HK1087329A

HK1087329A - Dc-sign blockers and their use for preventing or treating viral infections

Info

Publication number: HK1087329A
Application number: HK06107571.6A
Authority: HK
Inventors: 阿里．阿玛拉; 费尔南多．阿伦萨纳-塞斯德多斯; 菲利普．德普雷斯; 让-路易．维雷利齐尔
Original assignee: 巴斯德研究院; 国家健康与医学研究院
Priority date: 2002-11-05
Filing date: 2003-11-05
Publication date: 2006-10-13

Description

DC-SIGN blockers and their use in preventing or treating viral infections

Background

Technical Field

The present invention relates to methods, uses and compositions for preventing or treating a disease in a mammal, wherein at least one symptom of the disease is mediated at least in part by the binding or interaction of an effector molecule (effector molecule) with the DC-SIGN receptor of the mammal to be treated. The effector molecule may be a molecule of a foreign organism. The foreign organism may be a virus.

The invention also relates to compositions and methods for identifying compositions that can be used to treat a disease in a mammal, wherein at least one symptom of the disease is mediated at least in part by the binding of an effector molecule to the DC-SIGN receptor of the mammal to be treated.

The invention also relates to compositions and methods for targeting subject molecules (subjects) to cells expressing the DC-SIGN receptor, such as dendritic cells. These compositions and methods are based on a targeting complex in which one or more subject molecules are covalently bound to one or more DC-SIGN blockers and the subject molecules are targeted to cells expressing the DC-SIGN receptor by binding of the DC-SIGN blocker to DC-SIGN of the targeting complex by the one or more DC-SIGN blockers of the targeting complex.

Background

Dengue is an acute febrile tropical disease and the virus causing the disease is an arbovirus, transmitted by mosquitoes. The carrier of the disease is the mosquito of the genus aedes, particularly aedes aegypti (Aedesaegypti), which most commonly gives its larvae to habitats or areas surrounding habitats. Disease-causing viruses were isolated in 1951 and have been classified into 4 different antigen types (DEN1, DEN2, DEN3 and DEN 4). The virus belongs to the genus Flaviviridae (genus Genusflavivirus) of the family Flaviviridae.

Over 20 million people live in disease endemic areas, and the annual population infected with the virus is considered to exceed 1 million. In particular, dengue fever, is treated in 500000 hospitalizations each year and causes death of tens of thousands of people each year, mostly children.

Clinical symptoms usually appear suddenly after incubation for 5 to 8 days, with mixed heat (DF dengue fever) with severe headache, lumbago, muscle and joint pain and chills. From day 3 to 5 of the spontaneous fever phase, congestive maculopapular begins to appear, which may last for 3 to 4 days (common dengue fever).

Severe infections can lead to the appearance of hemorrhagic syndromes (DHF or dengue hemorrhagic fever) characterized by increased vascular permeability and abnormal blood clotting. Although the disease in most patients usually begins to improve within a week, it can be fatal if hypovolemic shock occurs (DSS or dengue shock syndrome). These complications may occur due to pre-existing immunity that is acquired upon initial infection with a different dengue virus (different serotype). In particular, two different types of serological responses were identified in individuals infected with dengue virus: individuals who have never had a flavivirus infection and who have not received immunization against another flavivirus (e.g., yellow fever virus, japanese encephalitis virus) develop a primary response (primary response) characterized by the slow production of antibodies specific for the virus causing the infection; individuals who have had an infection with a flavivirus (e.g., other dengue serotypes) or who have been immunized against another flavivirus will develop a secondary response (secondary response) characterized by rapid antibody production.

The infectious agent is dengue virus, which belongs to the Flaviviridae family, yellow fever virus and Japanese encephalitis virus also belong to the Flaviviridae family (T.P.Monath et al, (1996) Flaviviviruses in B.N.fields, D.M.Knipe, P.M.Howly et al (eds.) "Fields Virology" Philadelphia: Lippincott raven Press Publishers). These viruses have a single-stranded positive polarity RNA (single-stranded RNA with positive polarity) that contains 11000 nucleotides and encodes a polyprotein of about 3400 amino acids. It is separated into three structural proteins and seven non-structural proteins, namely NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5, by viral and intracellular protease cleavage both after co-translation and after translation. The NS1 nonstructural protein was first identified in 1970 by P.K. Russel et al (J.Immunol., (1970), 105, 838-. This glycoprotein is highly conserved in the flavivirus genus (t.p. monath has been mentioned), in particular among the 4 dengue virus serotypes, in one intracellular and one extracellular form. This intracellular form is thought to be involved in the early stages of viral replication (Hall R.A. et al, J.Virol. (1999), 73, 10272-; RiceC.M. et al, J.Virol., (1997), 71, 291-. NS1 protein dimerizes before being transported to the plasma membrane. In mammalian cells (but not insect cells), a portion of the NS1 protein is released into the extracellular medium, which may be in the form of a soluble protein first, or in the form of microparticles second. If it is in soluble form, the protein is in the form of an oligomer, in particular a pentamer or a hexamer (Crooks A.J. et al J.Chrom. (1990), 502, 59-68 and J.Gen.Virol. (1994), 75, 3453-.

At present, no specific treatment exists, and only symptomatic treatment is carried out on patients. For common dengue fever, treatment is based on administration of analgesics and antipyretics. For DHF, treatment includes infusion to replenish plasma loss, and correct electrolyte disturbances and induce diuresis.

There is no commercial vaccine against dengue virus. On the other hand, the results of protective assays carried out by N.Bhamaravati et al with attenuated strains of 4 Dengue virus serotypes are unsatisfactory (Dengue and Dengue halorhagic carver (1997), 367-. Thus, prevention is only based on viral-based vectors, which combine destruction of larvae and spraying with "adulticides".

The pathogenesis of severe dengue fever (DEN) virus infection is not fully understood. Significant T cell activation was observed in severe DEN disease. In dengue hemorrhagic fever and/or dengue shock syndrome, elevated levels of various cytokines and chemokines are found. Macrophages have long been considered to be an important component of the pathogenesis of DEN. As highlighted by Palucka, immature human Dendritic Cells (DCs), unlike other leukocytes, tend to tolerate DEN infection (Wu et al, nat. Med.6: 816, 2000; for review see: Palucka, nat. Med.6: 748, 2000). Unlike monocytes/macrophages, specific antibodies do not enhance DEN virus infection (Marovich et al, JID Syrnp. Proc.6: 219, 2001). Immature DCs can mature in response to DEN virus infection. Following DC infection, upregulation of the surface markers B7-1, B7-2, HLA-DR CD11B and DC83, and cytokine production were observed. There is increasing evidence that DEN infection can induce functional maturation of DCs. This infection causes the up-regulation of surface markers B7-1, B7-2, HLA-DR CD11B and DC83 and stimulates cytokine production (Ho et al, Immunology 166: 1499, 2001). Immature DC exposed to DEN virus produce TNF- α, which can interfere with endothelial cell function.

Dendritic Cells (DCs) are professional Antigen Presenting Cells (APCs) that are involved in eliciting T cell-dependent immune responses due to their high expression of MHC and costimulatory molecules. Myeloid DCs are distributed throughout the body in an immature state and have high antigen uptake and processing ability. Once activated by inflammatory stimuli or infectious agents, DCs undergo a maturation process, migrate to lymphoid organs, and acquire the ability to activate naive (naive) T lymphocytes.

An important issue is which DC-specific molecule is utilized by DEN viruses as an incoming receptor. The human DC-specific adhesion receptor DC-SIGN (ICAM-grabing non-integrin or CD-209) is a type II integral membrane protein (integral protein) and is of particular interest because its expression is mainly restricted to immature DC. DC-SIGN has been found to be a ligand for ICAM-3, which enables transient interactions between DC-T cells, thereby promoting primary immune responses (Geijtenbeek et al, Nature 1: 353, 2000). DC-SIGN appears to be a crucial mediator of the migration and T cell interaction capacity exhibited by immature myeloid monocyte-derived DCs during maturation. DC-SIGN expression is IL-4 dependent and is negatively regulated by IFN- γ, TGF- β, and anti-inflammatory agents (Relloso et al, J.Immunol.168: 2634, 2002). DC-SIGN polymorphisms may also explain why some patients develop protective immunity while others do not.

DC-SIGN is a C-type lectin with a single carbohydrate recognition domain that interacts in a calcium-dependent manner with proteins with mannose or galactose side chains (Drickammer, Curr. Opin.1 mmnol. 13: 585, 1999). DC-SIGN is now thought to bind to high mannose oligosaccharides on viral glycoproteins and thus capture enveloped viruses (Feinberg et al, Science 294: 2163, 2001). For example, DC-SIGN binds to the HIV envelope glycoprotein gp120(Geijtenbeek et al, Cell, 100: 587, 2000) and thereby mediates rapid internalization of intact HIV into a non-lysosomal structure (Kwon et al, Immunity, 16: 135, 2002).

There is a need to develop methods and compositions that modulate the specific binding of effector molecules to DC-SIGN receptors, such as DC-SIGN receptors on mammalian dendritic cells. Such methods and compositions are needed, for example, for the prevention and treatment of diseases, such as viral infections, e.g., dengue virus infection. In this regard, there is a need to identify cellular proteins involved in viral attachment and/or fusion. In addition, there is a need for methods and compositions that are capable of specifically targeting cells expressing the DC-SIGN receptor, such as dendritic cells or alveolar macrophages, to facilitate therapy or diagnosis.

Disclosure of Invention

The present inventors sought to determine whether the DC-specific adhesion receptor DC-SIGN could promote DEN virus infection of human DC cells. The data presented herein show that DC-SIGN specific antibodies have a blocking effect on DEN-1 virus infection. These results establish a novel function of DC-SIGN as a dengue virus binding protein, probably through interaction with the E glycoprotein. The process of DC-SIGN mediated dengue virus infection of DC provides a new mechanism for designing antiviral compounds.

Thus, the present invention identifies DC-SIGN as a receptor involved in the binding of viruses other than HIV to dendritic cells. The invention also provides various novel methods, uses and compositions for treating mammalian diseases, including viral infections.

It is a first object of the present invention to provide a method of preventing or treating a disease in a mammal, wherein at least one symptom of the disease is mediated at least in part by the binding of an effector molecule to a DC-SIGN receptor of the mammal to be treated, and wherein the method comprises administering to the mammal a DC-SIGN modulator (modulator) in an amount sufficient to modulate the binding of the effector molecule to the DC-SIGN receptor sufficiently, thereby preventing or treating the disease.

