WO2003093415A2 - Methods useful in treatment and prevention of hiv infection - Google Patents

Abstract

A method for preventing or treating HIV infection comprising administering to an individual in need thereof an effective amount of a Langerin antagonist is provided. Also provided are methods for identifying compounds which interfere with the infectivity of HIV and compounds identified by those methods.

Description

METHODS USEFUL IN TREATMENT AND PREVENTION OF HIN INFECTION

FIELD OF THE INVENTION

The present invention relates to methods of preventing HIN infection comprising blocking the interaction between an HIN envelope glycoprotein and human Langerin.

BACKGROUND OF THE INVENTION

Dendritic cells (DC) are antigen-presenting cells that are required for initiation of a specific immune response. See, e.g., Banchereau et al., 1998, Nature 392:242-52. One type of DC is exemplified by Langerhans cells (LC), immature DC cells that reside in non- lymphoid tissue, such as the epidermis, and whose primary function is to capture antigen. See, e.g., Steinman et al., 1995, J Exp. Med. 182:283-288.

Antigen capture is achieved primarily through specialized surface-membrane endocytic structures or through macropinocytosis, thus permitting Langerhans cells to concentrate solutes which are present in large volumes of fluid. See, e.g., Sallusto et al., 1995, J. Exp. Med. 182:389-400. Concomitant with processing of antigen in specialized organelles of the endocytic pathway, DC, such as Langerhans cells, migrate to secondary lymphoid tissue and undergo a number of phenotypic modifications. These maturation events ultimately translate into highly efficient presentation of processed antigen, by appropriate MHC molecules of the DC, to T cells.

In particular, the maturation process of the Langerhans cell includes loss of adhesion receptors such as E-cadherin, and the disappearance of Birbeck granules (BG), which are characteristic for LC. Conversely, upon acquisition of antigen-presentation function, costimulatory receptors such as the CD80 and CD86 molecules are upregulated on LC to permit T cell activation. Maturation events can be reconstituted in vitro by TNF-α and CD40-ligand which mimic, respectively, the response to pro-inflammatory cytokines following encounter with pathogen, and the response to contact with T cells in secondary lymphoid tissue. See, e.g., Caux et al., 1996, J. Exp. Med. 184:695-706. Recently, human Langerin has been identified as a novel transmembrane C-type lectin specifically expressed by Langerhans cells (Valladeau et al., 2000, Immunity 12:71-81). Langerin functions as an endocytic receptor routing extracellular ligands into Birbeck granules, the unique organelles of Langerhans cells. In addition, Langerin induces the formation of Birbeck granules by membrane zippering (Valladeau et al., 2000, Immunity 12:71-81). Langerin is thus a candidate to target antigen selectively to Langerhans cells in vivo, with potential applications to boost immune responses against weak antigens. The full genomic sequences of human and mouse Langerin and antibodies to the molecule are disclosed in WO 00/18803, published April 6, 2000.

The molecule DC SIGN, a C-type lectin with considerable homology to Langerin, was recently demonstrated to be expressed on dendritic cells in mucosal tissues and to bind the human immunodeficiency virus (HIV) envelope glycoprotein gpl20 (Geijtenbeek et al., 2000, Cell 100:587-597; WO 01/64752). Furthermore, DC-SIGN promotes efficient viral infection of cells bearing CD4 and chemokine receptors. It is thus proposed that DC-SIGN captures HIN-1 in the periphery and permits its transport to T cell areas of secondary lymphoid organs, resulting in productive infection (Geijtenbeek et al., 2000, Cell 100:587- 597;WO 01/64752).

Natural transmission of HIV infection requires the dissemination of virus from sites of infection at mucosal surfaces to T cell zones in secondary lymphoid organs, where extensive viral replication occurs in CD4+ T cells (Fauci et al., 1996, Nature 384:529-534). Immature dendritic cells in the skin and mucosal surfaces are believed to represent the first cells targeted by HIV, that subsequently migrate to draining lymph nodes and transmit the virus to CD4+ T cells during the process of antigen presentation (Grouard et al., 1997, Curr. Opin Immunol. 9(4):563-567). Entry of HIN-1 into immature dendritic cells has been reported to proceed in a CD4- independent mechanism, suggesting the involvement of other receptors (Blauvelt et al., 1997, J. Clin. Invest. 100:2043-2053). Notably, DC-SIGN is absent from mucosal epithelial surfaces where initial contact between virus and the immune system is expected following sexual exposure.

Therefore, there is a need to identify the protein or proteins involved in the mediation of HIV from the mucosal surfaces to the T cell areas of secondary lymphoid organs. There is also a need for methods of identifying agents that will interfere with this mediation. SUMMARY OF THE INVENTION

The present invention meets this need by identifying a DC receptor which plays a part in HIN-1 recognition in Langerhans cells of mucosal tissues. The invention is based, in part, upon the discovery that human Langerin binds the HIN-1 glycoprotein gp 120 in a dose- dependent manner, and that binding is inhibited by anti-Langerin neutralizing monoclonal antibodies. Thus, the invention provides a method for preventing or treating HIN infection comprising administering to an individual in need thereof an effective amount of an agent which modulates the Langerin/gpl20 interaction. In preferred embodiments, the agent which modulates the Langerin/gpl20 interaction is a Langerin antagonist. The invention also provides new intervention strategies and novel approaches toward prevention and therapy against HIV infection.

In preferred embodiments, the Langerin antagonist is a binding composition, such as an antibody or antibody fragment. In other preferred embodiments, the Langerin antagonist is a small molecule, or a nucleotide sequence included in a gene delivery vector.

In certain embodiments, the Langerin antagonist is administered intravenously, intradermally, intramuscularly, subcutaneously, or topically.

The methods of the invention may further comprise administering an effective amount of one or more other agents which prevent HIN binding or entry. In certain embodiments, the agent preventing HIN binding or entry is a chemokine receptor antagonist. Preferably, the chemokine receptor antagonist is an antagonist of CCR5 or CXCR4. In preferred embodiments, the chemokine receptor antagonist is an antibody or antibody fragment, a soluble natural ligand, a small molecule or a nucleotide sequence included in a gene delivery vector.

In certain embodiments, the agent preventing HIN entry is an antagonist of the HIV fusogenic element gp41. Preferably, the HIN gp41 antagonist is an antibody or antibody fragment, a soluble natural ligand, a small molecule, or a nucleotide sequence included in a gene delivery vector.

In further embodiments of the invention, an agent which inhibits Langerhans cell maturation may also be advantageously administered. Preferably, the agent which inhibits

Langerhans cell maturation is a CD40 antagonist or a RANK antagonist. More preferably, the CD40 antagonist or RANK antagomst is an antibody or antibody fragment, a soluble natural ligand, or a small molecule.

The methods of the invention may further comprise administration of an effective amount of an agent which modulates the DC-SIGN/gpl20 interaction in combination with the Langerin antagonist. Preferably, the agent which modulates the DC-SIGN/gpl20 interaction is a DC-SIGN antagonist or a gp 120 antagonist. More preferably, the DC-SIGN antagonist is in the form of an antibody or antibody fragment, soluble natural ligand, or small molecule.

Also provided are methods of identifying compounds that interfere with the productive infectivity of HIV. One such embodiment comprises contacting a first cell which expresses Langerin with HIN in the presence of a test compound. A second cell, permissive to HIN productive infection, is added to the system, and the effect of the test compound on HIV replication capacity is tested. The present invention further provides the compounds identified by these methods.

DETAILED DESCRIPTION OF THE INVENTION

All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety including all figures, graphs, and drawings.

General

The present invention provides methods for inhibiting the infectivity of HIV in an individual comprising administering to said individual an effective amount of an agent which modulates a Langerin/gpl20 interaction, in particular a Langerin antagomst. The invention is based, in part, on the discovery that recombinant gpl20 envelope protein of HIN- 1 binds to human Langerin both in transfected cells and in Langerhans cells. Furthermore, Langerin is an endocytic receptor that induces the formation of Birbeck granules, and routing of extracellular material into these organelles particular to Langerhans cells. The expected consequences of these findings is that HIN virus will enter Langerhans cells via Langerin, and subsequently be transported within Birbeck granules as the Langerhans cells migrate from mucosal epithelia to draining lymph nodes. Within the lymph node environment, virus contained in immigrant Langerhans cells would subsequently be transmitted to infect CD4+ T lymphocytes during the process of antigen presentation where the two cell types are in intimate contact. This discovery indicates that the interaction between Langerin and gpl20 is an important target for therapeutic intervention and vaccine development. This, in turn, allows the construction of drug screening assays to identify compounds that can interfere with these processes and thereby prevent or treat AIDS.

First, the inventors have shown that 293 cells transfected with human Langerin cDNA bind iodinated recombinant gpl20 from HIV-1 in a dose-dependent manner (see Example 20). The inventors next extended these results to recombinant gpl20 protein preparations from MN (XX4 tropic) JRFL (R5 tropic) and SF-2 (X4RR5 tropic) HIN-1 strains. Binding of gpl20 to cells transfected with Langerin cDΝA was of comparable magnitude to that observed with DC-SIGΝ, used as a positive control. Using biotin-labeled gpl20, binding was confirmed on HeLa cells transfected to stably express human Langerin (Example 20). Furthermore, binding was inhibited by mannan (a ligand of the extracellular lectin domain of Langerin), and by several monoclonal antibodies (mAbs) directed against Langerin (Example 21). Further mapping of the binding was performed using a truncated form of Langerin (Example 22). Together, the results demonstrate that gpl20 binds Langerin in its carbohydrate-recognition domain (CRD), and the specificity of the interaction as determined by neutralizing mAbs to Langerin. Finally, gpl20 was also found to efficiently and specifically bind to Langerin on Langerhans cells obtained in vitro from cultures of CD34+ progenitors (Example 23). The binding was independent of CD4 and of DC-SIGΝ. Together, these data indicate that Langerin is a gpl20-binding protein. It is expected that Langerin binds and internalizes HIN-1 viral particles for subsequent transmission to CD4+ T cells. Agents which antagonize Langerin will therefore be useful for inhibiting infection by HIV-1.

