M. bovis BCG by immature DC by ligating the ManLAM mycobacterial cell wall component.

Example 7. Mycobacteria and ManLAM are internalized by DC-SIGN and directed to lysosomes.

Recently, we have demonstrated that DC-SIGN can act as an antigen receptor that internalizes the antigens and directs them to the lysosomal compartments for MHC Class II presentation. Therefore, the fate of the M. bovis BCG captured by immature DCs was followed by immunofluorescence analyzes. Immature DCs were incubated with FITC-conjugated M. bovis BCG for 2 hours and both DC-SIGN and Lamp-1 were revealed (Figure 9a). The observed colocalization of DC-SIGN with FITC-conjugated M. bovis BCG further supports a role for DC-SIGN in mycobacterial capture and internalization (Figure 9a). Phagocyte mycobacteria are directed to lysosomes, as internalized FITC-conjugated mycobacteria are placed with the revelation of Lamp-1 (Figure 9a). Similarly, ManLAM was also captured and internalized by DC-SIGN in immature DC, as ManLAM's revelation placed it with DC-SIGN (Figure 9a) while AraLAM was not internalized by DC (results not shown). . Internalized ManLAM was placed with the LAMP-1 / CD107a lysosomal marker in immature DC (Figure 9a) indicating that internalized ManLAM is directed to the lysosomes. Thus, both the entire mycobacteria and the ManLAM cell wall component are similarly internalized by immature DC through DC-SIGN, which supports the findings that DC-SIGN interacts with ManLAM in mycobacteria.

Recently, we have demonstrated that binding of soluble ligands or antibodies to DC-SIGN triggers the internalization of the DC-SIGN-ligand complex to the final endosomes / lysosomes, and results in down-regulation of DC-SIGN from the surface.¹⁰. Therefore, we investigated whether DCSIGN is internalized upon ManLAM binding by measuring the expression of DC-SIGN on cell surfaces following ManLAM binding using a DCSIGN specific antibody. In fact, binding ManLAM, but not AraLAM, to DC-SIGN results in down-regulation of DC-SIGN (Figure 9b), demonstrating that DC-SIGN in DC binds ManLAM and mediates ManLAM internalization for lysosomes. CD107a⁺. Furthermore, other mycobacterial receptors such as MR, CD11b and CD11c were not down-regulated (Figure 9b). Thus, binding of ManLAM to DCSIGN triggers the internalization of the DCSIGN / ManLAM complex, and could enable ManLAM antigen processing by DC.

Example 8. ManLAM alters cytokine production by DC through DC-SIGN M. mycobacteria macrophages in

ManLAM is present not only as a component of the mycobacterial cell wall but is also secreted from phagosomes after ingestion of tuberculosis.³¹. Potentially, infected macrophages can influence bystander immune cells and modulate the immune response through ManLAM secretion. Cytokine IL-10 is a potent immunosuppressive factor induced in macrophages by some decreasing and promoting responses to its influence of intracellular to immunological host bacteria binding.

Qibribivencia. We investigated from ManLAM to DC-SIGN in the production of IL-10 by DC. ManLAM alone did not induce IL-10 production by immature DC (Figure 10a). Surprisingly, ManLAM, but not AraLAM, strongly induced IL-10 production by DC when simultaneously receiving an activation signal, such as LPS (Figure 10a). This IL-10 induction was completely inhibited by DCSIGN-specific antibodies at the level of LPS-activated DC alone (Figure 10a). Observations that only ManLAM could induce IL-10 production, which could be blocked by DC-SIGN specific antibodies, indicates that IL-10 induction is specific for the ManLAM / DC-SIGN interaction. Antibodies against DC-SIGN alone do not induce LPS-activated DC production (Figure 10a), binding of DC-SIGN alone is not sufficient for IL-10 induction. Thus, binding of ManLAM to DC-SIGN triggers intracellular signals that induce IL-10 production by DC, indicating that mycobacteria target DC-SIGN to suppress the immune response and promote its survival in the host. Both immature and LPS-activated DC alone from IL-10 alone demonstrated that or in combination with ManLAM produced very low amounts of IL-12p70 (&lt;5 pg / mL). Infection of immature DC with M. BCG bovis induced a strong IL-10 production that was not inhibited by antibodies against DC-SIGN (Figure 10b). No differences were observed in the presence of ManLAM (Figure 10b). These results suggest that mycobacteria induce IL-10 production by direct infection as well as ManLAM segregation.