It is another object of the invention to provide a method of preventing or treating a disease in a mammal, wherein at least one symptom of the disease is mediated at least in part by the binding of an effector molecule to the DC-SIGN receptor of the mammal to be treated, and wherein the method comprises administering to the mammal an amount of a DC-SIGN blocker sufficient to inhibit the binding of the effector molecule to the DC-SIGN receptor, thereby preventing or treating the disease.

In some embodiments, the DC-SIGN blocker is a blocking derivative of the effector molecule. In other embodiments, the DC-SIGN blocker is an antibody.

Embodiments of the invention in which the DC-SIGN blocker is an antibody include embodiments in which the antibody specifically binds DC-SIGN, and embodiments in which the antibody specifically binds an effector molecule.

In some embodiments, the DC-SIGN blocker is a mannosylated molecule (glycosylated molecule) that binds to the DC-SIGN receptor. The mannosylated molecule may be mannan (mannan).

It is a further object of the present invention to provide a method for preventing or treating a viral infection in a mammal, wherein the viral infection is mediated at least in part by the binding of a viral effector molecule to the DC-SIGN receptor of the mammal to be treated, wherein the method comprises administering to the mammal an amount of a DC-SIGN modulator sufficient to modulate the binding of the viral effector molecule to the DC-SIGN receptor, thereby preventing or treating the viral infection.

It is another object of the present invention to provide a method for preventing or treating a viral infection in a mammal, wherein the viral infection is mediated at least in part by the binding of a viral effector molecule to the DC-SIGN receptor of the mammal to be treated, wherein the method comprises administering to the mammal an amount of a DC-SIGN blocker sufficient to inhibit the binding of the viral effector molecule to the DC-SIGN receptor, thereby preventing or treating the viral infection.

In some embodiments of the methods of the invention, the DC-SIGN blocker comprises a binding moiety of the viral effector molecule. In other embodiments, the DC-SIGN blocker comprises a binding moiety of a viral envelope glycoprotein. In other embodiments, the DC-SIGN blocker is an antibody. The antibody may specifically bind DC-SIGN or specifically bind a viral effector molecule. In other embodiments, the DC-SIGN blocker is a mannosylated molecule that binds to the DC-SIGN receptor. The mannosylated molecule may be mannan.

Embodiments of the invention in which the DC-SIGN blocker is an antibody include some wherein: the antibody is a monoclonal antibody; the mammal is a human and the antibody is a humanized monoclonal antibody; the antibody specifically binds DC-SIGN; the monoclonal antibody is Mab1 B10.2.6; the antibody specifically binds to a viral effector molecule; and a binding moiety for which an antibody specifically binds to the viral effector molecule.

In a further embodiment of the method of the invention, the viral effector molecule is a molecular component of the viral envelope. In a particular embodiment, the molecular component of the viral envelope is an envelope glycoprotein.

In a further embodiment of the method of the invention, the DC-SIGN blocker comprises a binding moiety of said viral effector molecule. In some embodiments of the invention where the viral effector molecule is a molecular component of the viral envelope, the DC-SIGN blocker used comprises a binding moiety for the envelope glycoprotein.

In a preferred aspect of the invention, the viral infection is a flaviviridae viral infection and the viral effector molecule is a flaviviridae viral effector molecule. In a more preferred embodiment, the viral infection is a dengue virus infection and the viral effector molecule is a dengue virus effector molecule. In a further preferred aspect, the mammal is a human. In some embodiments, the dengue virus effector molecule is a molecular component of a dengue virus envelope. In another embodiment, the molecular component of the dengue virus envelope is a dengue virus envelope glycoprotein. In another embodiment, the dengue virus envelope glycoprotein is dengue virus E glycoprotein.

In embodiments of the invention where the viral infection is a dengue viral infection and the viral effector molecule is a dengue viral effector molecule, some embodiments are included wherein: the DC-SIGN blocker comprises a binding moiety for a dengue virus effector molecule; the DC-SIGN blocker comprises a binding moiety of dengue virus E glycoprotein; DC-SIGN blockers are recombinantly produced proteins; and the DC-SIGN blocker is an antibody. In embodiments where the DC-SIGN blocker is an antibody, some embodiments are included wherein: the antibody is a monoclonal antibody; the mammal is a human and the monoclonal antibody is humanized; the antibody specifically binds DC-SIGN; the monoclonal antibody is Mab1B10.2.6; and the antibody specifically binds to a dengue virus effector molecule. Among the embodiments in which the antibody specifically binds to a dengue virus effector molecule is an embodiment in which the dengue virus effector molecule is dengue virus E glycoprotein.

In yet another aspect the invention provides a method for preventing or treating an HIV or SIV infection in a human or simian, wherein the method comprises administering to the human or simian an amount of a DC-SIGN modulator sufficient to modulate the binding of HIV or SIV to a DC-SIGN receptor on dendritic cells of the human or simian, thereby preventing or treating the HIV or SIV infection.

In another aspect the present invention provides a method for preventing or treating HIV or SIV infection in a human or simian, wherein the method comprises administering to the human or simian an amount of a DC-SIGN blocker sufficient to substantially inhibit binding of HIV or SIV to a DC-SIGN receptor on dendritic cells of the human or simian, thereby preventing or treating HIV or SIV infection. In a preferred embodiment, the DC-SIGN blocker comprises a binding moiety for the E glycoprotein of dengue virus. In another preferred embodiment, HIV infection in humans is prevented or treated.

In yet another aspect the invention provides a method for preventing or treating inflammation in a mammal caused by specific binding of ICAM-3 on T cells of the mammal to a DC-SIGN receptor on dendritic cells of the mammal, wherein the method comprises administering to the mammal an amount of a DC-SIGN modulator sufficient to modulate the binding of ICAM-3 on T cells of the mammal to the DC-SIGN receptor on dendritic cells of the mammal, thereby preventing or treating inflammation.

In another aspect the invention provides a method for preventing or treating inflammation in a mammal caused by the specific binding of ICAM-3 on T cells of the mammal to DC-SIGN receptors on dendritic cells of the mammal, wherein the method comprises administering to the mammal an amount of a DC-SIGN blocker sufficient to substantially inhibit the binding of ICAM-3 on T cells of the mammal to DC-SIGN receptors on dendritic cells of the mammal, thereby preventing or treating inflammation. In a preferred embodiment, the DC-SIGN blocker comprises a binding moiety for the E glycoprotein of dengue virus. In another preferred embodiment, the mammal is a human.

Another object of the invention is:

-use of an amount of a DC-SIGN modulator sufficient to modulate binding of an effector molecule to a DC-SIGN receptor, in the preparation of a medicament for preventing or treating a disease in a mammal, wherein at least one symptom of the disease is mediated at least in part by binding of the effector molecule to the DC-SIGN receptor of the mammal to be treated.

-use of a DC-SIGN blocker in an amount sufficient to substantially inhibit binding of an effector molecule to the DC-SIGN receptor for the preparation of a medicament for the prevention or treatment of a disease in a mammal, wherein at least one symptom of the disease is mediated at least in part by binding of the effector molecule to the DC-SIGN receptor of the mammal to be treated.

-use of a DC-SIGN modulator in an amount sufficient to modulate binding of a viral effector molecule to a DC-SIGN receptor in the manufacture of a medicament for preventing or treating a viral infection in a mammal, wherein the viral infection is mediated at least in part by binding of the viral effector molecule to the DC-SIGN receptor of the mammal to be treated.

-use of a DC-SIGN blocker in an amount sufficient to substantially inhibit binding of a viral effector molecule to a DC-SIGN receptor in the manufacture of a medicament for preventing or treating a viral infection in a mammal, wherein the viral infection is mediated at least in part by binding of the viral effector molecule to the DC-SIGN receptor of the mammal to be treated.

-use of a DC-SIGN modulator in an amount sufficient to modulate the binding of HIV or SIV to a DC-SIGN receptor on a dendritic cell of a human or simian in the manufacture of a medicament for the prevention or treatment of HIV or SIV infection of said human or simian.

-the use of a DC-SIGN blocker in an amount sufficient to substantially inhibit the binding of HIV or SIV to a DC-SIGN receptor on a dendritic cell of a human or simian for the preparation of a medicament for the prevention or treatment of HIV or SIV infection of said human or simian.

-use of a DC-SIGN modulator in an amount sufficient to modulate sufficiently the binding of ICAM-3 on T cells of a mammal to a DC-SIGN receptor on dendritic cells of the mammal for the preparation of a medicament for preventing or treating inflammation in a mammal caused by the specific binding of ICAM-3 on T cells of a mammal to a DC-SIGN receptor on dendritic cells of a mammal.

-use of a DC-SIGN blocker in an amount sufficient to substantially inhibit binding of ICAM-3 on T cells of a mammal to DC-SIGN receptors on dendritic cells of the mammal in the preparation of a medicament for preventing or treating inflammation in the mammal caused by specific binding of ICAM-3 on T cells of the mammal to DC-SIGN receptors on dendritic cells of the mammal. In a preferred embodiment, the DC-SIGN blocker comprises a binding moiety for the E glycoprotein of dengue virus. In another preferred embodiment, the mammal is a human.

The preferred embodiments listed for the above process are suitable for these uses.

In yet another aspect the present invention provides a pharmaceutical composition comprising:

a) a DC-SIGN modulator, and

b) at least one pharmaceutically acceptable excipient;

wherein the DC-SIGN modulator is present in the composition at a therapeutically achievable concentration.

In another aspect the present invention provides a pharmaceutical composition comprising:

c) a DC-SIGN blocker, and

d) at least one pharmaceutically acceptable excipient;

wherein the DC-SIGN blocker is present in the composition at a therapeutic concentration that is attainable.

In some embodiments of the pharmaceutical composition, the DC-SIGN blocker is a derivative of a viral effector molecule. In one embodiment, the DC-SIGN blocker comprises a binding moiety for a dengue virus effector molecule. In another embodiment, the dengue virus effector molecule is dengue virus E glycoprotein.

In some other embodiments of the pharmaceutical composition, the DC-SIGN blocker is an antibody. Embodiments in which the DC-SIGN blocker is an antibody include some embodiments in which: the antibody is a monoclonal antibody; the monoclonal antibody is humanized; the antibody specifically binds DC-SIGN; the monoclonal antibody is Mab1 B10.2.6; the antibody specifically binds to a viral effector molecule; or an antibody specifically binds to a binding moiety of the viral effector molecule.