Various terms are used in the specification, which are defined as follows:

As used herein, and unless otherwise specified, the terms "agent", "potential drug",

"compound", "test compound" or "potential compound" are used interchangeably, and refer to chemicals which potentially have a use as an inhibitor of HIN infection and/or for use in treatment/prevention of AIDS. Therefore, such "agents", "potential drugs", "compounds", "test compounds" or "potential compounds" may be used, as described herein, in drug assays and drug screens and the like.

The term "effective amount" is used herein to mean an amount sufficient to reduce by at least about 15 percent, preferably by at least 15 percent, preferably by at least 50 percent, more preferably by at least 90 percent, and most preferably prevent, a clinically significant deficit in the activity, function and response of the host. Alternatively, an effective amount is sufficient to cause an improvement in a clinically significant condition in the host.

A molecule is "antigenic" when it is capable of specifically interacting with an antigen recognition molecule of the immune system, such as an immunoglobulin (antibody) or T cell antigen receptor. An antigenic polypeptide contains at least about 5, and preferably at least about 10, amino acids. An antigenic portion of a molecule can be that portion that is immunodominant for antibody or T cell receptor recognition, or it can be a portion used to generate an antibody to the molecule by conjugating the antigenic portion to a carrier molecule for immunization. A molecule that is antigenic need not be itself immunogenic. i.e., capable of eliciting an immune response without a carrier.

Administration "in combination" refers to both simultaneous and sequential administration. The agents can be delivered or administered at the same site or a different site and can be administered at the same time or at different times during the course of a common treatment schedule. In the latter case, the two agents are administered sufficiently close in time to achieve the intended effect.

In a specific embodiment, the term "about" means within 20%, preferably within 10%, and more preferably within 5%.

I. LANGERIN NUCLEIC ACIDS AND PROTEINS

The present invention contemplates the use of the nucleic acids encoding Langerin (e.g. encoding the human protein having the amino acid sequence defined in SEQ ID NO: 2 of WO 00/18803) and encoding fragments thereof including chimeric/fusion proteins.

The natural Langerin protein mediates various physiological responses leading to biological or physiological responses in target cells. The Langerin gene and protein were first described in WO 00/18803, published April 6, 2000, which is expressly incorporated herein by reference. Langerhans DC cells are responsible for antigen presentation, e.g., presentation of haptens in hypersensitivity reactions. Langerin is a 40 kDa N-glycosylated protein. Immunoprecipitation from DC extracts with the antibody DCGM4 (also disclosed in WO 00/18803) and subsequent elution with SDS-PAGE sample buffer yielded a homogeneous band of 40-42 kDa molecular mass. Two dimensional gel electrophoresis analysis confirmed the molecular mass of the molecule and indicated a pi of 5.2-5.5. Finally, Langerin is a glycoprotein, and most of the carbohydrate constituents were removed by N-glycosylase treatment. The Langerin protein is a type II membrane lectin, with a calcium-dependent carbohydrate recognition domain.

II. ANTAGONISTS

A. Antibodies

Antibodies can be raised to the various mammalian, e.g., primate Langerin proteins and fragments thereof, both in naturally occurring native forms and in their recombinant forms. Denatured antigen detection can also be useful in, e.g., Western analysis. Anti- idiotypic antibodies are also contemplated, which would be useful as agonists or antagonists of a natural Langerin protein or an antibody.

Antibodies, including binding fragments and single chain versions, against predetermined fragments or the whole of the protein can be raised by immunization of animals. Monoclonal antibodies are prepared from cells secreting the desired antibody.

These antibodies can be screened for antagonistic activity. These monoclonal antibodies will usually bind with at least a Kpj of about 1 mM, more usually at least about 300 μM, typically at least about lOOμM, more typically at least about 30 μM, preferably at least about 10 μM, and more preferably at least about 3 μM or better.

In some instances, it is desirable to prepare monoclonal antibodies from various mammalian hosts, such as mice, rodents, primates, humans, etc. Description of techniques for preparing such monoclonal antibodies may be found in, e.g., Stites, et al. (eds.) Basic and Clinical Immunology (4th ed.), Lange Medical Publications, Los Altos, CA, and references cited therein; Harlow and Lane (1988) Antibodies: A Laboratory Manual. CSH Press; Goding (1986) Monoclonal Antibodies: Principles and Practice (2d ed.) Academic Press, New York; and particularly in Kohler and Milstein (1975) in Nature 256: 495-497, which discusses one method of generating monoclonal antibodies. Each of these references is incorporated herein by reference. Summarized briefly, this method involves injecting an animal with an immunogen. The animal is then sacrificed and cells taken from its spleen, which are then fused with myeloma cells. The result is a hybrid cell or "hybridoma" that is capable of reproducing in vitro. The population of hybridomas is then screened to isolate individual clones, each of which secrete a single antibody species to the immunogen. In this manner, the individual antibody species obtained are the products of immortalized and cloned single B cells from the immune animal generated in response to a specific site recognized on the immunogenic substance.

Other suitable techniques involve in vitro exposure of lymphocytes to the antigenic polypeptides or, alternatively, selection of libraries of antibodies in phage or similar vectors. See, Huse, et al. (1989) "Generation of a Large Combinatorial Library of the Immunoglobulin Repertoire in Phage Lambda," Science 246:1275-1281; and Ward, et al. (1989) Nature 341 :544-546, each of which is hereby incorporated herein by reference. The polypeptides and antibodies of the present invention may be used with or without modification, including chimeric or humanized antibodies. Frequently, the polypeptides and antibodies will be labeled by joining, either covalently or non-covalently, a substance which provides for a detectable signal. A wide variety of labels and conjugation techniques are known and are reported extensively in both the scientific and patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties, magnetic particles, and the like. Patents teaching the use of such labels include U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. Also, recombinant immunoglobulins may be produced, see Cabilly, U.S. Patent No. 4,816,567; or made in transgenic mice, see Mendez, et al. (1997) Nature Genetics 15:146-156. These patents are incorporated herein by reference.

In a specific embodiment, antibodies that agonize or antagonize the activity of Langerin can be generated. Such antibodies, when conjugated with a toxin or radioactive element, can be used to target HiN-facilitative cells for destruction. Thus, cells harboring

HIN, particularly in its dormant phase, can be destroyed with antibodies when such antibodies are specific for Langerin, gpl20, or Langerin gpl20 complex.

In addition to antibodies, binding compositions or agents may also be used in the methods of the invention. A binding composition or agent refers to molecules that bind with specificity to Langerin protein, e.g., in a ligand-receptor type fashion, an antibody-antigen interaction, or compounds, e.g., proteins which specifically associate with Langerin protein, e.g., in a natural physiologically relevant protein-protein interaction, either covalent or non- covalent. The molecule may be a polymer, or chemical reagent. This implies both binding affinity and binding specificity or selectivity. These binding compositions typically bind to a Langerin protein with high affinity, e.g. at least about 100 nM, usually better than about 30 nM, preferably better than about 10 nM, and more preferably at better than about 3 nM. A functional analog may be a protein with structural modifications, or may be a wholly unrelated molecule, e.g., which has a molecular shape which interacts with the appropriate binding determinants. The proteins may serve as antagonists of a receptor, see, e.g., Goodman, et al. (eds. 1990) Goodman & Gilman's: The Pharmacological Bases of Therapeutics (8th ed.) Pergamon Press, Tarrytown, N.Y. Assays can be developed to identify molecules that antagonize the binding of HIV-1 to Langerin. A typical assay would consist of screening a compound library for molecules that inhibit the binding of labeled soluble recombinant Langerin to immobilized gpl20.

Soluble fragments of the Langerin antibodies may also be used in the methods of the invention. Solubility of a polypeptide or fragment depends upon the environment and the polypeptide. Many parameters affect polypeptide solubility, including temperature, electrolyte environment, size and molecular characteristics of the polypeptide, and nature of the solvent. Typically, the temperature at which the polypeptide is used ranges from about 4° C to about 65° C. Usually the temperature at use is greater than about 18° C and more usually greater than about 22° C. For therapeutic purposes, the temperature will usually be body temperature, typically about 37° C for humans, though under certain situations the temperature may be raised or lowered in situ or in vitro.

B. Functional Variants

The blocking of physiological response mediated by Langerin proteins may result from the inhibition of binding of the molecules to a natural binding partner, e.g., through competitive inhibition. Thus, in vitro assays of the present invention will often use isolated protein, membranes from cells expressing a recombinant membrane associated Langerin protein, soluble fragments comprising binding segments, or fragments attached to solid phase substrates. These assays will also allow for the diagnostic determination of the effects of either binding segment mutations and modifications, or protein mutations and modifications, e.g., analogs. In particular, Langerin is expressed on immature DC , but the molecule is lost after DC cell maturation.