Example 9. ManLAM inhibits TLR4-mediated DC activation through DC-SIGN

Immature DCs are highly effective in antigen capture and processing, while mature DCs are specialized in activating naive T cells required for cellular immune responses. Immature DC matures in response to specific 'danger' signals such as bacterial components (LPS) or inflammatory cytokines (TNFα, PGE2). We investigated the effect of ManLAM on DC maturation. Neither ManLAM nor AraLAM induced DC maturation, as both ManLAM and AraLAM, in contrast to the TLR4-triggering LPS, do not positively regulate the expression of CD80, CD83, CD86 or HLA-DR activation markers. (Figure 11a).

Acute mycobacterial infections represent sites of inflammation that attract and induce CD maturation through the presence of maturation components. Therefore, we investigated the effect of ManLAM and AraLAM in combination with the bacterial stimulator LPS. The interaction of the Toll-4 receptor (TLR4) with the LPS generates intracellular signaling, most especially through the NFkB transcription factor, which results in activation / maturation of DC. In fact, CD effectively matures in the presence of LPS alone (Figure 11b). Surprisingly, this LPS-induced activation is inhibited in the presence of ManLAM, as the expression levels of CD80, CD83 and CD86 activation markers were considerably lower than those of LPS-activated DC (Figure 11b). The observed inhibition of DC activation / maturation is specific for ManLAM, as AraLAM did not inhibit DC activation (Figure 11b). This is further supported by the ability of antibodies against DCSIGN, which inhibit ManLAM binding, to completely restore LPS-induced maturation in the presence of ManLAM (Figure 11b). These results indicate that ManLAM binding to DC-SIGN generates intracellular signals that interfere with TLR4-mediated DC activation. Moreover, this process is specific for the ManLAMDC-SIGN interaction and binding of DC-SIGN alone is not sufficient since DC-SIGN antibodies do not block LPS-induced activation / maturation of DC (Figure 11b). .

Example 10. BCG-induced maturation of M. bovis is inhibited by ManLAM.

Both M. tuberculosis and M. bovis BCG are capable of inducing DC maturation through their cell wall components through TLR2 and TLR4-mediated signaling.^33-36 . In fact, infection of immature DC by M. bovis BCG results in DC maturation, as demonstrated by increased expression of MHC class II and CD80, CD83, and CD86 costimulatory molecules following M. bovis BCG infection (Figure 12a ). Next, we investigated whether binding of ManLAM to DC-SIGN prevented M. bovis BCG-induced DC maturation, since immature DC attracted to mycobacterial sites of infection will find secreted ManLAM and intact mycobacteria. Surprisingly, M. bovis BCG-induced DC maturation is strongly inhibited by ManLAM (Figure 12b). The expression of MHC class II, CD80, CD83 and CD86 in M. bovis BCG-infected DC in the presence of ManLAM was considerably lower than in M. bovis BCG-infected DC (Figure 12b). In addition, maturation was almost completely restored when DCs were preincubated with the DC-SIGN blocking specific antibody (Figure 12b), demonstrating that the interaction of ManLAM with DC-SIGN prevents BCG maturation of DC from M. bovis. In addition, AraLAM did not block the maturation of DC by M. bovis BCG (Figure 11c), since the costimulatory molecules, CD80, CD83 and CD86, are expressed at similar levels in infected and treated infected DC. with AraLAM. This indicates that the DC-SIGN-ManLAM interaction blocks LPS-induced DC maturation as well as M. bovis BCG.

Example 11. Soluble S. mansoni egg antigens (SEA) bind to human immature dendritic (DC) cells through interaction with DC-SIGN.

Since DCs are instrumental in coordinating Thl-Th2 responses, we looked for a DC-expressed cell surface receptor that would interact with S. mansoni's SEA. To detect the binding of SEA to human immature DC, a fluorescent bead adhesion assay was developed. Fluorescent beads were pre-coated with monoclonal antibodies (MAbs) against SEA glycan antigens and then used to capture SEA. Conjugated beads were allowed to interact with DC. SEA is a glycoprotein mixture containing many immunogenic glycan antigens (3, 36). The major glycan antigens present in the SEA, and the recognition of these antigens by the antiglican MAbs, are depicted in Figure 13. To capture the SEA in fluorescent beads, we used MAbs against