In yet another aspect the invention provides a method of identifying a DC-SIGN modulator, wherein the method comprises:

a) determining a baseline binding value by:

i. providing cultured cells comprising a DC-SIGN receptor;

exposing said cultured cells to a labeled binding moiety for a sufficient period of time to allow binding to reach equilibrium; and

determining the extent of binding of said labelled viral effector molecule binding moiety to said cultured cells, thereby determining a baseline binding value;

b) determining the test agent binding value by:

i. providing cultured cells comprising a DC-SIGN receptor;

exposing said cultured cells to a labeled viral effector molecule binding moiety in the presence of a test substance for a sufficient period of time to allow binding to reach equilibrium; and

determining the extent of binding of said labeled viral effector molecule binding moiety to said cultured cells, thereby determining a test agent binding value; and

c) determining a test substance binding modulation value for the test substance by dividing the test substance binding value by the baseline binding value,

wherein a test agent binding inhibition value representing about 95% modulation of binding of said viral effector molecule to dendritic cells by said test agent indicates that said test agent is an agent that sufficiently modulates binding of a viral effector molecule to a DC-SIGN receptor.

In a preferred aspect the present invention provides a method of identifying a DC-SIGN blocker, wherein the method comprises:

a) determining a baseline binding value by:

i. providing cultured cells comprising a DC-SIGN receptor;

b) determining the test agent binding value by:

i. providing cultured cells comprising a DC-SIGN receptor;

c) determining a test agent binding inhibition value for the test agent by dividing the test agent binding value by the baseline binding value,

wherein a test agent binding inhibition value representing about 95% inhibition of binding of the viral effector molecule to dendritic cells by the test agent indicates that the test agent is an agent that substantially inhibits binding of the viral effector molecule to the DC-SIGN receptor.

Methods of identifying DC-SIGN blockers include some embodiments wherein: the cultured cells are DCs; the cultured cells are THP-1 cells; the viral effector molecule is a dengue virus effector molecule; and the dengue virus effector molecule is dengue virus E glycoprotein.

In yet another aspect the invention provides an isolated DC-SIGN blocker identified by the method of identifying DC-SIGN blockers described above.

In another aspect the invention provides a method of targeting a subject molecule to cells expressing a DC-SIGN receptor by exposing the cells to a targeting complex, wherein the targeting complex comprises the subject molecule and a DC-SIGN blocker, and wherein the exposure is performed under conditions such that the DC-SIGN blocker binds DC-SIGN on cells expressing the DC-SIGN receptor, thereby targeting the subject molecule to cells expressing the DC-SIGN receptor.

Methods of targeting a subject molecule to cells expressing the DC-SIGN receptor include some embodiments wherein: the DC-SIGN blocker is an antibody; the DC-SIGN blocker is a monoclonal antibody; the subject molecule is a protein; the subject molecule is an antibody; the subject molecule is labeled; exposure is carried out in vivo; and exposure is performed in vitro.

Drawings

The invention is described in more detail below with reference to the attached drawing figures, wherein:

FIG. 1 shows infection of ex vivo (ex-vivo) human DCs with DEN-1 virus. DCs infected with the DEN-1 strain FGA/NA d1d (5Ap61 FFU/cell) were fixed with 3% PFA (in PBS) 40h post infection and increased in permeability with 0.1% Triton X-100 (in PBS). Intracellular DEN proteins were visualized by direct immunofluorescence with anti-DEN-1 virus HMAF and nuclei stained with Hoechst 33258. DEN virus-infected DCs (viral antigens) and chromatin condensation (apoptotic nuclei) were observed by fluorescence. Apoptotic DCs are shown (arrows).

FIG. 2 shows apoptotic DNA fragmentation of DEN-1 virus infected DCs. DEN antigen (viral antigen) in infected DCs was detected by indirect immunofluorescence as described in the legend to FIG. 1 and apoptosis (TUNEL) of infected DCs was simultaneously detected by TUNEL. TUNEL positive cells were observed by fluorescence. TUNEL positive cells are shown (arrow). Low (a) or high (B) magnification.

FIG. 3 shows that anti-DC-SIGN Mab1B10.2.6 blocks DEN-1 virus infection of human DCs. Prior to infection, DCs were incubated with either anti-DC-SIGN Mab 110 (20. mu.g/ml) or anti-DEN E Mab9D12 (dilution 1: 50) (Desprs et al Virology, 196: 209-219, 1993) for 20 minutes. In the presence of Mab, antibody-treated DCs were infected with the DEN-1 strain FGA/NA d1d for 2 hours. Viral antigens were detected by indirect immunofluorescence as shown in the legend to figure 1.DC show the percentage of infected DCs 42 hours after infection.

FIG. 4 shows flavivirus infections of THP-1 and THP-1/DC-SIGN cells. After 40 hours of infection, viral antigens were detected in cells infected with DEN-1 strain FGA/NA did (5AP61 FFU/cell), YF strain 17D-204(50 VEROFFU/cell) or WN strain IS-98-ST1(5AP61 FFU/cell) by indirect immunofluorescence as shown in the legend of FIG. 1. Viral antigens were visualized as anti-DEN-1 virus HMAF (AB anti-DEN-1), anti-YF virus HMAF (AB anti-YF), or anti-WN virus HMAF (Ab anti-WN). (A) Mock-infected THP-1 cells (top) after 40 hours of infection, or THP-1 cells infected with flavivirus (bottom) and infected THP-1/DC cells (bottom) (m.o.i., multiplicity of infection). (B) The percentage of infected cells is shown. Values represent the mean soil SD of triplicate tests.

FIG. 5 shows that mannan, EDTA and antibody-specific DC-SIGN are sufficient for DEN-1 virus to infect THP-1/DC/SIGN cells. Prior to infection, THP-1/DC/SIGN cells were incubated with either Mab9D12 (diluted 1: 50), Mab BD12.5 (20. mu.g/ml), Mab1B10.2.6 (20. mu.g/ml), EDTA (5mM), mannan (20. mu.g/ml), or sham treatment (control). The treated cells were infected with DEN-1 strain FGA/NA d1d (5AP61 FFU/cell) for 2 hours in the presence of the reagent. Viral antigens were detected by indirect immunofluorescence as shown in the legend to figure 1. The percentage of infected cells 48 hours after infection is shown. Values represent mean ± SD of triplicate detections.

FIG. 6 shows DEN-1 virus infecting a clone of THP-1 cells expressing a mutant form of DC-SIGN. At 40 hours post-infection, viral antigens were detected by indirect immunofluorescence in THP-1. delta.35 cell clones (THP-1/DC-SIGN mutation 35) infected with the DEN-1 strain FGH/NA did (5AP61 FFU/cell) as depicted in the legend of FIG. 1.

FIG. 7 shows a comparison of DC-SIGN expressing THP-1 cell clones (THP/DC-SIGN cells) infected with DEN-1, DEN-2, DEN-3 and DEN-4 viruses with THP-1 cells.

Detailed Description

The present invention relates to a method of preventing or treating a disease in a mammal, wherein at least one symptom of the disease is mediated at least in part by the binding of an effector molecule to the DC-SIGN receptor of the mammal to be treated. The method comprises administering to the mammal an amount of a DC-SIGN blocker sufficient to substantially inhibit binding of an effector molecule to the DC-SIGN receptor, thereby preventing or treating the disease.

In the present invention, "mammal" refers to any animal of the class mammalia. Non-limiting examples of mammals include: humans and apes; pets such as dogs, cats, mink, and guinea pigs; livestock, such as pigs, cattle, horses, sheep, goats, and camels; and zoo animals such as bears, zebras, elephants, and buffalos. The mammal is preferably a human.

As used herein, a "disease" is any pathological condition in a mammal that is caused by, for example, an infection, genetic defect, or material exposed to the environment. The methods and compositions of the invention are useful for preventing or treating a disease characterized by at least one symptom of the disease mediated at least in part by binding of an effector molecule to a DC-SIGN receptor on a cell of a mammal, such as a dendritic cell or alveolar macrophage. Specific examples of such diseases include viral infections. One specific example of a viral infection that may be treated by the methods of the present invention is dengue virus infection in humans.

The human "DC-SIGN receptor" is genetically defined to mean DC-SIGN (Curtis et al, 1992) and/or DC-SIGNR (Pohimann et al, 2001), and/or an analog of DC-SIGN or DC-SIGNR. One skilled in the art will recognize that in some cases it may be preferable or even necessary to use one or the other of these forms of the DC-SIGN receptor. One skilled in the art will recognize that human DC-SIGN protein can be obtained in a variety of ways. For example, human DC-SIGN can be purified from human dendritic cells obtained from an autologous source, such as from human blood, or from an autologous source, such as human dendritic cells generated from human dendritic cell precursor cells in tissue culture. Human DC-SIGN may also be expressed using recombinant systems, using cultured dendritic cells as hosts or using a suitable xenogenic (heterologous) cell line, for example COS-7 or HeLa cells, or bacteria such as E.coli.

For non-human mammals, "DC-SIGN receptor" refers to a human DC-SIGN receptor homolog (homologues). One skilled in the art will recognize that such proteins can be identified in any of a variety of ways. These include expression cloning, polymerase chain reaction using degenerate (degenerate) oligonucleotide primers, and low stringency screening of bacterial or phage libraries.

Dendritic cells are a qualitatively different population of morphologically similar cell types in lymphoid or non-lymphoid tissues. Dendritic cells function as antigen presenting cells, which efficiently capture antigen in peripheral tissues and process it to form MHC-peptide complexes. Dendritic cells are also involved in early activation of non-MHC-restricted γ δ and CDI-restricted T cells specific for various mycobacterial glycoproteins, including CAM (Kaufmann, 2001 and Moody, et al, 2000). Upon antigen uptake, these immature dendritic cells acquire a specific capacity to migrate from the periphery to the T cell region of the secondary lymphoid organs. Dendritic cells convert antigens from foreign cells and infectious microorganisms into short peptides that bind to membrane proteins of the Major Histocompatibility Complex (MHC). These MHC peptide complexes are formed within the cell but are ultimately presented on the plasma membrane where they act as ligands for antigen-specific T Cell Receptors (TCRs). In addition to forming TCR ligands, dendritic cells perform a number of other functions that make them immune-controlling in several links (Steinman, 2000).

Alveolar macrophages and dendritic cells are examples of cells that express the DC-SIGN receptor. Endothelial cells are examples of cells expressing DC-SIGNR.

It will be understood by those skilled in the art that dendritic cells can be obtained from in vivo sources such as mammalian blood, or grown in vitro by culturing dendritic cell precursor cells under appropriate conditions. Dendritic cell precursor cells include monocytes prepared as in example 2.