This invention also contemplates the use of competitive drug screening assays, e.g., where neutralizing antibodies to Langerin or binding partner fragments compete with a test compound for binding to the protein. In this manner, the antibodies can be used to detect the presence of any polypeptide which shares one or more antigenic binding sites of the protein and can also be used to occupy binding sites on the protein that might otherwise interact with a binding partner. Additionally, neutralizing antibodies against the Langerin protein and soluble fragments of the antigen which contain a high affinity binding site, can be used to inhibit antigen function in cells or tissues, e.g., cells or tissues experiencing abnormal or undesired physiology. "Derivatives" of the Langerin antigens, and of antibodies, include amino acid sequence mutants, glycosylation variants, and covalent or aggregate conjugates with other chemical moieties. Covalent derivatives can be prepared by linkage of functionalities to groups which are found in the Langerin amino acid side chains or at the N- or C- termini, by means which are well known in the art. These derivatives can include, without limitation, aliphatic esters or amides of the carboxyl terminus, or of residues containing carboxyl side chains, O-acyl derivatives of hydroxyl group-containing residues, and N-acyl derivatives of the amino terminal amino acid or amino-group containing residues, e.g., lysine or arginine. Acyl groups are selected from the group of alkyl-moieties including C3 to C18 normal alkyl, thereby forming alkanoyl aroyl species. Covalent attachment to carrier proteins may be important when immunogenic moieties are haptens.

In particular, glycosylation alterations are included, e.g., made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing, or in further processing steps. Particularly preferred means for accomplishing this are by exposing the polypeptide to glycosylating enzymes derived from cells which normally provide such processing, e.g., mammalian glycosylation enzymes. Deglycosylation enzymes are also contemplated. Also embraced are versions of the same primary amino acid sequence which have other minor modifications, including phosphorylated amino acid residues, e.g., phosphotyrosine, phosphoserine, or phosphothreonine.

The phosphoramidite method described by Beaucage and Carruthers (1981) Terra.

Letts. 22:1859-1862, will produce suitable synthetic DNA fragments. A double stranded fragment will often be obtained either by synthesizing the complementary strand and annealing the strand together under appropriate conditions or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.

Such polypeptides may also have amino acid residues which have been chemically modified by phosphorylation, sulfonation, biotinylation, or the addition or removal of other moieties, particularly those which have molecular shapes similar to phosphate groups. In some embodiments, the modifications will be useful labeling reagents, or serve as purification targets, e.g., affinity ligands. This invention also contemplates the use of derivatives of the Langerin proteins other than variations in amino acid sequence or glycosylation. Such derivatives may involve covalent or aggregative association with chemical moieties. These derivatives generally fall into the three classes: (1) salts, (2) side chain and terminal residue covalent modifications, and (3) adsorption complexes, for example with cell membranes. Such covalent or aggregative derivatives are useful as immunogens, as reagents in immunoassays, or in purification methods such as for affinity purification of antigens or other binding proteins. For example, a Langerin molecule can be immobilized by covalent bonding to a solid support such as cyanogen bromide-activated Sepharose, by methods which are well known in the art, or adsorbed onto polyolefin surfaces, with or without glutaraldehyde cross-linking, for use in the assay or purification of anti-Langerin protein antibodies or other binding partner. Langerin molecules can also be labeled with a detectable group, for example radioiodinated by the chloramine T procedure, covalently bound to rare earth chelates, or conjugated to another fluorescent moiety for use in diagnostic assays. Purification of Langerin protein may be effected by immobilized antibodies or binding partners.

In addition to the antibodies and mimetics described herein, other antagonists which can be used in the invention include small molecules antagonists, antisense nucleotide sequences, or nucleotide sequences included in gene delivery vectors such as adenoviral or retroviral vectors that are shown in a binding or functional assay to inhibit the activation of the receptor. It is well known in the art how to screen for small molecules which specifically bind a given target, such as a receptor. See, e.g., Meetings on High Throughput Screening, International Business Communications, Southborough, MA 01772-1749.

Recombinant Langerin antagonists can be purified and then administered to a patient.

These reagents can be combined for therapeutic use with additional active ingredients, e.g., in conventional pharmaceutically acceptable carriers or diluents, along with physiologically innocuous stabilizers and excipients. These combinations can be sterile, e.g., filtered, and placed into dosage forms as by lyophilization in dosage vials or storage in stabilized aqueous preparations. This invention also contemplates the use of antibodies or binding fragments thereof which are not complement binding.

Using Langerin or fragments thereof to screen for binding partner or for compounds having binding affinity to Langerin antigen can be performed including the isolation of associated compounds. Subsequent biological assays can then be utilized to determine if a putative ligand or binding agent can provide competitive binding, which can block intrinsic stimulating activity. Langerin fragments can be used as a blocker or antagonist in that it blocks the activity of ligand or binding agent. This invention further contemplates the therapeutic use of antibodies to Langerin as antagonists. This approach will be particularly useful with other Langerin protein species variants and other members of the family.

π. COMBINATION THERAPY

Additional agents which prevent HIN binding or entry may also be administered according to the methods of the invention. Examples of agents which prevent HIN binding include antagonists of chemokine-receptors such as CCR5 or CXCR4. Langerhans cells are known to express both of these HIV coreceptors (Kawamura et al., 2001, Eur. J. Immunol. 31: 360-368; Tchou et al., 2001, J. Leukoc. Biol. 70: 313-321). Small-molecule antagonists of CCR5 or CXCR4 might therefore prove to be effective components of HIV-1 combination therapies (Blair et al., 2000, Drug Development Today 5: 183-194).

Examples of agents which prevent HIV entry include antagonists of the HIV fusogenic element gρ41. Binding of HIV to its receptors induces conformational changes in gp41, unmasking the fusion peptide and facilitating its insertion into the host cell lipid bilayer (Chan et al., 1998, Cell, 93: 681-684). The structure of the fusion-active hairpin of gρ41 reveals prominent contacts between the C-helices and the Ν-peptide coiled-coil, and deep cavities within hydrophobic grooves formed by the Ν-terminal elements, which are potentially useful targets for inhibiting HIN-induced fusion (Chan et al, 1998, Cell, 93: 681- 684; Chan, et al., 1997, Cell, 89: 263-273).

Antagonists of the DC-SIGΝ/gpl20 interaction may also be further administered as part of the methods of the invention. Several examples of DC-SIGN antagonists, gpl20 antagonists, and antagonists of the DC-SIGN/gpl20 interaction are disclosed in WO 01/64752, published September 7, 2001, which is hereby expressly incorporated herein by reference. Such antagonists may be in the form of an antibody, soluble natural ligand, or small molecule.

Also, agents inhibiting Langerhans cell maturation may be advantageously administered. Such agents could be useful as a second barrier to prevent possible transmission by Langerhans cells that may have escaped the Langerin antagonist, and that consequently could harbor virus upon reaching lymph nodes draining the site of contamination. An agent inhibiting Langerhans cell maturation would prevent their expression of costimulatory molecules and cytokines necessary to recruit CD4+ T cells that may become infected. Examples of such agents (in the form of an antibody, soluble natural ligand, or small molecule) include an antagonist of CD40 (Caux, et al., 1994 J. Exp. Med., 180: 1263-1272 ) or of RANK (Anderson, et al., 1997, Nature, 390: 175-179).

The quantities of reagents necessary for effective therapy will depend upon many different factors, including means of administration, target site, reagent physiological life, pharmacological life, physiological state of the patient, and other medicants administered. Thus, treatment dosages should be titrated to optimize safety and efficacy. Typically, dosages used in vitro may provide useful guidance in the amounts useful for in situ administration of these reagents. Animal testing of effective doses for treatment of particular disorders will provide further predictive indication of human dosage. The actual dosage of reagent, formulation or composition that modulates an immunological disorder depends on many factors, including the size and health of an organism, however, one of ordinary skill in the art can use teachings describing methods and techniques for determining clinical dosages. See, e.g., Spilker (1984) Guide to Clinical Studies and Developing Protocols. Raven Press Books, Ltd., New York, esp. pp. 7-13, 54-60; Spilker (1991) Guide to Clinical Trials. Raven Press, Ltd., New York, esp. pp. 93-101; Craig and Stitzel (eds. 1986) Modern Pharmacology, 2d ed., Little, Brown and Co., Boston, esp. pp. 127-33; Speight (ed. 1987) Avery's Drug Treatment: Principles and Practice of Clinical Pharmacology and Therapeutics, 3d ed., Williams and Wilkins, Baltimore, esp. pp. 50-56; Tallarida, et al. (1988) Principles of General Pharmacology. Springer-Nerlag, New York, esp. pp. 18-20; Gilman, et al. (eds.) Goodman and Gilman's: The Pharmacological Bases of Therapeutics, latest ed., Pergamon Press; Remington's Pharmaceutical Sciences, latest ed., Mack Publishing Co., Easton, Penn.; and Rich, et al. (1998) Clinical Immunology: Principles and Practice Vols. I & II, Mosby, St. Louis, Mo., each of which is hereby incorporated herein by reference in its entirety including all drawings and figures. Methods for administration are discussed therein and below, e.g., for oral, intravenous, intraperitoneal, or intramuscular administration, transdermal diffusion, and others. Pharmaceutically acceptable carriers will include water, saline, buffers, and other compounds described, e.g., in the Merck Index. Merck & Co., Rahway, New Jersey. Because of the likely high affinity binding, or turnover numbers, between a putative ligand or binding agent and its binding partner, low dosages of these reagents would be initially expected to be effective. Thus, dosage ranges would ordinarily be expected to be in amounts lower than 1 mM concentrations, typically less than about 10 μM concentrations, usually less than about 100 nM, preferably less than about 10 pM (picomolar), and most preferably less than about 1 fM (femtomolar), with an appropriate carrier. Slow release formulations, or slow release apparatus will often be utilized for continuous administration.