GalNAch1,4GlcNAc (LDN) and GalNAch1,4 (FucD1, 2FucDl, 3) GlcNAc (LDN-DF) (Figure 13), both epitopes that occur in many glycoconjugates within the SEA and are absent in DC. Strong SEA binding to DC was observed with SEA-coated fluorescent beads which were coupled via anti-LDN MAb as well as anti-LDN-DF MAb (Figure 14b). SEA binding to DC was comparable in strength to the binding of HIV-1 gp120 to DC. Since DC-SIGN of type C lectins and MR, which are expressed by immature DC (Figure 14a), are potential receptors for glycan antigen recognition, we investigated whether antibodies directed against CRD of lectins of type C of these molecules, or hapten mannan could inhibit the binding of SEA to DC. SEA binding to DC was strongly blocked by both the anti-DC-SIGN MAb AZN-D1, which binds to DC-SIGN CRD, and by mannan, but not by anti-MR clone 19 MAb (Figure 14b). These data indicate that DC-SIGN is not only a recipient of DC pathogens for HIV1, but also for Schistosome SEA.

Example 12. A subtraction of SEA contains high affinity ligands for DC-SIGN.

In order to further analyze the binding properties of DC-SIGN to S. mansoni SEA, we investigated the binding of soluble chimeric DC-SIGN-Fc to SEA. In an ELISA-based assay, DC-SIGN-Fc showed efficient binding to SEA coated wells (Figure 15a). DC-SIGN-Fc binding to SEA is mediated by DC-SIGN CRD, as the interaction was completely inhibited by the anti-DC-SIGN antibody AZND1, or EGTA which removes Ca ions.²⁺ which are essential for carbohydrate binding. Binding of DC-SIGN-Fc to SEA is likely to be of high affinity as binding to very low SEA coating concentrations was observed (Figure 16). Since SEA contains many different glycoproteins, we investigated whether a subset of glycoproteins within SEA interact with DC-SIGN. SEA glycoproteins were separated by SDSPAGE and analyzed by Western blot with DC-SIGNFc and Le-reactive anti-glycan antibodies.^x and LDN-DF, respectively. Among the proteins present in SEA, a major protein of approximately 70-80 kD, and two high molecular weight secondary proteins interacted with soluble DC-SIGN-Fc (Figure 17). Notably, some apparent molecular weight glycoproteins similar to those reacting with DC-SIGN-Fc have been bound to Le glycan specific MAbs.^x and LDN-DF.

Example 13. DC-SIGN binds to α3-fucosylated glycans.

Many SEA glycoproteins are strongly fucosylated (3, 36). Since it has been described that DC-SIGN may exhibit binding to mannose and fucose, Western transfer of SEA has suggested that Le glycans^x and / or LDN-DF are present in SEA proteins with apparent MM similar to DC-SIGN binding SEA proteins, we explored whether SEA defucosylation affects their recognition by DCSIGN-Fc. Treatment of SEA with Xantomonas 01,3 / 1,4fucosidase resulted in a 50% decrease in anti-Le MAb reactivity.^x for SEA, while no loss in reactivity was observed as expected with the anti-LDN-DF Mab. The LDN-DF epitope contains terminal α2-linked fucose. Thus, the results indicate that glycosidase treatment specifically removed the part of the α3-fucose units present in SEA. Loss in 3/4 SEA fucose residues after treatment with Xantomonas al, 3 / al, 4 fucosidase also resulted in a 25% loss of DC-SIGN-Fc binding (data not shown). These data suggest that A1, 3 / A1, 4-linked fucose residues may be important for the binding of soluble DC-SIGN-Fc to SEA.

Next, we investigated whether one or more of the antigens of SEA Le^x, Fucosylated LDNF or LDN-DF (see Figure 13) may act as ligand (s) for DC-SIGN in SEA. We analyzed the potential of DC-SIGN-Fc to bind to neoglycoproteins containing these glycan antigens by ELISA. Results show that DC-SIGN-Fc strongly interacts with HSA-Le neoglycoprotein^x and to a lesser extent with BSA-LDNF, which both have a terminal α3-fucose. In contrast, DC-SIGN-Fc does not bind to BSA-LDN-DF in which α3-fucose is terminated with α2-fucose (Figure 18). Binding is fucose dependent as no binding has been observed to neoglycoproteins having Galpl, 4GlcNAc (LN) or GalNAc (81.4GlcNAc (LDN).^x and LDNF by DCSIGN is mediated through its CRD since binding was inhibited by anti-DC-SIGN and EDTA. In conclusion, these results indicate that DC-SIGN strongly recognizes α3-fucosylated trisaccharide Le^x and most likely interact with a3-fucosylated glycans in SEA such as Le^xand / or LDNF.