An "effector molecule" is any molecule that specifically binds to a DC-SIGN receptor on a mammalian cell, such as a dendritic cell or alveolar macrophage of a mammal, and thereby mediates a symptom associated with a disease in the mammal. Examples of effector molecules are ligands present on the virus that bind to receptors of mammalian cells and thereby facilitate entry of the virus into the cells of the mammal. For the case where the effector molecule is a ligand on a virus, the effector molecule may be referred to as a "viral effector molecule". Examples of such ligands include gp120 of HIV and glycoprotein E of dengue virus, which binds to a DC-SIGN receptor on cells such as human dendritic cells or alveolar macrophages, and in the case of dengue virus, may thereby facilitate entry of the virus into cells expressing DC-SIGN. The dengue virus E glycoprotein is thus a "dengue virus effector molecule". Other types of effector molecules are endogenous ligands of mammals. Such ligands include ligands that bind to other cell surfaces as well as soluble ligands that can localize in the extracellular space of a particular tissue or in the systemic circulation.

"symptoms" refer to any pathological manifestations of the disease to be treated. If binding (reduction or increase) of the modulated effector molecule to the DC-SIGN receptor results in a detectable reduction in the occurrence of the symptoms or their severity or both, the symptoms are caused at least in part by binding of the effector molecule to the DC-SIGN receptor on dendritic cells of the mammal to be treated. In a preferred embodiment of the invention, the symptoms disappear or are prevented from appearing after decreasing the binding of effector molecules to the DC-SIGN receptor.

An effector molecule is said to "specifically bind" to a DC-SIGN receptor of a cell such as a dendritic cell or alveolar macrophage of a mammal to be treated if binding of the effector molecule to the DC-SIGN receptor is not competitively inhibited by the presence of an unrelated molecule such as fetal bovine serum, but is inhibited by a DC-SIGN antibody such as 1B10.2.6 and/or another effector molecule.

An example of an effector molecule that specifically binds to a DC-SIGN receptor on a cell, such as a dendritic cell or alveolar macrophage of a mammal to be treated, is dengue virus E glycoprotein. Binding of E glycoprotein on the surface of dengue virus to DC-SIGN could not be inhibited by 0.2% bovine serum albumin as shown in FIGS. 3 and 5. However, this binding was inhibited by DC-SIGN specific antibodies, as shown in FIGS. 3 and 5, or by soluble mannan added to the medium, as shown in FIG. 5, or by EDTA added to the medium, as shown in FIG. 5.

It will be appreciated by those skilled in the art that these assays can also be used to identify effector molecules that specifically bind to DC-SIGN receptors on other cells, for example, dendritic cells of the mammal to be treated. It will also be apparent to those skilled in the art that other equivalent test methods may be used in place of those specifically mentioned in the examples.

Once an effector molecule is known to specifically bind to the DC-SIGN receptor, the binding of the effector molecule to DC-SIGN can simply be referred to as "binding". One skilled in the art will also appreciate that this binding is specific. In this regard, "modulation" of the binding may be discussed. Modulation may include "suppression" or "enhancement".

"modulation" refers to modulation, including the induction of a change in a property of a molecule. In the present invention, "modulation" refers to modulation and alteration of the binding of an effector molecule to its receptor. The effect of this modulation is to inhibit binding or enhance binding or to exert other regulatory controls.

In the present invention, "inhibition" of binding refers to a reduction in the total amount of effector molecules bound to DC-SIGN over a fixed period of time. Inhibition of binding of effector molecules may be achieved by providing DC-SIGN blockers. A "DC-SIGN blocker" is any molecule that significantly inhibits the binding of a particular effector molecule at a concentration at which the effector molecule specifically binds DC-SIGN. In a preferred embodiment, the DC-SIGN blocker used is a monoclonal antibody that specifically binds DC-SIGN. In another preferred embodiment, the DC-SIGN blocker used comprises a binding moiety for dengue virus E glycoprotein.

In the present invention, "enhancement" of binding refers to increasing the total amount of effector molecules bound to DC-SIGN over a fixed period of time. Enhancement of binding of effector molecules can be achieved by providing a DC-SIGN enhancer (enhancer). A "DC-SIGN enhancer" is any molecule that significantly increases the binding of a particular effector molecule at a concentration at which the effector molecule specifically binds DC-SIGN.

"binding moiety" refers to a portion of a molecule that substantially retains the ability to bind a second molecule when other portions of the molecule are removed or modified, or when the portion (binding moiety) is placed in a heterologous molecule. For example, for an effector molecule as defined herein, a binding moiety for the effector molecule may be defined. A binding moiety of an effector molecule is a portion of the effector molecule that substantially retains the ability to bind DC-SIGN when other portions of the molecule are removed or modified, or when the portion (binding moiety) is placed in a heterologous molecule. In this regard, one skilled in the art can define "substantially retained" based on the particular characteristics of the binding moiety sought.

By "substantially inhibit" is meant greater than 80% inhibition, greater than 90% inhibition, greater than 95% inhibition, or greater than 99% inhibition. In a preferred embodiment of the invention, the inhibition of binding is up to about 90%.

"inhibition" is measured by comparing the extent of binding of effector molecules to DC-SIGN in the presence of a DC-SIGN blocker with the extent of binding of effector molecules to DC-SIGN in the absence of a DC-SIGN blocker. The ratio of the degree of binding in the presence of the DC-SIGN blocker to the degree of binding in the absence of the DC-SIGN blocker is then determined. Thus the percentage inhibition is proportional to the reduction in the amount of binding. For example, a ratio of 0.1 indicates a 90% reduction in binding.

The term "treating" refers to administering a therapy to an individual who has exhibited at least one symptom of a disease. Such individuals include individuals diagnosed as having a known disease.

The term "prevention" refers to the administration of prophylactic therapy to individuals who may ultimately develop the disease but who are not currently suffering from it (i.e., those in need of prophylactic measures). Such individuals may be identified on the basis of risk factors known to be associated with the subsequent occurrence of the disease.

The term "therapeutic benefit" refers to an improvement in at least one symptom of a disease, a slowing of disease progression (e.g., a slowing of progression of the severity of at least one symptom of a disease), or a cessation of progression of at least one symptom of a disease. The therapeutic benefit is determined by comparing disease symptoms before and after administration of the DC-SIGN blocker.

The "antibody" refers to any antibody that can be prepared by any technique known in the art. Suitable antibodies can be obtained by immunizing a host animal with a peptide comprising all or part of the target protein. Suitable host animals include mice, rats, sheep, goats, hamsters, rabbits, and the like. The source of the protein immunogen may be mouse, human, rat, monkey, or a microorganism such as a bacterium or virus, and the like. The host animal is usually of a different species from the immunogen, e.g., a mouse immunized with a human protein, etc.

The immunogen may comprise the complete protein or a fragment or derivative thereof. Preferred immunogens comprise all or part of a subject protein in which the residues contain post-translational modifications, such as glycosylation, which the native target protein possesses. Immunogens comprising extracellular domains can be produced by a variety of routes known in the art, such as expression of cloned genes using conventional recombinant methods, isolation from tumor cell culture supernatants, and the like.

To produce polyclonal antibodies, the first step is to immunize a host cell with a target protein, wherein the target protein is preferably in a substantially purified form, containing less than about 1% contamination. The immunogen may comprise the entire target protein or a fragment or derivative thereof. To enhance the immune response of the host animal, the target protein may be combined with an adjuvant, where suitable adjuvants include alum, dextran, sulfate, large polymeric anions, oil and water emulsions, such as freund's adjuvant, freund's complete adjuvant, and the like. The target protein may also be conjugated to a synthetic carrier protein or a synthetic antigen. Various hosts can be immunized to produce the polyclonal antibodies. Such hosts include rabbits, guinea pigs, rodents such as mice, rats, sheep, goats, and the like. The target protein is typically administered to the host by the intradermal route in one initial dose, followed by one or more booster doses, typically at least 2. After immunization, the blood of the host is collected and the serum is subsequently separated from the blood cells. The Ig fraction in the resulting antiserum can be further separated by known methods, such as ammonium salt fractionation, DEAE chromatography, and the like.

Monoclonal antibodies are produced by conventional techniques. Generally, the spleen and/or lymph nodes of the immunized host animal provide a source of plasma cells. Hybridoma cells are produced by immortalizing plasma cells by fusion with myeloma cells. Culture supernatants from individual hybridomas are screened using standard techniques to identify those hybridomas that produce antibodies with the desired specificity. Suitable animals for generating monoclonal antibodies to proteins of humans include mice, rats, hamsters, and the like. To generate antibodies against mouse proteins, the animals are typically hamsters, guinea pigs, rabbits, and the like. Antibodies can be purified from hybridoma cell supernatants or ascites fluids by conventional techniques, e.g., by affinity chromatography, using a protein of the invention bound to an insoluble support protein a sepharose, and the like.

The antibodies produced may be single chain, rather than the usual multimeric structure. Jost et al (1994) J.B.C.269: 26267-73 and others describe single chain antibodies. The DNA sequences encoding the heavy chain variable region and the light chain variable region are linked to a spacer region that encodes at least 4 small neutral amino acid residues, including glycine and/or serine. The proteins encoded by such fusions are capable of assembling a functional variable domain that retains the specificity and avidity of the original antibody.

Also provided are "artificial" antibodies, such as antibodies and antibody fragments produced and screened in vitro. In some embodiments, such antibodies are displayed on the surface of a bacteriophage or other viral particle. In many embodiments, such artificial antibodies are fusion proteins formed with viral or phage structural proteins, including the M13 gene III protein. Methods for producing such artificial antibodies are well known to those skilled in the art. See, for example, U.S. patent nos.: 5,516,637; 5,223,409; 5,658,727, respectively; 5,667,988, respectively; 5,498,538, respectively; 5,403,484; 5,571,698; and 5,625,033.

For in vivo use, particularly for injection into humans, it is desirable to reduce the antigenicity of the antibody. The immune response of the recipient to the blocking agent may potentially shorten the time for effective treatment. Methods for humanizing antibodies are known in the art. Humanized antibodies may be the product of an animal having a transgene in a human immunoglobulin constant region gene (see, e.g., international patent applications WO 90/10077 and WO 90/04036). Alternatively, the desired antibody may be produced by genetic engineering, i.e. by replacing CH1, CH2, CH3, the hinge domain and/or the framework domain by the corresponding human sequence by recombinant DNA technology (see WO 92/02190).