Langerin antagonists, agents preventing HIN binding or entry, and agents inhibiting Langerhans cell maturation may be administered directly to the host to be treated or, depending on the size of the compounds, it may be desirable to conjugate them to carrier proteins such as ovalbumin or serum albumin prior to their administration. Therapeutic formulations may be administered in many conventional dosage formulations. While it is possible for the active ingredient to be administered alone, it is preferable to present it as a pharmaceutical formulation. Formulations comprise at least one active ingredient, as defined above, together with one or more acceptable carriers thereof. Each carrier must be both pharmaceutically and physiologically acceptable in the sense of being compatible with the other ingredients and not injurious to the patient. Formulations include those suitable for oral, rectal, nasal, or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration. The formulations may conveniently be presented in unit dosage form and may be prepared by methods well known in the art of pharmacy. See, e.g., Gilman, et al. (eds. 1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics, 8^th Ed., Pergamon Press; and Remington's Pharmaceutical Sciences, current ed., Mack Publishing Co., Easton, Penn.; Avis, et al. (eds. 1993) Pharmaceutical Dosage Forms: Parenteral Medications Dekker, ΝY; Lieberman, et al. (eds. 1990) Pharmaceutical Dosage Forms: Tablets Dekker, ΝY; and Lieberman, et al. (eds. 1990) Pharmaceutical Dosage Forms: Disperse Systems Dekker, ΝY. The therapy of this invention may be combined with or used in association with other therapeutic agents.

III. SCREENING METHODS

The present invention contemplates methods for identifying agonists and antagonists of HIN entry using various screening assays known in the art. Such agonists or antagonists can competitively inhibit HIN binding to Langerin. Any screening technique known in the art can be used to screen for antagonists of the Langerin/gpl20 association. The present invention contemplates screens for small molecules, as well as screens for natural ligands that bind to and antagonize such activity in vivo.

A typical method for identifying a compound that interferes with the binding and entry of HIV comprises contacting Langerin with gpl20 in the presence of a test compound, wherein a Langerin-gpl20 complex forms in the absence of the test compound. For example, one assay utilizes a cell line engineered by transfection to stably express Langerin at its cell surface. Test compounds are added into the system and unbound compound washed away. A preparation of gpl20 radiolabeled with ¹²⁵I or with a fluorophore is added into the assay, and excess (unbound) gpl20 washed away. The amount of bound gpl20 is quantitated in the presence and absence of test compounds. A test compound inhibiting binding between Langerin and gpl20 results in a diminished signal. This assay can alternatively be performed with recombinant Langerin protein coated onto microwell plates.

In another type of assay, Langerin and gp 120 are each labeled with fluorophores with suitably overlapping spectra, such that physical interaction between the two proteins results in fluorescence resonance energy transfer (FRET). Test compounds are added to the labeled Langerin preparation and fluorescence is measured following subsequent addition of labeled gpl20. If binding of Langerin and gpl20 is unaffected by test compound, FRET will occur, typically resulting in green fluorescence emission. A test compound blocking the binding of Langerin and gpl20 will disrupt FRET by causing an increase in the distance between the two fluorophores, thus typically resulting in blue fluorescence emission. This assay configuration would make use of Langerin and gpl20 on solid-phase support (ie inert polymer beads). FRET technology is suitable for ultra-high-throughput screening on an industrial scale (Mere et al, 1999, Drug Discovery Today, 4: 363-369).

Test compounds inhibiting binding in the two previously described assays are highly likely to inhibit entry of gpl20 and HIN subsequent to binding to Langerin. This is confirmed

19S in an internalization assay wherein accumulation of labeled gpl20 (typically I radiolabeled) is quantitated in Langerin-positive cells at 37°C in the presence or absence of compound.

Further assays which may be developed to identify compounds that interfere with the productive infection of HIN comprise contacting a first cell which expresses Langerin with HIV infectious particles in the presence of a test compound, wherein the addition of a second cell allows productive HIN replication in the absence of test compound. A typical assay consists of sequential addition of test compound followed by HIN infectious particles at 37°C to cells expressing cell surface Langerin. A second cell type is subsequently added into the system. This second cell type is permissive for HIN replication (typically a T cell). HIV replication is quantitated (ie by determination of HIV p24 protein synthesis) in the presence or absence of test compounds. Compounds inhibiting Langerin-mediated HIV replication in permissive cells (trαn-.-infection) are identified by reduced p24 levels. EXAMPLES

The invention can be illustrated by way of the following non-limiting examples.

Example 1

Dendritic Cells

Collection and Purification of cord blood CD34+ HPC

Umbilical cord blood samples were obtained according to standard institutional guidelines for human samples. Cells bearing CD34 antigen were isolated from mononuclear fractions by positive selection with Minimacs separation columns (Miltenyi Biotec, Bergish Gladbach, Germany), using an anti-CD34 mAb (Immu-133.3, Immunotech, Marseille, France) and goat anti-mouse IgG-coated microbeads (Miltenyi Biotec). In all experiments, isolated cells were 80-99% CD34⁺ as judged by staining with an anti-CD34 mAb.

Hematopoietic factors

Recombinant human (rh) GM-CSF (specific activity: 2 x 10^ U/mg) was used at 100 ng/ml (200 U/ml); rhTNF-α (specific activity: 2 x 10⁷ U/mg) was used at 2.5 ng/ml (50 U/ml); rhSCF (specific activity: 4 x 10^ U/mg) was used at 25 ng/ml; rhIL-4 (specific activity: 10 U/mg) was used at 5 ng/ml; and rhTGF-βl (R&D) was used at 1 ng/ml.

Dendritic cell generation from CD34⁺ HPC

Cultures of CD34⁺ HPC were established as described in Caux, et al. (1992) Nature 392:258-261, in the presence of GM-CSF, SCF, and TNF-α, in endotoxin-free medium consisting of RPMI 1640 (Gibco BRL, Gaithersburg, MD) supplemented with 10% heat- inactivated fetal bovine serum (FBS; Flow Laboratories, Irvine, UK), 10 mM Hepes, 2 mM L-glutamine, 5 x 10"^ M 2-mercaptoethanol and gentamicin (80 μg/ml) (referred to as complete medium).

CD34⁺ cells were seeded in 25 to 75 cm^ culture flasks (Corning, New York; NY) at 2 x 10^ cells/ml. Optimal conditions were maintained by splitting cultures at day 4 with medium containing fresh GM-CSF and TNF-α. In some experiments, TNF-α was replaced at day 7 by TGF-β, to generate cells with properties typical of Langerhans cells (e.g. presence of Birbeck granules as visualized by electron microscopy). In other experiments, DC were activated at day 9 or day 12 with murine Ltk' fibroblast L cells stably transfected with the human CD40 ligand (L) gene (cell line established by Dr. C. van Kooten (van Kooten, et al. (1994) Eur J Immunol 24:787-92). Briefly, 10⁵ irradiated CD40L L cells (7,500 rads) were seeded together with 5 10⁵ CD34-derived DC in the presence of GM-CSF. Isolation of epidermal cells

Epidermal cell suspensions were obtained from normal skin of patients undergoing reconstructive plastic surgery of the breast. Skin was split-cut with a keratome set and the dermo-epidermal slices treated for 18 h at 4° C with 0.05% trypsin (Sigma) in Hank's balanced salt solution without Ca^ ^" and Mg2⁺ (Seromed, Biochrom KG, Berlin, FRG). The epidermis was detached from the dermis with fine forceps. Epidermal sheet cell suspensions were obtained by subsequent tissue dislocation and filtration through sterile gauze. Enrichment of Langerhans cells was obtained by density gradient centrifugation on Lymphoprep (Nycomed Pharma, Oslo, Norway).

Example 2

Generation and Characterization of anti-Langerin Monoclonal Antibody DCGM4

BALB/c mice (Iffa Credo, Les Oncins, France) were immunized with three intraperitoneal injections of CD34-derived DC (10^ cells) with Freund's adjuvant (Sigma Chemical Co., St. Louis, MO). Three days after the final injection, splenocytes were fused with the murine myeloma cell line SP2, using polyethylene glycol-1000 (Sigma). Hybrid cells were placed in 96-well Falcon tissue culture plates (Falcon, Lincoln Park, NJ) and fed with DMEM F12 (Life Technologies, Gaithersburg, MD) supplemented with streptomycin (100 μg/ml), penicillin (100 U/ml), glutamine (2 mM), 10% horse serum (Life Technologies), 1% culture medium additive (CRTS, Lyon, France), 10"^ M azaserine (Sigma), and 5 x 10"^ M hypoxanthine. Supernatants were screened for reactivity with CD34-derived DC and three unrelated cell types, namely peripheral blood polynuclear cells, T lymphocytes activated with PHA, and the myeloid cell line KG1 (ATCC; Rockville, MD). Supernatant from one hybridoma, designated DCGM4, was found to react only with a minor subset of DC and not with the other cell types of the differential screening. The hybridoma was cloned by limiting dilution and ascites were produced in BALB/c mice. MAb DCGM4 was purified by anion-exchange chromatography on DEAE A50 (Pharmacia Biotech, Uppsala, Sweden) and coupled with fluorescein and biotin using standard procedures. Mab DCGM4 was found to be of IgGl/κ isotype as determined by ELISA using a mouse hybridoma subtyping kit (Boehringer-Mannheim, Mannheim, Germany). Finally, early experiments indicating that LC stained positive, we termed Langerin the antigen recognized by mAb DCGM4. Example 3

Generation and Characterization of anti-Langerin Monoclonal Antibodies 817G7 / 2164/ 9, 823G1 / 2131 / 47, 822D3 / 2128 / 32, and 824E1/ 2134 / 62

The overall protocol for antibody generation was identical to that used for the generation of mAb DCGM4 (described above), except that COP5 fibroblasts transfected with human Langerin cDNA were used for immunization of Balb/c mice and for screening of the hybridoma supernatants (selection of antibodies reacting exclusively with Langerin but not with mock-transfected COP5 cells). Epitope-mapping was performed by flow-cytometry (FACScalibur, Becton-Dickinson), by analysis of mAb reactivity with COP5 cells expressing either wild-type Langerin or a form of Langerin (D4 construct) lacking the entire lectin CRD domain at its C-terminus.