Example 14. The amino acid residue Val within DC-SIGN is crucial for binding DC-SIGN to SEA and Le^x DC-SIGN type C lectin domain binds two Ca ions²⁺ and amino acid residues in close contact with Ca²⁺ at site 2 (Glu³⁴⁷ , Asn³⁴⁹, Glu³⁵⁴ and Asn³⁶⁵) or with Ca²⁺at site 1 (Asp³²⁰ , Glu³²⁴ , Asn³⁵⁰ and Asp³⁵⁵) are essential for ligand binding³⁷. Since the above results indicated that α3-fucosylated trisaccharide Le^x Since SEA can act as a ligand for DC-SIGN, we investigated the binding properties of these antigens to K562 cell transfectors expressing mutated forms of DC-SIGN. The mutation from E to Ala, or E to Gin in DC-SIGN (Figure 19a) resulted in complete loss of interaction with SEA and Le^xindicating that binding to these antigens is mediated through the primary ligand binding site and is Ca-dependent²⁺ (Figure 19b). This is similar to the results described for binding DC-SIGN to HIV-1 gp120.³⁷.

It has recently been shown that a specific mutation in the DC-SIGN CRD (V³⁵¹G) allows binding of HIV-1 gp120, but disallows binding to ICAM-3, a T-cell ligand that has previously been shown to interact with DC-SIGN. Our results indicate that the DC-SIGN VG mutant does not bind to SEA or Le^xwhile continuing to observe the binding to HIV-1, indicating that Vai³⁵¹ essential for connecting to SEA and Le^xbut not to HIV-1 (Figure 19b). To determine if the SEA binding site overlaps with the ICAM-3 or HIV-1 binding sites in DC-SIGN, we explored whether SEA inhibits the interaction of DC-SIGN with ICAM-3 and HIV-gp120. 1 in a DC binding assay. The results demonstrate that SEA can inhibit the interaction between DC and ICAM-3 as effectively as anti-DC-SIGN MAb, while binding of DC-SIGN to HIV gp120 was only partially inhibited by SEA (Figure 19c). . These results demonstrate that the SEA binding site in DCSIGN may resemble the ICAM-3 binding site and may partially overlap the HIV-1 gp120 binding site.

Example 15. L-SIGN Does Not Interact with DC-SIGN Binding to SEA or Lewis X

L-SIGN, an adhesion receptor that resembles DC-SIGN in recognizing HIV ICAM-2, ICAM-3 and gp120, contains a CRD that is almost identical to DC-SIGN, and both receptors recognize N high mannose type glycans. Our above results demonstrated a new binding activity of DC-SIGN to fucosylated glycans and showed that Vai in DC-SIGN is essential for its binding activity. At the corresponding position in the L-SIGN CRD is a Being instead of Vai (Figure 19a), raising the question that L-SIGN can recognize SEA and Le^x. L-SIGN binding to these antigens was investigated by an adhesion assay where fluorescent beads containing SEA and Le^x-PAA, respectively, were incubated with transfected K562 cells expressing recombinant DC-SIGN and L-SIGN (Figure 19e). K562 cells expressing L-SIGN (KL-SIGN) do not interact with SEA or Le^x-PAA, but showed interaction with HIV-1 gp120. DC-SIGN expressing K562 cells (K-DC-SIGN) bound to SEA and Le^xas expected (Figure 19b). These data show that the two highly related receptors DC-SIGN and L-SIGN have carbohydrate binding specificity and pathogen recognition characteristics.