The use of Ig cDNAs for the construction of chimeric immunoglobulin genes is known to those skilled in the art (Liu et al (1987) P.N.A.S.84: 3439 and (1987) J.Immunol.139: 3521). mRNA is isolated from hybridomas or other antibody-producing cells and used to produce cDNA. The desired cDNA can be amplified by polymerase chain reaction using specific primers (U.S. Pat. Nos. 4,683,195 and 4,683,202). Alternatively, libraries are constructed and screened to isolate the desired sequence. The DNA sequence encoding the variable region of the antibody is then fused to the human constant region sequence. The sequence of the human constant region genes can be found in Kabat et al (1991) Sequences of Proteins of Immunological Interest, N.I.H.publication No. 91-3242. The human C region gene can be easily obtained from a known clone. The isotype can be selected for the desired effector function, such as fixed complement, or antibody-dependent cytotoxic activity. Preferred isotypes are IgG1, IgG3, and IgG 4. Human light chain constant regions κ or λ may be used. Then, the chimeric humanized antibody can be expressed by a conventional method.

In another embodiment, the antibody may be a fully human antibody. For example, a xenogenic antibody identical to a human antibody can be used. Allogeneic human antibodies refer to antibodies identical to human antibodies, i.e., they are fully human antibodies, except that they are produced by a non-human host that has been genetically engineered to express human antibodies. See, e.g., WO 98/50433; WO98, 24893 and WO 99/53049, the disclosures of which are incorporated herein by reference.

Antibody fragments such as Fv, F (ab')₂And Fab can be prepared by cleaving the intact protein, for example by protease or chemical cleavage. Alternatively, a truncated gene can be designed. For example, encoding F (ab')₂The chimeric gene of a portion of the fragment may comprise a DNA sequence encoding the CH1 domain and hinge region of the H chain, followed by a translation stop codon to produce a truncated molecule.

The consensus sequences of the H and LJ regions can be used to design oligonucleotides for use as primers to introduce useful restriction sites within the J region for subsequent ligation of V region fragments to human C region fragments. The C region cDNA can be modified by site-directed mutagenesis to place a restriction site at a similar position in the human sequence.

Expression vectors include plasmids, retroviruses, YACs, episomes from EBV, and the like. One convenient vector is one that encodes a fully functional human CH or CL immunoglobulin sequence, with appropriate restriction sites engineered so that any VH or VL sequence can be readily inserted and expressed. In such vectors, processing typically occurs between a processing donor site within the inserted J region and a processing acceptor site preceding the human C region, and also occurs in a processing region located within the human CH exon. Polyadenylation and transcription termination occur at natural chromosomal sites downstream of the coding region. The resulting chimeric antibody can be linked to any strong promoter, including the reverse transcribed LTR, such as the SV-40 early promoter (Okayama et al (1983) mol.cell.Bio.3: 280), Rous sarcoma virus LTR (Gorman et al (1982) P.N.A.S.79: 6777), and Moloney murine leukemia virus LTR (Grosschedl et al (1985) Cell 41: 885); native Ig promoters, and the like.

An example of a disease that can be prevented or treated using the present invention is dengue virus infection. The results presented in the examples demonstrate for the first time the role of DC-SIGN in the binding of dengue virus to human dendritic cells.

The results described herein, including those described in the examples, show that highly purified DEN-1 virus carrying mosquito N-linked oligosaccharides is able to replicate in human DCs and produce progeny virus. Apoptotic cell death was observed in DEN-1 virus infected DCs. The C-lectin molecule DC-SIGN is expressed on the surface of DC. The experiments described herein attempted to determine whether the DC-specific adhesion receptor DC-SIGN has the ability to facilitate DEN virus infection of human DCs. The results indicate that DC-SIGN specific antibodies have a blocking effect on DEN-1 virus infection. Thus, a novel function of DC-SIGN was identified, namely a binding protein for DEN virus, possibly by interaction with the E glycoprotein. The DC-SIGN mediated DEN virus infection process on DC provides a new mechanism for designing antiviral compounds.

In accordance with these results, the present invention provides a method for preventing or treating a disease in a mammal, wherein at least one symptom of the disease is mediated at least in part by the binding of an effector molecule to the DC-SIGN receptor of the mammal to be treated, and wherein the method comprises administering to the mammal an amount of a DC-SIGN blocker sufficient to substantially inhibit the binding of the effector molecule to the DC-SIGN receptor, thereby preventing or treating the disease.

In some embodiments, the DC-SIGN blocker is a mannosylated molecule that binds to the DC-SIGN receptor. The mannosylated molecule may be mannan.

The invention also provides a method for preventing or treating a viral infection in a mammal, wherein the viral infection is mediated at least in part by the binding of a viral effector molecule to the DC-SIGN receptor of the mammal to be treated, wherein the method comprises administering to the mammal an amount of a DC-SIGN blocker sufficient to substantially inhibit the binding of the viral effector molecule to the DC-SIGN receptor, thereby preventing or treating the viral infection.

In a preferred embodiment of the invention, the effector molecule is identical to the DC-SIGN blocker. In a second preferred embodiment, the effector molecule is different from the DC-SIGN blocker.

Interestingly, both dengue virus and HIV (as well as SIV) can bind DC-SIGN. Binding of HIV to dendritic cells is mediated by binding of the gp120 glycoprotein of HIV to DC-SIGN. Gp120 is thus a viral effector molecule. The present invention thus provides a method for the prevention and treatment of HIV infection. In particular, it is an object of the present invention to provide a method for preventing or treating HIV or SIV infection in a human or simian. The methods comprise administering to the human or simian an amount of a DC-SIGN blocker sufficient to substantially inhibit the interaction of HIV or SIV with a DC-SIGN receptor on dendritic cells of the human or simian, thereby preventing or treating HIV or SIV infection.

DC-SIGN is also thought to have a crucial role in mediating the loose adhesion known to occur between dendritic cells and T cells in the absence of foreign antigens. This adhesion is believed to provide the necessary opportunity for the TCR to scan the surface of dendritic cells and identify the presence of minute quantities of TCR ligands, and thus be activated by such ligands. To this end, the interaction between DC-SIGN on dendritic cells and ICAM-3 on T cells is likely to be crucial for the process of T cell activation and stimulation. This model suggests that DC-SIGN-ICAM-3 interaction may play a role in mediating and/or potentiating other T cell stimulatory effects of dendritic cells.

To this end, DC-SIGN blockers may be effective anti-inflammatory agents by blocking the interaction of ICAM-3 effector molecules with DC-SIGN. Accordingly, the present invention also provides a method for preventing or treating inflammation in a mammal, which is caused by interaction of ICAM-3 on T cells of the mammal and DC-SIGN receptors on dendritic cells of the mammal. The method comprises administering to the mammal an amount of a DC-SIGN blocker sufficient to substantially inhibit the interaction of ICAM-3 on T cells of the mammal with DC-SIGN receptors on dendritic cells of the mammal, thereby preventing or treating inflammation.

The invention also provides the use of a DC-SIGN modulator in an amount sufficient to substantially modulate the binding of an effector molecule to a DC-SIGN receptor for the manufacture of a medicament for the prevention or treatment of a disease in a mammal, wherein at least one symptom of the disease is mediated at least in part by the binding of the effector molecule to the DC-SIGN receptor of the mammal to be treated.

The invention also provides the use of a DC-SIGN blocker in an amount sufficient to substantially inhibit the binding of an effector molecule to the DC-SIGN receptor for the manufacture of a medicament for the prevention or treatment of a disease in a mammal, wherein at least one symptom of the disease is mediated at least in part by the binding of the effector molecule to the DC-SIGN receptor of the mammal to be treated. In some embodiments, the DC-SIGN blocker is a blocking derivative of the effector molecule. In another embodiment, the DC-SIGN blocker is an antibody. In another embodiment, the antibody specifically binds DC-SIGN. In other embodiments, the antibody specifically binds to an effector molecule.

In other embodiments, the DC-SIGN blocker is a mannosylated molecule that binds to the DC-SIGN receptor; the mannosylated molecule is preferably mannan.

The invention also provides the use of a DC-SIGN modulator in an amount sufficient to modulate substantially the binding of a viral effector molecule to a DC-SIGN receptor for the manufacture of a medicament for preventing or treating a viral infection in a mammal, wherein the viral infection is mediated at least in part by the binding of the viral effector molecule to the DC-SIGN receptor of the mammal to be treated.

The invention also provides the use of a DC-SIGN blocker in an amount sufficient to substantially inhibit binding of a viral effector molecule to a DC-SIGN receptor for the manufacture of a medicament for preventing or treating a viral infection in a mammal, wherein the viral infection is mediated at least in part by binding of the viral effector molecule to the DC-SIGN receptor of the mammal to be treated. In some embodiments of this use, the viral effector molecule is a molecular component of the viral envelope. In other embodiments, the molecular component of the viral envelope is an envelope glycoprotein.

In a further embodiment, the DC-SIGN blocker comprises a binding moiety of said viral effector molecule. In other embodiments, the DC-SIGN blocker comprises a binding moiety of the envelope glycoprotein. In other embodiments, the DC-SIGN blocker is an antibody. The antibody is a monoclonal antibody. In other embodiments, the mammal is a human and the monoclonal antibody is humanized. The antibody may specifically bind DC-SIGN or specifically bind a viral effector molecule. In further embodiments, the antibody specifically binds to a binding moiety of said viral effector molecule. In addition, DC-SIGN blockers are mannosylated molecules that bind to the DC-SIGN receptor. The mannosylated molecule may be mannan.

In a preferred embodiment, the viral infection is a flaviviridae viral infection and the viral effector molecule is an effector molecule of a flaviviridae virus. In a more preferred embodiment, the flaviviridae infection is a dengue virus infection and the flaviviridae effector molecule is a dengue effector molecule. In a more preferred aspect, the mammal is a human. In some embodiments, the dengue virus effector molecule is a molecular component of a dengue virus envelope. In a further embodiment, the molecular component of the dengue virus envelope is a dengue virus envelope glycoprotein. In another embodiment, the dengue virus envelope glycoprotein is dengue virus E glycoprotein.

In embodiments of the use wherein the viral infection is dengue viral infection and the viral effector molecule is dengue viral effector molecule, some embodiments are included wherein: the DC-SIGN blocker comprises a binding moiety for a dengue virus effector molecule; the DC-SIGN blocker comprises a binding moiety of dengue virus E glycoprotein; DC-SIGN blockers are recombinantly produced proteins; and the DC-SIGN blocker is an antibody. In embodiments where the DC-SIGN blocker is an antibody, some embodiments are included wherein: the antibody is a monoclonal antibody; the mammal is a human and the monoclonal antibody is humanized; the antibody specifically binds DC-SIGN; the monoclonal antibody is Mab1810.2.6; and the antibody specifically binds to a dengue virus effector molecule. Among the embodiments in which the antibody specifically binds to a dengue virus effector molecule is an embodiment in which the dengue virus effector molecule is dengue virus E glycoprotein.