Example 4

Gpl20 preparations, Binding assays, and Antibodies for flow-cytometry

Recombinant preparations of gpl20 envelope protein from various strains of HIV-1 were purchased (Immunodiagnostics, Woburn, MA) and radiolabeled with I. Commercially available preparations of gpl20 directly labeled with biotin (Immunodiagnostics, Woburn, MA) were also employed.

Following incubation and extensive washes, binding of gpl20 to various cell types was analyzed either by measuring radioactivity in a γ-counter (revealing ¹²⁵I-gpl20), or by flow-cytometry after addition of flurochrome-labeled streptavidin (revealing biotinylated-gp 120).

For single cell staining, cells were labeled using the following mAbs: anti-Langerin mAbs (DCGM4; 817G7; 823G1; 822D3; 824E1; 903G13), anti-E-cadherin (SHE 79.7; Takara, Shiga, Japan), anti-MHC class II (HLA-DR) (Becton Dickinson), all revealed by FITC-conjugated goat anti-mouse immunoglobulin (Dako, Glostrup, Denmark). For double staining, cells were labeled with mAb DCGM4 revealed by phycoerythrin(PE)-conjugated goat anti-mouse immunoglobulin (Dako), and after saturation in 5% mouse serum, with FITC-conjugated anti-CD la (Ortho, Raritan, NJ). Negative controls were performed with unrelated murine mAbs. Fluorescence was determined with a FACSCAN flow-cytometer (Becton Dickinson). For intracytoplasmic phenotyping, cells were stained in PBS, 0.3% Saponin (Sigma) and 5% BSA, using the same procedure. Example 5 Biochemistry

Proteins were extracted from CD34-derived DC supplemented with TGF-β by addition, to a frozen pellet of 100 μl 10⁷ cells, of 50 mM Tris-HCL pH 8 buffer with 150 mM NaCl, 5 mM EDTA, 1% Triton XI 00 and protease inhibitor (complete Mini, Boehringer Mannheim). After 1 h at 4° C, samples were centrifuged to remove cellular debris. Superaatants were then incubated for 1 h at 4° C with mAb DCGM4 covalently linked to Dynabeads M-450 Sheep anti-mouse magnetic beads (Dynal, Oslo, Norway). Beads were washed with extraction buffer by using a Dynal magnetic particle-concentrator and boiled in the presence of 50 μl SDS-PAGE sample buffer or resuspended in 100 μl of 0.5 M Glycine, 0.15 M NaCl pH 2.3 for 4 minutes. Then, supernatant was neutralized with 3.5 μl of saturated Tris solution. SDS-PAGE analysis was performed with a PhastSystem in a 10-15% gradient gel (Pharmacia Biotech), and gels were stained with Coomassie R250. 2-D analysis was performed on a Multiphor II flat bed system with Immobiline DryStrip pH 3-10 and Excelgel SDS 8-18% for the second dimension (Pharmacia Biotech), and gels were silver stained. Dialyzed samples of proteins eluted from IgG linked to Dynabeads were digested with N-glycosidase F (Boehringer-Mannheim) at 37° C overnight. Five microliters of original samples or 10 microliters of digested samples were deposited on nitrocellulose, and treated with DIG glycan detection kit (Boeliringer-Mannheim).

Example 6 Purification of Langerin

Langerin expressing cells are used for large scale protein extraction. The protein is gently solubilized, and the resulting Langerin is purified using standard methods of protein purification. Chromatographic methods are used, and immunoaffinity techniques can be applied. The Langerin protein is followed by SDS PAGE and/or immunoassays. Diagnostic methods are used to ensure that the protein is substantially pure.

Purified protein is used for protein microsequencing. See, e.g., Matsudaira (ed. 1993) A Practical Guide to Protein and Peptide Purification for Microsequencing. Academic Press, San Diego, CA. Sequence data is used to search sequence databases, e.g., GENBANK, to find natural genes encoding the Langerin. Alternatively, the sequence data is useful for isolating a nucleic acid encoding Langerin using, e.g., degenerate PCR primers, etc. Protein will also be used to raise additional antibodies. Such antibodies may be polyclonal or monoclonal. The protein can be used to assay and determine titers and affinity. Standard methods of immunization are available, as described above.

Example 7

Internalization Assay

CD34-derived DC supplemented with TGF-β (yielding Langerhans-type DC) were generated as detailed above and internalization was performed as described (Cella, et al. (1997) J. Exp. Med. 185:1743-51). One aliquot of cells was fixed with RPMI, 0.1% glutaraldehyde for 5 min. at room temperature and another aliquot was used without fixation. Both samples were stained with mAb DCGM4 or mAb DCGM1 (generated by Applicant, and recognizing the macrophage mannose receptor) for 40 min. on ice, and incubated with biotin-labeled F(ab')2 goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, PA) for 1 h on ice. Cells were then placed in a 37°C water-bath for various time periods, cooled on ice and stained with PE-conjugated streptavidin (Becton Dickinson). After washing, cells were analyzed by FACS. The measure of internalization is given by the percentage decrease of cell-surface median fluorescence intensity (MFI) as compared to control samples kept at 4° C. The percentage decrease of MFI observed in fixed cells was taken as measure of the off-rate of the antibody at 37° C. Linear regression analysis of the plots of logio (percentage of median fluorescence) vs. time was performed and rates of ingestion (k) and half-life (tι/2) of membrane-bound complexes were calculated from the slope (m) of the resulting straight line using the relationships k(%/min)=-2,303m x 100, and ti/2 (min) = log2/m (Leslie,EJI

1980). In this experiment, mAb DCGM1 was used as positive control for receptor-mediated endocytosis.

Example 8 Immunohistology

Microscope slides of acetone-fixed cryocut tissue sections or cell cytospin preparations were incubated with mAbs for 60 min., and subsequently with biotinylated sheep anti-mouse Ig (The Binding Site, Birmingham, UK) for 30 min. Following incubation with streptavidin coupled to alkaline phosphatase (Biosource, CA, USA) for 30 min., enzyme activity was developed using Fast Red substrate (Dako). Double staining, with mouse IgGl antibody DCGM4 and IgG2b anti-CD la (immunotech) were revealed by sheep anti-mouse IgGl (The Binding Site) followed by mouse anti-alkaline phosphatase-alkaline phosphatase complexes (Dako) (APAAP technique), and biotinylated sheep anti-mouse IgG2b (The Binding Site) followed by ExtrAvidin-peroxydase (Sigma). The binding of goat anti-sIgD- biotin and DCGM4-biotin (for double staining with Lag) were directly revealed by ExtrAvidin-peroxidase. Alkaline phosphatase activity and peroxidase activity were respectively demonstrated using Fast Blue substrate (Sigma) and 3-amino-ethylcarbazole (Sigma).

Example 9 Electron Microscopy

Langerhans cell-enriched epidermal cell suspensions were incubated with control mouse IgGl (Sigma), anti-CD la (DMC1 mAb), or DCGM4 for 1 h at 4°C. After washing, cells were incubated with a goat anti-mouse IgG conjugated with colloidal gold particles of 5 nM (GAM-nM) (Amersham, Les Ulis, France) for 30 min. at 4°C. Cells were either fixed immediately for 18 hours with 2% glutaraldehyde in cacodylate buffer, followed by washing for at least 24 h in cacodylate buffer with sucrose, or warmed up to 37°C or room temperature before fixation. Samples were post-fixed for 1 hour with 1% osmium in cacodylate buffer with sucrose, dehydrated, and embedded in Epoxy resin. Ultrathin sections were post-stained with uranyl acetate and lead citrate, and examined on a JEOL 1200 EX electron microscope (CMEABG, Universite de Lyon, Lyon, France). A quantitative evaluation of cell-surface antigen density was performed according to Lafferty, et al. (1981) J. Histochem Cytochem 29:49-56. The number of gold granules bound along the cell membrane was counted and total circumference of the cell was measured on micrographs with a minimop morphometric analyzer (Zeiss). Results were expressed as number of gold granules per 100 μm of cell membrane. For each group of cells, counts were obtained from at least 20-30 cell sections. Mean and standard deviation were subsequently determined.

In another series of experiments, COP5 fibroblasts were transfected either with plasmid cDNA encoding human Langerin or with a mock plasmid cDNA. The COP5 cells were subsequently prepared for electron microscopy as described above, and examined on the JEOL 1200EX instrument for the presence of Birbeck granules (BG).

Example 10 Confocal Microscopy

Intracellular immuno fluorescence staining was performed as previously described by

Winzler, et al. (1997) J. Exp. Med. 185:317-28. Cells on polylysine coated coverslips and fixed for 15 min. with 4% paraformaldehyde, were washed in 10 mM glycine, then permeabilized with 0.5% saponin, 0.2% BSA for 30 min. Coverslips were incubated for 30 min. at room temperature with anti-LAMP-1 (Pharmingen, San Diego, CA), anti-HLA-DR (Becton Dickinson) or anti-Lag (Kashishara, et al. J Invest Dermatol 87:602-607) at a final concentration of 5 μg/ml in permeabilization medium. After three washes, cells were incubated for 30 min. with secondary labeled antibody (donkey anti-mouse coupled to Texas- red (Vector Laboratories, Burlingame, CA)), washed, and incubated with mouse preimmune serum for 30 min., washed again, post-fixed with 2% paraformaldehyde, and finally incubated for 30 min. with mAb DCGM4 coupled to fluorescein (FITC). After washing, coverslips were mounted onto glass slides with fluoromount (Southern Biotechnology Associates Inc., Birmingham, AL). Confocal microscopy was performed using Confocal Laser Scanning Microscope TCS 4D (Leica Lasertechnik GmbH, Heidelberg, Germany) interfaced with an argon/krypton ion laser and with fluorescence filters and detectors allowing to simultaneously record FITC and Texas-red markers (Rovere, et al.,1998, Proc. Nat. Acad. Sci. USA 95:1067-10721

Example 11

Langerin is Selectively Expressed on Langerhans-Type Immature Dendritic Cells

CD34+ HPC cultured with a combination of GM-CSF and TNF-α for 12 days differentiate into CDla⁺ DC (Caux et al., 1996, J Exp Med 184:695-706). The expression of Langerin during such cultures was investigated. Langerin is expressed by a subset of CDla⁺ dendritic cells derived from cord blood CD34⁺ HPC. Kinetics of CD la and Langerin expression during culture of CD34 ^" HPC in GM-CSF plus TNF-α was determined at various time points, cells were recovered and double-labeled using anti-CD la-FITC and DCGM4 plus anti-mouse IgG-PE. The results are representative of 5 experiments.