Example 16. New Carbohydrate Specificity Predicts and Confirms New Cellular Counterstructures for DC-SIGN; DC-SIGN recognizes Le carbohydrates^x on CD66a present in granulocytes to mediate DCgranulocyte interactions

DC-SIGN is known to interact with ICAM-2 and ICAM-3, however the glycan ligands on these molecules have not yet been identified. 0 Le group blood antigen^x(CD15) is expressed by gastric mucosal epithelial cells, and polymorphonuclear leukocytes (granulocytes) (Figure 20a); In fact, DC-SIGN-Fc coated beads are strongly bound to granulocytes (Figure 20b). In addition, DC-SIGN-Fc also binds strongly to granulocytes (Figure 21). Binding is specific for DC-SIGN as it can be inhibited by anti-DC-SIGN antibodies, mannan and EGTA calcium depletion, but also by an anti-Le^xindicating that cellular interaction is dependent on Le^x(Figures 20 and 21). Furthermore, an antibody against Le^xspecifically reduces the observed adhesion of DC-SIGN to CD66a, CD66e and CDllb by demonstrating that carbohydrates Le^x actually participate in the interaction (Figures 20, 54, 59D). Other glycan structures such as Le^Y him^B may also be involved since the observed blog is not complete. This demonstrates that granulocytes express a cell surface glycoprotein which expresses a glycan structure that is recognized by DC-SIGN.

In addition, we predict that based on the DC-SIGN carbohydrate recognition standard (Figure 1), DC-SIGN can also mediate the binding of DC to tumor cells since Le expression^Y is increased in many carcinomas including ovarian, pancreatic, o / prostate, breast, colon, and nonpegular lung cells, while sulfo-Le^The It is present in certain tumors that express mucins. Thus, these results indicate that the recognition of distinct carbohydrate structures by DC-SIGN may allow DC-mediated cell adhesion to T cells, endothelial cells, PMNs as well as tumor cells.

Immunoprecipitation studies have shown that DC-SIGN-Fc does not bind to granulocyte DC-SIGN, ICAM-2 or ICAM-3 cell counterstructures but does bind to a new 66kD protein glycoprotein is only present in granulocytes (Figure 22a). The fact that DCSIGN binds carbohydrates Le^x and the fact that granulocytes express high levels of CD66a, a cell surface receptor that has Le^x with a molecular weight of 66kD (Figure 22b and c) led us to examine the binding of CD66a-Fc to DC-SIGN-expressing immature and DC-SIGN-expressing transfectants. Strong CD66a binding was observed with DC-SIGN expressed by DC and DC-SIGN transfectors (Figure 23c), similar to that observed for ICAM-3 binding (Figure 23b). Uncoated beads do not interact with these cells (Figure 23a). The interaction of CD66a with DC-SIGN appeared to be stronger than the previously described ICAM-3-DC-SIGN interaction. These data indicate that CD66a is a novel cellular counter-structure of DC-SIGN and may play an important role in mediating DC-granulocyte cell interactions that play a role in innate immune responses.

Example 17. DC-SIGN binds tightly to a subset of cells in peripheral blood.

To determine whether DC-SIGN preferentially interacts with naive T cells expressing high levels of ICAM-3, as previously postulated, we first investigated the binding of DC-SIGN to the total PBL cell population. Surprisingly, DC-SIGN-Fc interacted with a population of CD3 ”cells that were CD56⁺ and belonged to the subset of NK cells (Figure 24). Moreover, the interaction was concentration dependent (Figure 25) and could be blocked with antibodies to DC-SIGN (Figures 24 and 25). NK CD56 cells⁺ in blood can be divided into two cell populations: NK CD5 6 cells^0bscuredCD16⁺and CD56^brightCD16 ”(Figure 26). DC-SIGN-Fc specifically bound to the NK CD5 6 population^obscuredCDl 6⁺ and the interaction could be inhibited by anti-DC-SIGN antibodies (Figure 26). ICAM-1-Fc did not interact with NK population

CD5 6^obscuredCDl 6⁺, demonstrating that the interaction of DC-SIGNFc is specific and not mediated by the Fc marker. Both NK cell populations were able to interact specifically with ICAM-1-Fc upon activation of their β2 integrin LFA-1 (Figure 26). Thus, these results indicate that DC interact with NK CD5 6 cells.^obscuredCDl 6⁺ through DC-SIGN. Surprisingly, both NK populations have similar levels of ICAM-2 and ICAM-3, indicating that DC-SIGN may bind other new ligands in the NK CD56 population.^obscuredCD16⁺. In fact, immunoprecipitation reveals that DC-SIGN-Fc binds, in addition to ICAM-2, also a molecule with a molecular weight of 166 kDa (Figure 27). This ligand could be CD166 or CD16, which is strongly glycosylated and is only expressed in NK cells.

Example 18. DC-SIGN mediates the interaction of DC-NK cells and is involved in the lysis of immature DC by NK cells.