The invention also provides the use of a DC-SIGN modulator in an amount sufficient to modulate the binding of HIV or SIV to a DC-SIGN receptor on a dendritic cell of a human or simian in the manufacture of a medicament for the prevention or treatment of HIV or SIV infection of said human or simian.

The invention also provides the use of a DC-SIGN blocker in an amount sufficient to substantially inhibit binding or interaction of HIV or SIV with a DC-SIGN receptor on a dendritic cell of a human or simian in the manufacture of a medicament for the prevention or treatment of HIV or SIV infection of said human or simian. In some embodiments, the DC-SIGN blocker comprises a binding moiety of dengue virus E glycoprotein. In a further embodiment, the human being is prevented or treated from HIV infection.

The invention also provides the use of a DC-SIGN modulator in an amount sufficient to substantially modulate the binding of ICAM-3 on T cells of a mammal to a DC-SIGN receptor on dendritic cells of the mammal in the preparation of a medicament for preventing or treating inflammation in the mammal caused by the specific binding of ICAM-3 on T cells of the mammal to a DC-SIGN receptor on dendritic cells of the mammal.

The invention also provides the use of a DC-SIGN blocker in an amount sufficient to substantially inhibit the binding or interaction of ICAM-3 on T cells of a mammal with a DC-SIGN receptor on dendritic cells of the mammal in the manufacture of a medicament for preventing or treating inflammation in the mammal caused by the specific binding of ICAM-3 on T cells of the mammal with a DC-SIGN receptor on dendritic cells of the mammal. In some embodiments, the DC-SIGN blocker comprises a binding moiety of dengue virus E glycoprotein. In other embodiments, the mammal is a human.

The invention also provides pharmaceutical compositions comprising a DC-SIGN blocker. Such compositions may be suitable for pharmaceutical use and administration to a patient. The compositions generally contain an achievable therapeutic concentration of purified DC-SIGN blocker and a pharmaceutically acceptable excipient. As used herein, "pharmaceutically acceptable excipients" include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, suitable for pharmaceutical administration. The use of such media and formulations for pharmaceutically active substances is known in the art. The compositions may also contain other active compounds to provide supplemental, additional, or enhanced therapeutic functions. The pharmaceutical composition may also be placed in a container, package, or dispenser along with instructions for administration.

The pharmaceutical compositions of the present invention are formulated to suit their route of administration. Methods of effecting administration are well known to those of ordinary skill in the art. Administration may be, for example, intravenous, intramuscular, subcutaneous, or by inhalation.

Solutions or suspensions for subcutaneous administration typically include one or more of the following ingredients: sterile diluents such as water for injection, saline solution, non-volatile oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants, such as ascorbic acid or sodium sulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates; and agents for regulating osmotic pressure, such as sodium chloride or glucose. The pH can be adjusted with acids or bases, for example with hydrochloric acid or sodium hydroxide. Such formulations may be enclosed in ampoules, disposable syringes or multi-dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injection include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, sterile water, Cremophor ELTM (BASF, Parsippany, NJ), or Phosphate Buffered Saline (PBS). In all cases, the composition must be sterile and should be fluid for ease of injection. It must be stable under the conditions of manufacture and storage and must be resistant to contamination by microorganisms such as bacteria and fungi during storage. The carrier can be a solvent or dispersant containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained by some means, such as by the use of a coating, such as lecithin, by the maintenance of the required particle size for the dispersion and by the use of surfactants. The action of microorganisms can be prevented by using various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, isotonic agents, for example sugars, polyalcohols such as mannitol, sorbitol, sodium chloride, can be added to the composition. Prolonged absorption of the injectable compositions can be brought about by the addition to the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile acid salts, fusidic acid derivatives, and the like. Transmucosal administration can be carried out using nasal sprays or suppositories. For transdermal administration, it is well known in the art to formulate the active compounds into ointments, salves, gels, or creams.

For inhalation administration, the composition containing the DC-SIGN blocker may be delivered in the form of an aerosol using a pressure vessel or dispenser with a propellant such as carbon dioxide or with a nebulizer.

In one embodiment, purified DC-SIGN blockers are prepared using a number of vehicles that prevent their rapid clearance from the body, such as controlled release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid may be used. Methods of preparing such formulations will be apparent to those skilled in the art. Raw materials are available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions containing LAM can also be used as pharmaceutically acceptable carriers and can be prepared according to methods known to those skilled in the art, as described in U.S. patent No.4,522,811.

Agents useful in therapy, such as growth factors (e.g., BMP, TGF- β, FGF, IGF), cytokines (e.g., interleukins and CDF), antibiotics, and any other therapeutic agent beneficial to the disease being treated, may optionally be added or administered simultaneously or sequentially with the DC-SIGN blocker.

It is particularly preferred to formulate the compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suitable as unitary dosages for the subject to be treated; each unit dose contains a predetermined amount of the active compound calculated to produce the desired therapeutic effect in association with the desired pharmaceutical carrier. The specification for the dosage unit forms of the invention are directly dependent upon the unique characteristics of the active compound and the particular therapeutic effect to be achieved, as well as the limitations inherent in the art of formulation, e.g., active compounds used to treat individuals.

Toxicity and therapeutic efficacy of compositions comprising DC-SIGN blockers can be determined by standard pharmaceutical procedures in cell culture or animal experiments, e.g., determining LD50 (the dose that causes death in 50% of the population) and ED50 (the dose that produces a therapeutic effect in 50% of the population). The ratio between toxic and effective therapeutic doses is the therapeutic index, which can be expressed as the ratio LD50/ED 50. DC-SIGN blockers with high therapeutic indices are preferred.

Data from cell culture analysis and animal experiments can be used to formulate a range of dosage for use in humans. The range of doses of such compounds is preferably a circulating concentration that includes ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration employed. For any DC-SIGN blocker used in the present invention, a therapeutically effective amount may be estimated initially from cell culture analysis. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes IC50 (i.e., the test concentration of DC-SIGN blocker that achieves half the maximal inhibition of symptoms) as determined in cell culture. Levels in plasma can be determined by, for example, high performance liquid chromatography. The effect of any particular dose can be monitored by suitable biological detection methods.

The targeting complexes of the invention comprise at least one DC-SIGN blocker molecule covalently bound to at least one subject molecule. In some embodiments, a DC-SIGN blocker molecule is covalently bound to a subject molecule. In other embodiments, more than one DC-SIGN blocker molecule may be covalently bound to a subject molecule. A plurality of DC-SIGN blocker molecules may each independently be covalently bound to the subject molecule; alternatively, one or more of the more than one DC-SIGN blocker molecules may be covalently bound only to one or more other DC-SIGN blocker molecules, wherein at least one blocker molecule is itself covalently bound to a subject molecule.

In other embodiments, a plurality of subject molecules are covalently bound to one DC-SIGN blocker molecule. The plurality of subject molecules may each independently be covalently bound to the DC-SIGN blocker molecule; alternatively, one or more of the more than one subject molecules may be covalently bound only to one or more other subject molecules, wherein at least one subject molecule is itself covalently bound to a DC-SIGN blocker molecule.

Additional embodiments of the invention employ more than one composition of DC-SIGN blockers of the various types described above. There is no limitation on the variety of such compositions that can be employed. It will be appreciated by those skilled in the art that the particular composition used will depend on a variety of factors, and thus the appropriate composition may be suitably selected for each use of the present invention.

The techniques for preparing the DC-SIGN blockers of the present invention are well known and widely used by those skilled in the art of biochemistry and therefore need not be described in detail herein. However, one skilled in the art will recognize that any suitable technique may be used to form a covalent bond between the subject molecule and the DC-SIGN blocker molecule.

The subject molecule may be any molecule of interest. Non-limiting examples include: small organic molecules, proteins, nucleic acids, carbohydrates, and lipids. One of ordinary skill in the art will appreciate that any known derivative and complex of one or more such molecules may also be used.

In case the subject molecule is a protein, nucleic acid, carbohydrate or lipid, said subject molecule may be derived from a natural source, i.e. purified from an organism comprising the molecule. Alternatively, the subject molecule may be obtained by recombination, i.e. from a recombinant organism that has been genetically engineered to produce the selected subject molecule. In some cases, the recombinant organism used to produce the recombinant subject molecule is one that, like the naturally occurring organism, contains a non-recombinant form of the subject molecule. In other cases, the subject molecule is one that does not naturally occur in the recombinant organism.

The subject molecules of the invention also include derivatives of small organic molecules, proteins, nucleic acids, carbohydrates, and lipids. Here, a derivative is a form of a small organic molecule, protein, nucleic acid, carbohydrate, or lipid that is modified from its native state by the addition, deletion, or alteration of one or more chemical reactive sites on the small organic molecule, protein, nucleic acid, carbohydrate, or lipid. Techniques for preparing derivatives of small organic molecules, proteins, nucleic acids, carbohydrates or lipids are well known and widely used by those skilled in the art of biochemistry and need not be described in detail herein.

In a preferred embodiment, the subject molecule is an antibody.

The subject molecule may also be an antigenic molecule. A molecule is antigenic if it is capable of specific interaction with an antigen recognition molecule of the immune system, such as an immunoglobulin (antibody) or T cell antibody receptor. An antigenic polypeptide contains at least about 5, and preferably at least about 10 amino acids. The antigenic portion of the molecule may be a portion that is immunodominant for recognition by antibodies or T cell receptors, or it may be a portion used to generate antibodies to the molecule, wherein the antigenic portion is conjugated to a carrier molecule used for immunization. An antigenic molecule need not be immunogenic in itself, by which is meant capable of eliciting an immune response without the need for a carrier.

The targeting complex of the invention can be exposed to cells expressing DC-SIGN, such as dendritic cells, in vivo or in vitro. In vivo exposure is achieved by administering a targeting complex in a pharmaceutical composition, which may be any suitable equivalent formulation described herein or known in the art. In this case, the targeting complex can bind to DC-SIGN on the surface of dendritic cells in vivo. In vitro exposure may occur by exposing in vitro grown dendritic cells to a targeting complex.

The following examples are presented to illustrate certain aspects of the invention. Those skilled in the art will recognize that many modifications and variations can be made without departing from the spirit and scope of the invention. Such modifications and variations are also intended to be included within the scope of the present invention. These examples are not intended to limit the invention in any way.