No staining was detected at day 0 or day 6, indicating that CD34⁺ HPC and their immediate progeny do not express Langerin. The antigen appeared at day 7, on a small subset of the CDla⁺ cells. Between day 7 to day 12, Langerin expression reached a maximum, staining between 15 to 35% of CDla⁺ cells. The dendritic nature of the Langerin-expressing cells in the cultures was confirmed on cytospin preparations of DCGM4+ FACS-sorted cells.

Caux et al., 1996, J Exp Med 184:695-706 have further shown that CD34+ HPC differentiate along two independent pathways from distinct precursor subsets, identified by mutually exclusive expression of CD la and CD 14 at early time points during the culture (day

5-7). When such precursors were separated by FACS-sorting at day 6 and cultured with GM- CSF and TNF-α for 6 more days, Langerin was found mostly expressed on the CD la-derived DC (40%) as compared to the CD14-derived DC (16%). The CDla-derived DC have been shown to display features that are associated with Langerhans cells, including the presence of Birbeck granules.

Next, the in situ distribution of Langerin was examined by immunohistological analysis of various human tissues. Langerin is selectively expressed by LC-like DC. Immunohistological analysis of Langerin expression was made on sections of skin, tonsil and lung. Within the epidermis, Langerin was only found on LC, which also stained with anti- Lag and anti-CD la antibodies. In skin, staining with mAb DCGM4, or mAb DCGM4 plus anti-Lag or anti-CD la, showed positive Langerin expression by LC. In tonsil, the Langerin- positive cells were found in the epithelium (e.g., on follicular mantle B cells). A few cells were occasionally stained in the T cell areas, but never observed in germinal centers Langerin^4" cells were also notably present in lung epithelium. In lung, mAb DCGM4 and counterstaining with hematoxylin showed LC-like Langerin⁺ cells only in bronchiolar epithelium. The Langerin^"1" DC showed dendritic morphology. No staining was detected with control mAbs. These results were representative of 5 experiments.

By contrast to CD34-derived DC, mAb DCGM4 did not react with DC obtained from peripheral blood monocytes cultured 6 days with a combination of GM-CSF and IL-4, thus, further confirming the restriction of Langerin expression. Likewise, Langerin was neither detected in ex-vivo purified DC isolated from peripheral blood, nor in germinal center DC isolated from tonsils.

Finally, mAb DCGM4 was analyzed for reactivity on a panel of different hematopoietic-derived cell types. Langerin was neither detected in ex-vivo isolated T lymphocytes, B lymphocytes, monocytes or granulocytes, nor in myeloid (HL60, KG1, U937, THP1) or lymphoid (Jurkat, JY, PREALP) cell lines.

Taken together, the above data indicate that Langerin expression is restricted to an immature DC compartment, and that it is subsequently lost upon DC maturation.

Example 12

Langerin Expression is Upregulated by TGF-β and Decreased Following CD40 Activation

Since mAb DCGM4 was found to react selectively with LC-like immature DC, it was investigated whether factors that influence DC maturation would affect the expression levels of Langerin. Studies in vitro and in vivo have shown that TGF-β plays an essential role in LC development. Therefore, the effect of TGF-β on Langerin expression by in vitro derived DC was evaluated.

To do so, cord blood CD34+ HPC were cultured for 12 days in GM-CSF and TNF-α in absence or presence of TGF-β from day 7 to day 12. Subsequently, the DC were cultured with L-cells transfected with CD40L for 2 days. Cells were processed for staining without or after pretreatment with 0.1% saponin, using mAbs revealed by FITC-conjugated anti-mouse Ig. The results are representative of more than 5 experiments.

CD34-derived DC were supplemented with TGF-β for the last three days of culture (day 9-12). This resulted in strong upregulation of Langerin expression. In addition to increasing the proportion of Langerin^"1" cells, TGF-β raised the mean number of surface- membrane molecules per cell (93 x 10^ instead of 33 x 10^ without TGF-β). The effect of TGF-β was predominantly exerted on the CD14-derived DC subset, normally devoid of Langerhans markers such as the Birbeck granule associated antigen Lag. Langerin expression is not restricted to the plasma membrane, but is also detected intracellulary following membrane permeabilization. Levels of intracellular Langerin were also markedly enhanced by TGF-β. It was also found that although TGF-β also increased the expression of the LC markers Lag and E-cadherin, Lag was never detected at the cell-surface.

Removal of TNF-α for the last three days of culture upregulated Langerin expression whether in the presence or absence of TGF-β. In line with this result, a strong decrease in Langerin surface-membrane expression was found associated with an increase of HLA-DR following activation with CD40L, a signal which triggers the maturation of DC including upregulation of costimulatory molecules. Altogether, these results confirm that TGF-β, which induces an LC phenotype, upregulates Langerin expression, whereas signals that trigger DC maturation decrease Langerin expression.

Example 13

Langerin is Associated with Birbeck Granules and Endocytic Structures

Since Langerin is expressed at the cell surface (as detected by FACS in the absence of membrane permeabilization), electron microscopy was performed on epidermal cell suspensions to analyze its precise distribution. Langerhans cells are easily recognizable in such suspensions by their folded nuclei, lack of keratin filaments, lack of desmosomes and melanosomes, and the presence of characteristic BG.

An epidermal cell suspension was obtained as described above. mAbs were revealed by 5 nm gold-labeled goat anti-mouse IgGl. CD la is homogeneously distributed at the cell surface, whereas Langerin is often associated with areas of membrane thickening. Cytomembrane sandwiching structures and coated pits were visualized upon staining with DCGM4 at 4°C. Upon incubation at 37°C, cytoplasmic gold particle-containing coated vesicles and Birbeck granules were seen. DCGM4 staining and gold particles were revealed on the luminal side of BG. These results were representative of 3 experiments on different skin samples.

Staining at 4°C with DCGM4 and 5 nm gold particles confirmed that Langerin is clearly associated with the cell surface in LC, although at a lower density than CD la (161.5 ± 97.1 versus 1589.1 ± 418.8 gold granules/ lOOμm membrane). In addition to single gold particles, a spontaneous clustering was observed with DCGM4, even though the antibody was ultracentrifuged. An isotype-matched control mouse IgGl did not bind to the LC (1.3 ± 3.4 granules/100 μm). No labeling of DCGM4 was observed on the keratinocytes or melanocytes present in the epidermal cell suspensions.

As compared to the rather homogeneous distribution of CD la, Langerin was not randomly distributed at the cell surface, but was often associated with particular areas of membrane thickening. Furthermore, DCGM4 induced typical endocytic coated pits at the cell membrane at 4° C. The number of coated pits was significantly enhanced (an average 3.6 times) by DCGM4, as compared to staining with anti-CDla or control mouse IgGl. Notably, the coated pits induced during DCGM4 staining contained gold particles. Furthermore, when cells were allowed to warm up before fixation, DCGM4 staining was observed inside coated vesicles already after 2 min. at 37° C. These data demonstrate that Langerin is associated with structures characteristic of early steps of receptor-mediated endocytosis.

Staining with DCGM4 at 4° C also resulted in the formation of cytomembrane sandwiching structures at the cell surface. Consistently, following DCGM4 at 4° C, gold labeled BG were seen in continuity with the cell membrane, and found inside the cytoplasm when cells were warmed up to 37° C for 2 min. The BG were labeled in their central striated lamella or in their bulb. Taken together, these results indicate that Langerin is associated with endocytic structures and can also gain access to Birbeck granules from the cell membrane.

Example 14 Langerin Mediates Rapid Internalization in DC

To further examine the role of Langerin in endocytosis, we analyzed its capacity to internalize DCGM4 as a ligand.

CD34-derived DC supplemented with TGF-β were labeled at 4° C with mAbs and subsequent F(ab')2 biotinylated secondary antibody. Cells were incubated at 37° C for time periods indicated, and internalization measured as decreased cell-surface-bound antibody determined by FACS analysis using PE-conjugated streptavidin. MAb DCGM4 is rapidly internalized at 37°C, with similar kinetics as an anti-mannose-receptor mAb (positive control). In fixed cells, no decrease of cell-surface fluorescence was detected. Results were analyzed as the percentage decrease of mean fluorescence intensity (MFI), as compared to control samples kept at 4° C.

It was found that the antibody was very rapidly internalized by DC at 37° C, but not at 4° C. Approximately 75% of surface-membrane bound DCGM4 was internalized already within one minute at 37°C, with similar kinetics to that of anti-mannose-receptor mAb

DCGM1, used as positive control for receptor-mediated endocytosis. In a representative experiment, the half-life (tι/2) of DCGM4 at the cytomembrane was calculated to be 4.5 minutes at 37° C, with an internalization rate of k=15.3%/min. The rapid disappearance of mAb DCGM4 from the cell-surface was not due to antibody dissociation, as no decrease in fluorescence was observed in glutaraldehyde-fixed DC incubated at 37° C. Finally, Langerin did not display a mannose-type receptor specificity, as DCGM4 failed to inhibit uptake of Dextran-FITC at 37° C and binding of the antibody was not inhibited by mannan at 4° C.