To investigate whether the interaction of DC-SIGN with NK cells is relevant in NK-mediated immune response, we investigated DC lysis by NK cells and DC maturation by NK cells; two processes that have recently been identified as playing an important role in innate immunity.

DC-SIGN did not inhibit NK cell maturation of DC (Figure 28) but was strongly involved in NK-mediated immature DC lysis (Figure 29), as antibodies against DC-SIGN inhibited NK-mediated lysis of DC. A.D. These data indicate that DC-SIGN regulates the interaction of immature DC with NK cells by binding a 166 kDa protein to NK CD56 cells.^obscuredCD16⁺. This DC-SIGN-NK cell interaction also strongly improves NK-mediated lysis of DC-SIGN transfected cell lines (Figure 30), while antibodies against DC-SIGN inhibit lysis. These results indicate that DC-SIGN may be important in the interaction of DC with NK cells and that inhibition of this interaction prevents NK-mediated lysis.

Example 19. DC-SIGN Interacts with Virus herpes simplex Type 1 and 2

Previous results have shown that DC-SIGN interacts specifically with HIV-1.³⁹ and it was recently published that DC-SIGN binds to Ebola Virus⁴⁰. Therefore, we investigated whether DC-SIGN and its counterpart L-SIGN can interact with other viruses, which contain glycosylated envelope proteins. Surprisingly, DC-SIGN-Fc binds tightly to herpes simplex Virus (HSV) -1 and -2, and this interaction is specifically inhibited by antibodies against DC-SIGN (Figure 31). Further analysis demonstrates that DC-SIGN interacts with HSV gB glycoprotein (Figure 32), as DC-SIGN expressed by DCs and transfectors strongly binds gB-coated beads. This interaction is blocked with anti-DC-SIGN antibodies (Figure 32). DC-SIGN is specifically expressed by DC and L-SIGN is expressed by liver sinus endothelial cells (LSEC) and some macrophage populations. These data indicate that both DC-SIGN and L-SIGN could be involved in the interaction of HSV with DC and LSEC, respectively. In fact, DC binds strongly to HSV-1 gB and interactions are mediated by DC-SIGN since antibodies against DC-SIGN block this interaction (Figure 32). Thus, the interaction of DC with HSV is mediated by DC-SIGN and this type C lectin could be important in DC infection by these viruses. Furthermore, these data could suggest that these viruses target DCSIGN in DC not only to infect these DCs but also to evade the immune response by modulating the function of DCs as we have demonstrated for mycobacteria and HIV-1.

Example 20

Materials and methods particular to this example and relevant to the experiments described in the legends of figures 33 to 66.

Bacterial Strains.

Clinical isolates of H. pylori from different geographical origins (the Netherlands, Canada, Poland, Italy and the People's Republic of China) have been previously described.⁵, as well as H. pylori strain NCTC 11637 and phase variants lc, k4, l, k5, l, 3a, and lb⁹. The mutant has been described

Pylori 4187E and generation of futA (HP0379 gene) and futB (HPO651 gene) annulled mutants¹⁰. H. pylori J223 clinical isolate was obtained from Dr. HP Wirth⁶. Positive and negative variants for Le^{x / y} of J223 were isolated as previously described⁹. Briefly, biopsy bacteria were inoculated into Dent plates (Columbia agar, 5% horse blood + Dent supplement), followed by a single pass in liquid phase (Brucella broth + 3% fetal calf serum) and distribution over solid medium. , after which colony transfer detection of LPS phase variants occurred⁹. Genotyping through

<td>analysis by</td><td colspan="2">Polymorphism</td><td>in</td><td>Length</td><td colspan="2">Fragment</td>

<td>Amplified</td><td>(AFLP)</td><td>was</td><td colspan="2">performed as</td><td>described</td><td>at</td>

<td>reference³⁹</td><td>with</td><td colspan="2">following</td><td>modifications:</td><td colspan="2">: In a volume</td>

<td>end of 20</td><td>mL, 20</td><td>ng from</td><td>DNA</td><td>chromosomal</td><td>purified</td><td>was</td>

<td>digested with</td><td>1 U of</td><td>EcoRI</td><td colspan="3">(Pharmacia, Uppsala, Sweden)</td><td>and 1</td>