Examples

Example 1: flavivirus virus

The DEN1 (DEN-1) strain FGA/NA d1d (GenBank accession No. AF226686) (Duarte dos Santos et al, Virology, 274: 292, 2000) and the West Nile (WN) strain IS-98-ST1(GenBank accession No. AF481864) (Mashimo et al, PNAS, 99: 11311, 2002) were prepared and purified from a mosquito Aedespsese pseudomonas aeruginosa AP61 monolayer as described previously (Despress et al, Virology, 196: 209, 1993) and virus titrations were performed on AP61 by the focus immunodetection assay (focus immunodetection assay, FIA). Yellow Fever (YF) virus vaccine strain 17D-204(STAMARIL, Pasteur vaccines, Lot E113) (GenBank accession number: X07755) was propagated 2 times in a monolayer of African green monkey kidney VERO cells, purified on a sucrose gradient and titrated on VERO. Infectious titer is expressed as Focus Forming Units (FFU).

Notably, the FGA/NA d1d E glycoprotein is at Asn₆₇Position and Asn₁₅₃Positions have 2N-linked glycosylation sites. Both N-glycosylation sites of DEN-1 glycoprotein E appear to be utilized during N-glycosylation (Courageot et al, J.Virol., 74: 564-572). The IS-98-ST 1E glycoprotein has a single N-linked glycosylation site and appears to be utilized (desspres, personal communication). Whereas the 17D-204E protein is not N-glycosylated. Flavivirus M protein is not glycosylated.

In these experiments, we tested DEN-1 and WN virion-associated E glycoproteins, which carry mature N-linked oligosaccharides from mosquito AP61 cells.

Example 2: human mononuclear cell

Human DCs from purified monocytes, the human monocyte cell line THP-1 (ATCCIB 202) and the cell clone THP/DC-SIGN expressing DC-SIGN were all from Ali Amara (immunology Virus) (Kwon et al, Immunity, 16: 135, 2002). Immature DC, THP-1 and THP/DC-SIGN cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated Fetal Calf Serum (FCS) (Eurobio, lot 160402), 2mM L-glutamine and the antibiotic Peni/Strepto.

DC were attached to poly-L-lysine (Sigma) -coated glass Lab-tek compartment (NalgeNunc International) (5X 10)⁴Cells/cm²). Adherence of THP-1 and THP/DC-SIGN cells to Poly-L-lysine treated glass Lab-tek compartments or polylysine treated 12-well plates (5X 10)⁴Cells/cm²)。

Example 3: viral infection

Cells were washed once with RPMI 1640, incubated with highly purified virus in RPMI 1640 supplemented with 0.2% bovine serum albumin (BSA, pH7.5) (Sigma) for 2 hours at 37 ℃ and placed in fresh medium supplemented with 2% FCS, 2mM L-glutamine and the antibiotic Peni/Strepto for 40 hours at 37 ℃.

Ascites Fluid (HMAF)9801 (C) using DEN-1 virus-specific hyperimmunized mice diluted 1: 50Strain), WN virus-specific HMAF 0801(IS-98-ST1 strain) or YF virus-specific HMAF 9803(FNV strain), by indirect immunofluorescence as described previously (desspres et al, j.virol., 70: 4090, 1996), the percentage of cells expressing viral antigens is determined.

Example 4: inhibition of DC-SIGN mediated viral binding

Adherent cells were incubated with EDTA (5mM), mannan (20. mu.g/ml), anti-DC-SIGNMab 1B10.2.6 (20. mu.g/ml), anti-LMCV Mab 12.5 (isotype control, 20. mu.g/ml), or DEN E-specific Mab9D12 (1: 50 dilution) in RPMI 1640 supplemented with 0.2% BSA at room temperature for 20 minutes followed by binding to the virus for 2 hours. 2 hours after infection, cells were washed with RPMI 1640 and incubated with RMPI 16402% FCS for 40 hours. anti-DC-SIGN Mab1B10.2.6 and anti-LMCV Mab 12.5 were from Ali Amara.

Example 5: immunofluorescence assay

Briefly, cells were fixed with 3% Paraformaldehyde (PFA) (in PBS) for 20 min at room temperature with 50mM NH₄Cl (in PBS) was incubated for 20 min and treated with 0.1% Triton X-100 (in PBS) for 5 min to increase permeability. Intracellular viral antigens were stained with anti-flavivirus HMAF. The secondary antibody was FITC conjugated goat anti-mouse igg (sigma). Cells were observed with a fluorescence microscope.

Example 6: in situ detection of apoptotic cells

To assess nuclear changes associated with apoptotic cell death, the PFA-fixed cells on the slides were treated with 0.1. mu.g/ml Hoechst33258(Sigma) in 0.1% citrate buffer (pH6.0) for 10 minutes at room temperature. Cells are considered apoptotic if the nucleus exhibits a limbus and chromatin condensation. Cells were observed with a fluorescence microscope.

Apoptosis-induced DNA fragmentation was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) (Despres et al, J.Virol., 70: 4090, 1996). With streptavidin-CY^TM3 conjugates (Jackson Immunoresearch) were subjected to TUNEL analysis. Cells were observed with a fluorescence microscope.

Example 7: DEN virus ex vivo infection of human DCs

We have determined whether the DEN-1 strain FGA/NA d1d replicates in DC. As determined by IF analysis with anti-DEN-1 HMAF, 5AP61 FFU/cell inoculation was required so that 50% of human DCs infected with DEN virus within 40 hours (fig. 1). At 48 hours post-infection, infectious particles aggregated in FGA/NA d1 d-infected DCs reached 9 (+ -3) x 10⁴APGlFFU/ml (50,000 DCs). Unlike DEN-1 virus, less than 1% of DCs were infected with either the WN strain IS-98-ST1(5AP61 FFU/cell m.o.i.) or the YF vaccine strain (50 VEROFFU/cell m.o.i.) 40 hours post infection (data not shown).

Infection of DCs with DEN-1 strain FGA/NA d1d caused apoptosis 40 hours after infection, as determined by Hoescht 33258 staining (FIG. 1) and the TUNEL method (FIGS. 2A & B).

Example 8: anti-DC-SIGN Mab1B10.2.6 blocking DEN-1 virus infection DC

We analyzed the effect of anti-DC-SIGN specific Mab1B10.2.6 on the infectivity of DEN-1 virus strain FGA/NA d1 d. FIG. 3 illustrates that 20. mu.g/ml Mab1B10.2.6 blocked infection of DC cells by FGA/NA d1d as determined by the IF method. As a positive control, anti-E Mab9D12 reduced the infectivity of DEN-1 virus by 70%. FGA/NA d1 d-infected DCs treated with Mab1B10.2.6 produced very low numbers of infectious particles (< 5AP61 FFU/ml).

Example 9: DEN virus infects human monocyte THP-1 and THP/DC-SIGN

Specific interaction between DC-SIGN and DEN viruses

We further investigated the specificity of the interaction between DC-SIGN and DEN viruses. For this purpose, human monocytic THP-1 cells were first infected with the DEN-1 strain FGA/NA d1 d. At m.o.i. 5AP61 FFU/cell, less than 1% of THP-1 appeared positive for DEN antigen as determined by IF analysis (fig. 4A & B). Similarly, YF virus vaccine strain 17D-204(m.o.i. 50 VEROFFU/cell) failed to replicate in the THP-1 cell line (FIGS. 4A & B). Whereas an m.o.i.was required to be 5AP61 FFU/cell to allow approximately 70% of the THP-1 cells to be infected with the WN strain IS-98-ST1 (FIGS. 4A & B).

Role of DC-SIGN in the infectivity of DEN-1 Virus on THP-1

To determine whether DC-SIGN confers infectivity of DEN-1 virus on THP-1, we tested a human monocytic THP cell line expressing DC-SIGN, THP/DC-SIGN (Kwon et al, Immunity, 16: 135, 2002). At m.o.i. 5AP61 FFU/cell, more than 50% of THP/DC-SIGN cells appeared positive for FGA/NA d1d antigen 48 hours post infection (fig. 4). At 96 hours post infection, the THP/DC-SIGN cells in which DEN-1 virus replicated died. Unlike DEN-1 virus, YF virus vaccine strain 17D-204 did not replicate in THP/DC-SIGN at m.o.i. up to 50 VEROFFU/cell. Also interestingly, DC-SIGN mediated enhanced infection of monocytes by WN virus (figure 4).

3. Effect of mannan, ETDA and DC-SIGN specific Mab1B10.2.6 on DC-SIGN mediated DEN viral binding

We tested the effect of mannan, ETDA and DC-SIGN specific Mab1B10.2.6 on DC-SIGN mediated DEN viral binding. In these experiments, Mab BD12.5 served as a negative control, while anti-E Mab9D12 served as a positive control. THP/DC-SIGN was infected with DEN-1 strain FGA/NAd1d at m.o.i. 5AP61 FFU/cell. When pre-incubated with either EDTA or DC-SIGN specific Mab1B10.2.6 with THP/DC-SIGN cells, the infectivity of DEN-1 virus was substantially eliminated (FIG. 5). At a dose of 201. mu.g/ml mannan reduced infectivity of FGA/NA d1d by 75%. Therefore, it is reasonable to assume that DC-SIGN can promote DEN virus infection of mononuclear cells.

The cytoplasmic tail of DC-SIGN contains 2 defined putative internalization motifs, 1 di-leucine-based motif (dileucine-based motif), and 1 tyrosine-based motif. Next we tested THP-1 cell clone A35 expressing a mutant form of DC-SIGN in which the cytoplasmic domain of the molecule is truncated (Kwon et al, Immunity, 16: 135, 2002). This truncation removed 35 amino acids including the dual leucine motif and the tyrosine-based motif. We found that DEN-1 virus retained most of the infectivity of THP-1 cell clone A35 (FIG. 6). Thus, the cytoplasmic tail did not function in DC-SIGN enhanced DEN-1 viral infection.

Comparison of interactions between DCs and different DEN Virus types

DEN virus [ DEN-1 strain FGA/NA d1d (French Guiana); DEN-2 strain Jam (Jamaica); DEN-3 strain PaH 881 (Thailand); DEN-4 strain 63632(Birmanie) ] infected THP/DC-SIGN cells, with a multiplicity of infection (MOI) of from 0.1 to 10AP61 FFU/cell. At 40 hours post-infection, the percentage (%) of DEN antigen-positive THP cells expressing DC-SIGN was determined by indirect immunofluorescence using specific anti-DEN HMAF (fig. 7). DEN antigen was not detected in THP cells infected with various strains of DEN virus.

The results are summarized in the following table.