These results are in accordance with the above electron microscopy analysis and demonstrate that Langerin is implicated in a rapid endocytosis process by DC.

Example 15

Triggering of Cell-Surface Langerin Results in Birbeck Granule Formation

As triggering of cell surface Langerin resulted in the formation of CMS and the detection of gold-labeled BG as visualized by electron microscopy, the potential role of Langerin cell surface in BG formation was further investigated. An epidermal cell suspension was obtained as described above. Cells were incubated with an excess of mAb DCGM4 or anti-CD la at 4°C, and processed immediately for electron microscopy, or left at room temperature for 5 min. before fixation. LC incubated with DCGM4 displayed a striking accumulation of Birbeck granules in the perinuclear region. The effect is predominantly observed at 4°C, as subsequent warming up results in vacuolization. Treatment with anti- CD la mAb failed to induce significant changes in the LC cytoplasm, whether in cells kept at 4°C or brought up to room temperature.

This treatment resulted in a considerable increase of densely packed BG in the LC cytoplasm, with a marked accumulation in the perinuclear region around the Golgi. The BG displayed an elongated, round, or irregularly-shaped expanded portion, in addition to their typical rod-shaped part. Moreover, the cytoplasm of DCGM4-treated LC was filled with numerous rounded or elongated vesicles.

When LC were warmed up to room temperature (5 min.) following incubation with excess DCGM4, increased fusion was observed between single and short rod-shaped BG and large vesicular components. Moreover, numerous vesicles of various sizes and shapes occupied a considerable volume of the LC cytoplasm.

In contrast to DCGM4, incubation of epidermal cell suspensions with an excess anti- CD la mAb only led to the formation of some small vesicles but no other significant changes in the LC cytoplasm. Notably, no accumulation of perinuclear BG was observed, even when cells were allowed to warm up before fixation. Similarly, incubation with a control mouse IgGl or with anti-E-cadherin mAb did not modify the BG granules.

Taken together, these data demonstrate that cell-surface Langerin actively participates in BG formation, resulting in their perinuclear accumulation.

Example 16

Transfection of Langerin cDNA induces the formation of Birbeck granules (BG) in fibroblasts

The above data indicated that Langerin induces BG formation by rapid internalization of the LC cytomembrane. To further address the role of Langerin in the process of membrane superimposition and zippering, Langerin cDNA was transfected into murine fibroblastic

COP5 cells. Strikingly, this resulted in a massive accumulation of superimposed membranes separated by a central leaflet typical of BG. The sandwiching process included cytomembrane structures open to the outside of the cell. Also, the nuclear and endoplasmic reticulum membranes were zippered, suggesting that Langerin can initiate membrane superimposition immediately after synthesis of the protein. In contrast, mitochondria, which have no direct relation with the synthetic machinery, never showed membrane zippering. COP5 cells transfected with cDNA encoding another type II lectin (asialoglycoprotein- receptor) did not display BG. These findings demonstrate that expression of the LC-restricted Langerin gene is sufficient to drive BG formation in unrelated cells.

Example 17 Langerin is a 40 kDa N-glycosylated Protein

Immunoprecipitation with DCGM4 from DC extracts and subsequent elution with SDS-PAGE sample buffer yielded a homogeneous band of 40-42 kDa molecular mass. SDS- PAGE analysis of immunopurified Langerin in non-reducing and reducing conditions was carried out. If DTT was omitted all along the purification steps, the profile was not modified on the gel, suggesting that Langerin is present at the cell membrane as a single chain or as an homodimer with non covalent association.

2-D gel electrophoresis analysis of immunopurified Langerin was conducted establishing the molecular mass of the molecule and indicating that Langerin had a pi of 5.2- 5.5. Finally, a dot-blot analysis of Langerin using (1) creatinase from E. coli as non- glycosylated control, (2) transferrin as positive N-glycosylated control, (3) N-glycosidase, (4) Langerin, and (5) N-glycosidase-treated Langerin, demonstrated that Langerin is a glycoprotein, and that most of the carbohydrate constituents were removed by N-glycosylase treatment.

Example 18

Isolating a Nucleic Acid Encoding Langerin

Numerous methods are available to isolate a gene encoding a purified protein, especially where antibodies which recognize the protein exist. One method is to determine methods for purification of the protein and subsequently to determine the peptide sequences. Given sufficient sequence information, and using redundant oligonucleotides, PCR or hybridization techniques will allow for isolation of genes encoding Langerin proteins.

Another alternative is to generate additional antibodies to Langerin proteins, which may be isolated by immunoaffinity methods using the DCGM4 antibodies. See above. These antibodies are applicable in "panning" techniques, such as described by Seed and Aruffo (1987) Proc. Nat'l Acad. Sci. USA 84:3365-3369. Phage expression techniques are also applicable to screen cDNA libraries derived from appropriate DC or T cell subpopulations enriched for Langerin expression. Glycosylation interference with antibody recognition will be generally less problematic in the phage selection systems. Cell sorting techniques on a mammalian expression library are applicable also.

Another method for screening an expression library is to use antibody to screen successive subpopulations of libraries. The following provides one method of screening using small populations of cells on slides stained by a specific labeling composition, e.g., an antibody.

For example, on day 0, precoat 2-chamber permanox slides with 1 ml per chamber of fibronectin, 10 ng/ml in PBS, for 30 min. at room temperature. Rinse once with PBS. Then plate COS cells at 2-3 x 10^ cells per chamber in 1.5 ml of growth media. Incubate overnight at 37° C.

On day 1 for each sample, prepare 0.5 ml of a solution of 66 μg/ml DEAE-dextran, 66 μM chloroquine, and 4 μg DNA in serum free DME. For each set, a positive control is prepared, e.g., of human IL-10-FLAG cDNA construct at 1 and 1/200 dilution, and a negative mock. Rinse cells with serum free DME. Add the DNA solution and incubate 5 h at 37° C. Remove the medium and add 0.5 ml 10% DMSO in DME for 2.5 min. Remove and wash once with DME. Add 1.5 ml growth medium and incubate overnight.

On day 2, change the medium. On days 3 or 4, the cells are fixed and stained. Rinse the cells twice with Hank's Buffered Saline Solution (HBSS) and fix in 4% paraformaldehyde (PFA)/glucose for 5 min. Wash 3X with HBSS. The slides may be stored at -80° C after all liquid is removed. For each chamber, 0.5 ml incubations are performed as follows. Add HBSS/saponin (0.1%) with 32 μl/ml of 1M N-1N3 for 20 min. Cells are then washed with HBSS/saponin IX. Soluble antibody, e.g., DCGM4, is added to cells and incubate for 30 min. Wash cells twice with HBSS/saponin. Add second antibody, e.g., Vector anti-mouse antibody, at 1/200 dilution, and incubate for 30 min. Prepare ELISA solution, e.g., Vector Elite ABC horseradish peroxidase solution, and preincubate for 30 min. Use, e.g., 1 drop of solution A (avidin) and 1 drop solution B (biotin) per 2.5 ml HBSS/saponin. Wash cells twice with HBSS/saponin. Add ABC HRP solution and incubate for 30 min. Wash cells twice with HBSS, second wash for 2 min., which closes cells. Then add Vector diaminobenzoic acid (DAB) for 5 to 10 min. Use 2 drops of buffer plus 4 drops DAB plus 2 drops of H2O2 per 5 ml of glass distilled water. Carefully remove chamber and rinse slide in water. Air dry for a few minutes, then add 1 drop of Crystal Mount and a cover slip. Bake for 5 min. at 85-90° C.

Alternatively, the Langerin proteins are used to affinity purify or sort out cells expressing the ligand. See, e.g., Sambrook, et al. (supra) or Ausubel, et al., (supra) both of which are incorporated herein by reference.

Example 19 Chromosomal Mapping of Human Langerin

Chromosomal localization was performed by radiation hybrid (RH) mapping (D.R. Cox et al., Science, 1990, 250: 245-250), using the Stanford G3 Radiation Hybrid panel (Research Genetics, Inc., Huntsville, AL). Briefly, two oligonucleotides (U863 and LI 180) used to amplify Langerin cDNA by PCR reaction were selected as they also amplfied a stretch of human genomic DNA. PCR reactions were carried out with U863 / LI 180 on the Stanford G3 panel of 83 clones covering the human genome. PCR data were submitted to the Stanford Human Genome Center Rhserver for mapping using the RHMAP statistical program (RHMAP).

Results from the RH Server indicated as closest matches the markers SHGC-58922 (LOD score: 8.67) and SHGC-12714 (LOD score 7.74). Both markers are located on chromosome 2, linked to the GDB locus D2S292 (SHGC source AFH203yb6: SHGC-1610 microsatellite marker). Genes mapping near the D2S292 locus include: transforming growth factor-alpha precursor (2pl3), dynactin (2pl3), gamma actin enteric smooth muscle form (2pl3), annexin IN (2pl3), MAD (2pl2-13), alpha-CPl, early growth response 4, nucleolysin TIA-1, protein tyrosine phosphatase P, protein kinase C substrate 80K-H, glutamine-fructose 6-phosphate transaminase, retinoic acid-responsive protein, RAB.IA, and pleckstrin. The results localize the gene of human Langerin to chromosome 2pl3, in the vicinity of the D2S292 locus.