U from Msel (New England Biolabs, Beverly, MA). The final volume was then increased to 30 mL by the addition of 50 pmol each of EcoRI adapter and Msel adapter, 1.2 U T4 DNA ligase (Pharmacia), 1 mM ATP and ligase buffer. The adapters were allowed to bind to the restriction fragments for 16 h at 167 ° C, after which the sample was diluted with distilled water to a final volume of 500 mL. The fluorescent Texas Red labeled EcoO primer (50-AGACTGCGTACCAATTC-30; Isogen Bioscience, Maarssen, The Netherlands) and the unlabeled MseO primer (50 GACGATGAGTCCTGAG-30) were used for DNA amplification in a GeneAmp 9700 thermocycler PCR system ( PerkinElmer) with 35 cycles:

denaturation (30 to 94 ° C), pairing (30 to 65 56 ° C), and extension of the DNA molecule (60 to 72 ° C). In the first 12 cycles, the pairing temperature was decreased by 0.7 ° C per cycle. Upon completion of the cycling program, each sample was analyzed according to the manufacturer's instructions on a Vistra 725 automated DNA sequencer (Amersham Life Science). AFLP fluorescent images were stored as TIFF files with Vistra 2 TIFF software (Amersham).

Monoclonal and glycoconjugate antibodies.

H. pylori strains and phase variants were serotyped as previously described.⁵ with the following monoclonal antibodies (mAbs): Hpl51, specific for Le^Y, 6H3, specific for Le^x monomeric and 4D2, specific for H type 1 (all three a generous offer from R. Negrini, General Hospital, Brescia, Italy). MAb 54.1F6A, specific for Lewis x polymeric (a generous offering from G. van Dam, Leiden, the Netherlands), NAM61-1A2, antigen specific i (a generous offering from D. Blanchard, Regional Blood Transfusion Service, Nantes, France)¹⁰. The AZND1 and AZN-D2 MAbs²¹ were used to block DC-SIGN and MAb Clone 17 (a generous offering from S. Gordon, University of Oxford, UK) was used to block the mannose receptor. Synthetic glycoconjugates (Syntesome, Munich, Germany) comprised mono- and oligosaccharides that were multivalently linked to a polyacrylamide carrier. Le antigens^x ceramide-linked dimerics, trimers and tetramers were a generous offering from RR Schmidt, University of Konstanz, Germany, and were synthesized as described⁴⁰.

Cells

Immature DC were generated by culturing monocytes in RPMI1640 / 10% FCS in the presence of IL-4 (500 U / mL, ScheringPlough, Kenilworth, NJ) and GM-CSF (800 U / mL, ScheringPlough) for 5-8 days.⁴¹. K-562 cells, K562-DC-SIGN cells and RAW 264.7 macrophages were cultured as described.⁴¹.

Bacterial binding

H. pylori cells were labeled with FITC. Cells (50,000) in 0.5% BSA TSM were preincubated with 20 µg / ml mAb, 200 µg / ml manane or 10 mM EDTA for 10 min at RT. FITC-labeled bacteria (10 bacteria / cell) were added and incubated for 45 min at 37 ° C. Samples were analyzed by flow cytometry.

Soluble DC-SIGN-Fc Adhesion Test.

Soluble DC-SIGN adhesion assay was performed by ELISA⁴². The antigens (3.75 x 10⁶ bacterial cells / well or 5 pg / mL in the case of LPS or glycoconjugated) were coated on ELISA plates and soluble DC-SIGN-Fc binding (1 Dg / mL in TSM) was determined by an anti91 ELISA.

Human Ig-Fc. When indicated, DC-SIGN-Fc was preincubated with 20 µg / ml mAb, 200 µg / ml mannan or 5 mM EDTA for 10 min at room temperature.

Immunohistochemistry

After informed consent, 2 antral biopsies were collected during gastroscopy. Tissue cryosections were fixed in acetone and incubated with AZN-D1 followed by anti-mouse-HRP. Developing was performed with the ABC-AP Vectastain and AEC kit (Vetor Laboratories, Burlingame, CA) and sections were stained with hematoxylin contrast. In parallel, the other biopsy was analyzed for H. pylori infection by incubation in urease medium for 12 h. No differences in morphology and revelation were observed between uninfected and infected individuals.