DEN virus infection of DC-SIGN expressing THP/DC-SIGN cells

Virus	MOI 0.1	MOI 1	MOI 10
Virus	MOI 0.1	MOI 1	MOI 10	DEN-1	25％	40％	50％
DEN-2	2.5％	6％	14％	DEN-1	25％	40％	50％
DEN-2	2.5％	6％	14％	DEN-3	27.5％	49％	70％
DEN-4	1.5％	2.5％	5％	DEN-3	27.5％	49％	70％

Preservation of

The Hela cell line entitled "Hela DC-SIGN Flap" was deposited at C.N.C.M., 28rue du Docteur Roux, 75724 Paris Cedex 15, France, accession number I-2949, 10/30.2002.

The DC-SIGN clone, entitled "DC-SIGN human clone 2", was deposited at C.N.C.M., 28rue du Docteur Roux, 75724 Paris Cedex 15, France, accession number I-2950, 10.30.2002.

A hybridoma designated "1 B10.2.6" was deposited at c.n.c.m., 28rue du Docteur Roux, 75724 paris Cedex 15, france, accession No. I-2951 on day 11, 7 of 2002.

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Claims

1. Use of a DC-SIGN modulator in an amount sufficient to modulate substantially the binding of an effector molecule to a DC-SIGN receptor in the manufacture of a medicament for the prevention or treatment of a disease in a mammal, wherein at least one symptom of the disease is mediated at least in part by the binding of the effector molecule to the DC-SIGN receptor of the mammal to be treated.

2. Use of a DC-SIGN blocker in an amount sufficient to substantially inhibit the binding of an effector molecule to the DC-SIGN receptor for the preparation of a medicament for the prevention or treatment of a disease in a mammal, wherein at least one symptom of the disease is mediated at least in part by the binding of the effector molecule to the DC-SIGN receptor of the mammal to be treated.

3. The use of claim 2, wherein the DC-SIGN blocker is a blocking derivative of the effector molecule.

4. The use of claim 2, wherein the DC-SIGN blocker is an antibody.

5. The use of claim 4, wherein the antibody specifically binds DC-SIGN.

6. The use of claim 4, wherein the antibody specifically binds to the effector molecule.

7. The use of claim 2, wherein the DC-SIGN blocker is a mannosylated molecule that binds to the DC-SIGN receptor.

8. The use of claim 7, wherein the mannosylated molecule is mannan.

9. Use of a DC-SIGN modulator in an amount sufficient to modulate substantially the binding of a viral effector molecule to a DC-SIGN receptor in the manufacture of a medicament for preventing or treating a viral infection in a mammal, wherein the viral infection is mediated at least in part by the binding of the viral effector molecule to the DC-SIGN receptor of the mammal to be treated.

10. Use of a DC-SIGN blocker in an amount sufficient to substantially inhibit binding of a viral effector molecule to a DC-SIGN receptor in the manufacture of a medicament for preventing or treating a viral infection in a mammal, wherein the viral infection is mediated at least in part by binding of the viral effector molecule to the DC-SIGN receptor of the mammal to be treated.

11. The use of claim 10, wherein the viral effector molecule is a molecular component of the viral envelope.

12. The use of claim 11, wherein the molecular component of the viral envelope is an envelope glycoprotein.

13. The use of claim 10, wherein the DC-SIGN blocker comprises a binding moiety of the viral effector molecule.

14. The use of claim 12, wherein the DC-SIGN blocker comprises a binding moiety of the envelope glycoprotein.

15. The use of claim 10, wherein the DC-SIGN blocker is an antibody.

16. The use of claim 15, wherein the antibody is a monoclonal antibody.

17. The use of claim 16, wherein the mammal is a human and the monoclonal antibody is humanized.

18. The use of claim 15, wherein the antibody specifically binds DC-SIGN.

19. The use of claim 15, wherein the antibody specifically binds to the viral effector molecule.

20. The use of claim 19, wherein the antibody specifically binds to a binding moiety of the viral effector molecule.

21. The use of claim 10, wherein the DC-SIGN blocker is a mannosylated molecule that binds to the DC-SIGN receptor.

22. The use of claim 21, wherein the mannosylated molecule is mannan.

23. Use according to claim 10, wherein the viral infection is a Flaviviridae (Flaviviridae) viral infection and the viral effector molecule is a Flaviviridae effector molecule.

24. The use of claim 23, wherein the mammal is a human.

25. The use of claim 23, wherein the flaviviridae infection is a dengue virus infection and the flaviviridae effector molecule is a dengue effector molecule.

26. The use of claim 25, wherein the dengue virus effector molecule is a molecular component of a dengue virus envelope.

27. The use of claim 26, wherein the molecular component of the dengue virus envelope is a dengue virus envelope glycoprotein.

28. The use of claim 27, wherein said dengue virus envelope glycoprotein is dengue virus E glycoprotein.

29. The use of claim 25, wherein the DC-SIGN blocker comprises a binding moiety for a dengue virus effector molecule.

30. The use of claim 28, wherein the DC-SIGN blocker comprises a binding moiety of dengue virus E glycoprotein.

31. The use of claim 30, wherein the DC-SIGN blocker is a recombinantly produced protein.

32. The use of claim 25 wherein the DC-SIGN blocker is an antibody.

33. The use of claim 32, wherein the antibody is a monoclonal antibody.

34. The use of claim 33, wherein the monoclonal antibody is humanized.

35. The use of claim 32, wherein the antibody specifically binds DC-SIGN.

36. The use of claim 32, wherein the antibody specifically binds to the dengue virus effector molecule.

37. The use of claim 36, wherein the dengue virus effector molecule is dengue virus E glycoprotein.

38. Use of a DC-SIGN modulator in an amount sufficient to modulate the binding of HIV or SIV to a DC-SIGN receptor on a dendritic cell of a human or simian in the preparation of a medicament for the prevention or treatment of HIV or SIV infection of said human or simian.

39. Use of a DC-SIGN blocker in an amount sufficient to substantially inhibit binding of HIV or SIV to a DC-SIGN receptor on a dendritic cell of a human or simian in the preparation of a medicament for preventing or treating infection by HIV or SIV in said human or simian.

40. The use of claim 39, wherein the DC-SIGN blocker comprises a binding moiety of dengue virus E glycoprotein.

41. The use of claim 39, wherein HIV infection in a human is prevented or treated.

42. Use of a DC-SIGN modulator in an amount sufficient to modulate the binding of ICAM-3 on T cells of a mammal to a DC-SIGN receptor on dendritic cells of the mammal sufficient to prevent or treat inflammation in the mammal caused by the specific binding of ICAM-3 on T cells of the mammal to a DC-SIGN receptor on dendritic cells of the mammal.

43. Use of a DC-SIGN blocker in an amount sufficient to substantially inhibit binding of ICAM-3 on T cells of a mammal to DC-SIGN receptors on dendritic cells of the mammal in the preparation of a medicament for preventing or treating inflammation in the mammal caused by specific binding of ICAM-3 on T cells of the mammal to DC-SIGN receptors on dendritic cells of the mammal.

44. The use of claim 43, wherein the DC-SIGN blocker comprises a binding moiety of dengue virus E glycoprotein.

45. The use of claim 43, wherein said mammal is a human.

46. A pharmaceutical composition comprising:

a) a DC-SIGN blocker, and

b) at least one pharmaceutically acceptable excipient;

47. The pharmaceutical composition of claim 46, wherein the DC-SIGN blocker is a derivative of a viral effector molecule.

48. The pharmaceutical composition of claim 46, wherein the DC-SIGN blocker comprises a binding moiety for a dengue virus effector molecule.

49. The pharmaceutical composition of claim 48, wherein said dengue virus effector molecule is dengue virus E glycoprotein.

50. The pharmaceutical composition of claim 46, wherein the DC-SIGN blocker is an antibody.

51. The pharmaceutical composition of claim 50, wherein the antibody is a monoclonal antibody.

52. The pharmaceutical composition of claim 51, wherein said monoclonal antibody is humanized.

53. The pharmaceutical composition of claim 50, wherein the antibody specifically binds DC-SIGN.

54. The pharmaceutical composition of claim 50, wherein the antibody specifically binds to a viral effector molecule.

55. The pharmaceutical composition of claim 54, wherein said antibody specifically binds to a binding moiety of said viral effector molecule.

56. A method for identifying a DC-SIGN modulator, wherein the method comprises:

a) determining a baseline binding value by:

i. providing cultured cells comprising a DC-SIGN receptor;

b) determining the test agent binding value by:

i. providing cultured cells comprising a DC-SIGN receptor;

wherein a test agent binding modulation value representing about 95% modulation of binding of said viral effector molecule to dendritic cells by said test agent indicates that said test agent is an agent that sufficiently modulates binding of viral effector molecules to DC-SIGN receptors.

57. A method for identifying a DC-SIGN blocker, wherein the method comprises:

a) determining a baseline binding value by:

i. providing cultured cells comprising a DC-SIGN receptor;

b) determining the test agent binding value by:

i. providing cultured cells comprising a DC-SIGN receptor;

c) determining a test agent binding inhibition value for the test agent by dividing the test agent binding value by the baseline binding value, wherein a test agent binding inhibition value representing an inhibition of approximately 95% of binding of the test agent to the viral effector molecule and dendritic cells indicates that the test agent is an agent that sufficiently inhibits binding of the viral effector molecule to the DC-SIGN receptor.

58. The method of claim 57, wherein the cultured cells are DCs.

59. The method of claim 57, wherein said cultured cells are THP-1 cells.

60. The method of claim 57, wherein said viral effector molecule is a dengue viral effector molecule.

61. The method of claim 60, wherein the dengue virus effector molecule is dengue virus E glycoprotein.

62. An isolated DC-SIGN blocker identified by the method of claim 57.

63. A method of targeting a subject molecule to cells expressing a DC-SIGN receptor by exposing the cells to a targeting complex, wherein the targeting complex comprises the subject molecule and a DC-SIGN blocker, wherein the exposure is performed under conditions such that the DC-SIGN blocker binds DC-SIGN on cells expressing the DC-SIGN receptor, thereby targeting the subject molecule to cells expressing the DC-SIGN receptor.

64. The method of claim 63, wherein the DC-SIGN blocker is an antibody.

65. The method of claim 64, wherein said antibody is a monoclonal antibody.

66. The method of claim 63, wherein the subject molecule is a protein.

67. The method of claim 63, wherein the subject molecule is an antibody.

68. The method of claim 63, wherein the subject molecule is labeled.

69. The method of claim 63, wherein said exposing occurs in vivo.

70. The method of claim 63, wherein said exposing occurs in vitro.

71. A pharmaceutical composition comprising:

a) a DC-SIGN modulator, and

b) at least one pharmaceutically acceptable excipient;