The localization of human Langerin will permit to survey immunological genetic disorders for a potential association with the chromosome 2pl3 region. Example 20

293 Cells Transfected with Human Langerin cDNA Bind gpl20 of HIN 1

293 cells were transiently transfected with plasmid cDΝA encoding human Langerin, human DC-SIGΝ, or with empty pCDΝA3 vector. Transfectants were incubated for one hour at 4°C with the indicated concentrations of I-labeled recombinant preparations of gpl20- from SF-2 (XX4R5 tropic) or MN (X4 tropic) strains of HIN-1. In parallel, dendritic cells (DC) prepared from human monocytes cultured in GM-CSF + IL-4 were incubated with the gpl20 preparations. Following extensive washes, bound radioactivity was measured in a γ-counter, and results expressed as mean cpm of triplicate points. The 293 cells transfected with human Langerin were observed to bind both SF-2 and MΝ I-radiolabeled gpl20 in a dose-dependent fashion (3ng, 12ng, and 48ng doses of gpl20 tested). Binding was of the same order of magnitude as that of ¹²⁵I-gpl20 to DC-SIGΝ transfected 293 cells. The findings were subsequently extended to gpl20 preparations from JRFL (R5 tropic) HIV-1 strain.

Example 21

Binding of gpl20 of HIN-1 to human Langerin is inhibited by mannan and by anti-Langerin monoclonal antibodies (mAbs)

HeLa cells were transfected with human Langerin cDΝA and a clone was selected that stably expresses Langerin protein as detected by staining with anti-Langerin mAb DCGM4. The HeLa-Langerin clone was preincubated for 15minutes at 4°C , either with mannan (a ligand for Langerin) at lOOμg/ml, with anti-DC-SIGΝ mAb 1B10 mAb (25μg/ml), or with 25μg/ml of the following anti-Langerin mAbs: 817G7 / 2164 / 9; 823G1 / 2131 / 47; 822D3 / 2128 / 32; 824E1 / 2134 / 62 (generation of the mAbs is described in Example 3). Subsequently, the cells were incubated in the presence or absence of recombinant gpl20 labeled with biotin (gpl20 from -TIB strain of HIV-1, purchased from Immunodiagnostics). After extensive washes, streptavidin conjugated to phycoerythrin (SAPE) was added, and fluorescence corresponding to binding of the gpl20 preparation was assessed by flow- cytometry. A strong signal was detected when the HeLa-Langerin cells were incubated with gpl20-biotin and SAPE, but not with SAPE alone. This result demonstrates binding of gpl20 to the HeLa-Langerin cells. Binding was not inhibited by preincubation with anti DC-SIGΝ mAb 1B10, nor with anti-Langerin mAb DCGM4. In contrast, gpl20 binding was abrogated following preincubation of the cells either with mannan or with the anti-Langerin mAbs 817G7 (2164 / 9), 823G1 (2131 / 47), 822D3 (2128 / 32), or 824E1 (2134 / 62) . These data demonstrate that binding of gpl20 to HeLa-Langerin cells is specific, and occurs via the lectin domain of Langerin which displays specificity for mannose residues. In addition to the mAbs described in Example 3 , a large panel of mAbs generated as above were also found to be directed against human Langerin and to inhibit the gpl20 / Langerin interaction (Table 1):

TABLE 1

Example 22

MAbs blocking binding of gpl20 of HIV-1 are directed against the lectin carbohydrate- recognition domain (CRD) of human Langerin

COP5 cells were transfected either with cDNA encoding wild-type human Langerin

(WT Langerin construct) or with cDNA encoding a form of human Langerin deleted for the entire C-terminal carbohydrate-recognition domain (CRD) (D4 Langerin construct). Cells were incubated with anti-Langerin mAbs or with unrelated control antibody, followed by a FITC-labeled anti-immunoglobulin reagent. Fluorescence was revealed by flow-cytometry analysis. As a positive control, anti-Langerin mAb 903G12 was found to react both with WT and D4 Langerin transfectants. In contrast, anti-Langerin mAbs 817G7 (2164 / 9) and 823 Gl (2131 / 47) reacted exclusively with the WT Langerin transfected cells. Unrelated control mAb reacted neither with WT or D4 transfected COP5 cells. These data demonstrate that mAbs 817G7 and 823G1 (described in Example 3) are directed against the sugar-binding CRD lectin domain of human Langerin. Our findings are consistent with the notion that mannosylated residues of gpl20 / HIN-1 bind to the CRD of Langerin which displays mannose specificity.

Example 23 Gpl20 binds to Langerhans cells in a Langerin-dependent manner

Human Langerhans cells were generated in vitro from umbilical cord blood CD34+ progenitors in the presence of TGFβl, as described in Example 1. Cells were preincubated in medium alone, in the presence of mannan or Ca++ chelators (EDTA, EGTA), or with mAbs directed against human Langerin, as described in Example 3 (mAbs 9 / 817G7 / 2164; 31 / 806G11 / 2180; 32 / 822D3 / 2128; 38 / 805H1 / 2141; 40 / 823C11 / 2170; 47 / 823G1 / 2131; 55 / 803A11 / 2173; and 62 / 824E1 / 2134) or non-related (mAbs 1B10, K15C, and 318). Following extensive washes, cells were incubated with biotin-labeled gpl20 (from HIN-1 strain ADA, purchased from Immunodiagnostics, Woburn, MA) and subsequently with SAPE (phycoerythrin). Cells were analyzed on a flow-cytometer, and results expressed as mean fluorescence intensity (MFI). Langerhans cells in medium alone detectably bind gpl20 as assessed by an MFI value of 50 in the flow-cytometry assay. Binding was not inhibited by preincubation with unrelated mAbs (1B10, K15C, 318). In contrast, preincubation with mannan, EDTA, EGTA, or several of the anti-Langerin mAbs tested (e.g; mAbs 9, 32, 47, and 62) strongly inhibited gp20 binding to the Langerhans cells. Of note, these same anti-Langerin mAbs also antagonized binding of gpl20 to HeLa-Langerin transfectants (Example 21). Epitopes recognized by mAbs 9 (817G7 / 2164) and 47 (823G1 / 2131) were mapped within the CRD of human Langerin (Example 22). These data demonstrate that Langerin accounts for binding of gpl20 /HIN to human Langerhans cells, and further illustrate the feasibility to antagonize this molecular interaction.

Many modifications and variations of this invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments described herein are offered by way of example only, and the invention is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.

Claims

WHAT IS CLAIMED IS:

1. A method for preventing HIN infection comprising administering to an individual in need thereof an effective amount of an agent which modulates a Langerin/gpl20 interaction.

2. The method of claim 1 wherein the agent which modulates a Langerin/gpl20 interaction is a Langerin antagonist or a gp 120 antagonist.

3. The method of claim 2 wherein the Langerin antagonist is a binding composition.

4. The method of claim 3 wherein the binding composition is an antibody or antibody fragment.

5. The method of claim 2 wherein the Langerin antagonist is a small molecule or a nucleotide sequence included in a gene delivery vector.

6. The method of claim 2, wherein the Langerin antagonist is administered intravenously, intramuscularly, subcutaneously or topically.

7. The method of claim 2, further comprising administration of an effective amount of an agent preventing HIN binding.

8. The method of claim 7, wherein the agent preventing HIN binding is a chemokine receptor antagonist.

9. The method of claim 8, wherein the chemokine receptor antagonist is a CCR5 antagonist or a CXCR4 antagonist.

10. The method of claim 9, wherein the CCR5 antagonist is: a) a soluble natural ligand;

b) a small molecule; c) a nucleotide sequence included in a gene delivery vector; or d) an antibody or antibody fragment.

11. The method of claim 9, wherein the CXCR4 antagonist is: a) a soluble natural ligand;

b) a small molecule;

c) a nucleotide sequence included in a gene delivery vector; or d) an antibody or antibody fragment.

12. The method of claim 2, further comprising administration of an effective amount of an agent preventing HIN entry.

13. The method of claim 12, wherein the agent preventing HIN entry is an HIN gp41 antagonist.

14. The method of claim 13, wherein the HIN gp41 antagonist is:

a) a soluble natural ligand; b) a small molecule; c) a nucleotide sequence included in a gene delivery vector; or d) an antibody or antibody fragment.

15. The method of claim 2, further comprising administration of an agent which inhibits Langerhans cell maturation.

16. The method of claim 15 wherein the agent which inhibits Langerhans cell maturation is a CD40 antagonist or a RANK antagonist.

17. The method of claim 16 wherein the CD40 antagonist is: a) a soluble natural ligand;

b) a small molecule; or c) an antibody or antibody fragment.

18. The method of claim 16 wherein the RANK antagonist is: a) a soluble natural ligand;

b) a small molecule, or c) an antibody or antibody fragment.

19. The method of claim 2, further comprising administration of an agent which inhibits a DC-SIGN/gρl20 interaction.

20. The method of claim 19, wherein the agent which inhibits a DC-SIGN/gpl20 interaction is a DC-SIGN antagonist or a gp 120 antagonist.

21. The method of claim 20 wherein the DC-SIGN antagonist is : a) a soluble natural ligand;

b) a small molecule, or c) an antibody or antibody fragment.

22. A method for identifying a compound that inhibits the binding and entry of HIV comprising:

a) contacting Langerin with gpl20 in the presence or absence of a test compound, wherein a Langerin-gpl20 complex forms in the absence of the test compound; b) removing unbound gpl20 and test compound from the first cell; and c) comparing the formation of a Langerin gpl20 complex in the presence and absence of the test compound; wherein a test compound is identified as a compound that interferes with the infectivity of HIN when the amount of Langerin gpl20 complex is less when the test compound is present in step (a) than when the test compound is absent in step (a).

23. The method of claim 22, further comprising:

a) contacting a second cell with the first cell; wherein the second cell is permissive for HIN replication susceptible to entry of vectors comprising the viral envelope protein; and b) determining the amount of vector that has entered the second cell; wherein a test compound is identified as a compound that interferes with the infectivity of HIN when the amount of vector entry is less when the test compound is present in step (a) then when it is absent in step (a).

24. The method of claim 23 wherein the second cell is a T cell or macrophage.

25. A compound identified by the method of claim 23.