DC activation and Thl / Th2 differentiation

In order to analyze maturation, DC (180,000 cells) were incubated with H. pylori for 1 h and a multiplicity of infection (MOI) of 1, 5, 10, 20, washed and cultured for 20 h. Cells were analyzed for maturation markers (CD80, CD86, CD83, HLADR) by flow cytometry and the supernatant was collected for cytochem ELISA. For T cell differentiation, DC were co-cultured with H. pylori (MOI of 10) for 2 days, washed and incubated with CD45RA +, CD4 + T cells (5,000 T cells / 20,000 DC). In parallel, CD were analyzed for the maturation markers described above and cytokine production following CD40L-transfected J558 stimulation (generous offer from P. Lane, University of Birmingham, UK) in the presence or absence of IFN-D. (1000 U / mL).

Quiescent T cells were again stimulated with PMA (10 ng / mL, Sigma-Aldrich, St. Louis, MO) and ionomycin (1 pg / mL, Sigma-Aldrich) for 6 h, the last 5 h in the presence of brefeldin A (10 pg / ml, Sigma-Aldrich). Single cell production of IL-4 and IFN-γ was determined by intracellular flow cytometry.

Results

DC-SIGN has been shown to have a carbohydrate specificity for mannose-containing carbohydrates, Lewis antigens and GlcNAc-containing structures (Figure 33, 34, 63-66). We have shown that the carbohydrate specificity of L-SIGN is different as it does not recognize Lewis antigen carbohydrate structures while recognizing structures with high mannose contents (Figures 33 and 34). The murine homologue mSIGNR1 recognizes, similar to DC-SIGN, high mannose and Lewis antigens, but also has specificity for sialylated Lewis antigens in contrast to DC-SIGN and L-SIGN (Figures 33 and 34). .

DC-SIGN is a particular type C lectin highly expressed in dendritic cells. Its function is to recognize antigens and process them and present them very efficiently in MHC class I and II molecules.

DC-SIGN has long been believed to be a pathogen recognition receptor that recognizes pathogens to activate the immune system, but as it now appears, the long list of pathogens targeting DC-SIGN all persist and escape. immunity by different mechanisms. We now have four examples of pathogens that target DC-SIGN to survive in the host.

1. HIV-1 and HCV target DC-SIGN to 'hide' within DC and escape intracellular routing to the lysosomal compartment.

2. ManLam secretion by mycobacteria targets DC-SIGN to induce negative modulation of TLR-induced DC maturation, and IL-10 induction.

3 Helicobacter pylori human gastric pathogen persists: Lipopolysaccharide phase variants modulate Thl / Th2 balance through interaction with the dendritic lectin cell DC-SIGN.

4 Lactobacilli target DC-SIGN in DC and induce regulatory T cells, inhibition of lactobacilli recognition by DC-SIGN reduces the regulatory state of T cells. Therefore, DC-SIGN is supposed to be a receptor that normally recognizes self-antigens to tolerate (Annual Review Immunology, 2004). We have demonstrated here that the lactobacilli approach of DC-SIGN can in fact induce regulatory T cells. In addition, it appears that DCSIGN may also recognize the CEA tumor antigen (CD66e) in particular Lewis X and Y. These tumor antigens may be secreted and suppress DC activation in a ManLam-like manner from tuberculosis mycobacteria.

The interaction of DC with granulocytes is elucidated by the fact that DC-SIGN recognizes CD66a and CD11b in granulocytes. In particular, the Lewis X antigen is recognized in these molecules. Thus, DC-SIGN acts as a cell adhesion receptor that mediates granulocyte cell interactions with DC. This cellular adhesive function between granulocytes and dendritic cells has never been described but it is likely that granulocyte-DC interaction is essential to bridge the innate immune response and adaptive immune response such that the granulocyte passes infectious agents to DC. building an appropriate immune response. In particular, granulocyte activates and matures DC, inducing up-regulation of cytokines and costimulatory molecules that are required for DC migration and initiation of the adaptive immune response. The interaction of CD with granulocytes is also observed in vivo for Crohn's disease. A new pathogen that interacts with DCSIGN in DC is Neisseria Meningitidis. Variant strains demonstrate that, in particular, an IgtB mutant strain interacts which contains a GlcNAc residue at the end position. Other experiments (Figure 66) demonstrate that DC-SIGN also recognizes GlcNac.

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39 Geijtenbeek 2000 as provided elsewhere in this list.

<td>40. Alvarez,</td><td>CPF.</td><td>Lasala, J. Carrillo,</td><td> 0.</td><td>Muniz, AL</td>

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